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Excitatory signal transduction mediated by inositol phospholipid metabolism in lobster (Panulirus argus) olfactory receptor neurons

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Title:
Excitatory signal transduction mediated by inositol phospholipid metabolism in lobster (Panulirus argus) olfactory receptor neurons
Creator:
Fadool, Debra Ann, 1962-
Publication Date:
Language:
English
Physical Description:
xii, 291 leaves : ill., photos ; 29 cm.

Subjects

Subjects / Keywords:
Antibodies ( jstor )
Cell membranes ( jstor )
Cultured cells ( jstor )
Inositols ( jstor )
Lobster culture ( jstor )
Lobsters ( jstor )
Neurons ( jstor )
Odors ( jstor )
Pain ( jstor )
Receptors ( jstor )
Dissertations, Academic -- Zoology -- UF
Zoology thesis Ph.D
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1993.
Bibliography:
Includes bibliographical references (leaves 263-288).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Debra Ann Fadool.

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University of Florida
Holding Location:
University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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028262524 ( ALEPH )
31234226 ( OCLC )
AKC7432 ( NOTIS )

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EXCITATORY SIGNAL TRANSDUCTION MEDIATED BY INOSITOL
PHOSPHOLIPID METABOLISM IN LOBSTER (PANULIRUS ARGUS)
OLFACTORY RECEPTOR NEURONS














By


DEBRA


ANN


FADOOL


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

































Copyright


1993


Debra


Ann


Fadool



































TO MY THREE


MEN













ACKNOWLEDGMENTS


deeply


thank


my husband,


Jim,


for


rearranging


his


schedule


seems


on a weekly


basis


to accommodate


research


demands


thank


him


sharing


excitement


and


frustration,


again


and


again,


and


especially


morning.


thank


him


listening


to all


ideas,


tutoring


me when


was


cluel


ess


, and


being


tremendous


father


even


when


time


was


tight.


thank


both


our


parents


extra


comforts


most


graduate


students


forego


. beds,


furniture,


clothes


, microwaves,


washing


cards.


machines


thank


, and


Nanny


those


Edith


little


and


inserts


Nanny


inside


Eula,


birthday


especially


thank


"Situ"


above


relieving


worries


and


caring


for


my family


when


was


working,


studying


attending


scientific


mee


tings


abroad.


thank


son,


Calvin,


never


complaining


about


crowded


living


quarters


or the


economic


condition


was


placed


in while


both

thank


myself


and


him


father


pestering


met

the


our e

heck


educational


goals.


me while


crunched


numbers


or composed


on the


computer:


"This


more


important


THAN


EVEN


DATA,


MAMA!"


thank


my baby,


Andy


, for


adding


entropy


to the


final


stretch


completion







thankful


to Uncle


Paul,


Tere


and


Peter


Lin,


Steve


Munger,


and


Carol


Diebel


carpooling


Jim


on Thursday


so I


could


spend


time


with


my boys


instead


hours


on the


road.


greatly


thank


my mentor,


Ache,


never


ending


tenacity


attempts


to develop


at challenging


my literacy.


me to


think


thank


critically


him


about


significance


data


statistical


, my


writing,


my presentations,


and


areas


that


comprise


expertise


of a research


scientist.


thank


him


encouraging


me always


appreciate


ones


to seek


he has


opportunities


directed


and


my way,


genuinely


including


wonderful


electrophysiological


room.


thank


friends


Whitney


and


Marine


Biological


Laboratories,


being


just


that.


You


know


who


you


are.


Especially


one


in Utah,


who


reassured


that


electronics


and


computers


were


easy,


who


was


a major


source


learning,


guidance


, jokes,


enthusiasm


during


my training.


Thanks


also


Gainesville


commuter


that


me have


couch


and


kitchen


table


a week


complete


my comprehensive


exams


in complete


hibernation.


don't


know


what


would


have


done


probably


slept)


hadn't


been


photocopy-night-before-partners.


thank


always


rese


archers


and


students


who


came


MBL


summer


1991


and


gave


their


time


excellence,


intellect,


enthusiasm


training








MacDonald,


Shirley


Metts


, Lynn


Milstead,


and


Jim


Netherton


always


helping


me finish


different


aspects


a project


under


time


constraints.


Sincere


thanks


to Leslie


VanEckeris


for


those


"Can


you


me a favor


Special


thanks


to Dr. M.


Greenberg


always


listening


and


caring,


and


carrying


a handkerchief.


Lastly


would


like


to thank


Wheatly


, my


Gainesville


connection,


who


allowed


me to assist


her


class


when


no one


else


was


willing


to take


risk.


thank


her


letting


me do


what


enjoy


most,


teaching.


Towards


that


end


thank


Dr. Anderson


letting


me lecture


his


department


Neuroscience


and


thank


Kelly


Jenkins


being


first


guinea


pig.


















TABLE OF CONTENTS

Page


ACKNOWLEDGMENTS ..... . iv


ABSTRACT. a . .


CHAPTERS


INTRODUCTION ..... ..


Amino Acid Receptors and
Aquatic Chemoreception............
Molecular Mechanisms of Signal
Transduction .. .. .......
Single-Channel Recording..............
Specific Aims .. ...... .. .. .........


SUSTAINED PRIMARY CELL CULTURE.........


Introduction.............
Materials and Methods....
Animals.............
Tissue Preparation..
Cell Cultures.......
Experimental Culture
Electrophysiology...
Solutions...........
Results........ ......


*.. .a .a a *. .. a. a
*. .. *. ..
*. .. S. *. ..*
. ..*. .S.* S


Condition


. a
*. .S
. S


Optimal Culture Conditions.
Morphology of Cell Types
and Neurite Outgrowth.
Electrophysiology........
Discussion .. ..


. .S S .
. *
*.. .*
. .S S .


*. S .
. S
. ..S


ODORANT SENSITIVITY IS NOT DEPENDENT
ON PROCESS FORMATION................... 64


Introduction...........
Materials and Methods..
Tissue Culture....
Electrophysiology.


. ~~ ~ .. S
*. ..S. S S
. .S a a. S. S S. .S
. .a.a a a.. a. a. a.. .








Results .. .. 70
Morphology: Neurite Outgrowth.... 70
Physiology: Electrical Properties 73
Physiology: Response to Odors.... 76
Discussion............ .... ..... 97

GTP-BINDING PROTEINS MEDIATE
ODOR-EVOKED CURRENTS................... 104


Introduction..........
Methods... .. ........
Animals..........
Tissue Culture...
Electrophysiology
Biochemistry.....
Solutions........
Results... .
Discussion............


. ............... 104
................. 107
... .............. 107
.. .108
. .. .. .. 108
a .a. a .a. 111
S. 112
. ................ 114
. ................ 131


IP3-ACTIVATED CHANNELS IN THE
PLASMA MEMBRANE. .. 137


Introduction ....
Results .. ... ....
Macroscopic Currents...
Unitary Currents.......
Immunochemistry and
Related Physiology
Discussion. ...
Experimental Procedures.....
Solutions .. ..........
Tissue Culture.........
Electrophysiology......
Immunochemistry........


. .. .. .. 137
. 139
S. .. .. 139
. .. .. .. 143

. 155
S. .. ... 164
. .. 171
. 171
. 172
.. .. ... 173
S. .. .. .. 175


ION SELECTIVITY AND MODULATION OF
IP3-ACTIVATED CHANNELS................. 178


Introduction..........
Materials and Methods..
Solutions..... ..
Animals .
Tissue Culture....
Electrophysiology.
Results...... ......


. ..... ... 178
. ............... 181
. ............... 181
.. .. .. 184
. ............... 184
S. .. .. 185
. .. 186


Effect of pH on IP3-activated
Channel Gating............... 186
Appearance of Modal Patterns
Differing in Gating Kinetics. 187
C. t -- -. C^ n .. S. ,









Pharmacology of Macroscopic
Odor-evoked Current &


IP3-gated Channels........... 205
Ionic Selectivity of
IP3-gated Channels........... 213


Discussion..... ........................
Gating Properties.................


Ionic Selectivity


& Pharmacology..


IP4-GATED CHANNELS IN NEURONS............ 234

Introduction........ 234
Results and Discussion................. 237


BIOGRAPHI CAL SKETCH. . 289


SUMMARY. .............


REFEREN CES. .. ................ .. .... .... ....














Abstract


Dissertation


The University
Requirements I


of Florida


Presented


to the


in Partial


Degree


Doctor


Graduate


School


Fulfillment


of Philosophy


EXCITATORY S
PHOSPHOLIPID


SIGNAL


TRANSDUCTION


METABOLISM
OLFACTORY


MEDIATED


IN LOBSTER


RECEPTOR


BY INOSITOL


NEURONS


Debra


Ann


Fadool


December


1993


Chairman:


Major


Barry


Department


. Ache
Zoology


Appropriate


primary


sustained


culture


conditions


were


developed


to study


signal


transduction


Panulirus


argus


olfact


ory


rec


eptor


neurons


(ORNs


Neurons


were


cultured


a modifi


ed Liebowitz


media


supplemented


with


salts,


vitamins


, L-glutamine


, low


dext


rose,


and


either


fetal


calf


serum


or lobster


stimuli,


sensitivity,


haemolymph.


degree


and


tuning


dual


nature


adequate


cells,


polarity


eshold


odor-evoke


currents


were


consistent


with


chemosensitivity


cultured


ORNs


being


olfactory.


The


magnitude


odor-evoked


currents


was


significantly


increase


or decr


eas


ed by


nonhydrolyzable


analogs


GTP


and


GDP,


respectively,


and


not


erturbed


5
no rt ii eel~ Cl


=1 r' n tNl~rtl ar~ t% '.r~ ~ Cl ,- Cl Cl r


*
-I Vfln~I ir, v',t-.'


(PANULIRUS


ARGUS)


_^ _- 1


',-l "1, 4 .-


-_l T"l f^







transduction.


An antibody


directed


against


immuno-


labelled


a 40.5 kDa


band


an enriched


membrane


preparation


of ORN


outer


dendrites


and,


along


with


an antibody


directed


against


selectively


decreased


odor-evoked


inward


current


within


10 mmn


initial


perfusion.


Inositol


,4,5-trisphosphate


(IP3)


selectively


evoked


an inward


current


in the


ORNs.


Application


to the


inside


face


cell-free


patches


of ORN


plasma


membrane


directly


gated


two


ion


channels


that


differed


conductance


, voltage


dependence,


and


dwell-time


kineti


cs.


An antibody

IP3 receptor


directed ac

recognized


;ainst


an intracellular,


a protein


similar


cerebellar


molecular


weight


mammalian


receptor


ORNs


and


was


found


increase


selectively


odor-evoked


inward


currents


and


-activated


unitary


Modulation


currents


channel


in the


gating


lobster


or ion


ORNs.


permeation


was


observed


both


IP3-gated


channels


in response


to elevated


[Ca]i.


Both


macroscopic


channels


odor-evoked


mimicked


inward


pharmacology


currents.


substitution


suggested


that


small-conductance


channels


were


nonselective


cations


and


that


large-conductance


channels


were


either


nonselective


between


and


Ca2"


were


selective


Ca2+


direct


metabolite


, inositol


I I I


tetrakisphosphate


IP4)


, gated


an ion


channel


that


differed







from


those


activated


IP3.


IP4-gated


channel


mutually


interacted


with


IP3-gated


channels


to alter


open


probability


channels.














CHAPTER


INTRODUCTION


Amino


Acid


Receptors


Acuatic


Chemorecet ion


The p

excitatory


ast de

amino


cade


acid


has shown

s (EAAs)


an explosive


important


interest


neurotransmitters


mammalian


central


nervous


system


(CNS)


Research


efforts


have


elucidated


synaptic


role


of EAAs,


EAA


subclasses


receptors


through


pharmacological


techniques,


mode


signal


transduction


, and


extent


to which


EAAs


are


involved


in brain


function.


To date,


EAAs


are


implicated


in a wide


range


of physiological


phenomena


including

processes,


processing


learning,


and


sensory


memory.


information,


Dysfunction


cognitive

receptor-


ligand


intera


action


subsequent


transduction


can


lead


neurological


disorders


such


as epilepsy,


spasti


city,


neurodegeneration


after


a stroke,


as well


as Huntington'


and


Alzheimer'


disease


(Croucher


et al. ,


1984;


Watkins


al. ,


1990


development


selective


agoni


and


antagonists


acids


distinct


in synaptic


EAAs


transmission


receptors


throughout


implicated


various


amino


regions


CNS/brain


and


offered


pharmacological


intervention


in treating


many


these


human


neuronal


diseases


(Lodge


and









Amino


CNS.


acid


Feeding


receptors


stimulants


are

for


not

many


limited

aquatic


mammalian


organisms,


both


sh and


invertebrates,


are


simple


hydrophilic


tissue


metabolites,


mainly


quaternary


ammonium


compounds,


nucleotides,


1990


amin


Chemical


acids


muscle


es, and

analysis


tissue


amino

found

the b


acids


Ache


19 of


lue


and


common


crab,


a typical


Carr,


amino

prey


organism


aquatic


lobster


organisms


(Carr


typically


et al.,


ssess


1984)


well


Consequently,


defined


chemosensory


structures


containing


primary


chemosensory


receptor


neurons


that


are


responsive


to micromolar


comolar


amount s


amino


acids


and


related


molecules.


Unlike


mammalian


brain


, where


amino


acid


receptors


are


buried

amino


and

acid


widely di

receptors


stributed


in aquatic


tissue


organisms


these


are


"external"

readily


accessible


and


often


highly


enriched


chemosensory


structures.


Large


decapod


crustaceans


such


as crabs


and


lobsters


have


such


a chemosensory


structure


on the


lateral


branch


first


antenna,


commonly


referred


as the


"olfactory"


organ


or antennule.


It consists


of a tuft


cuticular


sensilla.


Each


sensillum


contains


an average


bipolar


receptor


neurons


(Grunert


and


Ache


, 1988),


elding


upwards


receptor


neurons


per


antennule


Figure


1-1)












O
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(U '- 030
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Ci5 cQ'q' 0
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S


CSN
c /










Odorant s


reach


dendrites


through


porous


cuticle


surrounding


sensilla.


somata


rec


eptor


neurons


are


clustered


base


of each


sensillum


and


axons


from


each


cluster


project


, without


synapsing


to the


CNS.


Olfactory


object


receptor


of considerable


neurons


(ORN


research


lobsters


(review,


Ache


have


and


been


Derby,.


1985)


The


ORNs


can


be studied


situ


(Ache


et al. ,


1989)


or harvested


to produce


a preparation


almost


pure


chemorec


eptive


neurons


tissue


culture


(Chapter


biochemi


stry


Chapter


4-5) ,


or molecular


biology


Trapido-


Rosenthal


et al.,


1993


Munger


and


Ache,


press


ckground


relevance


to the


present


project


that


amino


acids


presented


mixtures


*e.


co-


eased)


typically


are


less


stimulatory


to the


neurons


, as measured


intensity


ectrophysiological


response


neuron,


than


when


presented


individually


and


responses


summed


This


phenomenon,


called


mixture


suppression,


potentially


result


seven


underlying


physiological


mechanisms


Ache


et al.,


1988)


Recently


was


shown


that


amino


acid


odorantss"


could


inhibit


as well


as excite


lobster


olfactory


neurons


and,


more


importantly,


that


these


two


processes


could


occur


same


neuron


Clintock


and


Ache,


1989b;


Michel


et al


., 1991;


Figure


1-2) .


In other



























Figure
excite


1-2.
the


Amino
lobster


acid


odorantss"


can


inhibit


olfactory receptor neuron


(CORN)


well


Shown


a single


ORN


configuration


that


(top


was


left


first
diagram)


recorded


in the


to monitor act


cell-attached
ion potential


increase


and decrease


in frequency under


stimulation by


excitatory odor mixture


(top


trace,


upper recording)


and an


inhibitor
The cell


odor


membrane


(top
Swas


trace,


lower recording)


then ruptured


to achieve


, respectively.


whole-


cell


recording


configuration


(top


right


diagram)


in which


the ORN
trace,
trace,


responded


with a membrane


upper recording)
lower recording)


and
unde


depolarization


then hyperpolarizatioi


r


same


stimulation


(bottom
n (bottom
i protocol.


This


singular experiment


transduction


pathway


demonstrated


that


amino acids was


more


present


than one
in these


neurons.


Taken


with


permission


from Michel


al.,


1991.































Mixture


-60 mV


Proline
5 sec


crn rI\/


- .A -









1991;


Michel


suggested


and


that


Ache,


more


in press).


than


one


This


transduction


finding


strongly


pathway


amino


acids


is present


in these


neurons.


What


these


pathways


were


remained


unknown.


Molecular


Mechanisms


of Signal


Transduction


Perhaps


classically


most


studied


and


hence


best


understood


transduction


pathway


in neurons


ligand


binds


to a receptor


prot


ein,


which


induces


activation


coupling,


an adenylate


then


cyclase


cyclic


enzyme


adenosine


through


G-protein


monophosphate


(cAMP)


end


product


acts


directly


or indirectly


on an ion


channel,


to induce


a generator


current.


When


commenced


doctoral


study,


olfactory


transduction,


defined


as the


mechanism


that


converts


a chemical


signal


(odor


into


electric


signal


(current)


was


well


perceived.


was


known


that


a preparation


enriched


olfactory


cilia


contained


an odor-activat


ed adenylate


cyclase


(Pace


al.,


1985)


suggesting


resemblance


that


that


olfactory


typical


man


transduction

y neurons.


had

Not


some

all


odorants


had


capacity


to evoke


significant


elevations


adenylate


cyclase,


suggesting


cAMP-mediated


transduction


was


not


sole mechanism.


Nonetheless,


a conductance


directly


gated


cyclic


nucleotides


(cAMP


cGMP)


was


local


zed


olfactory


cilia


(Nakamura


and


Gold,


1987


whi


soon









Soon


after


an olfactory


specific


G-protein,


was


cloned


and


localized


ciliary


layer


of rat


ORNs


(Jones


and


Reed


, 1990)


fact


that


cholera


toxin


ribosylated


prote


outer


dendritic


segments


lobster


ORNs,


implicat


a GTP


-dependent


protein


in at


least


one


tran


al. ,


sduction


1990


pro


. Yet


cesses


lobster,


in these


complex


neurons


odor


(McClintock


mixtures


identify

of cAMP


stimulatory


McClintock


odor


et al.,


molecules


1989


failed


Hence,


to alter

lobster


levels

ORNs,


data


did


support


CAMP


candidate


G-protein


linked


second


messenger.


Several


other


molecular


mechanisms


had


been


implicated


in signal


transduction


other


systems


, providing


me useful


avenues


investigation


commencement


my study.


They


included


following:


Amino


acids


could


directly


gate


channels


where


channel


was


contained


within


receptor


prot


ein


or where


channel


receptor


were


inherently


two


different


proteins


coupled


directly


or through


a G-protein


Codina


al. ,


1987;


Vogt


et al.


1990


Amino


acids


could


activate


a guanylate


cyclase


increase


cGMP


levels


to dire


ctly


activate


a channel


indirectly


activate


a channel


phosphorylation


via


protein


kinase


(PKC)


(Vogt


et al.,


1990


Golf,









stimulation


of cell-surface


amino


acid


receptors


hydrolyzes


a membrane


bound


inositol


phospholipid,


which


produces


two


second


messengers--neutral


membrane


bound


diacylglyceral


(DAG)


and


water


soluble


inos


itol


,4,5-triphosphate


(IP3)


DAG


remains


in the


plane


membrane


Nishizuka,


1984,


1988)


to activate


which


turn


could


activate


channel


diffuses


into


cytosol


releasing


Ca2+


from


internal


stores


, causing


Ca2


wave


oscillations,


and


activating


Ca2


channels


(Berridge


and


Irvine,


1989)


could


additionally


activate


a channel


directly


(Kuno


and


Gardener,


1987)


Inositol


,3,4,5-tetrakisphosphate


IP4)


direct


metabolite


IP3,


may


act


as an additional


messenger


Irvine,


1990


difficulty


in studying


olfactory


transduction


Panulirus


argus


resided


in the


fact


that


transduction


machinery


was


hypothesized


to lie


in the


outer


dendritic


membrane


neuron,


as was


true


analogous


structure


in vertebrates


(above


section;


Nakamura


and


Gold,


1987;


Kurahashi,


1989


Firestein


et al.,


1990


. The


thin


diameter


seals


this


virtually


process


impossible.


made


Recording


direct


from


electrode


neuron


more


accessible


soma


depended


upon


recording


transduction


current


from


as far


as 1000


pm away


and


often


produced


an inadequate


voltage-clamp.


hypothe


sized


that









properties,


then


perhaps


cells


would


assume


a form


that


was


more


amendable


voltage-clamping


and


studying


transductio

electrical


If lobster


properties


ORNs

their


vitro


mimicked


counterparts


in situ,


then


cultured


cells


would


an excellent


model


studying


elements


of olfactory


signal


transduction,


particularly


a cell


that


was


suspected


of having


multiple


mechanisms.


Single-Channel


Recording


should


remember


that


our


living


systems


are


essentially


watery


saline


systems


that,


through


a variety


electrodes,

unknown).


are

This


coupled

section


to physical

provides an


instruments"


Elementary


Author


background


electrophysiological


techniques


as a preface


experimental


results


of Chapters


2-7.


Electrophysiology


a powerful


tool


to probe


molecular


events


occurring


specialized

standard ap


structures


proach


along


is to detect


excitable


and


membranes.


measure


bioelectrical


potentials--yet


majority


of bioelectrical


potentials


are


so small


that


a sensitive


apparatus


required


to detect


them.


For


crustacean


olfactory


receptor


neurons


Panulirus


argus)


instruments


which


can


detect


a few


to 100


device


are


that


required.


couples


Electrode


biological


is a general


preparation


term


to electrical


instrumentation


recording


from


lobster


ORNs,










to permit


recordings


from


small,


8-15


diameter


cells.


Good


electrical


contact


must


exist


between


electrode


and


cell.


For


an unknown


reason


first


discovered


Ling


and


Gerard


(1949)


glass


electrode


forms


exce


llent


sea


with


lipids


nerve


cell-membrane


that


current


pulse,


once


converted


to voltage


headstage


current-to-voltage


converter,


is received


amplifier.


The


general


function


an amplifier


increase


voltage


of a bioelectrical


signal


so that


can


be displayed


or further


processed


a read-out


device,


such


as a cathode


ray


oscilloscope


(CRO),


a strip


chart


recorder,


or a computer.


On either


side


an excitable


membrane


there


are


varying


concentrations


ionic


spec


ies.


The


primary


species


are


and


This


electrical


and


chemical


gradient,


cell


membrane,


established


is defined


semi-permeable


as the


nature


potential


difference.


Ionic


flow


across


cell membrane


confined


to specialized


protein


pores


called


channels


(Figure


1-3)


Channels


lobster


ORNs


are


"gated"


or opened,


other


neurons,


in response


one


two


applied


stimuli:


ligands


such


as odors


, second


mess


enger


molecules


, or


neurotransmitters


and


voltage


as an electrical


signal


stimuli. T


e movement


r ions


through


these


channels


, K




















Figure
bilayer
channel
Channel
neurons
(1) vol
molecul
regions
adjacent
just io
neurons
channel
gated c
channel
channel
channel
structu
voltage


the
reg
ext
sel
a g
fas
hyp
exya


charge 1
voltage
electric
potent


(TOP)


es, a
of c
t cel
nic s
but
s are
hanne
s of
s (Ch
s by
rally
-gate
nnel
throu
1 med
vity
ion
-off
tical
, to
migra
sens
cal c
al.


1U
fi


Ionic


ned
an
ter
ons
2)
eur
ap
I '


, wn
cies
defi
ompr
of
homo
ter
fini
hese
chan
otei
whi
m to
iter


over
bindi
, whi
confo
tion,
or wh


.1.
i

n
i


flow


across


to specializ
he phospholi
ORNs are "ga
to one of t


as
o


ds
smi
ion
low
als
ye
f 4


uch
ters
betw
pass
hyp
in
homo


terogene
ous doma
all into
of their
nnels ar
S(BOTT
monstrat
he ions
internal
ch conta
of other
actions,
n be ope
onal cha
nd bindi
s suscep
a result


r

i


charges


M


as o
. G
een
age
othe
lobs
gene
s do
s.
he f
gati
more
;) A


s i
ond
cy
ns
ba


a g
ned
nges
ng,
tibl
of


cell


ei
ay
o
ie
s,
ju


the
of
siz
ter
ous
mai
Cur
ami
ng
cl
two
ma
t ci
pla
reg:
d o


e to mov
a change


n
e&"


po


pene
d st
sec
ncti
ell
rger
in
RNs.
omai


, an
usly
of
ture
ely
imen
fea
rent
or'
n th
mole
cute


ed
db
emp


phospholipid


res
(MI
d, a
imul
ond


on
mem
mo
olf
V
ns,
d g
IP
lig
, a
rel
sio
tur
fr
vic
at
cul
dlv


c
b


calle
DDLE)
s in
i:
messe


other

nger


channel
ranes


lecules t
actory
ioltage-ga
ligand-
ap-juncti
3-gated
and-gated
though
ated to
nal view
es: A po
om the
e-versa,
preference


ar


size


a


response,
ectrical
ure. and


han

ted

on




of
re

a
es
nd

for

a


ement of
in membrane


Modified


with


permission


from


Hille,


1992.


(
(


I


i




i



q
I
q


)
I
:]


(









ion flow

1 /""6 1 /'"1 /^"* /"1 /^-*\ /^^1 /~^- i/~^ / ^1^ /" ^S /"1
N- s'' 1-% d- ^N 'N \ 'N t% i%" .'^ 4'^ Z^ "N #'N^


Voltage-
gated


Ligand-
gated


Gap
junction


:bil


ayer~
'i^^


voltage
sensor


selective----
filter


pore


gate


+I
*- *-I*









voltage-clamp


recording,


a feedback


amplifier


measures


difference


between


a set


potential


(command


voltage)


and


potential


differential


generated


is converted


stimulate


ed cell.


to a current


voltage


given


back


ORN


to maintain


potential


that


was


fixed


on the


amplifier


(hence


term


voltage-CLAMP;


Figure


The


amount


of current


given


back


to the


ORN


used


as a measure


of what


was


received


oelectric


signal


from


cell


some


instances


, a single


electrode


operates


as the


voltage


sensing


as well


as current


ecting


device.


larger


cell


one


can


use


two


separate


electrodes,


one


each


function


Lobster


ORNs


are


big


enough


placement


ectrodes,


one


electrode


flip-flop


between


functions


voltage


-clamp


method


was


first


developed


Cole


(1949)


and


Hodgkin


et al


(1952)


use


with


famous


squid


giant


fact


currents


controlled


axon.


that


measured


voltage,


usefulness


much


from


than


easier


an area


when


clamp


to obtain


of membrane


voltage


stems


information


with


from


about


a uniform,


is changing


freely


with


time


and


between


diff


erent


regions


membrane.


voltage-clamp


was


modifi


ed by


Neher


Sakmann


(1976)


recent


Noble


laureates


in medi


cine,


investigating


basic


properties


channels


In order


to directly




























Figure


membrane
the prim
electric
relative
different
clamp me
of a cha
feedback
set pote
amount g
amount o
is given
fixed on
used a V


potential


1-4.


th
ary
al
ly
ce,
asu
nne
am
nti
ive]:
f v


On either


ere
ion
and
impe
clo
res


are
ic s
chem
rmea
set
the
he b
ier
iffe
the
ae i


to t
ampli


h
f
t


in lobste


re
s
s
e
ie
ro
r


side


-60
ow o
elec
BA)
nce
timu
convy
ORN
r


cie
ing
ien
er
in
har
c s
sur
mma
ed
ed
mai
mos


a biological


s


approximate
ORNs.


of va
+*
Na, KC
estab
etermi
obster
e thro
gnal -
s the
d volt
ell as
oa cu
tain t
exper
Sthe


trying


, Ca
lish
nes
ORN
ugh
cur
diff
age;
vol
rren
he p


2+
ed


excitable
concentration


and
by t


he pot
The


t
t
0


he
ent
ren
Vc)
age
in
ten


iments,
resting


pro


ential
voltag
tein po
The
between
i the
This gi
ment wh
1 that
ommonly


e-
re


ven
ich
was


membrane





17






Voltage-Clamp

---B

Vc -60 mV rest




N -L a
SNaNa
NaK

""-- U K--









restricting


size


recorded


membrane


to a small


patch.


They


then


electrically


isolated


membrane


patch


from


rest


cell


sealing


glass


micropipette


name


tigh


patch-clamp


tly onto

recording


the

was


membrane

derived


This


Figure


how


1-5)


was


only


accident


that


they


discovered


slightly


negative


pressure


or suction


created


a molecular


contact


between


pipette


and


plasma


membrane,


which


improved


seal


resistance


into


gigaohm


range


(Neher


et al .,


1978)


Horn


and


Patlak


(1980)


Hamill


and


Sakmann


(1981)


and


Hamill

removed


achieve

their


from


(1981

cells


excised


natural


discovered


retracting


patches.


environment,


Here


that

the


patches


glass


patches


either


with


could


pipette


removed

internal


from

(inside-


configuration)


or external


side


(outside-out


configuration)


membrane


facing


outside


bath,


allowing


optimal


control


solution


changes


from


either


face


membrane


cell-attached


recording


Although


retaining


configuration


cell


permits


measurements


a process


with


least


disturbance


intact


cell,


sometimes


more


experimental


control


is required.


Alternatively,


strong


suction


can


be applied


while


pipette


patch


still


and


attached


create


cell


a whole-cell


membrane


configuration,


to rupture


whereby









can


be controlled,


and


cell


left


otherwise


intact


Figure


1-5)


have


used


each


these


pipette


configurations


studies

attached


towards


a different


configuration,


gain:


a bath


applied


While

agonist


that


cell-

evokes


channel


activity


generally


infers


a cellular


mechanism


requiring


a second


messenger


molecule.


Membrane


impermeant


probes


can


be readily


applied


cytoplasmic


face


membrane


in the


inside


-out


configuration.


While


in the


outside-out


configuration,


a ligand


applied


to the


bath


that


evokes


channel


activity


strongly


suggests


a directly


gated


channel


mechanism.


also


took


advantage


of a modification


inside-out


configuration


Chapter


called


"patch


cramming"


as described


Kramer


1990


where


one


takes


an inside-out


configured


patch


containing


a channel


erest


and


inserts


patch


pipette


into


a second,


recipient


cell.


channel


activity


patch


while


inside


recipient


cell


can


then


used


as a probe


to detect


changes


in intracellular


second


messenger

baseline


change

clamp


production


odor


from


single


in a living


responsivity


unstimulated


ORNs


sequentially


cell


a neuron,


states,


In order


and


chose


or to rely


acquire


to monitor


to either

on the di


voltage-


ffusional


properties


solutions


over


time


backfilling


electrodes




















Figure


1-5.


TOP)


Diagram


a neuron


an on-ce


cell-att
measuremr
of the i
pipette
whole-ce
cell int
control
somewhat
macrosco
ionic fl
In lobst
efflux o
evoked i
excitato
from neu
excised
environm
configur
of the m
control
I common
order to
inositol
channel
of the p
event (0
unitary
ion pass


upward
as an


d
ou


ched
nts
tact
s st
1 co
rior
d by
inta
S


c
of
c
il


configuration. This
I .... i


a n
ell.
1 at


nfigur
and t.
the i
ct. T


pic cur
ux from
er ORNs
f catio
nward c
ry odor
rons by
patches
.ent, ei
ation)
embrane
of the
ly used
apply
1,4,5-
By co
atch pi


) and i


rent


th
a


the
or
fa
sol
th
the
tri
nve
pet
s d


current 1
ing in th
election
toward one


4-


n


e
n
(
e3
s]
t:
P<
r
e:
c;
ul
e
s
S]
ni
t
i
VE
i(
al


acura
Stron
tached
nation,
he memb
nrivestig
he meas
, compr
channel
odor-e
an inhi
nt is d
ponse.
racing
catches
with t
external
ing the
tions b
inside
membran
phospha


ti
e)
sp
el
op:
n


on,
is
lay
aw
pos
uni


pro
g s
to
whe
ran
ato
ure
ise
is
vok
bit
ue
(BO
th
can
he
si
ou
ath
-ou
e i


te


an
def
ed a
ay f
ite
tary


channel


cess
uctio
the c


re
e
r,
d
d
co
ed
or
to
TT
e
b
in
de
ts
in
t
mp
(I
on
ne
a
om
ir
cu


by
pot
an
cur
of
nta
Sou
y o
in
OM)
gla
e r
ter
(o
ide
g e
con
erm
P0


d


e
r


configuration p
with the least d
n can be applied
ell membrane to
the ionic milieu
ential can now b
d the cell is le
rent is defined
the total summat
ined in the whol
toward current is
dor response. A
flux of cations;
Membranes can
ss pipette to ac
moved from thei
nal (inside-out
utside-out confi
bath, allowing
either face of th
figuration (as s
eant second mess
to the inside f
ssing into the c
s an inward open
wnward deflection
e closed state (
ion is displayed


rent


eve


l and


erm
ist
wh
cre
of


its
urbance
ile the
ate a
the


on o
cel
due
odo
an


f
1.
to
r-


all

an


be removed
hieve
r natural

guration)
optimal
e channel.
hown) in
enger,
ace of a
ell (out
channel
n in
C). An
as an


is described


event


Modified


with


permission


from


Hille,


1992.


.


*


I























Patch


Clamp


on cell


outward


inward


whole cell


on cell


outward


rapid pull


p


inside-out


_-_--_ inward









The

behavior


analysis

1 details


of single


channel


passage


events


of a sing


describes

le ion SD


ecies


across


phospholipid


bilayer


a neuron.


An excellent


source


recording


and


analysis


currents


from


single


ion


channels


is Wonderlin


et al.


1990


Two


excellent


reviews


noble


laureates


that


first


recorded


unitary


channel


currents,


behavior,


provides


information


as discussed


on the


next


theory


three


pages


dissertation


, and


can


found


and


Cooper


(1990


Neher


(1992b),


and


Sakmann


(1992


When


quantifying


behavior


of a single


molecule,


channel


protein,


one


generally


calculates


magnitude,


duration,


and


order


channel


events;


of which


are


random


variables


and


none


which


can


inferred


observing


raw


data.


The


information


contained


channel


events


must


come


from


measurement


their


distributions;


statistics.


The


current


thinking


is that


randomness


thermal


motions


underlies


dwell


time


of a channel.


is postulated


that


bonds


channel


protein


are


vibrating,


bending,


and


stretching


on a picosecond


time


scale


to achieve


an open


or closed


conformation


once


an energy


barrier


is surmounted.


The


probability


surmounting


such


energy


barriers


an open


state


(Pr open


been


found


to be dependent


upon


one


more


natural


stimuli


channel


question:


membrane


J.









intracellular


ion


concentration;


or state


modulation,


phosphorylation.


first


typically

unitary c


random


calculated


current


variable,


based


amplitude:


event


magnitude


on one


a point


-by-point


, is


tributions

amplitude


histogram


or an event


amplitude


histogram.


In the


former,


sampled


channel


events


are


binned


into


assigned


current


levels,


chann


regardless


In the


state


latter,


(open


a histogram


or closed)


is constructed


containing


only


amplitudes


events


greater


than


times


rise


time


rise)


duration


event.


event


amplitude


within


ending


then


a window


trise


measured


beginning


before


averaging


rise


end


after


event.


current


opening


This


level


and


window


corresponds


to the


"flat"


region


event


and


excludes


regions


that


may


distorted


finite


rise


recording


instruments


In both


types


of distributions


mean


and


variance


current


magnitude


can


be determined


fitting


histogram


with


a Gau


ssian


curve.


An X-Y


plot


mean


current


as a function


of membrane


potential


(voltage)


can


be used


to generate


slope


conductance


channel,


a measure


degree


permeation.


greater


conductance


of a channel,


faster


ions


are


flowing


through


pore


channel.


Generally,


- 1









interaction


with


charged


amino


acids


inside


channel


pore


or between


each


other.


chose


use


point-by-point


amplitude


histogram


my analysis


because


number


channels


a patch


can


detected


with


this


distribution,


Propen


can


be easily


calculated,


and


presence


voltage-dependence


can


determined.


In a point-by-point


amplitude


histogram,


the


number


of peaks


minus


one


is generally


number


channels.


peaks


correspond


an integer


function,


then


one


likely


to be


recording


multiple


openings


identical


channels.


peaks


correspond


variable


amplitudes,


then


one


is likely


to be recording


heterogeneous


multiple


channels.


probability


opening


Propen,


defined


total


time


a channel


spends


open


state


divided


length


recording


amplitude


integration


histogram


area


is used


under


as an index


peaks


time.


presence


voltage-dependent


channel


opening


can


determined


plotting


Propen


as a


function


membrane


voltage.


Deviation


from


a zero


slope


would


indicate


voltage-dependent


channel


gating.


second


random


variable


duration,


exponential


distribution


of random


dwell


times.


One


measures


average


time


(dwell)


channel


spends


open


and








out


that


state.


mean dwell


times


are


thus


defined as


and


1/i where C


closed)


(open)


movement


between


0 and


C states


requires


differential


to describe


behavior,


called


probability distribution


function,


which defines


intervals


between


random channel


events


The


derivative,


f(t)


, of


this


function


s called a


or a


probability density function,


which


function


used


measured open


or closed dwell


times


of a channel.


Dwell


times


are


reported


form of


a histogram,


where


each


open or


closed


event


binned into a


dwell


time of


certain duration,


and


histogram provides


the exponential


mean dwell


time,


(f(t)


usually reported


msec.


number of


exponential


components


required


to fit


distribution


number


open or


closed states


channel.


Most

multiple


membrane

states, w


patches


whichh albeit


contain multiple


complicates


channels


and


analysis


channel


behavior,


provides


information about


third random variable,


order


channel


events.


Channel


bursting,


latency to


first


opening,


change


in mode


kinetic


state,


hibernation,


subconductance,


cooperativity,


and steady-state


flickering are


examples


complex


channel


behavior


that


can provide


rich knowledge


about


biolocrv


of a channel.


t------









Specific


Aims


addressed


doctoral


five


study.


maj or


questions


resolution


during

each a


course


question


helped


shape


subsequent


avenue


investigation


as the


following


sugge


sts,


namely:


Can


lobster


Panulirus


argus


olfactory


receptor


neurons


sustained


Are


primary


cell


voltage-activated


neurons


altered


culture?


odor-activated


cell


properties


culture?


Do GTP-binding


proteins


(G-prot


eins)


link


binding


odorant


receptor


to the


generation


an odor-evoked


current?


Are


odor-evoked


currents


elicited


direct


activation


an odorant


receptor


an appropriate


odor


molecule


or are


currents


evoked


through


a second


messenger


casc


ade?


What


membrane


primary


-gated


charge


channels


carrier


mediating


plasma


excitatory


transduction


(inward


currents


and


can


they


be activated


other


metabolites


in the


inositol


phospholipid


pathway?















SUSTAINED


CHAPTER
PRIMARY


CELL


CULTURE


Introduction


Dissociated


neurons


in primary


culture


are


providing


useful


models


a growing


number


of neurobiological


studies


(Benda


et al.,


1975;


Sebben


et al. ,


1990


Olfactory


receptor


neurons


in many


animals


are


long


, thin


bipolar


neurons


that


terminate


distally


a highly


branched


arbor


of cilia


or in an outer


dendritic


branch


(Steinbrecht,


1969


that


is suspected


to be


site


chemosensory


transduction


(Lowe


and


Gold,


1990


Studying


physiology


transduction


direc


ching


thin


cilia


or outer


dendritic


branch


een


poss


ible


(Nakamura


and


Gold,


1987


, Hatt


and


Zufall


1990


technically


difficult.


In most


cells.


instances,


In amphibians


necessary


, some


to work


olfactory


with


intact


receptor


cells


contract


(Fire


when


stein


dissociated


Werblin,


from


1989)


olfactory


, thereby


epithelium


facilitating


physiological


analysis


transduction


allowing


effective


NOTE: Th
reprinted


and


B.W.


is


chapter


with


Ache. 1


b


missionn
.991. Su


een a
from


stained


accepted
Fadool


for pu
, D.A.,


primary


iblication


W.C


culture


and


. Michel,


lobster









space


clamping


to characterize


odor-activated


currents


and


allowing


drugs


introduced


into


soma


through


patch


electrode


to diffuse


to the


cilia.


In other


animal


however,


dissociated


receptor


neurons


retain


their


diffuse


morphology


and


are


less


amenable


to physiological


analysis


transduction.


transduction


In these


would


instances


be facilitated


, physiological


, by


analysis


placing


cells


culture,


were


possible


to obtain


morphologically


more


compact


cells


that


still


retained


their


responsiveness


to odors.


Lobster


dendrite


olfactory


soma


neurons


distances


have


reported


one

for


longest


olfactory


receptor


cells


in any


organism


ca.


Grunert


and


Ache


, 1988)


In order


to study


physiology


transduction


these


cells,


would


be particularly


useful


cells


could


sustained


primary


culture


in a morphologically


more


compact

been de


form.


signed


Most

for m


tissue


.ammalian


culture

systems


protocols, however,

(especially human,


have

rat,


mouse,

insect


and


rabbit


tissue


are


Although


now


techniques


commercially


available


culturing

, culturing


techniques


other


invertebrate


nervous


tissues


are


less


well


established.


In particular


, crustacean


tissue


culture


just


initial


stages


of development


(Fainzilber


al. ,


1989)


Only


recently


have


techniques


culturing









adipose


tissue


(Van


Beek


et al.,


1987


, proprioceptor


organs


Hartman


et al.


, 1989


ovarian


tissues


(Fainzilber


al.,


1989)


been


reported


using


crustacean


species.


above


protoc


reported


widely


varying


conditions


suggesting


that


a systematic


test


culture


parameters


may


necessary


In order


each


to better


crustacean


study


species.


electrophysiological


properties

I developed


lobster


techniques


olfactory


receptor


to sustain


neurons,


cells


therefore,


primary


culture.


In this


chapter


nine


culture


parameters


are


described


that


were


systematically


tested


to establish


an in


vitro


model


which


most


closely


approximated


osmolarity


and


salt


composition


lobster' s


physiological


fluid;


haemolymph;


approximat


ed known


physical


parameters,


such


temperature;


effects


and


on nerve


which


included


outgrowth.


supplements


In this


chapter


known

are


to have

reported


conditions


that


allow


cells


harvested


from


lobster


olfactory


organs


(antennules)


survive


primary


culture


days.


Cultured


cells


are


more


compact


than


cells


vi Vo,


and


most


importantly,


are


electrically


excitable


and


odor


sensitive


that


they


bear


physiological


markers


of olfactory


neurons.


potential


these


cultured


cells


have


studying


physiological


process


olfactory


transduction


is discussed.









Methods


Animals


Specimens


Caribbean


spiny


lobster,


Panulirus


argus,


were


collected


from


Florida


Keys


and


maintained


in an open


circulating


sea


water


system.


Animals


were


mixed


diet


frozen


fish,


squid,


and


shrimp.


Tissue


Preparation


The o

antennular


factory organ

filament) was


(distal

excised


third

from


intermolt


ateral

animals


Figure


saline


2-1,


(PS,


A-C)


see


and


washed


solutions


Listerine


containing


penic


in Panulirus


illin,


streptomycin


sulfate,


and


amphotericin


as Fungizone


(Gibco;


AbAm)


The


organ


was


into


sections


three


annuli


long,


hemisected,


and


transferred


to fresh


PS + AbAm.


Soma


clusters


olfactory


(aesthetasc


receptor


cells,


which


from


washed


literally


fill


hemisection


repetitively


lumen


with


with


organ,


a sterile


PS + AbAm,


gauge


and


were


syringe


transferred


removed


needle


to fresh


PS + AbAm. Re

contamination,


hpetitive


washes


presumably


from


were


critical


epiphytes


to eliminate


on the


exoskeleton.


isolated


soma


clusters


were


then


incubated


50 min


rpm


10 ml


on an orbital


AbAm


shaker


containing


.2 micron


mg papain


filter-sterilized:


and


cysteine.













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Cell


Cultures


Proteolytic digestion was


enzyme


stopped by replacing the


solution with modified low glucose L-15 media


L-15


Stock,


50 ml


1.6X normal


concentration of


dextrose,


0.029 g L-glutamine,


and


.01%


gentamicin)


Cells


were


dissociated by trituration


using a


sterile


gauge


needle


and plated on poly-D-lysine hydrobromide


300-


53,000)


coated glass


mm coverslips


.5-5


gg/cm2)


cell


density of


12 x 10


cells/ml


(per


cm2 well)


Cells


were


placed


the dark and allowed


to adhere


to the


coverslips


hr without


agitation.


After this period,


fetal


calf


serum


FCS)


or 10%


lobster


haemolymph was


added


to each well.


In experiments


requiring


haemolymph


supplementation,


the blood extraction procedures of


Fadool


et al.,


1988


were


followed.


Haemolymph osmolarity was


measured in


samples


by freezing point


depression


(Osmette #2007,


Precision Systems,


Only one-half


media


was


changed


every fourth day to allow accumulation


any required neurotrophic


at saturation humidity

(Billups-Rothenberg) i


factors.


in a modular


inside a


Cells were


incubator


low-range


maintained


chamber


temperature


incubator


(Hotpack)


24C.


Experimental


Culture Conditions


Nine


culture


parameters were


systematically varied over









to 280C)


, (3)


humidity


(60%


to complete


saturation)


HEPES


buff


ering,


serum


supplementation


hr after


plating,


basic


minimal


essential


BME)


vitamin,


L-glutamine


, and


nerve


growth


factor


NGF


-7s)


supplementation,


substrate


glass,


plastic,


poly-D-


lysine


, laminin


, collagen


, or haemolymph


clots


length


of proteolyti


digestion


to 60 min)


, and


duration


animal


holding


to 8


Cell


counts


were


made


daily


a permanently


marked


eld


view


each


well


a 24


well


plate.


The


effect


of supplementing


olfa


ctory


neuron


culture


media


with


media


preconditioned


with


lobster


brain


tissue


was


measured


a separate


series


of experiments.


Either


entire brain

isolated from


or the

the a


olfactory


interior


and


region


accessory

of cold


neuropils


anestheti


were


zed


lobsters.


Tissues


were


rinsed


5% AbAm


volumes


and


then


diced


into


fine


pieces


a small


volume


modified


media.


Whole


tissue


slices


were


portioned


into


1 ml


modified


media


, supplemented


with


, and


continually


agitated


50 RPM


on an orbital


shaker.


Conditioned


media


was


collected


after


and


60 hr and


used


to supplement


olfactory


neuron


cultures


media:


conditioned


media


and


media


: 30


conditioned


media)


concentrations.


vitro


brain


tissue









ElectroDhvsioloqv


cells


configuration,


were


using


patch-clamped


an integrating


in the


whole-cell


patch-clamp


amplifier


Dagan


3900


. The


signal


was


filtered


with


a low


pass


Bessel


filter


digitally


samrnpl


ed during


odor


stimulation


every


msec.


Acqui


sition


and


subsequent


storage


and


analysis


data


was


done


using


pCLAMP


software


(Axon


Instruments)


Neurons


were


viewed


40X


magnification


under


Hoffman


optics.


Patch


electrodes


mm O.D


diameter


. boralex


glass


approximat


were

1.0


fire

um (B


polished


ubble


to a tip


number


Mittman


1978


High


resistance


sea


14 GQ)


were


formed


applying


gentle


suction


lumen


pipette.


Odorants


(pip


ette


concentration


10-3M)


were


delivered


cells from


a multibarrel


glass


micropipette


(Frederick


haer


& Co.)


coupled


to a pressurized


valve


temrn


(120


msec


pulses;,


Picospritzer;


General


Valve


Co.)


via


a 6-


way


rotary


valve


(Figure


2-1D)


Solutions


All


salts


used


in preparing


Panulirus


saline


and


modified

Chemical


Liebowitz


NGF-7s


Media


from


were


mouse


obtained


submaxillary


from


Sigma


glands


was


obtained


from


Boehringer


Mannheim.


patch


electrode


solution


consisted


NaCI,


11 EGTA,


10 HEPES,


et al ,









MgCl2,


CaCl,


Na2SO4,


HEPES,


and


glucose;


adjusted


7.4


with


IN NaOH.


Modified


Media


was


prepared


normal


follows:


concentration


50 ml Liebowitz


of PS,


Stock,


dextrose,


50 ml of


.026


glutamine,


1% BME


basic


minimal


essential)


vitamins


Sigma)


and


.01%


gentamicin.


Solutions


substances


tested


as odors


were


either


a 100


-fold


dilution


complex


mixture


prepared


from


TetraMarin


(TET)


commercially


available


flake


fish


food,


made


into


an aqueous


extract


homogenization


dry


flakes


into


saline,


followed


low


speed


centrifugation


remove


particulates


solutions


then


filtration


taurine,


betaine,


with


Whatman


ascorbic


10-3M


acid,


proline


glycine,


cyst


modified


eine,

L15 n


AMP


redia


, TMAO,

Odor


or arginine


concentrations


, prepared


are


daily


reported


pipette


concentration


concentration


reaching


and


absolute


cell.


absolute


test


concentration


was


estimated


to be


91.5% of


pipette


concentration,


based


on a method


determining


stimulus


concentration


using


steady


-state


K+ permeability


neurons


(Firestein


and


Werblin,


1989


Results


Optimal


Culture


Conditions


Optimal


culture


conditions


were


determined


based


upon









saturation humidity than at


humidity,


where


cell


densities were


percent,


reduced 1


respectively,


wk after plating by


in comparison with 100%


and 21

saturated


controls.


28C)


three


the cells


temperatures


survived longest


tested


at 24C


(20,


(Figure


and


2-2A)


Survival


24C was


significantly


longer than at


the next


best


temperature


(20C)


Cells did not adhere


to untreated glass


covers lips,


collagen or


laminin substrates,


and adhered with little


neurite outgrowth


or dishes


to commercial


(Corning and Falcon


plastic


#1008,


tissue


respective


culture wells

ly). Cells


that


did not


adhere


to an appropriate


substrate,


or as a


population


commence neurite


extension,


died within


36 hr.


Cells


sprouted processes with optimal


survival


when


plated


on poly-D-lysine


coated glass


coverslips


or when grown on


haemolymph clots


(Figure


2-2B)


Cells


that


adhered


poly-D-lysine


plating


substrate or


absence of


clots did


FCS.


immediately after


Process outgrowth and/or


extension was


observed as early as


10 min after plating.


Cells


did not


adhere uniformly to


substrate


when FCS was


provided


upon plating


(vs.


to 12 hr later)


nor when


cells


were


kept


under room light


or continually agitated.


Cells


survived equally well


between


and 1082 mOsm,


highest


four osmotic


conditions


tested.


Survival













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haemolymph


osmolarity


intermolt


animals


was


found


to be


1079


requirement


mOsm

for ]


(SEM,


N=4)


vitamins


Cells


demonstrated


, L-glutamine,


a marked


and


supplementation


(Figure


2-2D)


Cell


survival


was


maximal


when


was


supplied


hr after


plating


(Figure


2-2D


Cell


den


sity


was


reduced


one


-half


when


cells


were


deprived


as short


as 12 hr


Figure


2-2D


- 3)


no cells survived


24 hr


of deprivation


(Figure


2-2D


- 4)


addition


or omission


of HEPES


(Figure


2-2D


NGF


(Figure


2-2D


had


no gross


effect


on either


longevity


or neurite


outgrowth


When


antennular


tissue


was


taken


from


animals


held


or longer


as opposed


to animals


held


no more


density


than


dropped


(Figure


one-third


culture


within


first


plating


hours


plating


, with


cells


extending


processes.


BME


vitamin


supplementation


increase


cell


survival


when


using


animals


held


less


than


(Figure


2-2D


Cells


could


be maintained


culture


to 23


days


using


derived


optimal


conditions.


Media


preconditioned


with


tissue,


added


amounts


to 3


or 50%


of normal


olfactory


neuron


culture


media,


no significant


effect


longevity


olfactory


neuron


cultures


compared


to control,


non-conditioned


cell


cultures


observed


23 days.


This


was


true


whether


media


was


conditioned


or 60 hr,









Morpholoqv


Cell


Tyvpes


and


Neurite


Outgrowth


Five


morphologically


distinct


types


"neuron-like"


cells


could


defined


based


on the


number


and


type


pro


cesses:


soma


only,


bud


, (3


unipolar,


bipolar,


and


multiprocess


Figure


Soma


only


cells


process


were


spherical


formation.


shape


Bud


cells


and


underwent


possessed


no apparent


a single


short


process,


less


than


long,


typically


wide,


which


did


not


branch


Unipolar


cell


bore


a single


long


process,


greater


than


in length,


often


unbranched


and


thin.


Bipolar


cells


always


possessed


equal


length


processes;


majority


produced t

arborous a

instances,


processes


threee

nd o


soma


, when


which

to five


were


unbranched.


processes


f nonuniform


diameters


present,


length

ranged


between


that

and

from


Multiprocess


cells


were prominently

width. In all


to 16


and


with


Mm long.


In all


cultures,


larger,


"non-neuron-like"


cells


with


somata


greater


than


in diameter


were


also


observed


and


comprised


approximately


5% of


a given


cultured


population.


larger


cells


were


either


fusiform


or flat


(Figure


These


larger


cells


could


selectively


removed


withholding


FCS


supplementation


12-24


hr after


plating.


Electrophvsiolocrv


smaller


cells


(8-16


tested


were


electrically

























Figure 2
olfactory


8-16


-3.


The morphology of


cultures.


(A-E)


um in diameter;


soma


cells observed


"Neuron-i ike"


only,


bud,


cell
C)


in lobster
types,
bipolar,


multiprocess,


cell


types,


and E)


unipolar.


greater than 20


(F-G)


pm in diameter;


"Non neuron-like"


fusiform and


flat.


Magnification


780X.








46










activated


population


concurrently


cells


ca -30


average


(Figure


peak


2-4


amplitude


voltage-activated


inward


and


outward


macros


copic


currents,


when


cells


were


stepped


from


rest)


to +3


, was


-322


(SEM)


and


(SEM)


resp


ectively.


ionic


basis


these


conductances


was


not


determined.


In contrast


smaller


diameter


cells,


larger


20 pm)


cells


were


measurably


elec


trically


exc


itable


, and


had


ohmic


current\voltage


relationships


over


range


of membrane


potentials


tested


from


mV to


This


chotomy


between


smaller


and


larger


cells


was


mirrored


sensitivity


two


types


of cells


to odors.


None


large


cells


tested


responded


to appli


ed odors


whether


odor


was


single


compound


or the


complex


mixture


(TET


In contrast,


28 of


(56%)


smaller


cells


, each


teste


d with


one


variety


of single


compounds


(taurine,


betaine,


ascorbi


acid,


proline


, glycine,


cyst


elne


, AMP


, TMAO,


or arginine)


responded


with


either


an inward


or outward


current


(Figure


. The


collective


cell


population


did


respond


same


single


compound.


latency


odor-evoked


current


was


phase


locked


to the


appli


cation


odor


stimulus.


half


-peak


duration


odor-evoked


current


was


approximately


to 4


Although


dose-response

























Figure
A) Whol
conditi
+30 mV
cell di
activat
current
similar


2-4.
-cell
ns.
n 10
plays
d aro
. Al


Voltage-ac
macroscopi
The cell wa
mV episodes
the conduct
und -30 mV
1 cells wit


IV relati


tivated


C
S

ta


current
held a
B) No
nces w
+ = in
"neuro


currents
ts under
t -60 mV
te that
which cha
ward cur
n-like"


a cultured


vol
and
the
ract
rent
morp


ta
Ss
IV
er


ge-c
tepp
plo
isti
l =o
locv


ORN.


amp
d to
of the
ally
outward
showed


onships




























1200

800

400

0


-400


-800o
-60


-50 -40 -30 -20 -10 0 10 20


Voltage (mV)























Figure 2-5. Response
dilution of TET extract
trace for each cell sho
Holding potential = -60
odorant pulse.


of cultured ORNs to A) a 1000X
and B) 103 M glycine. Control
ws the response to a L15 media blank.
mV. Arrow = start of 120 msec














50 p


250


40


ms


pA


750


ms


A









to 10-2M.


An odor-evoked


current


could


be distinguished


from


background


noise


at a 10-M


odorant


concentration


one


cell


ted


threshold.


Eight


ten


cells


tested


(80%)


with


three


to five


odors


, res


ponded


to at


least


one


odorant.


None


these


cells


responded


to all


five


compounds;,


typically


they


responded


to only


one


or two.


When


cells


responded


more


than


one


compound,


magnitude


response


vari


across


ective


tested


compounds


and


also


between


different


cells


ted.


instance


did


identical


application


of only


culture


media


from


one


barrel


stimulus


delivery


pipette


elicit


any


measurable


current.


Similar


control


(flat)


traces


were


recorded


when


picospritzer


pressure


was


turned


off,


and


continuous


cell


baseline


current


was


monitored.


Discussion


Under


optimal


conditions


temperature;


humidity;


osmolarity;


appropriate


substrate;


serum,


sugar,


vitamin


and


amino


acid


supplementation,


olfactory


neurons


could


sustained


days


primary


culture,


with


continual


neurite


outgrowth


during


this


riod.


culture


consisted


fundamentally


different


types


cells


that


could


initially


classified


according


soma


diameter.


Based


upon


their


selective


responsiveness


to odors


and


current,


smaller


diameter


cells


to 16


would


appear


to be









and


Ache,


1988)


, the


cultured


system


is developed


from


enormously


enriched


starting


population


of olfactory


neurons,


with


proportionately


neurons


of other


types


i.e.


non-olfactory


chemoreceptors


and


mechanoreceptors)


Given


an average


of 3


receptor


neurons


per


soma


cluster


and


an average


of 15


soma


clusters


per


annulus


(Grfnert


Ache,


1988)


, the


annuli


used


per


culture


should


yield


neurons.


Based


upon


hemocytometer


counts,


estimate


that


neurons


are


harvested


and


plated


per


culture


, which


indicates


an approximate


total


yield


percent.


Some


loss


would


expect


ed from


mechanical


dissociation,


pipette


transfer,


initial


AbAm


washes,


and


removal


soma


clusters


from


connective


tissue


adjoining


cutic


le during


isolation.


Based


upon


yield


alone,


plausible


to conclude


that


a large


percentage


small


cells


are


olfactory


neurons.


Further


evidence,


however,


is required


to definitively


prove


that


these


cells


originate


from


olfactory


(aesthetasc)


sensilla.


Biologically


relevant


odors


aquatic


animals


are


small


ecules


such


amino


acids


used


this


study


(Carr


et al. ,


1984


Since


many


these


molecules


also


have


broad


biological


activity


on cells


(Carr


et al.,


1984


a systematic


study


of odor


responsiveness


cultured


cells


compares


that


of cells


situ


necessary









sensitivity


in cultured


cells


does


demonstrate


a degree


selectivity.


Both


types


of odor-evoked


currents


are


recorded


wide


culture


range


complex


and


aquatic


mixture.


cells


stimuli;,


Although


display


single


onset


odor


sensitivity


odorants


latency


and


as well


duration


response


cannot


strictly


compared


between


cultured


cells


and


that


intact


non-cultured


cells,


to the


different


odorant


delivery


systems


required


each


type


recording,


odor


response


kinetics


each


system


are


qualitatively


similar


(Michel,


unpublished


data)


The


cultured


cells


also


display


discrimination


properties


exemplary


evoked


of olfactory


curre


neurons


in as much


is concentration


dependent


the

and


magnitude

currents


are


not


evoked


every


stimuli


that


is presented.


The


percentage


total


cells


responding


to a single


presented


odorant


is greater


than


half;


a percentage


which


increases


as cells

odorants


are


sequentially


or with


a complex


presented

mixture.


with

This


a variety


degree


selectivity


to odors


would


expected


stances


were


activating


odor


receptors


and


receptors


some


more


generalized


function,


such


as modulation


sensitivity


These


data


suggest


that


each


cell


responding


to specific


odors


that


likelihood


applying


appropriate


stimuli


increases


when


cells


are









"neuron-like"


cells


are


only


olfactory,


that


they


retain


their


ability


to respond


to odor


compounds


selectively.


somewhat


surprising


that


only


poly-D-lysine


provided


a suitable


substrate


neurite


outgrowth


olfactory


to Con


neurons.

or uncoated


Poly-D-lysine


plastic


was


(Falcon


found

001)


to be


and


inferior


inferior


uncoated


Primaria


dishes


(Falcon


801)


or polyornithine


culturing


chicken


(Gonzales


et al.,


1985


and


insect


(Stengl


and


Hildebrand,


1990)


olfactory


receptor


cells,


respectively.


Neonatal


olfactory


cultures


maintained


poly-D-lysine


(Ronnett


also


et al. ,


displayed


1991)


poor


Krenz


plating


Fischer


effi


ency


1988


, found


only


to 50%


survival


crayfish


stomatogastric


ganglia


neurons


(1990


when


plated


, however,


did


onto


find


poly-D-lysine.


extensive


Graf


neurite


Cooke


outgrowth


stomatogastric


neurons


from


lobster


or crab


when


plated


onto


poly-D-lysine,


uncoated


Primaria


they

dishes


also found


(Falcon


cell


3801


attachment


cultures


albeit


from


same


family


(lobster)


, plastic


or uncoated


glass


substrates


prevent


ed attachment


cells


and


resulted


in cell


death.


could


argue


that


neurons


from


sensory


organs


may


have


similar


substrate


requirements.


Little


neurite


outgrowth,


however,


is seen


in retinal


ganglion


cell









fibroblasts


process


(Drazba


outgrowth


and


was


Lemmon,


observed


1990


While


neonatal


minimal

olfactory


neurons


plated


on poly-D-lysine,


significant


neurite


outgrowth


was


obtained


when


laminin


was


appli


to poly-


ornithine


in my


treated


cultures,


slides


laminin


(Ronnett


provided


et al


1991


highest


Although,


initial


plating


density


in terms


of survival


total


number


cells


2 hr

and


after


sub


plating,


sequently


cells


died.


failed


to adhere


observation


that


this

cells


substrate

died


within


hr if


they


did


not


adhere


an appropriate


substrate


and,


as a population,


exte


neurites,


consistent


with


Cooke


et al.


(1989)


findings


that


cultured


crab


or lobster


peptidergic


neurons


had


to adhere


substrate

substratum


for

for


support

neuron


and


outgrowth.


survival


and


Thus,


process


optimal


outgrowth


not


necessarily


genus


specific,


may


be dependent


on type


nervous


tissue,


and


may


even


vary


among


neurons


cultured


exc


lusively


from


different


sensory


organs.


observation


that


supplementation


after


plating


yields


even


greater


cell densities


than


suppli


immediate


ely


upon


plating


, suggests


that


an initial


period


neuronal-substrate


contact


without


may


important,


perhaps


to prevent


FCS-induced


aggregate


formation.


This


finding


in accordance


with


Sebben


et al.


(1990)


who


find









non-neuronal


cell


death


during


deprivation;


in their


work,


72 hours


selective


after


mortality


plating. Although

non-neuronal cells


in my system,


could


achieved


achieve


only


after


my goals


with


24 hours,


lobster


was


antennular


necessary


cultures.


larger


non


-neuronal


cells


only


comprised


about


5% of a given


culture,


did


proliferate


to confluency


to affect


survival


smaller,


neuronal


cell


types,


nor


did


their


presence


interfere


with


my electrophysiological


measurements.


While


excellent


clotted


substrate


lobster


serum


in terms


(haemolymph


survival


and


provided


neurite


extension,


was


conducive


to electrical


recordings.


Serum


and,


presumably,


proteins


in the


haemolymph


substratum


interfered


with


formation


high


resistant


gigaohm


seals


required


patch-clamp


recordings.


In contrast


cells


cultured


with


FCS,


which


could


be rinsed


with


serum-


free


media


prior


to recording


to alleviate


this


difficulty,


haemolymph


could


never


suffi


ciently


washed


from


cell


surfaces.


Many


sensory


neurons


have


biological


requirement


nerve


growth


factor


(NGF)


which


regulates


survival,


development,


maintenance


these


neurons


vertebrate


systems


(Johnson


et al.,


1986;,


Lindsay


et al.


, 1990;


and









explained


fact


that


cells


were


always


serum


supplemented.


many


undetermined


neurotrophic


factors

Hence,


which


any


could


addition


sustain

1 effect


olfactory n

(increased


eurons


in culture.


longevity,


neurite


outgrowth,


or electrical


excitability


from


NGF


-7s might


have


been


masked


factors


contained


PCS.


There


are


many


conceivable


reasons


why


cultures


derived


from


tissues


of recently


captured


animals


survived


twice


long


those


derived


from


animals


held


captivity


weeks


or more.


animals


may


have


been


as healthy


those


their


natural


environment,


even


though


food


quality,


water


turnover,


and


cleanliness


aquarla


were


carefully


monitored.


Certainly,


other


rese


archers


studying


various


aspects


of olfaction


well


as those


home


laboratory)


have


discovered


desen


sitization


neurons


in catfish


and


salamander


when


animals


were


held


little


as 2


weeks


Caprio


and


Firest


ein,


pers.


comm


While


remaining


tested


parameters


collectively


improved


pivotal


longevity,


cell


individually,


survival.


no single


optimal


factor


osmolarities


appeared


(965,


989,


and


1082


mOsm)


were


congruous


with


measured


osmolarity


(1079


mOsm)


of haemolymph.


The


temperature


which


gave


greatest


cell


survival


(24C


was


lower


than










study


was


based


largely


on behavioral


repertoire,


frequency


of molting


, and


growth


rate;


and


variation


found


between


tested


temperatures


using


these


indices


was


minimal


survival


within


a temperature


range


to 2


9C,


respect


lively


actual


temperature


optima


physiological


processes


in culture


may


not


precisely


coincide.


requirement


high


humidity


may


have


acted


indirectly


preventing


changes


in osmolarity,


although


less


than


saturated


humidity


evaporative


losses


were


minimal.


Measured


osmolarity


change


in media


over


any


one


week


time


was


no more


than


20-30


mOsm


60-80%


saturation.


There


appe


ared,


then,


to be a broad


range


suboptimal


conditions


(Figure


2-2


which


were


suitable


cell


maintenance


apparent


new


growth


indexed


neurite


extension)


one


two


other


report


in which


associated


crustacean


neurons


were


cultured


Cooke


et al.,


1989;


Graf


and


Cooke,


1990) ,


outgrowth


was


observed


in a simple


medium


only


physiological


saline


and


glucose


Although


both


work


theirs


use


cells from


congeneric


lobsters,


type


neurons


cultured


in each


study


differed.


Secretory


neurons

may be


from


able


X-organ-sinus


use


existing


gland


membrane


(Cooke

from s


et al.,


tored


1989)


granules


actively


synthesize


proteins


or regulate


transport









preconditioned


media,


had


a strong


dependence


(among


other


noted


factors)


haemolymph


on neurotrophic


The


regenerative


factors

nature o


supplied


f olfactory


in serum


neurons,


as primary


receptor


neurons,


may


require


strict


presence


specific


physiological


factors


continual


neurite


outgrowth,


and


subsequent


viability


culture.


findings


appear


to be


more


analogous


to the


other


reported


work


crustacean


neuron


cultures,


that


of Krentz


et al


(1990


who


also


found


serum


supplementation


(5-10%


necessary


sustained


growth.


Although


appropriate


target


organs


have


been


known


influence


proliferation


and


direction


neurite


outgrowth


culture


(Coughlin,


1975;


Pollack


al. ,


1981


, this


appears


to be


case


growing


olfactory


neurons


and


since


chicken


lobster


(Gonzales


olfactory


et al.,


neuron


1985


cultures


or of


can


lobster,


sustained


without


presence


lobes


preconditioned


media


had


no affect


on viability


or neurite


outgrowth.


This


finding


in contrast


to cultured


insect


olfactory


receptor


neurons


that


have


been


reported


to fail


within


2d in


absence


conditioned


media


from


either


non-neuronal


antennal


cells,


extracellular


fluid


from


antennae,


or from


hormone


20-hydroxyecdysone)


(Stengl


and


Hildebrand,


1990


possible


that


insect


olfactory


neurons









from


differentiated


adult


cells


and


embryonic


cells,


was


large


case


insect.


transplants


Graziadei,


1983


olfactory


, however,


In intact


organs


appropriate


organ


(Morrison


targets


cultures


Monti


may


influential


promoting


olfactory


neuron


growth.


Perhaps,


culture


systems


rat,


chicken,


and


lobster


may


have


allowed


sufficient


cell-cell


interaction


targets


to be effective,


since


they


were


plated


density


(range


x 10


s to 1


x 106


cells/ml


Two


recent


culture


systems


olfactory


receptor


cells,


one


a continual


clonal


cell


line


from


olfactory


epithelium


Coon


et al.,


1989


and


another


isolated


olfactory


neurons


(Ronnett


et al. ,


1991)


show


increased


CAMP


levels


in response


to odor


stimulation,


being


based


biochemistry


not


electrophysiological


recordings,


did


give


information


on the


odor


specificity


single


cells.


high


survival,


electrical


exc


itability,


and


odor


responsiveness


that


find


in cultures


of single


lobster


olfactory


receptor


cells


also


een


reported


in monolayer


cultures


isolat


olfactory


tissue


(Pixley


and


Pun,


1990


Identical


to cultured


lobster


olfactory


neurons,


neurons


from


rat


evoke


a fast


inward


current,


followed


outward


current,


when


depolarized


Upon


application


of odorant


mixtures


single


compounds


were


not









contrast,


cultured


lobster


olfactory


neurons


produced


either


outward


or inward


currents


in response


to odorant


mixtures


and


to single


compounds;


a finding


that


presumably


corresponds


previously


noted


depolarizing


and


hyperpolari


zing


receptor


potentials


lobster


olfactory


receptor


cells


in situ


(McClintock


Ache,


1989b)


This


may


imply


a functional


difference


between


lobster


and


olfactory


cells


their


ability


to produce


currents


of both


polarities,


may


also


reflect


limited


odors


tested


on the


olfactory


neurons.


Odor-evoked


decre


ases


in action


potential


frequency


that


may


reflect


an underlying


outward


current


have


been


reported


olfactory


receptor


cells


in another


vertebrate


(Dionne,


1990)


Panulirus


argus


olfactory


receptor


neurons


can


now


considered


crustacean


among


cells


a small,


More


growing


importantly,


number


they


of culturable


join


phylogenetically


diverse


group


of olfactory


receptor


neurons


that


can


maintain


odor


sensitivity


culture.


Since


lobster


olfactory


receptor


cells


culture


are


morphologically


effective


compact,


space-clamp


should


recording


possible


to obtain


odor-activated


currents;


drug

and


introduction

extended, res


and


incubation


pectively.


periods


can


Moreover,


be simplified


cells


are


directly


accessible


electrophysiological


recordings.









should


provide


a useful


model


future


studies


olfactory


transduction


as exemplified


Chapters


to 7.













CHAPTER


ODOR


SENSITIVITY


IS NOT


DEPENDENT


ON PROCESS


FORMATION


Introduction


small


size


and


thin,


elongated


morphology


olfactory


receptor


neurons


(ORNs)


was


long


an imp


ediment


understanding


olfactory


transduction.


advent


of patch-


clamp


recording


ameliorated


this


situation


and


facilitated


progress


toward


under


standing


olfactory


transduction


(reviews


: Anholt,


1991;


Firestein,


1991


Central


this


effort


surrounding


been


ability


epithelium


to dissociate


order


to study


ORNs


them


from


their


directly


or in


sustained


primary


culture


Dissociated


ORNs


are


not


only


accessible


patching,


they


often


assume


a more


compact


form


than


their


counterparts


in situ


that


allows


reasonable


space


clamp


and


facilitates


diffusion


of membrane


impermeant


probes


from


electrode


site


transduction.


While


transduction


thought


occur


in the


cilia


(outer


dendrites,


in invertebrates)


ORNs


.g.,


Kurahashi,


1989


Firestein


et al.,


1990;


Lowe


and


Gold,


NOTE


This


chapter


been


accepted


publication


and


reprinted


and


B.W.


with
Ache.


permission


1993


Odor


from


Fadool


D.A.


sensitivity


W.C


lobst


. Michel,
er olfactory


ry-cpn1i- nr)


n1c 1 -n a


-I -1 *nr~a'rnrn9ni-


*nmrnnpscs


fnrmsl nn _









1991)


, at


least


some


elements


transduction


cascade


are


confined


cilia.


Specifically,


cAMP-gated


cation


channels


that


are


effectors


in the


transduction


cascade


in amphibian


ORNs


also


occur


on the


dendrite


and


soma


cells,


although


lower


densities


than


cilia


Indeed


Firestein


this


al. ,


1991;


variability


Zufall


density


et al


was


1991a)


exploited


to obtain


favorable


channel


density


recording


ibid.


Previously,


Chapter


preliminary


evidence


was


reported


that


cultured


lobster


ORNs


respond


to odors


independently


whether


cells


had


sprouted


processes.


This


observation


raises


interesting


possibility


that,


vitro,


elements


transduction


cascade


may


expressed


and


inserted


into


soma


ORNs


prior


independent


process


formation.


Given


ease


patching


soma


compared


to the


extremely


thin


cilia


(outer


dendrites)


and


ability


to culture


ORNs


vertebrates


(Coon


et al. ,


1989;


Pixley


and


Pun,


1990;


Calof


and


Chikarai


shi,


1991


Ronnett


et al.,


1991)


and


other


invertebrates


(Stengl


et al. ,


1989;


Zufall


et al.,


1991b)


such


a phenomenon


could


be of general


utility


studying


olfactory


transduction.


Without


normal


polarity


cell,


however,


must


be establi


shed


that


applied


"odors"


are


activating


what









particularly


important


when


studying


ORNs


from


aquatic


animals


such


stimuli


as fish


many


lobsters.


aquatic


animals


Adequate


are


olfactory


blood-born


components


prey,


compounds


such


as amino


acids,


amines


and


nucleotides


(review


: Carr,


1990)


These


types


compounds


could


expected


to activate


cells


neurotransmitters


or neuromodulators.


In order


to establish


utility


of cultured


lobster


ORNs


analysis


transduction


mechanisms,


will


this


chapter,


provide


functional


evidence


that


cultured


ster


ORNs


with


no or


varying


numbers


processes


are


morphologies


same


type


cell


and


that


odor-evoked


properties


cultured


cells


reflect


those


of lobster


ORNs


in situ.


Methods


Tissue


Culture


distinct


clusters


ORNs


were


dissected


from


aesthetasc


antennular


(olfa


filament


ctory)


sensilla


(olfactory


organ)


on the


lateral


of adult


specimens


Caribbean


spiny


lobster,


Panulirus


argus.


The


clusters


were


enzymatically


dissociated,


and


resulting


cells


sustained


primary


culture


as described


previously


Chapter


Bri


efly


, the


isolated


clust


ers


were


incubated


for


min


rpm


on an orbital


shaker


in 0


micron


filter-sterilized


solution


mg papain


and









(Gibco)


Proteolytic


digestion


was


stopped


replacing


enzyme


solution


with


glucose


L-15


media


supplemented


with


L-glutamine


, dextrose,


fetal


calf


serum,


and


BME


vitamins.


coated


Cells


glass


were


coverslips.


immediately


Cells


plated


were


on poly-d-lysine-


maintained


saturation

Rothenberg


humidity


24C.


a modular


Neurite


incubator


outgrowth


chamber


in individual


(Billups-

li cells


was


recorded


on a TL Panasonic


6050


time-lapse


video


cassette


recorder.


Images


were


later


captured


and


subsequently


analyzed


using


Image


analyst


software.


Electrophvsioloqv


Voltage-


odor-activated


currents


were


recorded


whole-cell


configuration


with


an integrating


patch-clamp


amplifier


(Dagan


3900)


analog


signal


was


filtered


and


digitally


sampled


every


msec.


Data


acquis


ition


and


subsequent


storage


analysis


digitized


records


were


done


with


pCLAMP


software


(Axon


Instruments)


Cells


were


viewed


40X


magnification


with


Hoffman


opti


cs.


Patch


electrodes,


pulled


from


mm O.D


. borosilicate


glass


were


fire


polished


to a tip


diameter


approximately


(Bubble


seals


number


to 14 GQ)


Mittma

were


al. ,


formed


1987)

applying


High


resistance


gentle


suction


lumen


pipette


upon


contact


with


cell.


experiments


, cells


were


voltage-clamped


at a holding









30 mV,


300ms


hyperpolarizing voltage


steps


into


cells


from the holding potential.


Each cell


was photographed


allow correlation of


soma size,


length of


process,


and cell


morphology with physiological


properties.


Odor Stimulation


Odors were


"spritzed"


on the


cells


120 msec


from a


7 barr

haer)


el


barrels used)


glass micropipette


coupled to a pressurized valve


system


(Frederick

(Picospritzer,


General


Valve)


In most


trials,


one


randomly


selected


barrel


was


filled with fluorescein


to permit


positioning


tip of


the pipette


relative


to the


cell


and


to assure


that


and its


delivered odorant


associated processes.


to odors was determined


six barrels contained


completely surrounded


magnitude of


to be


the odor.


independent


Dilution of


cell


response


of which of


the odor


between


pipette


and


cell


surface,


an average


distance of


two cell


diameters,


was


estimated


to be


approximately 9%,


based on


calculated K


permeability


method of


Firest


ein and Werblin


(1989)


Odor concentrations


are


reported as


pipette concentration and are not


corrected for this dilution.


The odors used were


solutions of:


an equimolar


mixture


that


included


(10-M)


betaine,


glycine,


lactic


acid,


taurine,


and


trimethylamine oxide,


referred


to as


S-1;









(Schmiedel-Jakob et


al.


, 1990)


and


diluted


1000


fold,


referred


to as TET;


and


single


substances


known


to be


effective odors


for the


lobster,


which included


(10 -3M)


adenosine monophosphate


(AMP)


, arginine,


ascorbic


acid,


betaine,


cysteine,


glycine,


histamine,


proline,


taurine,


and


trimethylamine oxide


(TMAO


. All


odorant


solutions


were


prepared daily


in modified L15


media and applied at


stated


concentration,


unless otherwise


noted.


The number of


different


odors


that


stimulated a given


cell


(the


response


spectrum)


was quantified using


breadth of


responsiveness metric of


Smith and


Travers


(1979)


Here,


the breadth of


responsiveness


defined


where


1=0


= a proportionality constant,


piLogp,


number of


odors


tested,


|pA|


absolute


current


(pA)


elicited


from


the nth


odor


and


expressed


a proportion


total


elicited from all


odors.


Solutions


Panulirus


saline


consisted of


(in mM


NaCI,


13.4


KCL,


MgC12,


Cad2


, 13


Na2SO4,


HEPES,


and


glucose;


Liebowitz


Modified L15


Stock,


Media


50 ml of


consisted


normal


50 ml


concentration


0.6g


dextrose,


0.026


g L-glutamine,


and


.01%


gentami-








30 NaCI,


11 EGTA,


10 HEPES,


1 CaC12,


K-acetate,


and


glucose;


pH 7.0.


salts were obtained from Sigma.


Results


Morpholoav:


Neurite Outcrrowth


The cultures


consisted largely of


five


morphological


types of


small


(8-16


um diameter soma)


"neuron-like"


cells,


described previously


(Fadool


al.,


1991b)


soma only,


bipolar,


soma with bud


multiprocess,


unipolar,


four of which were


used in


present


chapter


(Figure


3-1A)


The processes


ranged


from 3


to 160


um long.


Each of


four morphological


types


were


present


as early as


hours post-plating.


Initially,


the predominant


form was


"soma


only"


, but


the proportion of


each morphological


type changed over time;


relative


proportion of

concomitant i


cells


increase


lacking processes decreased,


in the proportion of


with a


cells with


processes


(Figure 3-1B)


To distinguish whether the change


in the


relative


proportion of


the morphological


types


reflected selective


loss of


cells


lacking processes


process proliferation,


or both,


"soma


only"


cells were


followed


individually with digital


time-lapse


imaging for


three


consecutive days


Forty-two


(20%)


starting


2 hr after plating.


cells died within


the observation


period,


indicating that


selective


loss of


"soma only"

























Figure
lobster
optics.
(d) mul
proport
data po
random
types.


3-1.
ORN
(a)
tipo
ion
int
fiell


A)
s obs
soma
lar.
of th
repre
ds of


Light micrographs
served under Hoffm
only, (b) unipol
Magnification 78
e four morphs ove
sents the incident
view, expressed


f
mo
(

d
of
a


our morphs of cul
adulation contrast
c) bipolar,
B) Changes in th
,ays in culture.
that morph in 10
percentage of all


tured


e
Each

four
















70
60
50
40
30
20
10-
0


Soma


Unipolar


Bipolar


Multi


-I I I I I
) 1 2 3 4 '


Time (days)










failed


to sprout


processes,


61 of


cells


sprouted


processes


throughout


observation


period,


becoming


uni-


and,


eventually,


multipolar.


latter


finding


supports


contention


that


four


morphological


types


"neuron-like"


cells


were


morphs


of a single


type


cell.


Phvsioloqv:


Electrical


Properties


That


a single


four


type


types


cell


"neuron-like"


was


cells


supported


were


finding


morphs


that


cells


tested


their


current-voltage


relationship


had


similar


voltage-activated


properties.


total


membrane


current


evoked


in a typical


cell by


depolari


zing


voltage-


steps


consisted


duration)


that


a transient


activated


inward


around


current


followed


usec


a much


larger


, prolonged


outward


current


that


activated


around


mV and


persisted


with


little


decay


throughout


msec


duration


pulse


(Figure


-2A)


The


magnitude


inward


and


outward


currents


was


independent


number


processes


on the


cell


(ANOVA,


- inward


currents


and


- outward


currents)


(Figure


-2B)


subsample


cells


, including


least


one


cell


each


morphological


type,


had


a mean


input


resistance


rest


GQ and


a membrane


time


constant


67.3


11.3


msec.


No detectable


equalizing


time


constant


could


measured


any


morphs,


including


morphs


with























Figure 3-2. Total
lobster ORNs.
A) Representative


inward current,


Macroscopic


volt
-60
not
(mea
bars
proc


age-steps
mV and st
leak subt
n SEM)
) current


esses


urrents
(lower
epped t
racted.
of the
s of 26


n=68


voltage-activated currents of


current-voltage


+ = outward cur
(upper traces)
traces) when t
o +30 mV in 10
B) Plot of t
inward (striped
8 cells grouped


soma only,


relationship of


rent. Inse
evoked by
he cell was
mV episodes
he maximum
bars) and
according


unipolar,


cultured


one


t:
depolariz
held at
Record
amplitude
outward (
to number


cell.


ing

s are

solid
of


69 bipolar,


multiprocess)


(







5000-

4000-


3000-

2000-

1000-

0

-1000-
-6


1200pA


2ms


-60 mV


0 -50 -40 -30 -20 -10 0


10 20 30


Voltage (mV)


1300-
1100-


<
{3..

c"


O)
-o
* -*
C
0)

C
a,
13


0


900-
700
500-
300-
100-
-100-
-300-
-500-









Phvsiolocrv:


Response


to Odors


cells


(including


cells


tested


above


their


electrical


properties


were


tested


their


ability


generate


a current


in response


to stimulation


with


one


five


different


odors.


The


odor


arrays


usually,


but


not


always,


included


complex


mixture,


TET.


Sixty-four


percent


cells


tested


responded


to at


least


one


odor.


This


percentage


increased


to 89%


when


cells


could


tested


with


least


three


different


odors


(n=182


Odors


evoked


a transient


current


that


rose


a maximum


over


several


hundred


msec


and


subsequently


declined


more


slowly


to rest


(Figure


The


current


could


be of either


polarity,


depending


on the


cell


and


odor


tested,


and


different


odors


could


evoke


currents


opposite


polarity


same


cell


(Figure


In cells


that


could


tested


with


least


three


different


odors


(n=182)


, the


odors


that


were


tested


evoked


only


inward


currents


in 48 cells


only


outward


currents


in 58 cells


(32%)


and


currents


of both


polarities


cells.


remaining


cells


did


not


respond


any


odors


tested.


Odor-evoked


currents


of both


polarities


were


assoc


iated


with


an increase


in membrane


conductance,


indicated


decrease


in input


resistance


, when


hyperpolarizing


voltage-


steps


were


injected


into


cells


prior


to and


during


odor-












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Figure


. Whol


a co
an
ulat
den


used


e-cell,


ter ORN s
th the od
rrent (up
was evok
SB) Inw
ase in co
ith TET.
0 mV. 300


to monitor


voltage


h
.0
[o
'p
;e
'a
n


membrane


*ing


in


stimul
trace
by sti
curre
ctance
Hiding
ec hyp
conduct


-clamp
crease
ated i
) nor
mulati
nt (up
(lowe
potent
erpola
stance.


rec


riz


ord
ndu
d c
ge
arr
tra
ace
-6


ings
ctan
urre
in c
ow)
ce)


voltage


from


ce
ture
ed
by


pul


ses


I
L

)




























B


100


400










to 0.7


GQ for


inward


current


(n=8


and


from


a mean


to 0


GQ for


outward


current


(n=4)


(paired


-test,


.05)


Concomitantly,


membrane


odor


time


stimulation


constant


from


, decreased


59.6


significantly


to 21.6


during


msec


inward


current


(n=14


from


to 34


msec


outward


current


(n=7) (paired


t-test,


.05)


latency


to the


onset


odor-evoked


currents,


measured


from


activation


spritzer,


ranged


from


msec


to > 1


sec


, but


typically


was


<100


msec


(Figure


Overall,


mean


latency


to onset


inward


current,


than


31.3


that


Statistic


msec


outward


< 0


.05)


(n=100


current,


To det


was


significantly


81.0


ermine


msec


this


longer


(n=121


difference


was


possibly


driven


between


cell


variation,


performed


paired


comparison


odor-evoked


currents


latency


of both


19 cells


polarities -


that

mean


supported

latency


in these

longer t


cells


han


inward


that


outward


current


was


current


significantly


(paired


t-test,


< 0


.05)


The


peak


amplitude


of odor-evoked


currents


of both


polarities


increased


with


concentration


odor


and


saturated


over


orders


of magnitude


(Figure


3-6


mean


slope


concentration-response


function


in the
















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outward


current.


Thresholds


were


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10-8


lowest


concentration


tested,


currents


either


polarity.


The


peak


amplitude


currents


of both


polarities


evoked


a single


concentration


10-3M


of odor


ranged,


typically,


from


5-85


(Figure


-7A)


average


magnitude


inward


current


was


significantly


greater


than


that


outward


current


+ 1.4 pA)


measured


across


cells


and


odors


(n=386;


Statistic


currents


.05)


evoked


The


odors


polarity


were


and


independent


magnitude


cell


morphology


six


different


odors.


results


for


two


odors,


proline


(n=lll


cells)


and


taurine


(n=102


cell


, are


shown


Figure


-7B,C.


peak


amplitude


odor-evoked


currents


of either


polarity


was


also


independent


length


process


in cells


bearing


processes


(n=55,


correlation


analysis,


r > 3


.86)


Figure


and


size


soma


cells


lacking


proce


sses


(n=60,


correlation


analysis


, r > 3


.86)


(Figure


3-8B)


Single


odors


activated


from


77 percent


cells


(Table


3-1)


. The


stimulatory


effectiveness


was:


betaine


> histamine


> glycine


> proline


> taurine


> AMP


TMAO


> ascorbate


> arginine


> cysteine.


An equimolar


mixture


five


compounds


(S-1


betaine


, taurine,


glycine,


























Figure 3
currents


Plots


in cultured


peak


lobster


amplitude


ORNs


of odor-evoked


as a function


cell


morphology.


Distribution


peak


amplitude


currents
common cc
currents;


evoked


in 3


)ncentration


solid


bars,


5 cells
(103 M)
outward


y single
Striped
currents.


odors
bars,


tested
inward


Arrows


denote


amplitude


(filled
currents


10-3


arrow)


both


M proline


according
negative,


inward


currents.


polarities
=111) and 1


to morphology.


outward


arrow)


(open
Plots
evoked


M taurine


Inward


and
peak


outward
amplitude


stimulation


currents


(n=102
are


with


)) grouped
denoted as


as positive


mean


0O-3


(n=



























0 50 100 150 200 250


Response Magnitude


(pA)


100-
80-

60-
40-
20-
0-
-20-
-40-
-60-
-80-

60-
40-
20-
0-
-20-
-40-
-60-


Taurine


Proline



-A-


*
-C
rA


I




Full Text

EXCITATORY SIGNAL TRANSDUCTION MEDIATED BY INOSITOL
PHOSPHOLIPID METABOLISM IN LOBSTER (PANULIRUS ARGUS)
OLFACTORY RECEPTOR NEURONS
By
DEBRA ANN FADOOL
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1993

Copyright 1993
by
Debra Ann Fadool

TO MY THREE MEN

ACKNOWLEDGMENTS
I deeply thank my husband, Jim, for rearranging his
schedule it seems on a weekly basis to accommodate my
research demands. I thank him for sharing the excitement
and the frustration, again and again, and especially at 2 or
3 in the morning. I thank him for listening to all my
ideas, tutoring me when I was clueless, and being a
tremendous father even when time was tight. I thank both
our parents for all the extra comforts most graduate
students forego . . . beds, furniture, clothes, microwaves,
washing machines, and those little inserts inside birthday
cards. I thank Nanny Edith and Nanny Eula, but especially
thank "Situ" above all for relieving my worries and for
caring for my family when I was working, studying or
attending scientific meetings abroad. I thank my son,
Calvin, for never complaining about the crowded living
quarters or the economic condition he was placed in while
both myself and his father met our educational goals. I
thank him too for pestering the heck out of me while I
crunched numbers or composed on the computer: "This is more
important THAN EVEN DATA, MAMA!" I thank my baby, Andy, for
adding entropy to the final stretch of the completion of my
degree; his little smiles make it all worth it. I am so
IV

thankful to Uncle Paul, Tere and Peter Lin, Steve Munger,
and Carol Diebel for carpooling Jim on Thursdays so I could
spend time with my boys instead of 3 hours on the road.
I greatly thank my mentor, Dr. Ache, for the never
ending attempts to develop my literacy. I thank him for his
tenacity at challenging me to think critically about the
significance of my data (no not the statistical!), my
writing, my presentations, and all the areas that comprise
the expertise of a research scientist. I thank him for
encouraging me always to seek opportunities and genuinely
appreciate all the ones he has directed my way, including my
wonderful electrophysiological room.
I thank all my friends at the Whitney and Marine
Biological Laboratories, for being just that. You know who
you are. Especially the one in Utah, who reassured me that
electronics and computers were easy, and who was a major
source of learning, guidance, jokes, and enthusiasm during
my training. Thanks also go to the Gainesville commuter
that let me have his couch and kitchen table for a week to
complete my comprehensive exams in complete hibernation. I
don't know what I would have done (probably not slept) if it
hadn't been for the photocopy-night-before-partners. I
thank always all the researchers and students who came to
the MBL the summer of 1991 and gave of their time,
excellence, intellect, and enthusiasm for the training
advancement of others. Thanks especially go to Louise
v

MacDonald, Shirley Metts, Lynn Milstead, and Jim Netherton
for always helping me finish different aspects of a project
under time constraints. Sincere thanks go to Leslie
VanEckeris for all those "Can you do me a favor?" Special
thanks go to Dr. M. Greenberg for always listening and
caring, and carrying a handkerchief.
Lastly I would like to thank Dr. Wheatly, my
Gainesville connection, who allowed me to assist her class
when no one else was willing to take the risk. I thank her
for letting me do what I enjoy most, teaching. Towards that
end I thank Dr. Anderson for letting me lecture in his
department in Neuroscience and thank Kelly Jenkins for being
my first guinea pig.
vi

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iv
ABSTRACT x
CHAPTERS
1 INTRODUCTION 1
Amino Acid Receptors and
Aquatic Chemoreception 1
Molecular Mechanisms of Signal
Transduction 8
Single-Channel Recording 11
Specific Aims 25
2 SUSTAINED PRIMARY CELL CULTURE 2 7
Introduction 27
Materials and Methods 30
Animals 30
Tissue Preparation 30
Cell Cultures 33
Experimental Culture Conditions... 33
Electrophysiology 3 5
Solutions 35
Results 36
Optimal Culture Conditions 36
Morphology of Cell Types
and Neurite Outgrowth 4 7
Electrophysiology 47
Discussion 52
3 ODORANT SENSITIVITY IS NOT DEPENDENT
ON PROCESS FORMATION 64
Introduction 64
Materials and Methods 66
Tissue Culture 66
Electrophysiology 67
Odor Stimulation 68
Solutions 69
Vll

Results 70
Morphology: Neurite Outgrowth.... 70
Physiology: Electrical Properties 73
Physiology: Response to Odors.... 76
Discussion 97
4 GTP-BINDING PROTEINS MEDIATE
ODOR-EVOKED CURRENTS 104
Introduction 104
Methods 107
Animals 107
Tissue Culture 108
Electrophysiology 108
Biochemistry Ill
Solutions 112
Results 114
Discussion 131
5 IP3-ACTIVATED CHANNELS IN THE
PLASMA MEMBRANE 137
Introduction 137
Results 139
Macroscopic Currents 13 9
Unitary Currents 143
Immunochemistry and
Related Physiology 155
Discussion 164
Experimental Procedures 171
Solutions 171
Tissue Culture 172
Electrophysiology 173
Immunochemistry 175
6 ION SELECTIVITY AND MODULATION OF
IP3-ACTIVATED CHANNELS 178
Introduction 178
Materials and Methods 181
Solutions 181
Animals 184
Tissue Culture 184
Electrophysiology 185
Results 186
Effect of pH on IP3-activated
Channel Gating 186
Appearance of Modal Patterns
Differing in Gating Kinetics. 187
Effect of Ca2+ on IP3-gated
Channel Gating 200
viii

205
Pharmacology of Macroscopic
Odor-evoked Current &
IP3-gated Channels
Ionic Selectivity of
IP3-gated Channels 213
Discussion 226
Gating Properties 226
Ionic Selectivity & Pharmacology.. 229
7 IP4-GATED CHANNELS IN NEURONS 234
Introduction 234
Results and Discussion 237
8 SUMMARY 252
REFERENCES 263
BIOGRAPHICAL SKETCH 289
IX

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
EXCITATORY SIGNAL TRANSDUCTION MEDIATED BY INOSITOL
PHOSPHOLIPID METABOLISM IN LOBSTER (PANULIRUS ARGUS)
OLFACTORY RECEPTOR NEURONS
By
Debra Ann Fadool
December 1993
Chairman: Barry W. Ache
Major Department: Zoology
Appropriate primary sustained culture conditions were
developed to study signal transduction in Panulirus argus
olfactory receptor neurons (ORNs). Neurons were cultured in
a modified Liebowitz media supplemented with salts,
vitamins, L-glutamine, low dextrose, and either fetal calf
serum or lobster haemolymph. The nature of the adequate
stimuli, the degree of tuning of the cells, the threshold of
sensitivity, and the dual polarity of the odor-evoked
currents were consistent with chemosensitivity in the
cultured ORNs being olfactory.
The magnitude of the odor-evoked currents was
significantly increased or decreased by nonhydrolyzable
analogs of GTP and GDP, respectively, and not perturbed by
pertussis and cholera toxins, implying that a class of
bacterial toxin-insensitive G-proteins was mediating signal
x

transduction. An antibody directed against Goa immuno-
labelled a 40.5 kDa band in an enriched membrane preparation
of ORN outer dendrites and, along with an antibody directed
against Gqa, selectively decreased the odor-evoked inward
current within 10 min of initial perfusion.
Inositol 1,4,5-trisphosphate (IP3) selectively evoked
an inward current in the ORNs. Application of IP3 to the
inside face of cell-free patches of ORN plasma membrane
directly gated two ion channels that differed in
conductance, voltage dependence, and dwell-time kinetics.
An antibody directed against an intracellular, cerebellar
IP3 receptor recognized a protein of similar molecular
weight to the mammalian receptor in the ORNs and was found
to increase selectively the odor-evoked inward currents and
IP3-activated unitary currents in the lobster ORNs.
Modulation of channel gating or ion permeation was
observed in both IP3-gated channels in response to elevated
pH or [Ca2+]i. Both channels mimicked the pharmacology of
the macroscopic odor-evoked inward currents. Ion
substitution suggested that the small-conductance channels
were nonselective for cations and that the large-conductance
channels were either nonselective between Na+ and Ca2+ or
were selective for Ca2+.
The direct metabolite of IP3, inositol 1,3,4,5-
tetrakisphosphate (IP4), gated an ion channel that differed
in conductance, density, kinetics, and voltage sensitivity
xi

channel
from those activated by IP3. The IP4-gated
mutually interacted with IP3-gated channels to alter the
open probability of the channels.
Xll

CHAPTER 1
INTRODUCTION
Amino Acid Receptors and Aquatic Chemoreceotion
The past decade has shown an explosive interest in
excitatory amino acids (EAAs) as important neurotransmitters
of the mammalian central nervous system (CNS). Research
efforts have elucidated the synaptic role of EAAs, the EAA
subclasses of receptors through pharmacological techniques,
the mode of signal transduction, and the extent to which
EAAs are involved in brain function. To date, EAAs are
implicated in a wide range of physiological phenomena
including the processing of sensory information, cognitive
processes, learning, and memory. Dysfunction of receptor-
ligand interaction and subsequent transduction can lead to
neurological disorders such as epilepsy, spasticity,
neurodegeneration after a stroke, as well as Huntington's
and Alzheimer's disease (Croucher et al., 1984; Watkins et
al., 1990). The development of selective agonists and
antagonists for distinct EAAs receptors has implicated amino
acids in synaptic transmission throughout various regions of
the CNS/brain and has offered pharmacological intervention
in treating many of these human neuronal diseases (Lodge and
Collingridge, 1990).
1

2
Amino acid receptors are not limited to the mammalian
CNS. Feeding stimulants for many aquatic organisms, both
fish and invertebrates, are simple hydrophilic tissue
metabolites, mainly quaternary ammonium compounds,
nucleotides, amines, and amino acids (e.g., Ache and Carr,
1990). Chemical analysis found 19 of the 20 common amino
acids in muscle tissue of the blue crab, a typical prey
organism for the lobster (Carr et al., 1984). Consequently,
aquatic organisms typically possess well defined
chemosensory structures containing primary chemosensory
receptor neurons that are responsive to micromolar to
picomolar amounts of amino acids and related molecules.
Unlike the mammalian brain, where amino acid receptors are
buried and widely distributed in tissue, these "external"
amino acid receptors in aquatic organisms are readily
accessible and often highly enriched in the chemosensory
structures.
Large decapod crustaceans such as crabs and lobsters
have such a chemosensory structure on the lateral branch of
the first antenna, commonly referred to as the "olfactory"
organ or antennule. It consists of a tuft of cuticular
sensilla. Each sensillum contains an average of 318 bipolar
receptor neurons (Grünert and Ache, 1988), yielding upwards
of 450,000 receptor neurons per antennule (Figure 1-1).
Outer dendrites of each bipolar cell branch extensively,
filling the distal 80% of the lumen of the sensillum.

Figure 1-1. (LEFT) Large decapod crustaceans such as crabs and lobsters have a
chemosensory structure on the lateral branch of the first antenna, commonly referred
to as the "olfactory" organ or antennule. (MIDDLE) The olfactory organ is divided
into rings called annuli. Each annulus contains of a tuft of innervated hairlike
cuticular structures called aesthetasc sensilla, which are arranged in a zig-zag like
pattern surmised favorable for sampling and clearing of odorants (Gleeson et al.,
1993). (RIGHT) Dendrites of each bipolar cell branch extensively, filling the distal
80% of the lumen of the sensillum. Odorants reach the dendrites, the presumed site
of signal transduction, through a porous cuticle surrounding the sensilla. The
somata of 318 bipolar receptor neurons are clustered at the base of each sensillum,
yielding upwards of 450,000 receptor neurons per antennule. The axons from each
cluster project, without synapsing, to the CNS.
Modified, with permission from Grünert and Ache, 1988.

400nm
Single
Aesthetasc
Sensillum

5
Odorants reach the dendrites through the porous cuticle
surrounding the sensilla. The somata of the receptor
neurons are clustered at the base of each sensillum and the
axons from each cluster project, without synapsing, to the
CNS.
Olfactory receptor neurons (ORNs) in lobsters have been
the object of considerable research (review, Ache and Derby,
1985). The ORNs can be studied in situ (Ache et al., 1989)
or harvested to produce a preparation of almost pure
chemoreceptive neurons for tissue culture (Chapter 2),
biochemistry (Chapter 4-5), or molecular biology (Trapido-
Rosenthal et al., 1993; Munger and Ache, in press).
Of background relevance to the present project is that
amino acids presented in mixtures (i.e., co-released)
typically are less stimulatory to the neurons, as measured
by intensity of the electrophysiological response of the
neuron, than when presented individually and the responses
summed. This phenomenon, called mixture suppression, is
potentially the result of several underlying physiological
mechanisms (Ache et al., 1988). Recently it was shown that
amino acid "odorants" could inhibit as well as excite
lobster olfactory neurons and, more importantly, that these
two processes could occur in the same neuron (McClintock and
Ache, 1989b; Michel et al., 1991; Figure 1-2). In other
words, the excitatory action of one amino acid can be
tempered by the inhibitory action of another (Michel et al.,

Figure 1-2. Amino acid "odorants" can inhibit as well as
excite the lobster olfactory receptor neuron (ORN). Shown
is a single ORN that was first recorded in the cell-attached
configuration (top left diagram) to monitor action potential
increase and decrease in frequency under stimulation by an
excitatory odor mixture (top trace, upper recording) and an
inhibitory odor (top trace, lower recording), respectively.
The cell membrane was then ruptured to achieve the whole¬
cell recording configuration (top right diagram), in which
the ORN responded with a membrane depolarization (bottom
trace, upper recording) and then hyperpolarization (bottom
trace, lower recording) under the same stimulation protocol.
This singular experiment demonstrated that more than one
transduction pathway for amino acids was present in these
neurons.
Taken with permission from Michel et al.,
1991.

7
Mixture
Proline
'iW*i^H»Kiil»rt wil'i
5 sec
-60 mV -
20 mV

8
1991; Michel and Ache, in press) . This finding strongly-
suggested that more than one transduction pathway for amino
acids is present in these neurons. What these pathways were
remained unknown.
Molecular Mechanisms of Signal Transduction
Perhaps classically the most studied and hence best
understood transduction pathway in neurons is (1) ligand
binds to a receptor protein, (2) which induces the
activation of an adenylate cyclase enzyme through G-protein
coupling, (3) then the cyclic adenosine monophosphate (cAMP)
end product acts directly or indirectly on an ion channel,
(4) to induce a generator current. When I commenced my
doctoral study, olfactory transduction, defined as the
mechanism that converts a chemical signal (odor) into an
electric signal (current) was not well perceived. It was
known that a preparation enriched in olfactory cilia
contained an odor-activated adenylate cyclase (Pace et al.,
1985), suggesting that olfactory transduction had some
resemblance to that typical of many neurons. Not all
odorants had the capacity to evoke significant elevations in
adenylate cyclase, suggesting cAMP-mediated transduction was
not the sole mechanism. Nonetheless, a conductance directly
gated by cyclic nucleotides (cAMP & cGMP) was localized to
the olfactory cilia (Nakamura and Gold, 1987), which soon
became established as the site of olfactory signal
transduction (Kurahashi, 1989; Firestein et al., 1990) .

9
Soon after, an olfactory specific G-protein, Golf, was cloned
and localized to the ciliary layer of rat ORNs (Jones and
Reed, 1990). The fact that cholera toxin ribosylated
proteins in the outer dendritic segments of lobster ORNs,
implicated a GTP-dependent protein in at least one of the
two transduction processes in these neurons (McClintock et
al., 1990). Yet in lobster, complex odor mixtures or
identified stimulatory odor molecules failed to alter levels
of cAMP (McClintock et al., 1989) . Hence, in lobster ORNs,
data did not support cAMP as the candidate G-protein linked
second messenger.
Several other molecular mechanisms had been implicated
in signal transduction in other systems, providing me useful
avenues of investigation at the commencement of my study.
They included the following:
(1) Amino acids could directly gate channels, where the
channel was contained within the receptor protein or where
the channel and receptor were inherently two different
proteins coupled directly or through a G-protein (Codina et
al. , 1987; Vogt et al., 1990).
(2) Amino acids could activate a guanylate cyclase to
increase cGMP levels to directly activate a channel or
indirectly activate a channel by phosphorylation via protein
kinase C (PKC) (Vogt et al., 1990).
(3) Amino acids could operate through an inositol phosphate
second messenger system (Berridge, 1989). In this scheme

10
stimulation of cell-surface amino acid receptors hydrolyzes
a membrane bound inositol phospholipid, which produces two
second messengers--neutral membrane bound diacylglyceral
(DAG) and water soluble inositol 1,4,5-triphosphate (IP3) .
DAG remains in the plane of the membrane (Nishizuka, 1984,
1988) to activate PKC which in turn could activate a
channel. IP3 diffuses into the cytosol releasing Ca2+ from
internal stores, causing Ca2+ wave oscillations, and
activating Ca2+ channels (Berridge and Irvine, 1989) . IP3
could additionally activate a channel directly (Kuno and
Gardener, 1987). Inositol 1,3,4,5-tetrakisphosphate (IP4),
the direct metabolite of IP3, may act as an additional
messenger (Irvine, 1990).
The difficulty in studying olfactory transduction in
Panulirus argus resided in the fact that the transduction
machinery was hypothesized to lie in the outer dendritic
membrane of the neuron, as was true for the analogous
structure in vertebrates (above section; Nakamura and Gold,
1987; Kurahashi, 1989; Firestein et al., 1990). The thin
diameter of this process (< 0.2 /xm) made direct electrode
seals virtually impossible. Recording from the neuron at
the more accessible soma depended upon recording a
transduction current from as far as 1000 /xm away and often
produced an inadequate voltage-clamp. I hypothesized that
if the lobster ORNs could be sustained in primary cell
culture without altering their voltage- or odor-evoked

11
properties, then perhaps the cells would assume a form that
was more amendable for voltage-clamping and for studying
transduction. If lobster ORNs in vitro mimicked the
electrical properties of their counterparts in situ, then
the cultured cells would be an excellent model for studying
the elements of olfactory signal transduction, particularly
in a cell that was suspected of having multiple mechanisms.
Single-Channel Recording
"We should remember that our living systems are
essentially watery saline systems that, through a variety of
electrodes, are coupled to physical instruments" (Author
unknown). This section provides an elementary background in
electrophysiological techniques as a preface to the
experimental results of Chapters 2-7. Electrophysiology is
a powerful tool to probe the molecular events occurring at
specialized structures along excitable membranes. The
standard approach is to detect and measure bioelectrical
potentials--yet by far the majority of bioelectrical
potentials are so small that a sensitive apparatus is
required to detect them. For crustacean olfactory receptor
neurons (Panulirus argus) instruments which can detect a few
to 100 pA are required. Electrode is a general term for a
device that couples the biological preparation to electrical
instrumentation. For recording from lobster ORNs, I
fabricated electrodes from boralex silicate glass. These
electrodes were fire polished to a final tip diameter of

12
1 /xm to permit recordings from the small, 8-15 /xm diameter
cells. Good electrical contact must exist between electrode
and cell. For an unknown reason first discovered by Ling
and Gerard (1949), the glass of the electrode forms an
excellent seal with the lipids in the nerve cell-membrane so
that the current pulse, once converted to voltage by a
headstage current-to-voltage converter, is received at an
amplifier. The general function of an amplifier is to
increase the voltage of a bioelectrical signal so that it
can be displayed or further processed by a read-out device,
such as a cathode ray oscilloscope (CRO), a strip chart
recorder, or a computer.
On either side of an excitable membrane there are
varying concentrations of ionic species. The primary
species are Na+, K+, Ca2+, and Cl". This electrical and
chemical gradient, established by the semi-permeable nature
of the cell membrane, is defined as the potential
difference. Ionic flow across the cell membrane is confined
to specialized protein pores called channels (Figure 1-3).
Channels in lobster ORNs are "gated" or opened, as in other
neurons, in response to one of two applied stimuli:
(1) ligands such as odors, second messenger molecules, or
neurotransmitters and (2) voltage as an electrical signal or
stimuli. The movement of ions through these channels is
essentially a flow of charge (or a current), which can be
measured by a technique called voltage-clamping. In

Figure 1-3. (TOP) Ionic flow across the cell phospholipid
bilayer is confined to specialized protein pores called
channels that span the phospholipid bilayer. (MIDDLE)
Channels in lobster ORNs are "gated" or opened, as in other
neurons, in response to one of two applied stimuli:
(1) voltage or (2) ligands such as odors, second messenger
molecules, and neurotransmitters. Gap junction channels,
regions of close apposition between the cell membranes of
adjacent cells, which allow passage of larger molecules than
just ionic species, are also hypothesized in olfactory
neurons but undefined as yet in lobster ORNs. Voltage-gated
channels are comprised of 4 homogeneous domains, ligand¬
gated channels of 5 heterogeneous domains, and gap-junction
channels of 6 homogeneous domains. Curiously IP3-gated
channels (Chapter 4) fall into the family of ligand-gated
channels by definition of their gating nature, although
structurally these channels are more closely related to
voltage-gated channels. (BOTTOM) A two-dimensional view of
the channel protein demonstrates its main features: A pore
region through which the ions conduct current from the
external medium to the internal cytoplasm or vice-versa, a
selectivity filter which contains a region that preferences
a given ion over that of others based on molecular size and
fast on-off binding reactions, a gate, reputedly
hypothetical, which can be opened or closed in response, for
example, to conformational changes induced by electrical
charge migration, ligand binding, pH or temperature, and a
voltage sensor which is susceptible to movement of
electrical charges as a result of a change in membrane
potential.
Modified with permission from Hille, 1992.

14
ion flow
Voltage- Ligand¬
gated gated
Gap
junction
gate

15
voltage-clamp recording, a feedback amplifier measures the
difference between a set potential (command voltage) and the
potential generated by the stimulated cell. This voltage
differential is converted to a current and given back to the
ORN to maintain the potential that was fixed on the
amplifier (hence the term voltage-CLAMP; Figure 1-4). The
amount of current given back to the ORN is used as a measure
of what was received as the bioelectric signal from the
cell. In some instances, a single electrode operates as the
voltage sensing as well as current injecting device. In
larger cells, one can use two separate electrodes, one for
each function. Lobster ORNs are not big enough for the
placement of two electrodes, so the one electrode has to
flip-flop between the two functions.
The voltage-clamp method was first developed by Cole
(1949) and Hodgkin et al. (1952) for use with the famous
squid giant axon. The usefulness of the clamp stems from
the fact that it is much easier to obtain information about
currents measured from an area of membrane with a uniform,
controlled voltage, than when the voltage is changing freely
with time and between different regions of the membrane.
The voltage-clamp was modified by Neher and Sakmann (1976) ,
recent Noble laureates in medicine, for investigating the
basic properties of ion channels. In order to directly
measure the elementary events (ion flux through single
channels) they achieved a reduction in background noise by

Figure 1-4. On either side of a biological excitable
membrane there are ionic species of varying concentration,
the primary ionic species being Na", K+, Ca2+, and Cl'. The
electrical and chemical gradient established by the
relatively impermeable barrier determines the potential
difference, close to -60 mV in lobster ORNs. The voltage-
clamp measures the flow of charge through the protein pore
of a channel; the bioelectric signal - current. The
feedback amplifier (FBA) measures the difference between the
set potential difference (command voltage; Vc) and the
amount given by the stimulated cell as voltage. This given
amount of voltage is converted to a current increment which
is given back to the ORN to maintain the potential that was
fixed on the amplifier. In most experiments, I commonly
used a Vc = -60 mV to approximate the resting membrane
potential in lobster ORNs.

17
Voltage-Clamp

18
restricting the size of the recorded membrane to a small
10 iim2 patch. They then electrically isolated the membrane
patch from the rest of the cell by sealing the glass
micropipette tightly onto the membrane. This is how the
name patch-clamp recording was derived (Figure 1-5). It was
only by accident that they discovered slightly negative
pressure or suction created a molecular contact between the
pipette and plasma membrane, which improved seal resistance
into the gigaohm range (Neher et al., 1978) .
Horn and Patlak (1980), Hamill and Sakmann (1981), and
Hamill et al. (1981), discovered that patches could be
removed from cells by retracting the glass pipette to
achieve excised patches. Here patches are removed from
their natural environment, either with the internal (inside-
out configuration) or external side (outside-out
configuration) of the membrane facing the outside bath,
allowing optimal control of solution changes from either
face of the membrane. Although retaining the cell in a
cell-attached recording configuration permits measurements
of a process with the least disturbance of the intact cell,
sometimes more experimental control is required.
Alternatively, strong suction can be applied while the
pipette is still attached to the cell membrane to rupture
the patch and create a whole-cell configuration, whereby the
ionic milieu of the cell interior and the membrane potential

19
can be controlled, and the cell is left otherwise intact
(Figure 1-5).
I have used each of these pipette configurations in my
studies towards a different gain: (1) While in the cell-
attached configuration, a bath applied agonist that evokes
channel activity generally infers a cellular mechanism
requiring a second messenger molecule. (2) Membrane
impermeant probes can be readily applied to the cytoplasmic
face of the membrane in the inside-out configuration.
(3) While in the outside-out configuration, a ligand
applied to the bath that evokes channel activity strongly
suggests a directly gated channel mechanism. I also took
advantage of a modification of the inside-out configuration
(Chapter 5) called "patch cramming" as described by Kramer
(1990), where one takes an inside-out configured patch
containing a channel of interest and inserts the patch
pipette into a second, recipient cell. The channel activity
of the patch while inside the recipient cell can then be
used as a probe to detect changes in intracellular second
messenger production in a living cell. In order to acquire
baseline odor responsivity of a neuron, and to monitor
changes from unstimulated states, I chose to either voltage-
clamp single ORNs sequentially or to rely on the diffusional
properties of solutions over time by backfilling electrodes
with test solutions and tip-filling them with control
solutions (Chapters 4-5).

Figure 1-5. (TOP) Diagram of a neuron in an on-cell or
cell-attached configuration. This configuration permits
measurements of a natural process with the least disturbance
of the intact cell. Strong suction can be applied while the
pipette is still attached to the cell membrane to create a
whole-cell configuration, whereby the ionic milieu of the
cell interior and the membrane potential can now be
controlled by the investigator, and the cell is left
somewhat intact. The measured current is defined as a
macroscopic current, comprised of the total summation of all
ionic flux from the channels contained in the whole cell.
In lobster ORNs, an odor-evoked outward current is due to an
efflux of cations; an inhibitory odor response. An odor-
evoked inward current is due to influx of cations; an
excitatory odor response. (BOTTOM) Membranes can be removed
from neurons by retracting the glass pipette to achieve
excised patches. Patches can be removed from their natural
environment, either with the internal (inside-out
configuration) or external side (outside-out configuration)
of the membrane facing the outside bath, allowing optimal
control of the solutions bathing either face of the channel.
I commonly used the inside-out configuration (as shown) in
order to apply the membrane impermeant second messenger,
inositol 1,4,5-trisphosphate (IP3) to the inside face of a
channel. By convention, an ion passing into the ceil (out
of the patch pipette) is defined as an inward open channel
event (0) and is displayed as a downward deflection in
unitary current level away from the closed state (C). An
ion passing in the opposite direction is displayed as an
upward deflection in unitary current level and is described
as an outward open channel event
Modified with permission from Hille, 1992.

21
Patch Clamp
inside-out

22
The analysis of single channel events describes the
behavioral details of the passage of a single ion species
across the phospholipid bilayer of a neuron. An excellent
source for the recording and analysis of currents from
single ion channels is Wonderlin et al. (1990). Two
excellent reviews by the noble laureates that first recorded
unitary currents, provides information on the theory of
channel behavior, as discussed in the next three pages of my
dissertation, and can be found Rae and Cooper (1990), Neher
(1992b), and Sakmann (1992). When quantifying the behavior
of a single molecule, the channel protein, one generally
calculates (1) the magnitude, (2) duration, and (3) order of
channel events; all of which are random variables and none
of which can be inferred by observing raw data. The
information contained in channel events must come from a
measurement of their distributions; statistics. The current
thinking is that randomness of thermal motions underlies the
dwell time of a channel. It is postulated that the bonds of
the channel protein are vibrating, bending, and stretching
on a picosecond time scale to achieve an open or closed
conformation once an energy barrier is surmounted. The
probability of surmounting such energy barriers to an open
state (Propen) has been found to be dependent upon one or
more natural stimuli for the channel in question: membrane
potential, applied voltage; temperature; sensory inputs;
second messenger, hormone, neurotransmitter, or

23
intracellular ion concentration; or state of modulation,
phosphorylation.
The first random variable, event magnitude, is
typically calculated based on one of two distributions of
unitary current amplitude: a point-by-point amplitude
histogram or an event amplitude histogram. In the former,
all sampled channel events are binned into assigned current
levels, regardless of the state (open or closed) of the
channel. In the latter, a histogram is constructed
containing only amplitudes of events greater than 2.5 times
the rise time (2.5 trise) duration of the event. The event
amplitude is then measured by averaging the current level
within a window beginning = 1 trise after the opening and
ending 1 trise before the end of the event. This window
corresponds to the "flat" region of the event and excludes
regions that may be distorted due to the finite trise of the
recording instruments. In both types of distributions the
mean and variance of the current magnitude can be determined
by fitting the histogram with a Gaussian curve. An X-Y plot
of the mean current as a function of membrane potential
(voltage) can be used to generate the slope conductance of
the channel, a measure of the degree of ion permeation. The
greater the conductance of a channel, the faster the ions
are flowing through the pore of the channel. Generally, low
conductance channels have a higher degree of ionic

24
interaction with charged amino acids inside the channel pore
or between each other.
I chose to use the point-by-point amplitude histogram
in my analysis because the number of channels in a patch can
be detected with this distribution, Propen can be easily
calculated, and presence of voltage-dependence can be
determined. In a point-by-point amplitude histogram, the
number of peaks minus one is generally the number of
channels. If the peaks correspond to an integer function,
then one is likely to be recording multiple openings of
identical channels. If the peaks correspond to variable
amplitudes, then one is likely to be recording
heterogeneous multiple channels. The probability of
opening, Propen, is defined as the total time a channel
spends in the open state divided by the length of the
recording. The integration of the area under the peaks in
the amplitude histogram is used as an index of time. The
presence of voltage-dependent channel opening can be
determined by plotting Propen as a function of membrane
voltage. Deviation from a zero slope would indicate
voltage-dependent channel gating.
The second random variable is duration, the exponential
distribution of random dwell times. One measures the
average time (dwell) the channel spends in the open and
closed state. The mean open time (tQ) or mean closed time
(tc) is dependent upon the rate constant (in sec"1) leading

25
out of that state. The mean dwell times are thus defined as
a
tc = 1/a ana tQ = 1/S where C (closed) , 0 (open) .
/3
The movement between the 0 and C states requires a
differential to describe its behavior, called the
probability distribution function, which defines the
intervals between the random channel events. The
derivative, f(t), of this function is called a pdf or a
probability density function, which is the function used to
fit measured open or closed dwell times of a channel. Dwell
times are reported in the form of a histogram, where each
open or closed event is binned into a dwell time of a
certain duration, and the exponential fit (f(t)) of the
histogram provides the mean dwell time, usually reported in
msec. The number of exponential components required to fit
the distribution = the number of open or closed states of
the channel.
Most membrane patches contain multiple channels and
multiple states, which albeit complicates the analysis of
channel behavior, provides a lot of information about the
third random variable, order of channel events. Channel
bursting, latency to first opening, change in mode or
kinetic state, hibernation, subconductance, cooperativity,
and steady-state flickering are all examples of complex
channel behavior that can provide rich knowledge about the
biology of a channel.

26
Specific Aims
I addressed five major questions during the course of
my doctoral study. The resolution of each question helped
shape the subsequent avenue of investigation as the
following suggests, namely:
1. Can lobster (Panulirus argus) olfactory receptor neurons
be sustained in primary cell culture?
2. Are the voltage-activated and odor-activated properties
of the neurons altered by cell culture?
3. Do GTP-binding proteins (G-proteins) link binding of an
odorant receptor to the generation of an odor-evoked
current?
4. Are odor-evoked currents elicited by direct activation
of an odorant receptor by an appropriate odor molecule
or are currents evoked through a second messenger
cascade?
5. What is the primary charge carrier of the plasma
membrane IP3-gated channels mediating excitatory
transduction (inward currents) and can they be activated
by other metabolites in the inositol phospholipid
pathway?

CHAPTER 2
SUSTAINED PRIMARY CELL CULTURE
Introduction
Dissociated neurons in primary culture are providing
useful models for a growing number of neurobiological
studies (Benda et al., 1975; Sebben et al., 1990) .
Olfactory receptor neurons in many animals are long, thin
bipolar neurons that terminate distally in a highly branched
arbor of cilia or in an outer dendritic branch (Steinbrecht,
1969) that is suspected to be the site of chemosensory
transduction (Lowe and Gold, 1990). Studying the physiology
of transduction by directly patching the thin cilia or outer
dendritic branches has been possible (Nakamura and Gold,
1987, Hatt and Zufall, 1990), but is technically difficult.
In most instances, it is necessary to work with intact
cells. In amphibians, some of the olfactory receptor cells
contract when dissociated from the olfactory epithelium
(Firestein and Werblin, 1989), thereby facilitating
physiological analysis of transduction by allowing effective
NOTE: This chapter has been accepted for publication and is
reprinted with permission from Fadool, D.A., W.C. Michel,
and B.W. Ache. 1991. Sustained primary culture of lobster
(Panulirus argus) olfactory receptor neurons. Tissue & Cell
23(5) : 719-732.
27

28
space clamping to characterize odor-activated currents and
by allowing drugs introduced into the soma through the patch
electrode to diffuse to the cilia. In other animals,
however, dissociated receptor neurons retain their diffuse
morphology and are less amenable to physiological analysis
of transduction. In these instances, physiological analysis
of transduction would be facilitated if, by placing the
cells in culture, it were possible to obtain morphologically
more compact cells that still retained their responsiveness
to odors.
Lobster olfactory neurons have one of the longest
dendrite to soma distances reported for olfactory receptor
cells in any organism (ca. 1 mm; Grünert and Ache, 1988) .
In order to study the physiology of transduction in these
cells, it would be particularly useful if the cells could be
sustained in primary culture in a morphologically more
compact form. Most tissue culture protocols, however, have
been designed for mammalian systems (especially human, rat,
mouse, and rabbit). Although techniques for culturing
insect tissue are now commercially available, culturing
techniques for other invertebrate nervous tissues are less
well established. In particular, crustacean tissue culture
is just in its initial stages of development (Fainzilber et
al., 1989). Only recently have techniques for culturing
stomatogastric ganglion (Graf and Cooke, 1990; Krenz and
Fischer, 1990), peptidergic neurons (Cooke et al., 1989),

29
adipose tissue (Van Beek et al., 1987), proprioceptor organs
(Hartman et al., 1989), and ovarian tissues (Fainzilber et
al., 1989) been reported using crustacean species. The
above protocols reported widely varying conditions
suggesting that a systematic test of culture parameters may
be necessary for each crustacean species.
In order to better study the electrophysiological
properties of lobster olfactory receptor neurons, therefore,
I developed techniques to sustain the cells in primary
culture. In this chapter nine culture parameters are
described that were systematically tested to establish an in
vitro model which most closely approximated the osmolarity
and salt composition of the lobster's physiological fluid;
haemolymph; approximated known physical parameters, such as
temperature; and which included supplements known to have
effects on nerve outgrowth. In this chapter are reported
the conditions that allow cells harvested from lobster
olfactory organs (antennules) to survive in primary culture
for 23 days. Cultured cells are more compact than cells in
vivo, and most importantly, are electrically excitable and
odor sensitive; that is, they bear the physiological markers
of olfactory neurons. The potential these cultured cells
have for studying the physiological process of olfactory
transduction is discussed.

30
Methods
Animals
Specimens of the Caribbean spiny lobster, Panulirus
argus, were collected from the Florida Keys and maintained
in an open circulating sea water system. Animals were fed a
mixed diet of frozen fish, squid, and shrimp.
Tissue Preparation
The olfactory organ (distal third of the lateral
antennular filament) was excised from intermolt animals
(Figure 2-1, A-C) and washed in 10% Listerine in Panulirus
saline (PS, see solutions) containing 1% penicillin,
streptomycin sulfate, and amphotericin B as Fungizone
(Gibco; AbAm). The organ was cut into sections three annuli
long, hemisected, and transferred to fresh PS + AbAm.
Soma clusters of the olfactory (aesthetasc) receptor cells,
which literally fill the lumen of the organ, were removed
from the hemisection with a sterile 26 gauge syringe needle,
washed repetitively with PS + AbAm, and transferred to fresh
PS + AbAm. Repetitive washes were critical to eliminate
contamination, presumably from epiphytes on the exoskeleton.
The isolated soma clusters were then incubated for 50 min at
80 rpm on an orbital shaker in 0.2 micron filter-sterilized:
10 ml PS + AbAm containing 2.5 mg papain and 12 mg L-
cysteine.

Figure 2-1. Procedures for establishing lobster olfactory receptor neuron (ORN)
cultures. A) The olfactory organ is comprised of chemosensory sensilla that are
located on the distal third of the lateral antennular filament. B) The organ is
excised from the spiny lobster, and cut into 3 annuli section, one of which is shown
in cross section. Note that the clusters of olfactory receptor cell somata literally
fill the lumen of the antennule once the section is hemisected and axons removed.
C) Clusters of olfactory receptor cell somata are then isolated, proteolytically
treated, mechanically dissociated, and plated on substrate coated coverslips for cell
culture. D) ORNs on coverslips are removed from culture for electrophysiological
recording. The odors are delivered to the cells by "spritzing" from a 6-chambered
multibarrel pipette, connected by way of tycon tubing to a rotary valve in series
with a picospritzer to deliver a 120 msec pulse of odorant onto the ORN. AbAm =
antibiotic-antimycotic 1% solution, PS = Panulirus saline, FCS = fetal calf serum,
NGF 7s = nerve growth factor 7s, BME 100X = basal minimum essential vitamins.

A
hair tuft
timed
pressure
source
0
lateral tilament
B
/
guard hair
sensilla
axons
0:0
hemisection
C
isolated somata
PROTEOLYTIC DIGESTION:
2.5 mg papain
12.0 mg L-cysteine
10 ml P S. + AbAm
MECHANICAL
DISSOCIATION
''
(oooooo
oooooo
oooooo
[poooco
MEDIA:
L15 Leibovitz
Panulirus salts
L-glutamine
dextrose
gentamycin
FCS
NGF 7s
BME 100X
CONDITIONS:
poly-d-lysine
24°C
saturation humidity
1080 mOsm
U)
to

33
Cell Cultures
Proteolytic digestion was stopped by replacing the
enzyme solution with modified low glucose L-15 media (50 ml
L-15 Stock, 50 ml of 1.6X normal concentration of PS, 0.6 g
dextrose, 0.029 g L-glutamine, and 0.01% gentamicin). Cells
were dissociated by trituration using a sterile 23 gauge
needle and plated on poly-D-lysine hydrobromide (MW 49,300-
53,000) coated glass 12 mm coverslips (2.5-5.0 /xg/cm2) at a
cell density of 12 x 104 cells/ml (per 2 cm2 well) . Cells
were placed in the dark and allowed to adhere to the
coverslips for 2 hr without agitation. After this period,
6% fetal calf serum (FCS) or 10% lobster haemolymph was
added to each well. In experiments requiring haemolymph
supplementation, the blood extraction procedures of Fadool
et al., 1988 were followed. Haemolymph osmolarity was
measured in 200 /x 1 samples by freezing point depression
(Osmette #2007, Precision Systems, MA). Only one-half of
the media was changed every fourth day to allow accumulation
of any required neurotrophic factors. Cells were maintained
at saturation humidity in a modular incubator chamber
(Billups-Rothenberg) inside a low-range temperature
incubator (Hotpack) at 24°C.
Experimental Culture Conditions
Nine culture parameters were systematically varied over
22 cultures and their effect on yield and longevity, if any,
noted: (1) osmolarity (917 to 1079 mOsm), (2) temperature

34
(20 to 28°C) , (3) humidity (60% to complete saturation),
(4) HEPES buffering, (5) serum supplementation at 2, 12, or
24 hr after plating, (6) basic minimal essential (BME)
vitamin, L-glutamine, and nerve growth factor 7s (NGF-7s)
supplementation, (7) substrate (glass, plastic, poly-D-
lysine, laminin, collagen, or haemolymph clots), (8) length
of proteolytic digestion (20 to 60 min), and (9) duration of
animal holding (1 to 8 wk). Cell counts were made daily of
a permanently marked field of view in each well of a 24 well
plate.
The effect of supplementing olfactory neuron culture
media with media preconditioned with lobster brain tissue
was measured in a separate series of experiments. Either
entire brain or the olfactory and accessory neuropils were
isolated from the anterior region of cold anesthetized
lobsters. Tissues were rinsed in 3 volumes of PS + 5% AbAm
and then diced into fine pieces in a small volume of
modified L15 media. Whole tissue slices were portioned into
1 ml modified L15 media, supplemented with 40 /¿I of FCS, and
continually agitated at 50 RPM on an orbital shaker.
Conditioned media was collected after 24 and 60 hr and used
to supplement olfactory neuron cultures at 50% (50 L15
media: 50 conditioned media) and 30% (70 L15 media: 30
conditioned media) concentrations. In vitro brain tissue
fragments were maintained for 2 wk by changing one-half
media every third day and re-supplementing with 35 /¿I FCS.

35
Electrophvsioloqy
The cells were patch-clamped in the whole-cell
configuration, using an integrating patch-clamp amplifier
(Dagan 3900) . The signal was filtered with a low pass
Bessel filter at 5 kHz and digitally sampled during odor
stimulation every 4 msec. Acquisition and subsequent
storage and analysis of the data was done using pCLAMP
software (Axon Instruments). Neurons were viewed at 40X
magnification under Hoffman optics. Patch electrodes of
1.8 mm O.D. boralex glass were fire polished to a tip
diameter of approximately 1.0 ¿im (Bubble number 4.8; Mittman
et al., 1978) . High resistance seals (Re = 8.0 to 14 GQ)
were formed by applying gentle suction to the lumen of the
pipette. Odorants (pipette concentration 10'3M) were
delivered to the cells from a multibarrel glass micropipette
(Frederick haer & Co.) coupled to a pressurized valve system
(120 msec pulses; Picospritzer; General Valve Co.) via a 6-
way rotary valve (Figure 2-ID).
Solutions
All salts used in preparing Panulirus saline and
modified Liebowitz L15 Media were obtained from Sigma
Chemical Co. NGF-7s from mouse submaxillary glands was
obtained from Boehringer Mannheim. The patch electrode
solution consisted of (in mM) 30 NaCl, 11 EGTA, 10 HEPES,
1 CaCl2, 180 K-acetate, and 696 glucose; pH adjusted to 7.0
with 5N KOH. PS consisted of (in mM): 458 NaCl, 13.4 KCL,

36
9.8 MgCl2, 13.6 CaCl2, 13.6 Na2S04, 3 HEPES, and 2 glucose;
pH adjusted to 7.4 with IN NaOH. Modified L15 Media was
prepared as follows: 50 ml Liebowitz L15 Stock, 50 ml of
1.6X normal concentration of PS, 0.6g dextrose, 0.026 g L-
glutamine, 1% BME (basic minimal essential) vitamins
(Sigma), and 0.01% gentamicin. Solutions of substances
tested as odors were either (1) a 100-fold dilution of a
complex mixture prepared from TetraMarin (TET), a
commercially available flake fish food, made into an aqueous
extract by homogenization of 2g dry flakes into 60 ml
saline, followed by low speed centrifugation to remove
particulates then filtration with Whatman #3, or (2) 1CT3M
solutions of taurine, betaine, ascorbic acid, proline,
glycine, cysteine, AMP, TMAO, or arginine, prepared daily in
the modified L15 media. Odor concentrations are reported as
the pipette concentration and not the absolute test
concentration reaching the cell. The absolute concentration
was estimated to be 91.5% of the pipette concentration,
based on a method for determining stimulus concentration
using the steady-state K+ permeability of neurons (Firestein
and Werblin, 1989).
Results
Optimal Culture Conditions
Optimal culture conditions were determined based upon
measurement of cell survival; time in days until 1/2 the
original plating density. Cells survived better at

37
saturation humidity than at 60 and 80% humidity, where cell
densities were reduced 1 wk after plating by 38 and 21
percent, respectively, in comparison with 100% saturated
controls. Of the three temperatures tested (20, 24, and
28°C), the cells survived longest at 24°C (Figure 2-2A).
Survival at 24°C was significantly longer than at the next
best temperature (20°C) .
Cells did not adhere to untreated glass coverslips,
collagen or laminin substrates, and adhered with little
neurite outgrowth to commercial plastic tissue culture wells
or dishes (Corning and Falcon #1008, respectively). Cells
that did not adhere to an appropriate substrate, or as a
population commence neurite extension, died within 36 hr.
Cells sprouted processes with optimal survival when plated
on poly-D-lysine coated glass coverslips or when grown on
haemolymph clots (Figure 2-2B). Cells that adhered to
poly-D-lysine substrate or clots did so immediately after
plating in the absence of FCS. Process outgrowth and/or
extension was observed as early as 10 min after plating.
Cells did not adhere uniformly to the substrate when FCS was
provided upon plating (vs. 2 to 12 hr later) nor when cells
were kept under room light or continually agitated.
Cells survived equally well between 965 and 1082 mOsm,
the highest of the four osmotic conditions tested. Survival
was reduced significantly at 920 mOsm, the lowest of the
four osmotic strengths tested (Figure 2-2C). Actual

Figure 2-2. Influence of A) temperature, B) substrate, C) osmolarity, and D)
supplementation on ORN longevity. Plotted data are mean survival of neurons (days ±
SEM) for 24 wells of at least 2 cultures per condition. Supplementation conditions
referred to in D) are: Experiment I - (1) FCS at 2hr + Nerve Growth Factor (NGF
7s), (2) FCS at 2 hr - NGF 7s, (3) FCS at 12 hr - NGF 7s, (4) FCS at 24 hr - NGF 7s.
Cells were maintained at 24°C in a humidified incubator; Experiment II - (5) + 3mM
HEPES, (6) - 3mM HEPES. Both treatments were tested in the presence of FCS. Cells
were maintained at 20°C at less than saturation humidity; Experiment III - (7) cells
cultured from animals held greater than 8 wk, (8) held less than 3 wk, (9) held less
than 3 wk + BME vitamin supplemented. Cells were maintained at 24°C at less than
saturation humidity. ORN = olfactory receptor neuron, FCS = fetal calf serum.

Temperature (°C)
1\2 Plating Density (Days)

1\2 Plating Density
C/)
03
Q
12
11-
10
9
8-
7-
6
5-
4-
3-
2-
1-
o-J
glass
plastic
Substrate
O

1\2 Plating Density (Days)
O
w
3
CD
Q.
03'
3
O
C/3
3
It’

1\2 Plating Density (Days)
Zb

43
haemolymph osmolarity of intermolt animals was found to be
1079 ± 37.5 mOsm (SEM, N=4). Cells demonstrated a marked
requirement for BME vitamins, L-glutamine, and FCS
supplementation (Figure 2-2D). Cell survival was maximal
when FCS was supplied 2 hr after plating (Figure 2-2D -
1,2). Cell density was reduced by one-half when cells were
deprived of FCS for as short as 12 hr (Figure 2-2D - 3); few
to no cells survived 24 hr of deprivation (Figure 2-2D - 4).
The addition or omission of HEPES (Figure 2-2D - 5,6) or
NGF-7s (Figure 2-2D - 1,2) had no gross effect on either
longevity or neurite outgrowth. When antennular tissue was
taken from animals held 8 wk or longer as opposed to animals
held no more than 3 wk (Figure 2-2D - 7,8), culture plating
density dropped by one-third within the first hours of
plating, with few cells extending processes. BME vitamin
supplementation increased cell survival when using animals
held less than 3 wk (Figure 2-2D - 9).
Cells could be maintained in culture up to 23 days,
using the derived optimal conditions. Media preconditioned
with CNS tissue, added in amounts up to 30 or 50% of normal
olfactory neuron culture media, had no significant effect on
longevity of olfactory neuron cultures compared to control,
non-conditioned cell cultures in the observed 23 days. This
was true whether the media was conditioned for 24 or 60 hr,
or whether it was derived from either whole brain, or the
isolated olfactory and accessory neuropils.

44
Morphology of Cell Types and Neurite Outgrowth
Five morphologically distinct types of "neuron-like"
cells could be defined based on the number and type of
processes: (1) soma only, (2) bud, (3) unipolar,
(4) bipolar, and (5) multiprocess (Figure 2-3). Soma only
cells were spherical in shape and underwent no apparent
process formation. Bud cells possessed a single short
process, less than 5 /xm long, typically 3-4 /xm wide, which
did not branch. Unipolar cells bore a single long process,
greater than 75 /xm in length, often unbranched and thin.
Bipolar cells always possessed two equal length processes;
the majority of which were unbranched. Multiprocess cells
produced three to five processes that were prominently
arborous and of nonuniform length and width. In all
instances, soma diameters ranged from 8 to 16 /xm with
processes, when present, between 3 and 160 /xm long. In all
cultures, larger, "non-neuron-like" cells with somata
greater than 20 /xm in diameter were also observed and
comprised approximately 5% of a given cultured population.
The larger cells were either fusiform or flat (Figure 2-3).
These larger cells could be selectively removed by
withholding FCS supplementation for 12-24 hr after plating.
Electrophvsiology
All smaller cells (8-16 /xm) tested were electrically
excitable; voltage elicited a transient inward current
followed by a sustained outward macroscopic current, which

Figure 2-3. The morphology of cells observed in lobster
olfactory cultures. (A-E) "Neuron-like" cell types,
8-16 /xm in diameter; A) soma only, B) bud, C) bipolar,
D) multiprocess, and E) unipolar. (F-G) "Non neuron-like"
cell types, greater than 20 ¿im in diameter; F) fusiform and
G) flat. Magnification 780X.


activated concurrently at ca -30 mV (Figure 2-4). In a
population of 50 cells the average peak amplitude of the
voltage-activated inward and outward macroscopic currents,
when the cells were stepped from -60 (rest) to +30 mV, was
-322.3 ± 27.9 pA (SEM) and 756.9 ± 30.0 pA (SEM)
respectively. The ionic basis for these conductances was
not determined. In contrast to the smaller diameter cells,
the larger (> 20 /xm) cells were not measurably electrically
excitable, and had ohmic current\voltage relationships over
the range of membrane potentials tested from -60 mV to
+30 mV (n= 11). This dichotomy between the smaller and
larger cells was mirrored in the sensitivity of the two
types of cells to odors. None of the large cells tested
responded to applied odors (n= 14), whether the odor was a
single compound or the complex mixture (TET). In contrast,
28 of 50 (56%) smaller cells, each tested with one of a
variety of single compounds (taurine, betaine, ascorbic
acid, proline, glycine, cysteine, AMP, TMAO, or arginine)
responded with either an inward or outward current (Figure
2-5). The collective cell population did not all respond to
the same single compound. The latency of the odor-evoked
current was phase locked to the application of the odor
stimulus. The half-peak duration of the odor-evoked current
was approximately 3 to 4 s. Although dose-response
relationship was not formally tested the amplitude of the
current increased with concentration over the range of 1CT6

Figure 2-4. Voltage-activated currents in a cultured ORN.
A) Whole-cell macroscopic currents under voltage-clamp
conditions. The cell was held at -60 mV and stepped to
+30 mV in 10 mV episodes. B) Note that the IV plot of the
cell displays the conductances which characteristically
activated around -30 mV ( + = inward current, â–¡ = outward
current). All cells with "neuron-like" morphology showed
similar IV relationships.

49
A
B

Figure 2-5. Response of cultured ORNs to A) a 1000X
dilution of TET extract and B) 10"3 M glycine. Control
trace for each cell shows the response to a L15 media blank.
Holding potential = -60 mV. Arrow = start of 120 msec
odorant pulse.

51
A
â–¼
40 pA
1— 750 ms

52
to 10'2M. An odor-evoked current could be distinguished
from background noise at a 10'8M odorant concentration in
one cell tested for threshold. Eight out of ten cells
tested (80%) with three to five odors, responded to at least
one odorant. None of these cells responded to all five
compounds; typically they responded to only one or two.
When cells responded to more than one compound, the
magnitude of the response varied across the effective tested
compounds and also between different cells tested. In no
instance did identical application of only culture media
from one barrel of the stimulus delivery pipette elicit any
measurable current. Similar control (flat) traces were
recorded when the picospritzer pressure was turned off, and
continuous cell baseline current was monitored.
Discussion
Under optimal conditions of temperature; humidity;
osmolarity; appropriate substrate; serum, sugar, vitamin,
and amino acid supplementation, olfactory neurons could be
sustained for 23 days in primary culture, with continual
neurite outgrowth during this period. The culture consisted
of two fundamentally different types of cells that could be
initially classified according the soma diameter. Based
upon their selective responsiveness to odors and current,
the smaller diameter cells (8 to 16 /¿m) would appear to be
neurons. Since the lobster chemosensory organ is almost
exclusively comprised of olfactory receptor cells (Grünert

53
and Ache, 1988), the cultured system is developed from an
enormously enriched starting population of olfactory
neurons, with proportionately few neurons of other types
(i.e. non-olfactory chemoreceptors and mechanoreceptors).
Given an average of 350 receptor neurons per soma cluster
and an average of 15 soma clusters per annulus (Grünert and
Ache, 1988), the 90 annuli used per culture should yield
472,500 neurons. Based upon hemocytometer counts, I
estimate that 288,000 neurons are harvested and plated per
culture, which indicates an approximate total yield of 61
percent. Some loss would be expected from mechanical
dissociation, pipette transfer, initial AbAm washes, and
removal of soma clusters from connective tissue adjoining
the cuticle during isolation. Based upon the yield alone,
it is plausible to conclude that a large percentage of the
small cells are olfactory neurons. Further evidence,
however, is required to definitively prove that these cells
originate from the olfactory (aesthetasc) sensilla.
Biologically relevant odors for aquatic animals are
small molecules such as the amino acids used in this study
(Carr et al., 1984) . Since many of these molecules also
have broad biological activity on cells (Carr et al., 1984),
a systematic study of odor responsiveness of cultured cells
as it compares to that of cells in situ is necessary in
order not to falsely confuse broad action of these molecules
with their actions as odorants. This initial test of odor

54
sensitivity in cultured cells does demonstrate a degree of
selectivity. Both types of odor-evoked currents are
recorded in culture and cells display odor sensitivity to a
wide range of aquatic stimuli; single odorants as well as a
complex mixture. Although onset latency and duration of
response cannot be strictly compared between cultured cells
and that of intact non-cultured cells, due to the different
odorant delivery systems required for each type of
recording, odor response kinetics in each system are
qualitatively similar (Michel, unpublished data). The
cultured cells also display discrimination properties
exemplary of olfactory neurons in as much as the magnitude
of evoked current is concentration dependent and currents
are not evoked by every stimuli that is presented. The
percentage of total cells responding to a single presented
odorant is greater than half; a percentage which increases
as cells are sequentially presented with a variety of
odorants or with a complex mixture. This degree of
selectivity to odors would be expected if the substances
were activating odor receptors and not receptors for some
more generalized function, such as modulation of
sensitivity. These data suggest that each cell is
responding to specific odors and that the likelihood of
applying the appropriate stimuli increases when cells are
screened with a number of compounds; three to five as
opposed to just one. I tentatively conclude that the

55
"neuron-like" cells are not only olfactory, but that they
retain their ability to respond to odor compounds
selectively.
It is somewhat surprising that only poly-D-lysine
provided a suitable substrate for neurite outgrowth of the
olfactory neurons. Poly-D-lysine was found to be inferior
to Con A or uncoated plastic (Falcon 3001) and inferior to
uncoated Primaria dishes (Falcon 3801) or polyornithine for
culturing chicken (Gonzales et al., 1985) and insect (Stengl
and Hildebrand, 1990) olfactory receptor cells,
respectively. Neonatal rat olfactory cultures maintained on
poly-D-lysine also displayed poor plating efficiency
(Ronnett et al., 1991). Krenz and Fischer (1988), found
only 40 to 50% survival of crayfish stomatogastric ganglia
neurons when plated onto poly-D-lysine. Graf and Cooke
(1990), however, did find extensive neurite outgrowth of
stomatogastric neurons from lobster or crab when plated onto
poly-D-lysine, but they also found cell attachment to
uncoated Primaria dishes (Falcon 3801) . In my cultures,
albeit from the same family (lobster), plastic or uncoated
glass substrates prevented attachment of cells and resulted
in cell death. One could argue that neurons from sensory
organs may have similar substrate requirements. Little
neurite outgrowth, however, is seen in retinal ganglion cell
cultures plated onto poly-D-lysine, while extensive
networking processes develop on substrates of laminin and

56
fibroblasts (Drazba and Lemmon, 1990). While minimal
process outgrowth was observed in neonatal rat olfactory-
neurons plated on poly-D-lysine, significant neurite
outgrowth was obtained when laminin was applied to poly¬
ornithine treated slides (Ronnett et al., 1991). Although,
in my cultures, laminin provided the highest initial plating
density in terms of survival of total number of cells at
2 hr after plating, cells failed to adhere to this substrate
and subsequently died. My observation that cells died
within 36 hr if they did not adhere to an appropriate
substrate and, as a population, extend neurites, is
consistent with Cooke et al. (1989) findings that cultured
crab or lobster peptidergic neurons had to adhere to a
substrate for support and outgrowth. Thus, the optimal
substratum for neuron survival and process outgrowth is not
necessarily genus specific, may be dependent on type of
nervous tissue, and may even vary among neurons cultured
exclusively from different sensory organs.
My observation that supplementation 2 hr after plating
yields even greater cell densities than if supplied
immediately upon plating, suggests that an initial period of
neuronal-substrate contact without FCS may be important,
perhaps to prevent FCS-induced aggregate formation. This
finding is in accordance with Sebben et al. (1990) who find
that both the amount and timing of serum introduction are
critical factors in long-term primary cultures. They report

57
non-neuronal cell death during FCS deprivation; in their
work, up to 72 hours after plating. Although, in my system,
selective mortality of the non-neuronal cells could be
achieved only after 24 hours, it was not necessary to
achieve my goals with lobster antennular cultures. The
larger non-neuronal cells only comprised about 5% of a given
culture, did not proliferate to confluency to affect
survival of the smaller, neuronal cell types, nor did their
presence interfere with my electrophysiological
measurements.
While clotted lobster serum (haemolymph) provided an
excellent substrate in terms of survival and neurite
extension, it was not conducive to electrical recordings.
Serum and, presumably, proteins in the haemolymph substratum
interfered with the formation of high resistant gigaohm
seals required for patch-clamp recordings. In contrast to
cells cultured with FCS, which could be rinsed with serum-
free L15 media prior to recording to alleviate this
difficulty, haemolymph could never be sufficiently washed
from cell surfaces.
Many sensory neurons have the biological requirement
for nerve growth factor (NGF) which regulates survival,
development, and maintenance of these neurons in vertebrate
systems (Johnson et al., 1986; Lindsay et al., 1990; and
Paves et al., 1990) . The fact that NGF-7s had no
significant effect on longevity or neurite outgrowth could

58
be explained by the fact that the cells were always serum
supplemented. FCS has many undetermined neurotrophic
factors which could sustain olfactory neurons in culture.
Hence, any additional effect (increased longevity, neurite
outgrowth, or electrical excitability) from NGF-7s might
have been masked by factors contained in the FCS.
There are many conceivable reasons why cultures derived
from tissues of recently captured animals survived up to
twice as long as those derived from animals held in
captivity for 8 weeks or more. The animals may not have
been as healthy as those in their natural environment, even
though food quality, water turnover, and cleanliness of
aquaria were carefully monitored. Certainly, other
researchers studying various aspects of olfaction (as well
as those in my home laboratory) have discovered
desensitization of neurons in catfish and salamander when
animals were held for as little as 2 weeks (Caprio and
Firestein, pers. comm.).
While the remaining tested parameters collectively
improved longevity, individually, no single factor appeared
pivotal for cell survival. The optimal osmolarities (965,
989, and 1082 mOsm) were congruous with the measured
osmolarity (1079 mOsm) of haemolymph. The temperature which
gave the greatest cell survival (24°C) , was lower than the
reported optimal environmental temperature for this species
(29-30°C; Lellis and Russel, 1990). Since the above cited

59
study was based largely on behavioral repertoire, frequency
of molting, and growth rate; and the variation found between
tested temperatures using these indices was minimal (95% to
99% survival within a temperature range of 25 to 29°C,
respectively), the actual temperature optima for
physiological processes in culture may not precisely
coincide. The requirement for high humidity may have acted
indirectly by preventing changes in osmolarity, although at
less than saturated humidity evaporative losses were
minimal. Measured osmolarity change in media over any one
week in time was no more than 20-30 mOsm at 60-80%
saturation. There appeared, then, to be a broad range of
suboptimal conditions (Figure 2-2) which were suitable for
cell maintenance but not for any apparent new growth
(indexed by neurite extension).
In one of the two other reports in which dissociated
crustacean neurons were cultured (Cooke et al., 1989; Graf
and Cooke, 1990), outgrowth was observed in a simple medium
of only physiological saline and glucose. Although both my
work and theirs used cells from congeneric lobsters, the
type of neurons cultured in each study differed. Secretory
neurons from the X-organ-sinus gland (Cooke et al., 1989)
may be able to use existing membrane from stored granules to
actively synthesize proteins or regulate transport
mechanisms for immediate neurite outgrowth. Lobster
olfactory neurons, in contrast, while not requiring

60
preconditioned media, had a strong dependence (among other
noted factors) on neurotrophic factors supplied in serum or
haemolymph. The regenerative nature of olfactory neurons,
as primary receptor neurons, may require the strict presence
of specific physiological factors for continual neurite
outgrowth, and subsequent viability in culture. My findings
appear to be more analogous to the other reported work on
crustacean neuron cultures, that of Krentz et al. (1990) who
also found serum supplementation (5-10% FCS) necessary for
sustained growth.
Although appropriate target organs have been known to
influence proliferation and direction of neurite outgrowth
in culture (Coughlin, 1975; Pollack et al., 1981), this
appears not to be the case for growing olfactory neurons of
rat and chicken (Gonzales et al., 1985), or of the lobster,
since lobster olfactory neuron cultures can be sustained
without the presence of the lobes and preconditioned media
had no affect on viability or neurite outgrowth. This
finding is in contrast to cultured insect olfactory receptor
neurons that have been reported to fail within 2d in the
absence of conditioned media (from either non-neuronal
antennal cells, extracellular fluid from antennae, or from
the hormone 20-hydroxyecdysone) (Stengl and Hildebrand,
1990). It is possible that the insect olfactory neurons
have more rigid culture requirements than that of rat,
chicken, or lobster, since the latter systems were developed

61
from differentiated adult cells and not embryonic cells, as
was the case for the insect. In intact organ cultures or in
large transplants of olfactory organs (Morrison and Monti
Graziadei, 1983), however, appropriate targets may be
influential in promoting olfactory neuron growth. Perhaps,
the culture systems of the rat, chicken, and lobster may
have not allowed sufficient cell-cell interaction for
targets to be effective, since they were all plated at low
density (range 3 x 105 to 1 x 106 cells/ml).
Two recent culture systems of rat olfactory receptor
cells, one a continual clonal cell line from olfactory
epithelium (Coon et al., 1989) and another of isolated
olfactory neurons (Ronnett et al., 1991) show increased cAMP
levels in response to odor stimulation, but being based on
biochemistry and not electrophysiological recordings, did
not give information on the odor specificity of single
cells. The high survival, electrical excitability, and odor
responsiveness that I find in cultures of single lobster
olfactory receptor cells has also been reported in monolayer
cultures of isolated rat olfactory tissue (Pixley and Pun,
1990). Identical to cultured lobster olfactory neurons,
neurons from rat evoke a fast inward current, followed by an
outward current, when depolarized to -30 mV. Upon
application of odorant mixtures (single compounds were not
tested), rat olfactory neurons produced only inward current
under voltage-clamp conditions (Pixley and Pun, 1990) . In

62
contrast, cultured lobster olfactory neurons produced either
outward or inward currents in response to odorant mixtures
and to single compounds; a finding that presumably
corresponds to the previously noted depolarizing and
hyperpolarizing receptor potentials in lobster olfactory
receptor cells in situ (McClintock and Ache, 1989b). This
may imply a functional difference between lobster and rat
olfactory cells in their ability to produce currents of both
polarities, but it may also reflect the limited set of odors
tested on the rat olfactory neurons. Odor-evoked decreases
in action potential frequency that may reflect an underlying
outward current have been reported in olfactory receptor
cells in another vertebrate (Dionne, 1990).
Panulirus argus olfactory receptor neurons can now be
considered among a small, yet growing number of culturable
crustacean cells. More importantly, they join a
phylogenetically diverse group of olfactory receptor neurons
that can maintain odor sensitivity in culture. Since
lobster olfactory receptor cells in culture are
morphologically compact, it should be possible to obtain an
effective space-clamp for recording odor-activated currents;
drug introduction and incubation periods can be simplified
and extended, respectively. Moreover, the cells are
directly accessible for electrophysiological recordings. In
light of these technical benefits, the cultured system

63
should provide a useful model for future studies of
olfactory transduction as exemplified in Chapters 3 to 7.

CHAPTER 3
ODOR SENSITIVITY IS NOT DEPENDENT ON PROCESS FORMATION
Introduction
The small size and thin, elongated morphology of
olfactory receptor neurons (ORNs) was long an impediment to
understanding olfactory transduction. The advent of patch-
clamp recording ameliorated this situation and facilitated
progress toward understanding olfactory transduction
(reviews: Anholt, 1991; Firestein, 1991). Central to this
effort has been the ability to dissociate ORNs from their
surrounding epithelium in order to study them directly or in
sustained primary culture. Dissociated ORNs are not only
accessible for patching, they often assume a more compact
form than their counterparts in situ that allows a
reasonable space clamp and facilitates diffusion of membrane
impermeant probes from the electrode to the site of
transduction. While transduction is thought to occur in the
cilia (outer dendrites, in invertebrates) of the ORNs (e.g.,
Kurahashi, 1989; Firestein et al., 1990; Lowe and Gold,
NOTE: This chapter has been accepted for publication and is
reprinted with permission from Fadool, D.A., W.C. Michel,
and B.W. Ache. 1993. Odor sensitivity of lobster olfactory
receptor neurons is independent of process formation. J.
exp. Biol. 174: 215-233.
64

65
1991), at least some elements of the transduction cascade
are not confined to the cilia. Specifically, cAMP-gated
cation channels that are the effectors in the transduction
cascade in amphibian ORNs also occur on the dendrite and
soma of the cells, although in lower densities than on the
cilia (Firestein et al., 1991; Zufall et al., 1991a).
Indeed, this variability in density was exploited to obtain
favorable channel density for recording (ibid.).
Previously, in Chapter 2, preliminary evidence was
reported that cultured lobster ORNs respond to odors
independently of whether the cells had sprouted processes.
This observation raises the interesting possibility that, in
vitro, all elements of the transduction cascade may be
expressed and inserted into the soma of ORNs prior to or
independent of process formation. Given the ease of
patching the soma compared to the extremely thin cilia
(outer dendrites) and the ability to culture ORNs in
vertebrates (Coon et al., 1989; Pixley and Pun, 1990; Calof
and Chikaraishi, 1991; Ronnett et al., 1991) and other
invertebrates (Stengl et al., 1989; Zufall et al., 1991b),
such a phenomenon could be of general utility for studying
olfactory transduction.
Without the normal polarity of the cell, however, it
must be established that applied "odors" are activating what
would otherwise be ciliary (dendritic) chemoreceptors. The
need to establish the adequacy of odor stimuli is

66
particularly important when studying ORNs from aquatic
animals such as fish and lobsters. Adequate olfactory
stimuli for many aquatic animals are the blood-born
components of prey, compounds such as amino acids, amines
and nucleotides (review: Carr, 1990). These types of
compounds could be expected to activate cells as
neurotransmitters or neuromodulators. In order to establish
the utility of cultured lobster ORNs for analysis of
transduction mechanisms, I will in this chapter, provide
functional evidence that cultured lobster ORNs with no or
varying numbers of processes are morphologies of the same
type of cell and that the odor-evoked properties of the
cultured cells reflect those of lobster ORNs in situ.
Methods
Tissue Culture
The distinct clusters of the ORNs were dissected from
the aesthetasc (olfactory) sensilla on the lateral
antennular filament (olfactory organ) of adult specimens of
the Caribbean spiny lobster, Panulirus argus. The clusters
were enzymatically dissociated, and the resulting cells
sustained in primary culture as described previously
(Chapter 2). Briefly, the isolated clusters were incubated
for 50 min at 80 rpm on an orbital shaker in 0.2 micron
filter-sterilized solution of 2.5 mg papain and 12 mg L-
cysteine in 10 ml Panulirus saline (PS) containing 1%
penicillin, streptomycin sulfate, and amphotericin B

67
(Gibco). Proteolytic digestion was stopped by replacing the
enzyme solution with low glucose L-15 media supplemented
with L-glutamine, dextrose, fetal calf serum, and BME
vitamins. Cells were immediately plated on poly-d-lysine-
coated glass coverslips. Cells were maintained at
saturation humidity in a modular incubator chamber (Billups-
Rothenberg) at 24°C. Neurite outgrowth in individual cells
was recorded on a TL Panasonic 6050 time-lapse video
cassette recorder. Images were later captured and
subsequently analyzed using Image 1 analysis software.
Electrophvsioloqy
Voltage- and odor-activated currents were recorded in
the whole-cell configuration with an integrating patch-clamp
amplifier (Dagan 3900). The analog signal was filtered at
5 kHz and digitally sampled every 4 msec. Data acquisition
and subsequent storage and analysis of the digitized records
were done with pCLAMP software (Axon Instruments). Cells
were viewed at 40X magnification with Hoffman optics. Patch
electrodes, pulled from 1.8 mm O.D. borosilicate glass, were
fire polished to a tip diameter of approximately 1.0 /xm
(Bubble number 4.8; Mittman et al., 1987). High resistance
seals (8.0 to 14 GQ) were formed by applying gentle suction
to the lumen of the pipette upon contact with the cell. In
all experiments, cells were voltage-clamped at a holding
membrane potential of -60 mV. Membrane resistance changes
were determined by injecting current sufficient to elicit

68
30 mV, 300ms hyperpolarizing voltage steps into the cells
from the holding potential. Each cell was photographed to
allow correlation of soma size, length of process, and cell
morphology with physiological properties.
Odor Stimulation
Odors were "spritzed" on the cells for 120 msec from a
7 barrel (6 barrels used) glass micropipette (Frederick
haer) coupled to a pressurized valve system (Picospritzer,
General Valve). In most trials, one randomly selected
barrel was filled with fluorescein to permit positioning of
the tip of the pipette relative to the cell and to assure
that the delivered odorant completely surrounded the cell
and its associated processes. The magnitude of the response
to odors was determined to be independent of which of the
six barrels contained the odor. Dilution of the odor
between the pipette and the cell surface, an average
distance of two cell diameters, was estimated to be
approximately 9%, based on the calculated K+ permeability
method of Firestein and Werblin (1989). Odor concentrations
are reported as the pipette concentration and are not
corrected for this dilution.
The odors used were solutions of: (1) an equimolar
mixture that included (10"3M): betaine, glycine, lactic
acid, taurine, and trimethylamine oxide, referred to as S-l;
(2) an aqueous extract of TetraMarin, a commercially
available fish food, prepared as described earlier

69
(Schmiedel-Jakob et al., 1990) and diluted 1000 fold,
referred to as TET; and (3) single substances known to be
effective odors for the lobster, which included (10'3M) :
adenosine monophosphate (AMP), arginine, ascorbic acid,
betaine, cysteine, glycine, histamine, proline, taurine, and
trimethylamine oxide (TMAO). All odorant solutions were
prepared daily in modified L15 media and applied at the
stated concentration, unless otherwise noted.
The number of different odors that stimulated a given
cell (the response spectrum) was quantified using the
breadth of responsiveness metric of Smith and Travers
(1979). Here, the breadth of responsiveness (H) is defined
as _n_
H = "K x PiL°5Pi
i = 0
where K = a proportionality constant, n = the number of
odors tested, pt = |pA| the absolute current (pA) elicited
from the nch odor and expressed as a proportion of total pA
elicited from all odors.
Solutions
Panulirus saline (PS) consisted of (in mM) 458 NaCl,
13.4 KCL, 9.8 MgCl2, 13.6 CaCl2, 13.6 Na2S04, 3 HEPES, and 2
glucose; pH 7.4. Modified L15 Media consisted of 50 ml
Liebowitz L15 Stock, 50 ml of 1.6X normal concentration of
PS, 0.6g dextrose, 0.026 g L-glutamine, and 0.01% gentami¬
cin. The patch electrode solution consisted of (in mM)

70
30 NaCl, 11 EGTA, 10 HEPES, 1 CaCl2/ 180 K-acetate, and 696
glucose; pH 7.0. All salts were obtained from Sigma.
Results
Morphology: Neurite Outgrowth
The cultures consisted largely of the five
morphological types of small (8-16 nm diameter soma)
"neuron-like" cells, described previously (Fadool et al.,
1991b): (1) soma only, (2) soma with bud, (3) unipolar,
(4) bipolar, or (5) multiprocess, four of which were used in
the present chapter (Figure 3-1A). The processes ranged
from 3 to 160 /¿m long. Each of the four morphological types
were present as early as two hours post-plating. Initially,
the predominant form was "soma only", but the proportion of
each morphological type changed over time; the relative
proportion of cells lacking processes decreased, with a
concomitant increase in the proportion of cells with
processes (Figure 3-IB). To distinguish whether the change
in the relative proportion of the morphological types
reflected selective loss of cells lacking processes or
process proliferation, or both, 218 "soma only" cells were
followed individually with digital time-lapse imaging for
three consecutive days starting 2 hr after plating.
Forty-two (20%) of the cells died within the observation
period, indicating that selective loss of "soma only"
contributed to the change in the relative proportion of the
cell types. While many of the 176 cells that persisted

Figure 3-1. A) Light micrographs of four morphs of cultured
lobster ORNs observed under Hoffman modulation contrast
optics, (a) soma only, (b) unipolar, (c) bipolar,
(d) multipolar. Magnification 780X. B) Changes in the
proportion of the four morphs over 9 days in culture. Each
data point represents the incidence of that morph in 10
random fields of view, expressed as a percentage of all four
types.

72

73
failed to sprout processes, 61 of the cells sprouted
processes throughout the observation period, becoming uni-,
bi- and, eventually, multipolar. The latter finding
supports my contention that the four morphological types of
"neuron-like" cells were morphs of a single type of cell.
Physiology: Electrical Properties
That the four types of "neuron-like" cells were morphs
of a single type of cell was supported by the finding that
268 cells tested for their current-voltage relationship had
similar voltage-activated properties. The total membrane
current evoked in a typical cell by depolarizing voltage-
steps consisted of a transient inward current (s 570 pisec
duration) that activated around -30 mV, followed by a much
larger, prolonged outward current that activated around
-20 mV and persisted with little decay throughout the
15 msec duration of the pulse (Figure 3-2A). The magnitude
of the inward and outward currents was independent of the
number of processes on the cell (ANOVA, p = 0.47 - inward
currents and p = 0.71 - outward currents) (Figure 3-2B). A
subsample of 12 cells, including at least one cell of each
morphological type, had a mean input resistance (RN) at rest
of 1.1 ± 0.2 GQ and a membrane time constant (r0) of 67.3 ±
11.3 msec. No detectable equalizing time constant could be
measured in any of the morphs, including morphs with
multiple processes.

Figure 3-2. Total voltage-activated currents of cultured
lobster ORNs.
A) Representative current-voltage relationship of one cell.
â–  = inward current, + = outward current. Inset:
Macroscopic currents (upper traces) evoked by depolarizing
voltage-steps (lower traces) when the cell was held at
-60 mV and stepped to +30 mV in 10 mV episodes. Records are
not leak subtracted. B) Plot of the maximum amplitude
(mean ± SEM) of the inward (striped bars) and outward (solid
bars) currents of 268 cells grouped according to number of
processes (n=68 soma only, 76 unipolar, 69 bipolar, 55
multiprocess).

Current Magnitude (pA) CD Current (pA)
A
75
-900
Soma Unipolar Bipolar Multiprocess

76
Physiology: Response to Odors
472 cells (including cells tested above for their
electrical properties) were tested for their ability to
generate a current in response to stimulation with one to
five different odors. The odor arrays usually, but not
always, included the complex mixture, TET. Sixty-four
percent of the cells tested responded to at least one odor.
This percentage increased to 89% when the cells could be
tested with at least three different odors (n=182). Odors
evoked a transient current that rose to a maximum over
several hundred msec and subsequently declined more slowly
to rest (Figure 3-3). The current could be of either
polarity, depending on the cell and the odor tested, and
different odors could evoke currents of opposite polarity in
the same cell (Figure 3-3). In cells that could be tested
with at least three different odors (n=182), the odors that
were tested evoked only inward currents in 48 cells (26%),
only outward currents in 58 cells (32%) and currents of both
polarities in 56 (31%) cells. The remaining 11% of the
cells did not respond to any of the odors tested.
Odor-evoked currents of both polarities were associated
with an increase in membrane conductance, as indicated by a
decrease in input resistance, when hyperpolarizing voltage-
steps were injected into the cells prior to and during odor-
stimulation (Figure 3-4). The input resistance (RN)
decreased significantly under odor stimulation from a mean

Figure 3-3. Whole-cell, voltage-clamp recording from a cultured lobster ORN in
response to spritzing the cell (arrow) with 10"3 M proline (top trace) , 10"3 M taurine
(second trace), TET (third trace) and culture medium only (bottom trace). Holding
potential, -60 mV for all traces.

r*yVv^1f^"V.AVV^»>v^v^^v4>.v,v*.-A/»J^guA»'^>WvAY',V^^'Y^'''-’l'V''^^t^A''V^',V'-r'uv'Ajv1fKv'^
50 pA
300 msec
Proline
Taurine
Tet
Control
00

Figure 3-4. Whole-cell, voltage-clamp recordings from a
cultured lobster ORN showing increased conductance
associated with the odor-stimulated inward current.
A) Neither current (upper trace) nor change in conductance
(lower trace) was evoked by stimulating (arrow) with culture
media control. B) Inward current (upper trace) associated
with an increase in conductance (lower trace) was evoked by
stimulating with TET. Holding potential, -60 mV. Solid
bars denote 30 mV, 300 msec hyperpolarizing voltage pulses
used to monitor membrane conductance.

80
A
\
B

81
of 1.1 ± 0.1 to 0.7 ± 0.1 GQ for the inward current (n=8)
and from a mean of 1.3 ± 0.6 to 0.7 ± 0.1 GQ for the outward
current (n=4) (paired t-test, p < 0.05). Concomitantly, the
membrane time constant (r0), decreased significantly during
odor stimulation from 59.6 ± 12.6 to 21.6 + 4.1 msec for the
inward current (n=14) and from 82.5 + 20.7 to 34.6 ± 9.5
msec for the outward current (n=7)(paired t-test, p < 0.05).
The latency to the onset of the odor-evoked currents,
measured from the activation of the spritzer, ranged from
<20 msec to > 1 sec, but typically was <100 msec (Figure
3-5). Overall, the mean latency to onset for the inward
current, 186.2 ± 31.3 msec (n=100), was significantly longer
than that for the outward current, 81.0 ± 10.6 msec (n=121)
(t' Statistic, p < 0.05). To determine if this difference
was possibly driven by between cell variation, I performed a
paired comparison of the latency in 19 cells that supported
odor-evoked currents of both polarities. The mean latency
in these cells for the inward current was not significantly
longer than that for the outward current (paired t-test,
p < 0.05).
The peak amplitude of odor-evoked currents of both
polarities increased with the concentration of the odor and
saturated over 3-4 orders of magnitude (Figure 3-6). The
mean slope of the concentration-response function in the
steepest region of the curve was 8.3 ± 1.7 pA/decade (n=3)
for the inward current and 1.3 ± 0.2 pA/decade (n=7) for the

Figure 3-5. Plot of the distribution of the latency of odor-evoked currents in
cultured lobster ORNs . Striped bars, inward currents (n=100); solid bars, outward
currents (n=121). Arrows denote the mean latency to onset of the inward (open arrow
and outward (filled arrow) currents, respectively.

30
25
20
15
10
5
0
0
t V
100 200 300 400
Onset Latency (msec)
iua
1200
00
u>

Figure 3-6. Dose response characteristics of cultured lobster ORNs. A) Whole-cell,
voltage-clamp recordings of outward currents in a cultured lobster ORN evoked by-
stimulating the cell (arrow) with decreasing concentrations of proline. Holding
potential, -60 mV. B) Plot of the peak inward current (X ± SEM) elicited in three
cells by taurine at the concentrations shown. C) Plot of the peak outward current
(X ± SEM) elicited in three other cells by proline at the concentrations shown.
Current magnitudes in B and C are normalized to that evoked by 10"3 M odor.

Mean Standardized Response
S 8
r ooi

86
outward current. Thresholds were at least 10'8 M, the
lowest concentration tested, for currents of either
polarity.
The peak amplitude of currents of both polarities
evoked by a single concentration (10'3M) of odor ranged,
typically, from 5-85 pA (Figure 3-7A). The average
magnitude of the inward current (39.2 + 3.0 pA) was
significantly greater than that of the outward current (20.1
± 1.4 pA) measured across all cells and odors (n=386; t'
Statistic, p < 0.05). The polarity and the magnitude of the
currents evoked by odors were independent of the cell
morphology for six different odors. The results for two of
the six odors, proline (n=lll cells) and taurine (n=102
cells), are shown in Figure 3-7B,C. The peak amplitude of
the odor-evoked currents of either polarity was also
independent of the length of the process in cells bearing
processes (n=55, correlation analysis, r > 3.86) (Figure 3-
8A) and the size of the soma in cells lacking processes
(n=60, correlation analysis, r > 3.86) (Figure 3-8B).
Single odors activated from 14 to 77 percent of the
cells (Table 3-1). The stimulatory effectiveness was:
betaine > histamine > glycine > proline > taurine > AMP >
TMAO > ascorbate > arginine > cysteine. An equimolar
mixture of five compounds (S-l: betaine, taurine, glycine,
TMAO, lactate) ranked intermediate in stimulatory
effectiveness. A complex mixture (TET) stimulated more of

Figure 3-7. Plots of the peak amplitude of odor-evoked
currents in cultured lobster ORNs as a function of cell
morphology. A) Distribution of the peak amplitude of the
currents evoked in 386 cells by single odors tested at a
common concentration (10'3 M). Striped bars, inward
currents; solid bars, outward currents. Arrows denote the
mean amplitude of the inward (open arrow) and outward
(filled arrow) currents. B) Plots of the peak amplitude of
currents of both polarities evoked by stimulation with
10'3 M proline (n=lll) and 10'3 M taurine (n=102) grouped
according to morphology. Inward currents are denoted as
negative, outward as positive.

Response Magnitude (pA) Percent of Total Observations
45
40
35
30
25
20
15
10
5
0
0 50 100 150 200 250
Response Magnitude (pA)
100
80
60
40
20
0
-20
-40
-60
-80
60
40
20
0
-20
-40
-60
-80
-100
-120
Soma Unipolar Bipolar Multiprocess
Proline
r3e
s S S
m
Taurine
* — _
-
88

Figure 3-8. Plots of the peak amplitude of odor-evoked
currents in cultured lobster ORNs. A) Amplitude as a
function of length of the longest process (if more than one
for 55 cells B) Amplitude as a function of diameter of the
soma in 60 cells lacking processes. The data set combines
currents evoked by 10"3 M proline, arginine, taurine, TMAO
and betaine as odors (1 cell, 1 odor). Inward currents in
both plots are denoted as negative, outward as positive.

Current Magnitude (pA) Current Magnitude (pA)
90
Process Length (micron)
Soma Diameter (micron)

91
Table 3-1. The percentage of cultured lobster olfactory
receptor cells responding to individual odors, a mixture of
5 odors, and a complex odor.
Compound
Cone
[mM]
# Cells
Tested
# Cells Responding
Inward Outward
% Responding
to Odor
Control
_
237
0
0
0
Betaine
1
13
0
10
77
Histamine
1
4
1
2
75
Proline
1
68
13
30
63
Taurine
1
58
24
12
62
Glycine
1
14
5
5
57
AMP
1
10
1
4
50
S-1 Mix
5
16
4
3
44
TMAO
1
34
7
7
41
Ascorbate
1
8
0
2
25
Arginine
1
17
3
1
24
Cysteine
1
14
0
2
14
Proline
100
54
9
25
63
Taurine
100
52
20
7
52
Arginine
100
21
2
8
48
TET
0.1*
4
3
1
100
TET
0.001'
52
19
12
60
* = relative concentration of stock solution where 0.1 = 10X dilution of Tetramarin and 0.001 =
1000X dilution of Tetramarin; TMAO = trimethylamine-oxide; S-1 Mix = betaine, TMAO, glycine,
taurine, lactate; Control = üebowitz L15 Media; AMP = adenosine monophosphate.

92
the cells than did any single compound or the 5 component
mixture, but this could be accounted for by concentration
since the concentration of TET relative to that of the pure
compounds is unknown. Culture medium itself, tested as a
control, stimulated none of the cells tested, even though it
contained some of the same amino acids as above as well as
L-glutamine. Some odors selectively activated either inward
or outward currents (Figure 3-9). Taurine elicited inward
currents twice as frequently as outward currents, while
proline showed the opposite tendency. Betaine and ascorbic
acid elicited strictly outward currents, although the number
of cells tested in each of these instances was not large.
In a separate experiment, 53 cells that were determined
to be odor responsive by their ability to respond to TET
were presented sequentially with the same five odors in
order to determine their response spectra or "tuning". The
cells varied in the number of odors they responded to, as
well as the magnitude and the polarity of the response to
any one odor (Figure 3-10A). The mean breadth of
responsiveness for the sample population was H = 0.31 ± 0.04
(Figure 3-10B). Repeating this analysis using fewer odors
or the same number of different odors yielded measures of H
between 0.2 and 0.3 (data not shown).

Figure 3-9. Plot of the percentage of cultured lobster ORNs that respond to various
odors with inward and outward currents. All odors tested at 10'3 M, with the
exception of TET, which was tested at 1000-fold dilution of stock. TMAO,
trimethylamine-oxide. AMP, adenosine monophosphate.

Percentage of Cells Responding
80 60 40 20 0 20 40
Outward Inward
taurine
(n=58)
TET
(n=52)
TMAO
(n=34)
proline
(n=68)
arginine
(n = 17)
AMP
(n = 10)
ascorbic acid
(n=8)
betaine
(n = 13)

Figure 3-10. Degree of odor tuning in cultured lobster
ORNs. A) Graph of the response profiles of 10 cultured
lobster ORNs to 5 odors tested at 10"3 M. Each horizontal
line depicts the responses of a different cell; the height
and direction of the bars on each line indicate the
amplitude and the polarity, respectively, of the odor-evoked
current (pA). Outward currents are denoted as positive
(upward bars); inward currents as negative (downward bars).
B) Plot of the breadth of responsiveness of 53 cells. Arrow
denotes the mean H value.

Breadth of Responsiveness (H)
Proportion of Neurons
Pro Tau
>
'X»
CTt
801

97
Discussion
The fact that the cells could be observed to sprout
processes in culture, together with their common active
electrical properties, strengthens the contention I proposed
in the previous chapter that the various types of "neuron¬
like" cells in the culture are morphs of one type of cell,
the ORN. This finding is consistent with the highly
enriched source of the cultured cells; the somata were
harvested from the lumen of the olfactory organ, which is
literally filled with grape-like clusters of the somata of
the ORNs (Grünert and Ache, 1988) .
Although most neurons, as well as non-neuronal cells,
are chemically irritable (e.g., Lerner et al., 1990), it is
reasonable to interpret the responses of the cultured ORNs
as olfactory. Most importantly, the cultured ORNs showed a
diversity of response profiles (Figure 3-10). Postsynaptic
or neuromodulatory receptors, in contrast, would be expected
to be much more homogeneously distributed across the
population of cells and show either the same or, if multiple
receptors, a restricted set of response profiles across
cells. For example, a histamine-gated chloride channel
implicated in modulating the output of lobster ORNs in situ
occurs on the soma of more than 95% of the cells (McClintock
and Ache, 1989a). Second, the average breadth of
responsiveness of the cultured ORNs (H = 0.31), approximated
that of lobster ORNs determined in situ with a similar, but

98
not identical, array of compounds (H = 0.2, Derby et al.,
1984) and did not differ significantly from that of lobster
ORNs determined in situ with the same array of compounds
(H = 0.35, Student's t-test, p < 0.05, pooled SD - M.
Wachowiak, unpublished data). Third, like in their
counterparts in situ, odors evoked both inward and outward
currents in the cultured ORNs, and different compounds could
evoke currents of opposite polarity in the same cell
(McClintock and Ache, 1989b). Odors that tended to
frequently elicit currents of one polarity in the cultured
cells, e.g., proline and outward currents, also did so in
lobster ORNs in situ (Schmiedel-Jakob et al., 1990; Michel
et al., 1991). That currents of opposite polarity were
associated with an increase in membrane permeability argues,
as occurs in situ (ibid.), that the odors were not up and
down-regulating a single conductance but, rather, were
regulating two distinct conductances through two parallel
transduction pathways. Last, the threshold and dynamic
range of the cultured ORNs matched that of lobster ORNs in
situ to the same or similar types of odor molecules
(Schmiedel-Jakob et al., 1989; W.C. Michel, unpublished
data). I conclude that most, and possibly all, of the
chemosensitivity evoked by cultured lobster ORNs is sensory,
i.e., is mediated by the same receptors and transduction
pathways found in the outer dendrites of lobster ORNs in
situ, and is not the result of activating synaptic,

99
neuromodulatory or other receptors that may occur on the
soma of lobster ORNs.
The inability of the culture medium to elicit responses
from the cells when applied as a control stimulus, even
though it contains amino acids shown to be odors (cysteine,
glycine, arginine) and others (e.g., glutamine) that are
potential odors, is not necessarily surprising. The cells
were maintained and tested in the same medium and would be
expected to adapt to continuous background stimulation, as
do their counterparts in vivo (Borroni and Atema, 1988) .
However, adaptation to continuous background stimulation
does shift the apparent threshold of the cells in vivo so
that recording in medium might have lowered the overall
sensitivity to those test stimuli in the medium (cysteine,
glycine, arginine). Indeed, the smallest percentage of
cells tested responded to cysteine and arginine; but, in
contrast, glycine was among the more effective odors tested
(Table 3-1). The remaining test odors, including those used
for threshold determininations, were not components of the
culture medium.
The overall magnitudes of the current evoked by odors
in cultured cells could differ appreciably from those in
situ due to possible differences in the number of channels
expressed in culture. It is interesting, therefore, that
the average magnitudes of the odor-evoked currents in the
cultured ORNs (39.2 ± 3.0 pA, inward; 20.1 ± 1.4 pA outward)

100
were within an order of magnitude of those evoked in lobster
ORNs in situ. A 10-fold greater concentration of TET than
that used in the present study elicited an average inward
current of 25.4 ± 5.1 pA, while the same concentration of
proline as that used in the present study elicited an
average outward current of 4.5 ± 0.6 pA in lobster ORNs in
situ - (Michel and Ache, in press). Odor-evoked currents of
dendritic origin in situ would be electrotonically
attenuated in the soma (where they were recorded), so the
actual magnitude of the current in the dendrite would
presumably be closer to that found in the cultured cells.
My results are consistent with the possibility that the
cultured cells express a full complement of odor-activated
channels.
The average amplitude of the odor-evoked inward
currents in lobster ORNs is an order of magnitude smaller
than those reported for salamander or rat ORNs (Kurahashi,
1989; Pixley and Pun, 1990; Firestein et al., 1991; Lowe and
Gold, 1991), but is consistent with the still limited
understanding of unitary currents that may underlie the
macroscopic current. Inositol 1,4,5-trisphosphate (IP3),
the suspected excitatory second messenger, activated 1-3
channels in most (49/56) cell-free patches taken from the
plasma membrane of the cultured cells (Fadool and Ache,
1992a). On the assumption that these IP3-activated channels
are transductory, there are an estimated 330 to 1990

101
channels per cell, given a pipette tip diameter of 0.5 - 1.0
/¿m and a total cell surface area of 380 ¡j.m2 (Fadool et al. ,
1991b) ; or 0.9 - 5.3 channels/ /im2 membrane. Taking the
smaller of the two IP3-activated unitary conductances
(2 pA - Fadool and Ache, 1992b), a macroscopic current of
average magnitude (39 pA; Figure 3-7) would require
activation of as few as 20 channels, or only 1-6% of the
available channels. Such a small fraction of recruited
channels is consistent with the presumed sensory function of
the cells (e.g. Firestein et al., 1991).
That the ability of the cultured cells to respond to
odors did not correlate with the number or even the presence
of processes, argues that the receptor sites for odors were
not necessarily confined to the processes. At least in
vertebrate ORNs, odor receptors are thought to be
selectively localized to the cilia. Deciliating the frog
olfactory epithelium with Triton X-100, for example,
completely abolishes the electro-olfactogram (Adamek et al.,
1984). Lowe and Gold (1991) confirmed this hypothesis by
focally stimulating the cilia of dissociated salamander
ORNs. Whether somatic sensitivity to odors in cultured
lobster ORNs is induced or is typical of mature lobster ORNs
in situ is unclear. Previous attempts to record responses
to odors in freshly isolated somata have been consistently
unsuccessful (e.g., Anderson and Ache, 1985), suggesting
that somatic sensitivity to odors in cultured ORNs may have

102
been induced by placing the cells in culture. This
conclusion would be consistent with the observation that
molluscan neurons raised in suspension culture to prevent
process formation insert postsynaptic receptors otherwise
normally confined to processes in the soma (Wong et al.,
1981). On the other hand, Hatt (1990) finds odor-gated
channels on the soma of freshly dissociated crayfish
chemosensory neurons; channels that have the same structure-
activity profiles and sensitivity of the intact cells.
Ligand-gated histamine receptors on the soma of lobster ORNs
resist the same enzymatic protocol used in the present study
(McClintock and Ache, 1989a). Therefore, I cannot eliminate
the possibility that the receptors in question normally
occur on the somata of mature lobster ORNs, but are
destroyed by the enzymatic treatment required to dissociate
the cells and fail to recover in the subsequent 4-6 hr over
which in situ recordings are usually made. It is known that
enzymatic digestion used to prepare cells for patch clamping
alters neurotransmitter responsiveness in invertebrate
neurons (Oyama et al., 1990) and eliminates odor
responsiveness in amphibian ORNs (Firestein and Werlin,
1989).
The latency to response was extremely brief, as short
as 20 msec with the majority of the responses occurring
within 125 msec of valve (spritzer) activation. These
values fall below the lower limit of the range of latencies

103
reported for dissociated salamander ORNs, the other system
for which comparable measurements are available (175-600
msec--Firestein et al., 1990). Both systems lack a mucus
barrier that would otherwise impede stimulus access to the
receptor sites, so the latencies presumably reflect the
actual time course of transduction. Breer et al. (1990)
recently reported that levels of two second messengers in
rat and insect ORNs, cyclic adenosine monophosphate and
inositol 1,4,5-trisphosphate, respectively, peak in response
to odor stimulation within 25-50 msec. Thus, while the
latency of the response of cultured lobster ORNs is
considerably shorter than that of salamander ORNs, it is
consistent with second messenger-mediated transduction.
Thus, in this chapter it was shown that lobster ORNs
not only survive in culture, but express the odor
sensitivity and selectivity of their counterparts in situ,
including the ability of odors to excite as well as inhibit
the cells. As the cultured cells are morphologically and,
presumably, electrotonically (as evidenced by the absence of
a measurable equalizing time constant) more compact than
their counterparts in situ, often consisting of only a
spherical soma, the ability to characterize the transduction
pathways and analyze potential interactions between them
should be greatly facilitated in vitro.

CHAPTER 4
GTP-BINDING PROTEINS MEDIATE ODOR-EVOKED CURRENTS
Introduction
Heterotrimeric G proteins are a highly homologous set
of proteins that relay an extracellular stimulus
(neurotransmitter, hormone, photon, odorant) from cell-
surface receptors to an effector (enzyme, ion channel)
(Gilman, 1987; Neer and Clampham, 1988; Birnbaumer, 1992;
Hille, 1992a, 1992b; Conklin and Bourne, 1993). To date
there are genes encoding 16 a subunits, 4 S subunits, and 5
gamma subunits, whose functions are incompletely known.
Phylogenic trees assign G proteins into three families, as,
aq, and cq, with similar functional correlates ascribed to
subunits within a family group (Birnbaumer, 1992) . The
coupling of receptor type to effector is not known in many
systems. Many of the criteria for the involvement of G
proteins (Gilman, 1987) in olfactory transduction (Bruch,
1990; Shepherd, 1991; Reed, 1992; Ronnett and Snyder, 1992)
have been satisfied: the requirement for GTP in the
initiation of an odorant response (Pace et al., 1985; Pace
NOTE: This chapter will be submitted to Chemical Senses and
is formatted to meet its style regulations.
104

105
and Lancet, 1986), provoked responses by nucleotide
analogues (Pace et al., 1985; Boekhoff and Breer, 1990;
Breer et al., 1990), ribosylation by bacterial toxins (Pace
et al., 1985; Bruch and Kalinoski, 1987; Boekhoff et al.,
1990a, 1990b; Breer, 1991), immunochemical localization of
individual G protein subtypes in the olfactory epithelium
(Anholt et al., 1987; Jones et al., 1988; Mania-Farnell and
Farbman, 1990), and finally molecular cloning of a highly
specific olfactory G protein (Jones and Reed, 1989) as well
as the discovery of putative olfactory receptors (Buck and
Axel, 1991; Selbie et al., 1992; Ngai et al., 1993; Raming
et al., 1993) that belong to a large superfamily of seven
transmembrane receptors coupled to GTP-binding proteins.
The presence of G proteins across a broad range of
species including toad, frog, rat, lobster, catfish, and
insects (Pace and Lancet, 1986; Anholt et al., 1987; Bruch
and Kalinoski, 1987; Jones and Reed, 1987, 1989; Jones et
al. , 1988; Boekhoff et al., 1990a; McClintock et al., 1990;
Olson et al., 1992) gives credence to a transductory role in
olfactory receptor neurons. Immunochemical, biochemical,
and molecular evidence for G protein-linked mediation of
olfactory signalling has in few instances been correlated
with function (Jones et al., 1988; Jones, 1989; Mania-
Farnell and Farbman, 1990; Raming et al., 1993).
Representatives of all three G protein families have been
localized in the olfactory epithelium, namely Golf, Gs, Go;

106
Git and Gall (Anholt et al. , 1987; Jones and Reed, 1987,
1989; Jones et al., 1988, Mania-Farnell and Farbman, 1990;
Shinohara et al., 1993; Abogadie and Bruch, in press; Rhoads
et al., in press), but coupling of G protein subtypes to
specific effector types remains incomplete. A direct method
would be to study the involvement and type of G proteins in
olfactory signalling by an electrophysiological approach,
where changes in transduction current can be monitored.
Lobster olfactory receptor neurons (ORNs) could provide an
advantageous model, where multiple mechanisms of signal
transduction are inferred by the dual polarity of odor-
evoked current responses in single cells (McClintock and
Ache, 1989b; Michel et al., 1992a; Fadool et al., 1993) and
the presence of potential target effectors, inositol 1,4,5-
trisphosphate (IP3) - and cyclic monophosphate (cAMP)-gated
ion channels, have been well studied (Fadool and Ache,
1992a; Michel and Ache, 1992) .
Although the production of cyclic nucleotide and
inositol phosphate second messengers can occur other than
via G protein-mediated activation of adenylyl cyclase and
phospholipase C (Wahl et al., 1989; Catt et al., 1991; Hille
et al., 1992a; Majerus, 1992), preliminary evidence in our
and other laboratories support the presence of G proteins in
various lobster tissues (Miwa et al., 1990; Taggart and
Landau, 1992) and in the olfactory organ of the lobster
(Fadool et al., 1991a; McClintock et al., 1992). In this

107
chapter, I now provide electrophysiological evidence for the
involvement of a bacterial toxin insensitive G-protein in
odor-evoked inward (excitatory) and outward (inhibitory)
currents of whole cell voltage-clamped lobster ORNs.
Secondly, through combined Western analysis and antibody
perfusion of voltage-clamped whole cells, I have identified
two candidate G protein subtypes of the cxi and aq families
linked functionally to the IP3-mediated excitatory currents
in these cells. As G proteins are a ubiquitous,
evolutionarily conserved family of proteins among the
multicellular eukarayotes (Pupillo et al., 1989; Simon et
al. , 1991), I feel my findings in this chapter can reveal
insight into the potential ways in which G protein-linked,
cell-surface receptors can be coupled to second messenger¬
gated ion channels in olfaction, as well as insight into the
identity of potential G proteins mediating inositol
phospholipid turnover in olfactory signalling of higher
animals.
Methods
Animals
Adult specimens of the Caribbean spiny lobster,
Panulirus argus, were collected in the Florida Keys and
maintained in the laboratory in a flow-through seawater
system. Animals were fed a mixed diet of frozen fish,
squid, and shrimp.

108
Tissue Culture
The distinct clusters of olfactory (aesthetasc)
receptor cells were dissected from the olfactory organ
(lateral antennular filament) of adult specimens of the
Caribbean spiny lobster, Panulirus argus. The clusters were
enzymatically dissociated, and the resulting cells sustained
in primary culture as described previously (Chapter 2).
Briefly, the isolated clusters were incubated for 50 min at
80 rpm on an orbital shaker in 0.2 micron filter-sterilized
solution of 2.5 mg papain and 12 mg L-cysteine in 10 ml
Panulirus saline (PS) containing 1% penicillin, streptomycin
sulfate, and amphotericin B (Gibco). Proteolytic digestion
was stopped by replacing the enzyme solution with low
glucose L-15 media supplemented with L-glutamine, dextrose,
fetal calf serum, and BME vitamins. Cells were immediately
plated on poly-d-lysine-coated glass coverslips. Cells were
maintained at saturation humidity in a modular incubator
chamber (Billups-Rothenberg) at 24°C.
Electrophvsioloqy
Patch electrodes were fabricated from 1.8 mm O.D.
borosilicate glass and fire polished to a tip diameter of
approximately 1.0 /xm (bubble number 4.8; Mittman et al. ,
1987). High resistance seals of between 8 and 14 GQ were
obtained. Cells were viewed at 40X magnification with
Hoffman optics. Signals were filtered at 5 kHz with a

109
low-pass bessel filter and were analyzed by pCLAMP software
(Axon Instruments).
Odor-activated currents were recorded in the whole-cell
configuration with an integrating patch-clamp amplifier
(Dagan 3900). In all experiments, cells were voltage-
clamped at a holding membrane potential of -60 mV. Odors
were "spritzed" on the cells from a seven barrel glass
micropipette (Frederick haer) coupled to a single
pressurized valve system (Picospritzer, General Valve) via a
6-way rotary valve as previously described (Chapter 2).
Nonhydrolyzable forms of a G-Protein activator (GTP) and
inhibitor (GDP), guanosine 5'-0-(3-thiotriphosphate)
(GTPGammaS) and guanosine 5'-0-(2-thiodiphosphate)
(GDPBetaS) respectively, were introduced to the cells
through the patch pipette. To observe changes in odor-
activated current in response to the above analogs,
individual cells were sequentially patched. This allowed
calculation of the initial odor responsivity, prior to the
addition of the analog in the second patch on the same cell.
In experiments using bacterial toxins, 1 /¿g/ml pertussis
(PTX) or 50 /xg/ml cholera toxin (CT) was added to the media
of 36 h cultured cells for 30 h; a protocol similar to that
used with other cultured systems (LaBelle and Murray, 1990;
Okajima and Ui, 1984; Lambert and Nahorski, 1990). The
magnitude of odor-activated currents of treated verses non-
treated cells was compared. In additional studies, to

110
insure PTX access into these cells, the active subunit of
the PTX holotoxin, the A Protomer, was introduced (10 /xg/ml;
PTX A) to cells directly through the patch pipette. Change
in odor-activated current at time 0 (control) was compared
with that of time 5-10 min (treated) after break through to
the whole-cell configuration.
In experiments where antibodies directed against
various G-protein subunits were perfused into whole cells,
patch electrodes were first tip-filled approximately 1 mm
with patch electrode solution (see solutions, PE) and then
backfilled with test antibody solution. Antibody solutions
were prepared by an initial 1:25 dilution in PE, followed by
brief vortexing, and then centrifugation for 30 min
(Eppendorf, #12 setting). This reduced the likelihood of
particulates blocking perfusion through the patch electrode.
The antibody supernatant was subsequently diluted in PE to a
1:100 working concentration. In previous studies utilizing
the same size pipette diameter, cell and antibody
preparation, and recording conditions, evidence of perfusion
of antibody generally occurred not later than 3 to 5 minutes
(Chapter 5). Odor-evoked currents were monitored every min
for up to 30 min so as to minimize adaptation or desensiti¬
zation. Pipettes backfilled with PE or with non-immune IgG
rabbit serum were used to monitor odor-evoked current over
time in the absence of antibody perfusion.

Ill
For statistical comparisons, significance was defined
at p < 0.05 for paired t-tests and at ot = 0.05 for analyses
of variance.
Biochemistry
Mouse cerebellum (3 animals), lobster brain
(3 animals), lobster olfactory receptor neuron (ORN) somata
(8 antennules), and ORN dendrites (24 antennules) were
isolated in chilled phosphate sucrose buffer (PSB). It was
not possible to obtain enough isolated protein from cultured
ORNs due to a high loss of tissue during removal of cells
from substrate and the large number of live animals (= 100)
required to test all G-protein probes. All dissected
tissues were homogenized in chilled homogenization buffer
(HB) 50 strokes by mortar and pestle (Wheaton, Size B) or
2 min by a Kontes tissue grinder in microcentrifuge tubes,
and then tip sonicated (Heat systems, Ultrasonics Inc.,
W-220) three times for 15 s at a #3 setting while iced.
Homogenates were centrifuged twice at 5,000 RPM (Eppendorf,
#12 setting), 4°C for 30 min. Combined supernatants were
then centrifuged (Beckman L70 Ultracentrifuge) 38,000 RPM
for 2.5 h at 4°C. The recovered pellets were resuspended in
HB by bath sonification (Heat systems, Ultrasonics Inc.,
W-225) three times for 15s at a #5 setting and frozen at
-80°C until use.
Protein was determined by a Bradford photometric assay.
20-25 pig of the respective membrane preparations were

112
separated at constant current (25 mA; 3 h) on a 10%
homogenous SDS electrophoretic gel (0.55 mm thick, 29:1
acrylamide/bis-acrylamide). Proteins were transferred to
nitrocellulose in normal blotting buffer (BB) at 0.5 mA for
4 h. Nitrocellulose membrane was soaked 2 x 10 min in
Tween-tris buffered saline (TTBS). Non-specific binding to
the membrane was blocked for 30 min with 2.5% Carnation dry
milk in TTBS (Blotto). Nitrocellulose membrane was then
incubated for 2.5h at RT with selected G-protein antibodies,
washed 2 x 10 min in TTBS to remove unbound antibody, and
reincubated for 2 h with a 1:250 dilution of peroxidase-
conjugated goat anti-rabbit secondary antibody (Boehringer
Mannheim). Electrophoretic bands were visualized by
4-chloro-l-naphthol color reagent activated by 30% H202 in
tris buffered saline (TBS) and 20% methanol.
Solutions
PS consisted of (in mM): 458 NaCl, 13.4 KCL,
9.8 MgCl2, 13.6 CaCl2, 13.6 Na2S04, 3 HEPES, and 2 glucose;
pH 7.4. Modified L15 Media consisted of: 50 ml Liebowitz
L15, 50 ml of 1.6X normal concentration of PS, 0.6g
dextrose, 0.026 g L-glutamine, and 0.01% gentamicin. PSB
consisted of (in mM): 10 phosphate and 250 sucrose; pH 7.3.
HB consisted of (in mM): 320 sucrose, 10 Trizma base,
50 KC1, and 1 EDTA. TBS consisted of (in mM): 50 Tris,
150 NaCl; pH 7.5. TTBS was made by adding 0.1% Tween-20 to
TBS. BB consisted of (in mM): 25 Tris, 0.1% SDS,

113
192 glycine, and 20% methanol. The patch electrode (PE)
solution consisted of (in mM): 30 NaCl, 11 EGTA, 10 HEPES,
1 CaCl2, 180 K-acetate, and 696 glucose; pH 7.0. GTPGammaS
(10'5M) and GDPBetaS (10~5M) were made daily in PE for
electrical recordings. Odorants consisted of either single
components (10‘3M) made daily in PS: taurine, betaine, 1-
proline, 1-arginine, 1-glycine, AMP, d-alanine or of a broad
spectrum odorant mixture, TetraMarin (TET; commercial flake
fish food, Tetra Werke, Melle, France), diluted 1000-fold in
PS. A stock extract of TET was made by mixing 2 gm dry
flakes in 60 ml saline, centrifuging the resulting
suspension at 1400 g, filtering it through Whatman #3 filter
paper, adjusting it to pH 7.4, and storing it frozen in 5 ml
aliquots. Antibodies directed against the various G-protein
subunits were diluted in Blotto at 1:250 or 1:1000 for
Western analysis and were diluted in PE at 1:100 for
electro-physiology. Anti-GiS and anti-GOQ, were purchased
from Upstate Biochemical and Dupont respectively.
Immunopurified anti-Golfa was a generous gift from Dr.
Richard Bruch. Anti-Gq (Z811-5DE), made to the common
carboxyl termini of ofq and axl (the amino terminal cysteine
and the last 12 amino acids, CILQLNLKEYNLV - Gutowski et
al., 1991), was a generous gift from Dr. Paul Sternweis.
Anti-Gq (E973), made to the amino acids 115-133 of the o;q
peptide sequence and anti-Gall (E976) , made to the amino
acids 160-172 of the o;i:L sequence (Strathmann and Simon,

114
1990), were generous gifts from Dr. John Exton. The G-
protein analogs, odorants, and all salts were obtained from
Sigma Chemical. PTX A, PTX, and CT were obtained from
Calbiochem.
Results
To test the involvement of GTP-binding proteins in odor
transduction, a total of 19 cells were sequentially patched,
incorporating either GTPGammaS or GDPBetaS in the second
seal of the same cell. The magnitude of the odor-evoked
inward (n=5) and outward current (n=5) was significantly
increased when 10'5M GTPGammaS was included in the pipette
during odor stimulation (paired t-test). In contrast, the
magnitude of each (n=5 inward, n=4 outward) was
significantly decreased when 1CT5M GDPBetaS was included
(paired t-test) (Figure 4-1). When the cultured cells were
preincubated with 1 /xg/ml PTX or 50 /xg/ml CT for 30 h or
when 10 ¿¿g/ml PTX A was introduced by way of the patch
pipette, the magnitude of odor-evoked inward or outward
current remained unchanged (one-way AITOVA, oi < 0.05,
n= 5-10) among treated and untreated cells (Figure 4-2).
The antibodies directed against GiE and Golfa
demonstrated no positive immunolabelling at the expected
reported molecular weights (arrows) in any of the lobster
membrane preparations (Figure 4-3). Positive
immunolabelling was observed in mouse cerebellum for anti-
GiS at 35 kDa. This antibody exhibited a high degree of

Figure 4-1. Histogram of the mean odor-evoked inward and
outward currents of voltage-clamped lobster olfactory
receptor neurons (ORNs) that were sequentially patched. The
magnitude of the odor-evoked current recorded with normal
patch solution was normalized (solid bar) and compared to
that recorded by the second patch on the same cell when
GTPGammaS (striped bar) or GDPBetaS (open bar) was included
in the patch pipette. Holding potential -60 mV. Number of
sequentially patched cells as noted. * = significant
difference, p < 0.05, paired t-test.

300
200
100
0
*
5
I
Inward Outward
Current Polarity

Figure 4-2. Histogram of the mean inward and outward odor-
evoked current of voltage-clamped lobster ORNs displayed as
a percentage function of the control group normalized to
100. Solid bar = untreated cells, striped bar = 1 ¡jlM
pertussis toxin (PTX) treated cells, open bar = 10 ¡J.M PTX A
Protomer (PTX A) treated cells, checkered bar = 50 /iM
cholera toxin (CT) treated cells. No significant difference
in treatment means (ANOVA, a s 0.05, n= 5-10 as noted).

Response Magnitude (%
118

Figure 4-3. Immunolabelling of membrane fractions from mouse cerebellum (M), lobster
brain (L), lobster dendrites (D), and lobster receptor cell soma (S) to antibodies
directed against G proteins classically coupled to adenylyl cyclase
activation/inactivation. Molecular weight standards (MW) as indicated. Arrow
denotes the expected reported molecular weight (see Figure 4-5) of the G protein
subtype being tested. Control 1 (Cl) = omission of primary antibody; Control 2 (C2)
= substitution of non-immune rabbit IgG for primary antibody. GiE 1:250, Golfa 1:1000.

kDa
208.1-
100.6-
71.2-
C1
43.5-
28.6-
MW M L D S
C2
M L D S
18.3-
1
M L D S" M L D S
120

121
non-selective staining at higher molecular weights (60-210
kDa) than those reported for any G-proteins. This was true
for all tissues, including that isolated from either mouse
or lobster. The antibody directed against Goa demonstrated
immunolabelling of a 40.5 kDa peptide in mouse cerebellum,
lobster brain, and lobster dendrite (Figure 4-4), close to
the expected, reported molecular weight (arrow) of 39 kDa
for this protein subunit. No immunolabelling at this
molecular weight was observed in extracts from lobster
somata. Again a degree of non-selective staining of high
molecular weight proteins was observed for this antibody.
Anti-Gq (E973) and anti-Gall (E976) displayed no positive
immunolabelling in any of the lobster tissues at the
expected, reported molecular weights for these protein
subunits (arrows), and only slight immunolabelling in mouse
cerebellum by anti-Gq (E973) at 42 kDa (Figure 4-4) . Anti
Gq (Z811-5DE) was not tested by Western analysis. In no
case were protein bands observed when the primary antibody
was omitted (Figure 4-3; Cl). Background staining of high
molecular weight proteins in both mouse and lobster was
observed when the primary antibody was replaced with non-
immune rabbit IgG (Figure 4-3; C2). A summary of all
Western analyses can be found in Figure 4-5.
Because the odor-evoked macroscopic inward and outward
currents in lobster ORNs are mediated by the second
messengers, IP3 and cAMP respectively (Fadool and Ache,

Figure 4-4. Immunolabelling of membrane fractions from mouse cerebellum (M), lobster
brain (L), lobster dendrites (D), and lobster receptor cell soma (S) to antibodies
directed against G proteins classically coupled to phospholipase C activation.
Molecular weight standards (MW) as indicated. Arrow denotes the expected reported
molecular weight (see Figure 4-5) of the G protein subtype being tested. G00(
Gqo (E973) , and Gn„ = 1:250.

123

Figure 4-5. G-protein subtypes localized in mouse and
lobster tissues and the theoretical G-protein linked
effectors in lobster olfactory receptor neurons (ORNs).
TOP: Summary table of the cross-reactivity of antibodies
directed against various G-protein subtypes as shown by the
Western analyses of Figures 4-3 and 4-4. "+" = positive
immunoreactive protein;"-" = no apparent immunoreactivity.
BOTTOM: Diagrammatic representation of all hypothesized
transduction cascades in lobster ORN based upon known
receptor-effector coupling reported in other systems.
Inhibitory cascade: Odor molecule (circle) binds to a cell-
surface receptor linked to either G± or Golf protein to
activate adenlyly cyclase (AC), which converts adenosine
trisphosphate (ATP) to cyclic monophosphate (cAMP), which
directly gates ( >) an ion channel evoking an outward
(inhibitory) transduction current. Excitatory cascade:
Odor molecule (square) binds to a cell-surface receptor
linked to either G0, Gq, or GX1 protein to activate
phospholipase C (PLC), which converts phosphoinositol 4,5-
bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3),
which directly gates ( >) an ion channel evoking an
inward (excitatory) transduction current.

125
Protein
Reported MW
(kDa)
Tissue
Mouse
Brain
Lobster
Brain
ORN
Dendrite
ORN
Somata
Gij3
35-36
+
-
-
long smear
Golf
43 & 52
-
-
-
-
Goa
39
+
+
+
-
Gq
42
+
-
-
-
G1 la
42
-
-
.
-
Inhibitory Cascade
Excitatory Cascade
outward
current
inward
current

126
1992a; Michel and Ache, 1992), binding of antibodies to the
G-proteins that target the enzymatic production of these
second messengers might be expected to perturb the
transduced current (Figure 4-5). In antibody perfusion
experiments, the perfusion of anti-GolfQ,, anti-Goa, anti-GiS,
anti-Gq (Z811-5DE) , anti-Gq (E973), or anti-Gall (E976) or the
perfusion of non-immune rabbit IgG or normal PE had no
effect on the magnitude of odor-evoked outward currents. In
contrast, binding of either anti-Gq (Z811-5DE) or anti-Goa,
completely blocked the odor-evoked inward current within
several minutes. It was not possible to reverse the effect
of the antibody block by inclusion of the peptide (in the
patch pipette) to which the antisera were raised because
this made the ORN membranes too unstable for reliable
recording. Cells perfused with non-immune rabbit IgG or
normal PE typically showed no decay in odor-evoked outward
current over the tested time interval but displayed a
rundown of odor-evoked inward currents over this same
period. Even in these control conditions, however, a
measurable inward current could be detected in many cells up
to 1 hr. An example of the odor-evoked responses observed
during perfusion of anti-G0 is shown in Figure 4-6.
The effect of antibody perfusion on the magnitude of
odor-evoked currents was fairly consistent across a sample
population (Figure 4-7). Binning the measured odor-evoked
currents into 5 minute intervals and normalizing an

Figure 4-6. Whole cell voltage-clamp recordings of odor-evoked inward (inward) and
outward (outward) currents as a function of time and perfusion treatment in lobster
olfactory receptor neurons (ORNs). Shown are current recordings from 2 ORN, the
first cell (left) was perfused with non-immune rabbit IgG (rabbit serum control) and
the second cell (right) was perfused with an antibody directed against the ot subunit
of G0 protein (anti-Goa) . Membrane potential = -60 mV; 1:100 dilution of serum or
antibody; arrow denotes application of the odor.

Rabbit Serum Control
r
v.
J
0 min
5
Inward
V i
w'V'
k.wvr^,~~*'
10
Anti-Goar
\
V
/
♦,k-v»'a-*.V‘*'~1^
128

Figure 4-7. Line graphs of the mean outward (A) and inward
(B) odor-evoked currents as a function of time and perfusion
treatment in whole-cell voltage-clamped lobster olfactory
receptor neurons (ORNs). Each data point represents the
mean (± SEM) odor-evoked current normalized to that at
breakthrough (BT) to the whole-cell configuration and binned
into the bracketed time intervals. Number of repetitions as
noted. * = significant difference across treatment
dimension (blocked factorial design two-way ANOVA, a; <
0.05). Membrane potential = -60 mV.
= serum, --a-- = patch solution, = anti-Golf,
-- + -- = anti-G0, X- - = anti-Gi, --x-- = anti-Gq

[1-4] [5-9] [10-14] [15-19] [20-24] [25-30]
Time (min)
00
Normalized Response Magnitude
OOOO -*â– 
o ho ^ b> bo -*â–  ho
[1-4] [5-9] [10-14] [15-19] [20-24] [25-30]
>
Normalized Response Magnitude
OOOO -*â– 
o ho o co —*■ ho
130

131
individual olfactory neuron response to its initial response
magnitude upon break through to the whole-cell
configuration, indicated that the binding of anti-Golfa,
anti-Goa, anti-Gifi, anti-Gq (Z811-5DE) , anti-Gq (E973), or
anti-Gall (E976) or the perfusion of non-immune rabbit IgG or
normal PE had no affect on the magnitude of odor-evoked
outward current across either the time or treatment group
dimension (blocked factorial design two-way ANOVA; Figure 4-
7A). In a similar analysis of odor-evoked inward currents,
all test perfusions, including controls, exhibited a
decrease in response magnitude across the time dimension,
but only those cells perfused with anti-Goa or anti-Gq (Z811-
5DE) showed a significant decrease in response magnitude
compared across treatment groups (blocked factorial design
two-way ANOVA; Figure 4-7B).
Discussion
The finding that nonhydrolyzable analogs of GTP and GDP
respectively increase and decrease both the macroscopic
odor-evoked inward (excitatory) and outward (inhibitory)
currents in lobster ORNs strongly supports a transductory
role for G proteins in both conductance pathways. Pertussis
and cholera toxin incubation of cultured ORNs did not
perturb the magnitude of the odor-evoked currents, implying
that the G protein(s) involved in transduction were of the
bacterial-toxin insensitive class. The ability of a G
protein to undergo bacterial toxin ADP-ribosylation is an

132
indication of its subtype: members of the cq family,
including Gai and Gao, can be ribosylated by PTX (Katada and
Ui, 1982; Codina et al., 1983; Bokoch et al., 1984; Neer et
al., 1984; Sternweis and Robinshaw, 1984); members of the
c/s, including Gas and Golfa, can be ribosylated by CT (Gill
and Meren, 1978); and members of the aq family, including
Gall and Gq, lack the recognition site for ribosylation (Pang
and Sternweis, 1990; Strathmann and Simon, 1990; Blank et
al., 1991; Sternweis et al., 1992). Since PTX is known to
alter odor-evoked second messenger production in rat and
insect olfactory ciliary preparations (Boekhoff et al.,
1990a) and ribosylates a 37/39 kDa doublet and 3 lower
molecular weight proteins in lobster aesthetasc membrane
preparations (McClintock et al., 1990), I questioned whether
the toxin was penetrating the ORN cell membrane, albeit at
micromolar concentrations and for 30 hours of incubation. I
eliminated this possibility by injecting the active subunit
of the PTX holoenzyme, the A Protomer (PTX A), into whole-
cells via the patch pipette, using similar tip diameters and
perfusion times documented for voltage-clamp studies of B5
snail neurons and frog sympathetic neurons (Elmslie, 1992;
Man-Son-Hing et al., 1992). Even though lobster aesthetasc
membranes contain a PTX-ribosylated protein(s) (McClintock
et al. , 1990) and a putative Gai subunit has been cloned
from lobster containing a potential cysteine ribosylation
recognition site (McClintock et al., 1992), my data support

133
functionally, bacterial toxin-insensitive G-proteins most
probably similar to those of the aq family. In contrast to
PTX, the ability for CT to ribosylate G proteins in lower
metazoans has been questionable, as these animals do not
possess an essential lipid component in their plasma
membrane to make such a modification (John Schetz, unpub
data). It is thus less clear whether the lack of CT
sensitive odor-evoked currents in lobster ORNs should be
truely classified as CT-insensitive.
Selective block of odor-evoked inward currents by the
binding of antibodies directed against Goa and Gqa is
consistent with the InsP3 mediation of excitation in these
neurons (Chapter 5). PLCSX is the reported effector for
both of these G protein subtypes (Review: Berstein et al.,
1992; Sternweis and Smrcka, 1992; Reports: Moriarty et
al., 1990; Blank et al., 1991; Smrcka et al., 1991; Taylor
et al., 1991), inferring that the lobster possesses a
similar isoform of this lipase to generate InsP3 from PIP2.
It is interesting to note that mRNA encoding G0 is enriched
in rat olfactory epithelium (Jones et al., 1990) and that G0
protein was suspected to be coupled to InsP3-mediated odor
detection based on bacterial-toxin sensitivity in this same
organism (Breer, 1990).
If both a Gqa- and a Goa-like G protein mediates InsP3
turnover in lobster ORNs, why the apparent redundancy? Most
recently are the report of two different G protein subtypes

134
mediating inositol phospholipid turnover in other systems
(Voyno-Yasenestskaya et al., 1989; Gerwins and Fredholm,
1992). Of particular interest are identified G protein
subtypes in smooth muscle, where both a toxin sensitive and
toxin insensitive protein are distinguished through separate
receptor activation by adenosine and bradykinin (Gerwins and
Fredholm, 1992) . The coexpression of Gqa and Goa subtypes
within single lobster ORNs could explain the apparent
bacterial-toxin insensitivity of the odor-evoked inward
currents: even though Goa is potentially ribosylated,
rendering the G protein of the transduction cascade in a
GDP-bound inactive state (Okajima and Ui, 1984), the active
odorant transduction cascade utilizing Gqa is unaffected and
no change in current magnitude is measurable.
The recognition of a 40.5 kDa protein by anti-Goa
specifically in the dendritic membrane preparation rather
than in the isolated somata of lobster ORNs, is in keeping
with the olfactory transduction elements being confined to
the cilia or the analogous outer dendritic membrane in
crustaceans (Nakamura and Gold, 1987; Kurahashi, 1989;
Firestein et al., 1990). The binding of an antibody to its
epitope does not confer perturbation of physiological
function. Only anti-Gqa (Z811-5DE) and not anti-Gq (E973)
had been previously demonstrated to target PLC activity, as
witnessed by its ability to attenuate the hormone stimulated
turnover of PIP2 (Gutowski et al. , 1991) . As anti-Goa was

135
commercially acquired, the mechanism of its discovered block
of excitatory currents is unknown. It cannot be excluded
that antibody binding of G0 acts to perturb a membrane-
delimited modulation of ion channels (Brown, 1991; Hille,
1992a), instead of perturbing the function of the enzyme
effector (PLC). Interfering with a mechanism that alters
the gating of K+ channels, such as that reported by Goal,
would be expected to target the odor-evoked outward currents
that are mediated by an underlying K+ conductance (Michel et
al., 1991) in these ORNs, however, these currents do not
change with any anti-G0 perfusion. If the lobster Goa
subunit was more like that of a Goa2 subtype, then binding of
anti-G0 could be suspected to interfere with a mechanism
that alters the gating of Ca2+ channels, such as that of the
74 pS InsP3-gated channel (Chapter 6), which would be
consistent with the observed block in odor-evoked inward
current.
Future experiments will certainly be directed at
sorting the complexity of the transduction elements in
lobster ORNs: neurons that contain more that one odor-
evoked conductance, G-protein mediated response, second
messenger, and even more than one type of ion channel gated
by a given second messenger. Earlier electrophysiological
findings (see Introduction), recent evidence for at least
two and maybe three second messenger-gated ion channels in
the outer dendrites of ORNs in situ (Ache et al., in press),

136
and biochemical cross-receptor binding studies (Michel et
al., 1992b) do not well support an emerging theory in the
chemoreceptive field of one receptor per olfactory receptor
cell (Lancet et al., 1993). Two different G-proteins
mediate inward odor-evoked currents and presumably a third
as yet unidentified G-protein must mediate the outward odor-
evoked currents. In a monoclonal exclusion model, as
proposed by Lancet et al. (1993), this would require a
single cell-surface receptor type to be linked to two or
three different G protein subtypes. Even greater levels of
complexity are probable when considering Sr subunit exchange
(Casey et al., 1988), potential functioning of ST dimers
(Birnbaumer, 1992; Taussig et al., 1993) and second
messenger related cross-talk (Bouvier, 1990). The potential
sorting of G protein-linked transduction cascades across
cells or the interaction of cascades within a cell may
provide an avenue for amplification and diversity in the
output of an ORN.

CHAPTER 5
IP3-ACTIVATED CHANNELS IN THE PLASMA MEMBRANE
Introduction
Adenosine 3',5'-cyclic monophosphate (cAMP) is now well
established as a second messenger in olfactory transduction
(reviews: Anholt, 1991; Firestein, 1991). Since odors
rapidly and transiently elevate levels of inositol 1,4,5-
trisphosphate (IP3) in the cilia/outer dendritic membranes
of olfactory receptor neurons (ORNs) in fish (Huque and
Bruch, 1986), rats and insects (Breer et al., 1990), IP3
must also be considered as an olfactory second messenger.
The relationship between phospholipid and cyclic
nucleotide second messengers in olfactory transduction is
still obscure. Odors that elevate IP3 in ciliary membrane
preparations of rat ORNs fail to elevate cAMP, and vice
versa (Boekhoff et al., 1990b; Breer and Boekhoff, 1991),
suggesting that the two second messengers mediate different,
odor-specific transduction pathways. Indeed, two distinct
transduction pathways can be predicted in lobster and
amphibian ORNs, where odors have been shown to suppress as
NOTE: This chapter has been accepted for publication and is
reprinted with permission from: Fadool, D.A. and B.W. Ache.
1992. IP3-activated channels in the plasma membrane of
lobster olfactory receptor cells. Neuron. 9: 907-918.
137

138
well as excite the cells via separate conductances
(McClintock and Ache, 1989b; Michel et al., 1991; Dionne,
1992) . In lobster ORNs, cAMP mediates an inhibitory-
transduction pathway that suppresses the output of the cell
(Michel and Ache, 1992b). Given that IP3 has been
implicated as an olfactory second messenger in at least one
other species of arthropod (Breer et al., 1990), IP3 is a
logical candidate to mediate excitation in the lobster, but
the excitatory transduction pathway in lobster ORNs is
unknown.
IP3 is known to release Ca2+ from non-mitochondrial
intracellular stores by binding to a receptor protein that
contains both an IP3 recognition site and a Ca2+ channel
(review: Ferris and Snyder, 1992). It is unclear whether
such IP3 receptors are associated with the plasma membrane
in neurons (Worley et al., 1987; Maeda et al., 1989, 1991;
Mignery et al., 1989; Ross et al., 1989), although IP3
receptors occur in the plasma membrane of lymphocytes (Kuno
and Gardener, 1987; Khan et al., 1992), mast cells (Penner
et al., 1988), and in transverse tubules (Viven and
Coronado, 1988) . Evidence is beginning to implicate what is
perhaps a novel type of IP3 receptor in the plasma membrane
of ORNs. IP3 activates a channel reconstituted from the
cilia of catfish ORNs (Restrepo et al., 1990). The cilia
are enriched in a 107 kDa protein that binds radiolabelled
IP3, but whose molecular weight and affinity for IP3 are

139
less than those reported for intracellular cerebellar IP3
receptors (Kalinoski et al., 1992). Preliminary evidence
localizes immunoreactivity of an antibody directed against
cerebellar IP3 receptors to the cilia of rat ORNs
(Cunningham et al., 1992). As the cilia of ORNs are devoid
of organelles, it could be assumed that the target of this
second messenger in olfactory neurons is a plasma membrane
IP3 receptor.
Here, in this chapter I report that IP3 mediates
excitation in cultured lobster ORNs by directly gating ion
channels in the plasma membrane. The study provides
functional evidence for channels activated by IP3 in the
plasma membrane of neurons.
Results
Macroscopic Currents
Introducing 2.4 x 10"5 M IP3 into the cells through the
patch pipette evoked a prolonged, inward current in 17 of 41
(42%) cells, with an average peak amplitude of 35.1 ± 10.4
pA (Figure 5-1A) . Without IP3 in the pipette, the cells
held a steady baseline over the test interval of 4 min.
These particular cells were not tested for their ability to
respond to odors, but the most effective odor I have been
able to test, an extract of fish food (TET - see
Experimental Procedures), excites approximately 37% of
cultured ORNs (see Chapter 3). The percentage of cells
activated by introducing IP3 through the pipette, therefore,

Figure 5-1. Inositol 1,4,5-trisphosphate (IP3)- and Odor-Evoked Macroscopic Currents
in Voltage-Clamped Cultured Lobster Olfactory Receptor Neurons (ORNs)
A) Whole-cell recording from an ORN that was sequentially patched with normal patch
solution, and then IP3, in the patch pipette. Holding potentials, -60 mV.
B) Whole-cell recording from an ORN that was sequentially patched with normal patch
solution, and then IP3, in the patch pipette and "spritzed" in each instance with
odors. 1st patch: Control, response to recording media. Odor, response to
TetraMarin (TET) . 2nd patch: IP3 + Odor, response to TET with 2.4 x 10'5 M IP3 in the
patch pipette. IP3 + Odor + RR, response to TET with IP3 in the patch pipette while
bathing the cell in 10 /xM ruthenium red (RR) . Holding potentials, -60 mV.
C) Plot of the mean peak amplitude of odor-evoked inward (n=20) and outward (n=35)
currents in the absence (solid bar) and presence (striped bar) of 10 ¿xM RR in the
bath. Response magnitudes in the absence of RR were normalized to 100%. * =
significant difference at p s 0.05, paired t-test. Holding potential, -60 mV.

Control
•wA-Y’"'. .»vv»«~V‘»*vVv***
xtñnVV
/irw
2.4 x 10-5M IP3
100 pA
50 see
B
IP3 + Odor + RR
/
Control
£»
K
,*ÍV* wv J
V
100 pA
Odor
IP3 + Odor
500 msec
C o 150
â– o
Current Polarity
H

142
is consistent with the percentage of cells that would be
expected to be excited by odors.
The polarity of the IP3-induced current matched the
polarity of the current induced by TET (Figure 5-IB).
Introducing 2.4 x 1CT5 M IP3 through the patch pipette
increased the magnitude of the TET-evoked inward current to
188 ± 12% of that evoked by TET without IP3 in the pipette
(n=4 sequentially patched cells) (Figure 5-1B). It was not
determined if higher concentrations of IP3 could saturate
the odor-evoked inward current. The inward current induced
by TET + IP3 in all four instances was substantially blocked
by bathing the cells with 10 /xM ruthenium red (RR - a drug
reported to block some IP3-gated conductances: Ehrlich and
Watras, 1988; Berridge, 1989), supporting a common origin of
the IP3- and odor-induced currents (Figure 5-1B).
The effect of RR was selective for the inward current
(Figure 5-1C) . Bathing the cells in 10 ¿xM RR significantly
reduced the peak amplitude of the inward current evoked by
TET, proline, or betaine from an average of 19.1 ± 4.0 to
4.8 ± 4.3 pA (n=20, paired t-test). The drug, however, had
no significant effect on the peak amplitude of the outward
current evoked by proline, betaine, glycine or taurine,
which averaged 16.4 + 2.6 pA before, and 15.5 ± 6.9 pA
after, bathing the cells in 10 ¿xM RR (n=35, paired t-test) .

143
Unitary Currents
TET transiently activated unitary currents in four of
21 cell-attached recordings (Figure 5-2). The mean
estimated chord conductance for the four channels was 86.7 ±
17.1 pS (Table 5-1) . The probability of being open (Propen)
for the four odor-activated channels increased from an
average of 0.02 ± 0.001 to 0.11 ± 0.03. The channels
characteristically had "flickery" kinetics and opened in
long bursts averaging 72.2 ± 53.6 msec. The mean open (tQ)
and closed (tc) times for the four odor-activated channels
were best fit by double exponentials (Table 5-1).
Applying 10'7 M IP3 to the inside face of 86 cell-free
patches activated unitary currents in 63 of the patches
within 100 msec of application. The patches typically
contained 1-3 channels, although 4 and 5 channels could be
resolved in two of the patches, respectively. The channels
were of two different types; in only one instance were both
types observed in a single patch of membrane. One type of
channel (Figure 5-3A) had a mean slope conductance of 73.7 ±
5.7 pS and reversed polarity at 2.4 ± 2.2 mV in symmetrical
solutions (n=12) (Figure 5-3B). The open probability
function closely followed a Gaussian distribution (Figure 5-
3C) . The Propen was between 0.04 and 0.05 from -90 mV to +60
mV (Figure 5-3B, inset) . The mean open (tQ) and closed (tc)
times were best fit by double exponentials (n=20)(Figure 5-
3D, Table 5-1).

Figure 5-2. Odor-evoked Unitary Currents in a Cell-Attached
Patch from a Cultured Lobster Olfactory Receptor Neuron
(ORN)
Control: Unitary currents recorded in response to
"spritzing" an ORN with normal patch solution to depolarize
it.
+ Odor: Unitary currents recorded in response to
"spritzing" an ORN with the odor TetraMarin (TET). In both
upper and lower traces, membrane potential, -60 mV. Records
filtered at 2 kHz. In this and subsequent figures, "C"
denotes closed state of the channel whereas "0" denotes open
state of the channel. Superposition in the open state of
multiple channels in a patch are numbered in subscript.

145
Control
c
o
¿uX*iW’ kiVf,¡‘V^v^'V-'V^r;f^/W^^^^^W1-'1
11 inr *j| i ]' if
v*^y
,w
'U
Odor

Table 5-1: Properties of odor- and IP3-activated channels in cultured lobster olfactory receptor
neurons
Property Large IP3 Channel Small 1P3 Channel Odor Channel
73.7 ± 5.7 pS
Conductance
Mean Open Time
Tau 1
Tau 2
Mean Closed Time
Tau 1
Tau 2
0.38 ± 0.07 msec
2.52 ± 0.43 msec
2.64 ± 0.19 msec
36.49 ± 6.80 msec
30.0 ± 1.6 pS
0.49 ± 0.06 msec
6.33 ± 1.64 msec
1.25 ± 0.24 msec
42.64 ± 15.90 msec
'86.7 ± 17.1 pS
0.43 ± 0.05 msec
5.18 ± 1.39 msec
2.85 ± 0.37 msec
35.78 ± 1.34 msec
’Denotes estimated chord conductance rather than slope conductance. See text for sample size of
each measure.

Figure 5-3. IP3-evoked, 74 pS Unitary Currents in Inside-
Out Patches of Membrane from Cultured Lobster Olfactory
Receptor Neurons
A) Basal current prior to (control) and after (IP3)
"spritzing" 1CT7 M IP3 on the internal face of a patch.
Membrane potential, -30 mV. Records filtered at 2 kHz. "C"
and "0" as defined in Fig. 5-2.
B) Plot of the current-voltage relation of the channel shown
in A). The current reversed near zero in symmetrical
solutions with a mean slope conductance of 73.7 ± 5.7 pS
(n=12). Voltage in mV on the abscissa and current in pA on
the ordinate. Inset: Probability of open state (Propen) of
the channel in A) at different membrane potentials.
C) Amplitude histogram of 9965 open-time events from the
membrane patch in A), fit by a Gaussian distribution with
mean and standard deviation of -1.87 ± 0.07 pA.
D) Plot of the open-time distribution of 2735 events of an
IP3-activated channel fit by a double exponential. tx =
0.38 ± 0.07 msec, r2 = 2.52 ± 0.43 msec (n=14). Closed time
distribution not shown.

148
A
Control
IP3
inn
v'Swi/*. v-<»
r,.
¡ I I r¡ i
[^.'•^V^»fA.*Vv M< 'v'^VW>
30 ms
1 pA
â– J^vi/Vr.
B
4
2
<
•
•
â–º
50 -40 -30 -20 -10
10 20 30 40
•
006
•
0 05
• ~2
004
•
£ 0 03
• -4
0 02
001
100 80 -60 -40 20 0
Voltage |mV|
Amplitude ipAl
2000-
20 40 60 80
Time (msl
50
20 40 60
100

149
The second type of channel (Figure 5-4A) had a mean
slope conductance of 30.0 ± 1.6 pS and reversed polarity at
30.2 ± 1.5 mV in symmetrical solutions (n=16) (Figure 5-4B).
The open probability function followed a Gaussian
distribution (Figure 5-4C), but less closely than that of
the larger conductance channel. The Propen of the channel
was voltage dependent and decreased from 0.5 to 0.1
between -40 and +40 mV (Figure 5-4B, inset). The mean open
(tQ) and closed (tc) times were best fit by double
exponentials (n=32)(Figure 5-4D, Table 5-1).
There was no significant difference in the distribution
of tG for the two types of IP3-activated channels and the
odor-activated channels (one-way Analysis of Variance;
ANOVA). However, there was a significant difference in the
distribution of tc for the two types of IP3-activated
channels compared to that of the odor-activated channels,
which could be attributed to the second exponent (r2) of the
smaller conductance channel (ANOVA, Student-Newman-Keuls).
The Propen of both types of IP3-activated channels (n=5,
each type) increased with the concentration of IP3 applied
to the patch, as would be expected if IP3 functioned as a
second messenger. The Propen for the larger conductance
channel increased from 0.04 ± 0.02 at 10~7 M to 0.25 ± 0.03
at 10'5 M, while the same parameter for the smaller
conductance channel increased from 0.06 ± 0.02 at 10"7 M to
0.28 ± 0.13 at 10~5 M. Concentrations outside of this range

Figure 5-4. IP3-evoked, 30 pS Unitary Currents in Inside-
Out Patches of Membrane from Cultured Lobster Olfactory
Receptor Neurons
A) Basal currents prior to (control) and after (IP3)
"spritzing" 10"7M IP3 on the internal face of a patch.
Membrane potential, -30 mV. Records filtered at 2 kHz. "C"
and "0" as defined in Fig. 5-2.
B) Plot of the current-voltage relation of the unitary
currents shown in A). The current reversed between 20-30 mV
in symmetrical solutions with a mean slope conductance of
30.0 ± 1.6 pS (n=16). Voltage in mV on the abscissa and
current in pA on the ordinate. Inset: Probability of open
state (Propen) of the channel in A) over different membrane
potentials.
C) Amplitude histogram of 3699 open-time events from the
membrane patch in A), fit by a Gaussian distribution with
mean and standard deviation of 0.65 ± 0.02 pA.
D) Plot of the open-time distribution of 1737 events of an
IP3-activated channel fitted by a double exponential. t1 =
0.49 ± 0.06 msec, r2 = 6.33 ± 1.64 msec (n=15). Closed time
distribution not shown.

151
A
Control
IP3
0.9
D
- 800
-600 ~
400 ®
v>
E
z
- 200
Amplitude (pA)
Time (ms)

152
were not tested. When membrane patches (n=5) containing the
larger conductance channel were stimulated repeatedly with
10'5 M IP3, so as to continuously expose the channel to the
ligand for 50 sec, the channel remained in the open state
42 ± 12% of the time and failed to desensitize. The smaller
conductance channel was not tested with continuous
stimulation.
Both the large (n=2) and the small (n=2) conductance
channels were blocked by 10 /xM RR, consistent with the
pharmacology of the macroscopic current (Figure 5-5A). The
blockade was partially reversible within 5 min of washout of
the drug. Although not tested on the macroscopic current,
both the larger (n=3) and the smaller (n=2) conductance
channels were also blocked by 2.5 /xM heparin, a drug known
to block intracellular IP3 receptors (Supattapone et al.,
1988; Frank and Fein, 1991) (Figure 5-5B). Heparin blockade
was fully reversible upon washout.
Channels of both types were insensitive to modulation
by ATP, unlike the 2-4 fold increases in Propen reported for
some IP3 receptors (Ehrlich and Watras, 1988). Co¬
presenting up to 50 mM ATP with 10"7 M IP3 failed to alter
the Propen (as reported above) of either the larger (n=3) or
the smaller (n=4) conductance channels (Signed-Rank Test).
In one trial, it was possible to successfully insert a
pipette with an inside-out patch containing the larger
conductance channel taken from one cell into a second cell

Figure 5-5. Ruthenium Red (RR) and Heparin Block of In¬
activated Channels in Inside-Out Patches from Cultured
Lobster Olfactory Receptor Neurons
A) Activity of a channel of the type shown in Fig. 5-4
induced by "spritzing" 10'7 M IP3 on the inside face of the
patch (upper three traces) is abolished when 10 ¡j.M RR is co¬
presented with IP3 (middle three traces) and partially
recovers (lower two traces) when RR is rinsed and IP3 re¬
presented. Membrane potential, -60 mV. Records filtered at
2 kHz. "C" and "0" as defined in Fig. 5-2.
B) Activity of a channel of the type shown in Fig. 5-3
induced by "spritzing" 10'7 M IP3 on the inside face of the
patch (upper three traces) is abolished when 2.5 ¿¿M heparin
is co-presented with IP3 (middle three traces), and recovers
(lower three traces) when heparin is rinsed and IP3 re¬
presented. Membrane potential, -60 mV. Records were
filtered at 2 kHz. "C" and "0" as defined in Fig. 5-2.

154
A
IP3 alone
c
o
B
IP3 alone
c
01
02
IP3 + RR
IP3 + heparin
c
IP3 alone
50 ms
1 pA
IP3 alone
40 ms
10 pA

155
(Figure 5-6). "Spritzing" the odor TET on the second,
recipient cell induced channel activity in the patch,
presumably, although not necessarily, due to elevation of
intracellular IP3, as illustrated in Figure 5-6. The
activity of the channel decreased over the subsequent
24 sec.
Immunochemistrv and Related Physiology
A polyclonal antibody raised against the 19 C-terminal
amino acids of a cDNA clone of a mouse cerebellar IP3
receptor (Mignery et al., 1989 - kindly supplied by Dr. P.
DeCamilli; Figure 5-7), immunolabelled a band greater than
200 kDa, as well as several lower molecular weight bands
(Figure 5-8A, left panel, left column). Only the
immunoreactivity of the greater that 200 kDa band was
incrementally blocked by preabsorbing the antibody with
increasing concentrations of a synthetic peptide patterned
after the original antigen PCD6 (data not shown). The
antibody also labeled a band greater than 200 kDa in
membranes isolated from cultured lobster ORNs (Figure 5-8A,
right panel, left column). No bands were observed in either
the mouse or lobster membrane preparation when the primary
antibody was replaced with non-immune rabbit serum (Figure
5-8A, both panels, right columns). It was not possible to
test pre-immune rabbit serum.
Introducing the antibody into the cell through the
patch pipette enhanced the odor (TET)-evoked inward

Figure 5-6. Odors Activate an IP3-gated Channel Inserted Into a Cultured Lobster
Olfactory Receptor Neuron (ORN)
Left: An inside-out patch taken from one ORN was confirmed to contain an IP3-gated
channel of the type shown in Fig. 5-3 by "spritzing" 1CT7 M IP3 on the inner face of
the patch. IP3 was applied at the beginning of the trace shown.
Right: The patch was subsequently "crammed" into a second ORN, which was
subsequently "spritzed" with TET (arrow). Application of the odor activates two
superimposed channels in the patch, suggesting that the odor elevates the
intracellular concentration of IP3. The peak concentration of IP3 in the cell is
presumably greater that 10'7 M, since it elicited superimposed channel openings;
10'7 M IP3 elicited only single channel openings in the calibration trial (left).
Activity of the channels declines over the subsequent 24 sec following odor
stimulation. Cartoon depicts the hypothesized mechanism of action: Odors (â– ) bind
to cell-surface receptors and activate the enzyme phospholipase C (PLC) through G-
protein (G) mediation to convert phosphatidylinositol 4,5-bisphosphate (PIP2) to
inositol 1,4,5-trisphosphate (IP3), which is detected by the IP3-gated channels in
the patch. No channel activity was observed prior to odor stimulation. Membrane
potential, -60 mV. Records filtered at 2 kHz. "C" and "O" as defined in Fig. 5-2.

157

Figure 5-7. A polyclonal antibody raised against the 19 ex¬
termina! amino acids (shaded portion) of a cDNA clone of a
mouse cerebellar IP3 receptor was used to localize the IP3
receptor protein in lobster by Western plot analysis and
used to perturb the magnitude of macroscopic odor-evoked
currents and the Propen and kinetics of IP3-gated channels by
electrophysiological analysis.
Modified, with permission from Ferris and Snyder, 1992.

159
from Ferris and Snyder, 1992.

Figure 5-8. Localization and Functional Selectivity of an IP3 Receptor in Cultured
Lobster Olfactory Receptor Neurons.
A) Immunoreactivity of membrane fractions from mouse cerebellum and cultured lobster
olfactory receptor neurons (culture) to an antibody directed against a cDNA clone of
a mammalian IP, receptor (first lane of each pair) and to nonimmune rabbit IgG
(second lane of each pair) . Molecular weight standards as indicated. Arrow denotes
antibody cross reactivity with a greater than 200 kDa protein in each preparation.
B) Upper traces: Whole-cell recording from a voltage-clamped cultured lobster
olfactory receptor neuron that was sequentially patched to determine the amplitude of
inward current evoked by the odor, TET (on at arrow) with (odor + antibody) and
without (odor) the antibody in A) in the patch pipette. The current increased over
time (as indicated) with antibody in the pipette. Membrane potential, -60 mV. Lower
traces: Same protocol applied to an outward current evoked by proline (on at arrow).
C) Unitary currents recorded in an inside-out patch, activated by "spritzing" 10"7 M
IP, on the inner face of the patch (IP,) are increased when the same concentration of
IP, is co-presented with the antibody in A) (IP, + Antibody). In each instance
shown,the records are representative of channel activity several seconds after
"spritzing". Membrane potential, -60 mV. Records filtered at 2 kHz. "C" and "O" as
defined in Fig. 5-2.
D) Amplitude histogram of the unitary currents recorded in (C), activated by
"spritzing" 10'7 M IP, on the inner face of the patch in the absence (IP,) and
presence (IP, + Antibody) of the antibody in A). The probability of open state
(Propen) , but not the conductance, increases in the presence of the antibody.
"Closed" and "Open" state current distributions of the channel are indicated with
arrows.

Mouse Culture
Brain
c
Odor
I
60 mV
20 pA
500 msec
Odor + Antibody
9 min
B IP3
r~~"
IP3 + Antibody
10 pA
161

162
current (Figure 5-8B, upper traces). The peak amplitude of
the odor-evoked current increased an average of 427 ± 48%
within 3 min after breakthrough over that evoked without the
antibody in the pipette (5 of 5 sequentially patched cells).
The effect of the antibody increased slightly over the
30 min following the second breakthrough, presumably
reflecting further diffusion of the antibody from the
pipette. The effect of the antibody was selective for the
inward current (Figure 5-8B, lower traces). Introducing the
antibody into the cell failed to visibly alter the odor
(proline, taurine, alanine)-evoked outward current (9 of 9
sequentially patched cells). Without antibody in the
pipette, odor (TET, proline, taurine, betaine)-evoked
currents of both polarities decreased up to 43% of their
initial magnitude over the 20-30 min following breakthrough.
To determine if the effect on the inward current was
specific for the antibody, another group of cells was
single-patched with pipettes that were tip-filled with
normal patch solution and backfilled with either heat
inactivated antibody (8 cells), rabbit serum (7 cells) or an
antibody raised against Golf (16 cells; a generous gift of R.
Bruch; this antibody does not recognize any protein in
lobster ORNs by Western blot analysis - unpublished data),
and the protein allowed to diffuse into the cell for up to
30 min. All three control proteins failed to visibly alter
the magnitude of the odor-evoked current of either polarity

163
from that observed without antibody in the pipette (arcsin
transformation of percentage data followed by Student's t-
test). Using this same technique, but backfilling with the
antibody enhances the odor (TET)-evoked inward current to
approximately the same extent observed in the sequentially
patched cells (375 ± 29 %, 4 cells), suggesting that the
control proteins were able to diffuse into the cells during
the experimental interval. In the absence of odor
stimulation, the antibody had no effect on the basal current
(data not shown).
The antibody increased the ability of 1CT7 M IP3 to
activate unitary currents in 4 of 5 cell-free patches
(Figure 5-8C) . The antibody increased the Propen of all
channels in the four patches (n = 2 small and 5 large) from
an average of 0.11 ± 0.06 to 0.41 ± 0.07 (Figure 5-8D). In
one patch that contained multiple channels (4 large), the
channels showed a marked increase in superposition in the
presence of the antibody; from 1-2 of the channels being
open 78% of the time to 3-4 of the channels being open 65%
of the time. The antibody selectively increased the
duration of the second exponent (r2) of the mean open time
(tD) from 0.84 ± 0.17 msec to 19.06 ± 9.66 msec; the other
kinetic parameters and the conductance were unaltered (n=4).
In the fifth patch, the antibody first decreased, and then
blocked, the activity of a large conductance channel.

164
Discussion
The ability of odors to increase channel activity in
cell-attached recordings argues strongly for the involvement
of a diffusible second messenger in the excitatory
transduction pathway. Second messenger mediation is also
consistent with evidence that the odor-evoked inward current
in lobster ORNs is affected by probes directed against GTP-
dependent proteins (see Chapter 4) that presumably would
link receptor activation to second messenger production.
Several findings argue that IP3 is the excitatory
second messenger. (1) IP3 selectively evokes macroscopic
inward currents, which would be expected to depolarize
(excite) the cells. (2) RR blocks both the odor-evoked
macroscopic current and the IP3-gated unitary current.
(3) An odor (TET) activates an IP3-gated channel inserted
into the cell, as would be expected if odor-binding elevated
the intracellular concentration of IP3. Although, in the
latter instance, I cannot exclude that the odor altered the
intracellular environment in some way other than elevating
IP3, preliminary findings suggest that altering
intracellular pH from 5-9 and elevating [Ca2+] ± up to 30 mM
fails to initiate channel activity in cell-free patches.
The ability of IP3 to elicit channel activity in cell-
free patches argues that IP3 acts directly on the channels
and not through activation of a protein kinase or some other
additional step in the transduction cascade, since any

165
soluble enzymes or substrates required for activation would
presumably be diluted below threshold concentrations in the
bath shortly after patch excision. I cannot conclude
directly that the IP3-gated channels observed in cell-free
patches are the same as the channels activated by odors in
cell-attached recordings. My finding that the unitary
currents in both recording configurations have similar mean
open times and that both the odor-evoked macroscopic current
and the IP3-gated channels observed in cell-free patches can
be blocked by RR argues that the IP3-gated channels observed
in cell-free patches are a component of an IP3-mediated
excitatory transduction cascade.
Finding channels on the soma of cultured ORNs that
presumably function on the dendritic processes of these
cells in situ, would be consistent with the finding that
cAMP-gated channels that mediate excitation in the cilia of
dissociated vertebrate ORNs also occur in low density on the
dendrite and soma (Nakamura and Gold, 1987; Firestein et
al., 1991). Whether the IP3-activated channels normally
occur on the soma of lobster ORNs or are inserted there
prior to relocation to the dendritic processes during
neurite outgrowth in culture remains to be tested.
I conclude that IP3 gates two different channels rather
than one channel with two different sub-conductance states,
as has been reported for IP3-activated channels in canine
cerebellum or aortic smooth muscle (Mayrleitner et al.,

166
1991; Watras et al., 1991). Save for one patch, the large
and small conductance channels occurred in different patches
of membrane. Differences in the voltage dependencies and
reversal potentials in symmetrical ionic conditions further
support the conclusion that IP3 gates two different
channels. Since two variably spliced mRNAs code for
intracellular IP3 receptors (e.g., mouse cerebellar IP3
receptor - Nakagawa et al. 1991), it would not be
inconsistent to have two subtypes of IP3-activated channels
expressed in lobster ORNs.
The lack of identical recording conditions makes it
difficult to compare the lobster olfactory channels with
other reported IP3-activated channels based on their
respective conductances (Enrich and Watras, 1988; Restrepo
et al., 1990; Maeda et al., 1991; Mayrleitner et al., 1991),
but the ability of RR and heparin to block the IP3-gated
channels in lobster ORNs is similar to the pharmacological
properties of intracellular IP3 receptors. RR inhibits IP3-
activated channels in skeletal muscle SR but not in aortic
smooth muscle (Enrich and Watras, 1988). Heparin, which is
thought to block the IP3-binding site (Supattapone et al. ,
1988), acts in a variety of cell types (Hill et al., 1987;
Worley et al, 1987; Ghosh et al., 1988; Kobayashi et al.,
1988; Komori and Bolton, 1990; Frank and Fein, 1991; Fisher
et al., 1992). The reversibility of the heparin block that
we observed with washing has also been reported in smooth

167
muscle (Mayrleitner et al., 1991) and demonstrates that
heparin, which is known to be cytotoxic at high
concentrations (Frank and Fein, 1991), did not damage the
membrane.
The IP3-activated channels in lobster ORNs differ from
intracellular IP3 receptors in their ATP dependency. While
ATP failed to alter the conductance or the mean open time of
the channels in lobster ORNs, it increases IP3-induced
macroscopic currents in aortic smooth muscle SR (Mayrleitner
et al. , 1991) and the Propen of intracellular IP3-gated
channels (Ehrich and Watras, 1988; Mayrleitner et al.,
1991). The ATP-driven Ca2+ pump postulated to be associated
with the IP3-release mechanism in other, non-sensory cells
(Ferris et al., 1990) may not be functional in sensory
transduction and therefore not present or active in the
lobster cells. Danoff et al. (1991) report mRNA coding for
IP3 receptors in rat and human that lack the SII region of
the channel protein that contains the ATP binding sites.
From my electrophysiological data alone, I cannot assign the
apparent lack of modulation by ATP to protein variation,
species differences or olfaction.
The IP3-activated channels in lobster ORNs presumably
share some structural homology with mammalian IP3 receptors,
since an antibody raised against a cDNA clone of a mammalian
IP3 receptor recognized a protein of similar size in the
lobster. The molecular weight (>200K) of the immunolabelled

168
band in lobster ORNs is consistent with the size of
intracellular IP3 receptors, that are proposed to be a
tetramer of noncovalently bound isomers with a
characteristic Mr of 260K by SDS-PAGE analysis (Mignery et
al., 1989; Fisher et al., 1992). That the antibody is
binding to a functionally relevant molecule is suggested by
the ability of the antibody to selectively increase odor-
evoked inward currents. The ability of the antibody to
selectively increase the Propen and t0 of IP3-activated
channels argues further that the antibody targets the
channel to augment the macroscopic current. The antibody
would not necessarily be expected to target the
transmembrane pore and block ion flow, as it was directed
against the 19-amino acid carboxyl terminus of the receptor
that DeCamilli et al. (1990) postulate is involved in Ca2+
sequestration. Indeed, a monoclonal antibody targeting a
different epitope of the C-terminus has been shown recently
to block IP3- and Ca2+-induced release of Ca2+ in fertilized
hamster eggs, suggesting that the C-terminus can regulate
channel gating (Miyazaki et al., 1992). Independent of
mechanism, the ability of the antibody to selectively
enhance odor-evoked inward currents independently of outward
currents provides further support that IP3 is the excitatory
second messenger.
Thus, from the data presented in this chapter, I
conclude that the IP3 receptors in cultured lobster ORNs are

169
in the plasma membrane since (1) a high percentage of cell-
free patches (63 of 86 successful seals) taken from the
plasma membrane are directly activated by IP3 and (2) an
antibody directed against a known IP3 receptor alters IP3-
activated unitary currents in cell-free patches of plasma
membrane. It is not likely that organelles closely apposed
to the plasma membrane would be frequently drawn into the
patch. Even if this occurred, the plasma membrane would
need to be disrupted in order to reseal onto the organelle,
and the organelle membrane would need to be recorded in the
outside-out configuration for IP3 to bind. Earlier reports
differ on whether IP3 receptors occur in the plasma membrane
of neurons. In studies of IP3 receptors in the cerebellum,
Maeda et al. (1989) localized the P400 (IP3) receptor to the
ER, postsynaptic densities and the plasma membrane, while
Ross et al. (1989) localized the receptor to the ER,
subplasmalemmal cisternae, and nuclear membrane, and NOT to
the plasma membrane. Immunogold labeling with the same
antibody used in this report failed to reveal an IP3
receptor in the plasma membrane of cerebellar neurons
(Mignery et al., 1989). Yet, my conclusion is consistent
with evidence emerging from studies of olfactory neurons.
Khan et al. (1992) identified a 260 kDa protein in the
plasma membrane of rat olfactory cilia that stained with
antiserum to a cerebellar IP3 receptor in Western blots.
Kalinoski et al. (1992) photoaffinity labeled a 107 kDa

170
protein in the plasma membrane of catfish olfactory cilia
which binds IP3. Restrepo et al. (1990) were able to
reconstitute an IP3-activated channel from a membrane
preparation of catfish olfactory cilia. To the extent that
the membrane preparations used in these studies are purely
ciliary, these findings argue for plasma membrane receptors
since the cilia of ORNs are devoid of organelles. Indeed,
Cunningham et al. (1992) have preliminary evidence that an
immunogold labelled antibody directed against a cerebellar
IP3 receptor labels rat ciliary membrane. The plasma
membrane IP3 receptors in olfactory receptor cells may
represent a new class of IP3 receptors that share structural
homology with intracellular IP3 receptors, but are localized
to the plasma membrane.
My ability to record IP3-activated unitary currents in
native plasma membrane of lobster ORNs and to tie these
unitary currents to odor-activated macroscopic inward
currents directly implicates plasma membrane IP3 receptors
in olfactory transduction. I thereby conclude that odors
excite lobster ORNs by increasing intracellular IP3, which
activates IP3-gated channels in the plasma membrane of the
cell and depolarizes the cell in a concentration dependent
manner. The IP3-mediated transduction pathway presumably
co-exists in some cells with a second, cAMP-mediated pathway
that hyperpolarizes the cell and modulates the magnitude of
excitation to reflect the particular ratio of excitatory and

171
inhibitory odor components in an odor blend (Michel et al.,
1991; Michel and Ache, in press). Having two, parallel
transduction pathways, one mediated by IP3 and the other by
cAMP, allows the lobster ORN to serve as an integrating
unit. The ability of olfactory receptor cells with parallel
transduction pathways to integrate information about the
composition of odor blends may be integral to odor coding,
as suggested by Reed (1992), and may explain why odors
activate both IP3- and cAMP-mediated second messenger
pathways in animals as phylogenetically diverse as
arthropods, fish and mammals, as noted in the Introduction.
Experimental Procedures
Solutions
Panulirus saline (PS) consisted of (in mM): 458 NaCl,
13.4 KCL, 9.8 MgCl2, 13.6 CaCl2, 13.6 Na2S04, 3 HEPES, and
2 glucose; pH 7.4. Modified L15 Media consisted of: 50 ml
Liebowitz L15 stock, 50 ml of 1.6 times the normal
concentration of PS, 0.6 g dextrose, 0.026 g L-glutamine,
and 0.01% gentamicin. Phosphate-sucrose buffer (PSB)
consisted of (mM): 10 Na2P04 and 250 sucrose; pH 7.3.
Homogenization buffer (HB) consisted of (mM): 320 sucrose,
10 Tris base, 50 KC1, and 1 EDTA. Tris buffered saline
(TBS) consisted of (mM): 50 Tris HCl, 150 NaCl; pH 7.5.
Tween-TBS (TTBS) was made by adding 0.1% Tween-20 to TBS.
Blotting buffer (BB) consisted of (mM): 25 Tris HCl,
192 glycine, plus 0.1% SDS and 10% methanol. Normal patch

172
solution consisted of (mM): 30 NaCl, 11 EGTA, 10 HEPES,
1 CaCl2, 180 K-acetate, and 696 glucose; pH 7.0. Ruthenium
red (10 fxm) and heparin (2.5 pirn) were prepared fresh daily.
Odors were an aqueous extract of TetraMarin, a
commercial flake fish food (Tetra Werke, Melle, FRG) and
solutions of d-alanine, betaine, glycine, 1-proline and
taurine; compounds known to be adequate stimuli for the
chemoreceptors of aquatic organisms such as lobsters (Carr,
1988). A stock extract of TetraMarin (TET) was made by
mixing 2 gm dry flakes in 60 ml saline, centrifuging the
resulting suspension at 1400 g, filtering it through Whatman
#3 filter paper, adjusting it to pH 7.4, and storing it
frozen in 5 ml aliquots. The stock extract was thawed and
diluted 1000-fold in either PS or culture media, as
appropriate, just prior to use and applied at that
concentration. The pure compounds were prepared fresh as
1CT3 M solutions in either PS or culture media, as
appropriate, adjusted to pH 7.4, as needed, and applied at
that concentration.
All drugs and chemicals were obtained from Sigma
Chemical Co, except for the culture media. Sources for the
culture media and supplements are listed in Fadool et al.
(1991b).
Tissue Culture
Distinct clusters of ORNs were dissected from the
olfactory organs (lateral antennular filaments) of adult

173
specimens of the Caribbean spiny lobster, Panulirus argus,
and enzymatically dissociated. The resulting cells were
sustained in primary culture as described previously (see
Chapter 2). Briefly, the isolated clusters were incubated
for 50 min at 80 rpm on an orbital shaker in 0.2 micron
filter-sterilized solution of 2.5 mg papain and 12 mg L-
cysteine in 10 ml Panulirus saline (PS) containing 1%
penicillin, streptomycin sulfate, and amphotericin B
(Gibco). Proteolytic digestion was stopped by replacing the
enzyme solution with low glucose L-15 media supplemented
with L-glutamine, dextrose, fetal calf serum, and BME
vitamins. Cells were immediately plated on poly-d-lysine-
coated glass coverslips. Cells were maintained at
saturation humidity in a modular incubator chamber (Billups-
Rothenberg) at 24°C.
Electrophysiology
The cultured cells were viewed at 40X magnification
with Hoffman optics for patch-clamp recording (Hamill et
al., 1981). Patch electrodes, fabricated from 1.8 mm O.D.
borosilicate glass and fire polished to a tip diameter of
approximately 1.0 /im (bubble number 4.8; Mittman et al. ,
1987), produced seal resistances between 8 and 14 GQ. The
electrodes were uncoated. Signals were amplified with an
integrating patch amplifier (Dagan 3900).
Macroscopic currents were recorded in the whole-cell
configuration at a holding potential of -60 mV, unless noted

174
otherwise, using normal patch solution in the pipette. The
analog records were filtered at 5 kHz and digitally sampled
every 4 msec (every 240 msec, in one noted instance) for
subsequent processing on an IBM-compatible computer using
pCLAMP software (Axon Instruments). The compact, spherical
shape of the cultured cells (see Chapter 2) facilitated
obtaining an effective space clamp on most cells. Odors and
membrane permeant probes were "spritzed" on the cells for
120 msec from a six barrel glass micropipette coupled to a
pressurized valve system (Picospritzer, General Valve). The
concentration of odors and probes delivered in this manner
are reported as the pipette concentration; no attempt was
made to correct for dilution. In order to compare the
effect of impermeant probes on cell activity, cells were
sequentially patched, first without, then with the probe in
the pipette in order to provide a within-cell control.
There was no noticeable effect on the response of
sequentially patched cells to odor or voltage stimulation in
the absence of any experimental manipulation.
Unitary currents were recorded in either the cell-
attached or (primarily) the cell-free, inside-out
configuration. The normal patch pipette solution was a
potassium acetate patch solution (see solutions); both faces
of the patch were exposed to symmetrical solutions. The
analog records were filtered at 2 kHz and recorded on
videotape. On playback, the records were sampled every

175
100 [isec for processing on an IBM-compatible computer, also
using pCLAMP software (Axon Instruments). Probes were
"spritzed" on the inner face of the patch for 550 msec from
the multibarreled pipette mentioned above.
All results are presented as the mean ± standard error
of the mean. Statistical significance was calculated at the
95% confidence level.
Immunochemistrv
Mouse cerebellum was isolated then diced in chilled
phosphate sucrose buffer (PSB). Thirty-six hour cultured
ORNs were scraped with a rubber policeman, centrifuged below
1,000 RPM for 2 min at 4°C, and removed from media without
disrupting the cells. Both tissues were individually
homogenized in chilled homogenization buffer (HB), either
with 50 strokes by mortar and pestle (Wheaton, Size B) or
2 min by a Kontes tissue grinder in microcentrifuge tubes,
and then tip sonicated (Heat systems, Ultrasonics Inc.,
W-220) three times for 15 s at a #3 setting while iced.
Homogenates were centrifuged twice at 5,000 RPM for 30 min
at 4°C. The respective supernatants were combined and
centrifuged (Beckman L70 Ultracentrifuge) at 38,000 RPM for
2.5 h at 4°C. The recovered pellets were resuspended in HB
by bath sonification (Heat systems, Ultrasonics Inc., W-225)
three times for 15s at a #5 setting and frozen at -80 °C
until use.

176
Protein was determined by a Bradford photometric assay.
Twenty to twenty-five micrograms of the membrane preparation
was separated at constant current (25 mA; 3 h) on a 5-15%
gradient SDS electrophoretic gel (0.55 mm, 29:1
acrylamide/bis-acrylamide). Proteins were transferred to
nitrocellulose in a modified blotting buffer (BB) at 0.5 mA
for 3.5 h. Nitrocellulose membrane was soaked twice for
10 min in Tween-tris buffered saline (TTBS). Non-specific
binding was blocked by incubating the membrane for 30 min
with 2.5% Carnation dry milk in TTBS. The nitrocellulose
membrane was then incubated for 15 h at 4°C with a
polyclonal antibody (1:200) raised against the 19 C-terminal
amino acids of a cDNA clone of a mouse cerebellar IP3
receptor (Mignery et al., 1989; Figure 5-7), washed twice
for 10 min in TTBS to remove unbound antibody, and
reincubated for 2 h with a 1:250 dilution of peroxidase-
conjugated goat anti-rabbit secondary antibody (Boehringer
Mannheim). Gels were visualized with 4-chloro-l-naphthol
color reagent activated by 30% H202 in tris buffered saline
(TBS) and 20% methanol.
In control experiments, membrane preparations (750 /¿g
total protein) were separated with a continuous front, 8%
gradient SDS electrophoretic gel. Proteins were transferred
to nitrocellulose, as previously, but were subsequently
incubated with preabsorbed antibody. The antibody was
preabsorbed by incubating the antibody for 30 min on ice

177
with 10:1, 1:1, 0.1:1, 0.01:1, and 0:1 molar ratios of the
peptide PCD6 (Mignery et al., 1989; the last 19C terminal
amino acids) to antibody. Following incubation, the samples
were centrifuged below 1,000 RPM for 30 min at 4°C to remove
particulates and antibody-peptide complexes. The respective
supernatants were applied as 200 /x 1 samples using a
miniblotter.

CHAPTER 6
ION SELECTIVITY AND MODULATION OF IP3-ACTIVATED CHANNELS
Introduction
Across a broad range of species, odor-evoked second
messenger molecules (Breer, Boekhoff, and Tareilus, 1990;
Ronnett, Cho, Hester, Wood, and Snyder, 1993; Ache, Hatt,
Breer, and Zufall, in press) directly gate ion channels in
olfactory receptor neurons (ORNs) to create a change in
membrane potential (Nakamura and Gold, 1987; Suzuki, 1990;
Restrepo, Miyamoto, Bryant, and Teeter, 1990; Firestein,
Zufall, and Shepherd, 1991; Kurahahi and Kaneko, 1991;
Zufall, Firestein, and Shepherd, 1991; Fadool and Ache,
1992a). Recent evidence strongly supports the inositol
phosphate pathway in olfactory transduction, in addition to
the earlier supported role of cyclic nucleotides (Reviews:
Firestein, 1992; Reed, 1992 ; Ronnett and Snyder, 1992) .
Odors rapidly and transiently elevate levels of InsP3 in
olfactory cilia of catfish (Fitzgerald, Restrepo, and
Bryant, in press) and rats (Breer, et al., 1990) and in the
outer dendrites of lobsters (Ache et al., in press);
NOTE: This chapter will be submitted to J. Gen. Physiology
and is formatted to meet its style regulations.
178

179
functionally equivalent structures that are the site of
transduction in olfactory receptor cells (Nakamura and Gold,
1987; Kurahashi, 1989; Firestein et al., 1990). InsP3
receptors can be reconstituted from olfactory cilia in
catfish (Restrepo et al., 1990) and immunocyto-chemically
localized to these same structures in rats (Cunningham,
Reed, Ryugo, Snyder, and Ronnett, 1992). Known blockers of
InsP3-gated channels in other systems block odor-evoked
currents in olfactory receptor cells in catfish and lobster
(Restrepo et al., 1990; Fadool and Ache, 1992a).
An olfactory specific InsP3-receptor has not yet been
cloned, but InsP3 receptors involved in olfactory
transduction clearly differ from cerebellar InsP3 receptors
mediating Ca2+ mobilization in the endoplasmic reticulum
(Review: Berridge, 1993). Most obvious is that olfactory
InsP3 receptors are plasma membrane receptors (Cunningham et
al., 1992; Fadool and Ache, 1992a; Khan, Steinter, Snyder,
1992) . The olfactory InsP3 receptor in catfish binds InsP3
and InsP4 with equal affinity (Kalinoski, Aldinger, Boyle,
Huque, Marecek, Prestwich, and Restrepo, 1992) in contrast
to the specificity of the cerebellar InsP3 receptor which
binds other metabolites, including InsP4, only at high
concentrations and with low affinity (e.g. Ferris, Huganir,
Supattapone, and Snyder, 1989). Functionally, however,
olfactory InsP3 channels in catfish, rat, and lobsters are
selectively gated by InsP3 (Restrepo et al. , 1990; Restrepo,

180
Teeter, Honda, Boyle, Marecek, Prestwich, and Kalinoski,
1992; Fadool and Ache, 1992a, 1993b). Unlike cerebellar
InsP3 receptors (Ferris, Huganir, and Snyder, 1990; Maeda,
Kawasaki, Nakade, Yokota, Taguchi, Kasai, and Mikoshiba,
1991; Watras, Bezprozvanny, and Ehrlich, 1991), olfactory
InsP3-activated channels are not modulated by ATP (Fadool
and Ache, 1992b), are blocked by both heparin and ruthenium
red (RR) (Restrepo et al., 1990; Fadool and Ache, 1992a),
and do not appear to have multiple conductance states
(Fadool and Ache, 1992a). Certain transduction elements
have been cloned and shown to be expressed in a highly
olfactory specific manner making the likelihood of an
olfactory specific InsP3 receptor highly probable. Examples
of such elements include the G-protein, Golf (Jones and Reed,
1989; Jones, 1990; Jones, Masters, Bourne, and Reed, 1990)
or the olfactory cyclic nucleotide gated channel (Dhallan,
Yau, Schrader, and Reed, 1990; Ludwig, Margalit, Eisman,
Lancet, and Kaupp, 1990; Goulding, Ngai, Kramer, Colicos,
Axel, Siegelbaum, and Chess, 1992).
There are two different InsP3 channels in the soma
membrane of cultured lobster ORNs, both of which potentially
are involved in olfactory transduction (Chapter 5). Lobster
ORNs in culture appear to express the elements of the
transduction cascade in the soma (Chapters 2-5). These
channels, which only rarely occur in the same patch of
membrane, differ in mean conductance (30 pS and 74 pS),

181
voltage sensitivity, and mean closed time (tc) kinetics, but
have similar pharmacology to RR and heparin, concentration
dependency, and similar altered gating when bound to an
antibody directed against the carboxyl terminus of a
cerebellar InsP3 receptor (Chapter 5). In this chapter I
further characterized the biophysical properties of the two
types of InsP3-gated channels in cultured lobster ORNs to
better understand any functional difference between the two
olfactory InsP3-gated channels, and the role of the inositol
phosphate pathway in olfactory transduction in general.
The data demonstrate that the large (74 pS)- and small
(30 pS)-conductance InsP3-gated channel respond similarly to
increased Ca2+ or pH; have analogous long open modal gating
patterns; comparable pharmacology with respect to TTX, TEA,
ruthenium red, and Co/Cd; and nearly identical ionic
selectivity, predominantly that of non-selective cation
channels. This chapter provides a comparison of the
olfactory InsP3-gated channels with the cerebellar InsP3-
gated channels. Speculation as to significance of the
apparent redundancy in having more than one channel gated by
the same second messenger is offered in the discussion.
Materials and Methods
Solutions
The compositions of Panulirus saline (PS), and patch
electrode (PE) and bath solutions are listed in Table 6-1.
Modified LI5 Media consisted of: 50 ml Liebowitz L15 media,

182
(in mM)
NaCl
EGTA
HEPES
CaCl2
CH3COOK
KC1
BaCh
MgCl2
CsCl2
ChCl2
Glucose
Control KA Patch
30
11
10
1
180
696
Ca Patches
30
10
0.001 to
30
180
595 to 699
Ba Patch
10
180
30
655
Panuliras saline
(PS)
460
10
13
13
10
Na IP3 Bath
480
20
0.1
1.7
NalOOCs Bath
380
20
0.1
100
1.7
Ca Bath
10
60
420

183
50 ml of 1.6X normal concentration of PS, 0.6g dextrose,
0.026 g L-glutamine, and 0.01% gentamicin. Tetrodotoxin
(TTX, 1 nM) as well as all control and modified InsP3
solutions were made up in PE as concentrated stock solutions
and stored frozen at -20°C until use. Single aliquots of
InsP3 were thawed and diluted in PE to a working
concentration of 2.4 x 10'7 M. Care was taken during
storage and use of InsP3 to minimize its exposure to light,
as its sensitivity to degradation is suspected. pH-modified
InsP3 solutions are estimates to within ± 0.5 pH unit based
upon litmus paper reaction. Ten micromolar ruthenium red
(RR) was prepared by grinding 0.55 mg (MW=551) of RR (=38%
dye content) into 10 ml PS until visibly reconstituted.
Solution was then filtered (0.2 /¿m) to remove any
particulates. As the precise dye content, molecular weight,
and solubility of this product are unknown, 10 /¿M RR
represents an upper limit estimate of concentration.
Thapsiagargin was stored in a concentrated EtOH solution
at -20°C until use, and then diluted with PS to a working
concentration of 8 x 1CT7 M. Tetraethylammonium (TEA, 5 mM)
and Co/Cd (5 mM each) were made as needed in either culture
media or PE as appropriate for whole-cell or single-channel
recording, respectively.
Odorants consisted of either single components (10'3 M)
made daily in PS: taurine, betaine, 1-proline, 1-arginine,
or 1-glycine, AMP, or a complex odorant mixture, TetraMarin

184
(TET), a marine fish food. A stock extract of TET was made
by mixing 2 gm dry flakes in 60 ml saline, centrifuging the
resulting suspension at 1400 g, filtering it through Whatman
#3 filter paper, adjusting it to pH 7.4, and storing it
frozen in 5 ml aliquots. The stock was thawed and diluted
1:1000 in either culture media or PE as appropriate for
whole-cell or single-channel recording, respectively.
Sources for the culture media and supplements are
listed in Chapter 2. All salts, amino acids, TEA, Co/Cd,
RR, and InsP3 were obtained from Sigma Chemical Company.
Thapsiagargin was purchased from Calbiochem.
Animals
Adult specimens of the Caribbean spiny lobster,
Panulirus argus, were collected in the Florida Keys and
maintained in the laboratory in a running seawater. Animals
were fed a mixed diet of frozen fish, squid, and shrimp.
Tissue Culture
The distinct clusters of olfactory (aesthetasc)
receptor neurons (ORNs) were dissected from the olfactory
organ (lateral antennular filament) of adult specimens of
the Caribbean spiny lobster, Panulirus argus. The clusters
were enzymatically dissociated, and the resulting cells
sustained in primary culture as described previously
(Chapters 2-5). Briefly, the isolated clusters were
incubated for 50 min at 80 rpm on an orbital shaker in
0.2 micron filter-sterilized solution of 2.5 mg papain and

185
12 mg L-cysteine in 10 ml Panulirus saline (PS) containing
1% penicillin, streptomycin sulfate, and amphotericin B
(Gibco). Proteolytic digestion was stopped by replacing the
enzyme solution with low glucose L-15 media supplemented
with L-glutamine, dextrose, fetal calf serum, and BME
vitamins. Cells were immediately plated on poly-d-lysine-
coated glass coverslips. Cells were maintained at
saturation humidity in a modular incubator chamber (Billups-
Rothenberg) at 24°C.
Electrophvsioloqy
Patch electrodes were fabricated from 1.8 mm O.D.
borosilicate glass and fire polished to a tip diameter of
approximately 1.0 /xm (bubble number 4.8; Mittman, Flaming,
Copenhagen, and Belgum, 1987). High resistance seals of
between 8 and 14 GQ were obtained. Cells were viewed at 40X
magnification with Hoffman optics.
Unitary currents were recorded from cell-free patches
of the soma plasma membrane in the inside-out configuration.
All patches were voltage-clamped at -60 mV holding
potential, unless noted otherwise. The unitary currents
were recorded with an integrating patch-clamp amplifier
(Dagan 3900), filtered at 2 kHz, and captured on videotape.
On playback, the analog records were sampled every 100 ¿xsec
for processing on an IBM-compatible computer, using pCLAMP
software (Axon Instruments). InsP3 solutions were
"spritzed" on the inner face of the patch for 550 msec from

186
a seven barrel glass micropipette (Frederick haer) coupled
to a single pressurized valve system (Picospritzer, General
Valve) via a 6-way rotary valve as previously described
(Chapter 2). The normal patch pipette solution was a
potassium acetate patch solution (KA patch, Table 1); both
faces of the patch were exposed to symmetrical solutions
unless noted otherwise.
Odor-activated macroscopic currents were recorded by
patch-clamping the ORNs in the whole-cell configuration and
were also held at -60 mV membrane potential. Odorants and
membrane permeant pharmacological probes were "spritzed" for
120 msec, as described, with the exception of the Ca2+-
ATPase pump inhibitor, thapsiagargin, which was applied to
the culture medium 2 hours prior to patch-clamp recording.
Macroscopic currents were filtered at 10 kHz and sampled
every 4 msec.
Data are reported as the mean ± the standard error of
the mean (SEM). For statistical comparisons, significance
was defined at the 95% confidence level.
Results
Effect of pH on InsP,-activated Channel Gating
To determine whether free hydroxide concentration could
affect the gating of InsP3-activated channels, pH modified
solutions of 2.4 x 10'7 M InsP3 were applied to the inside
face of 6 cell-free patches of ORN membrane. Both channels
were pH insensitive between pH 6 and pH 8. The open

187
probability (Propen) of the 30 pS InsP3-gated channel then
increased between pH 8 and 11 from 0.05 ± 0.03 to 0.62 ±
0.08; a change of approximately 12 fold (n=4). The 74 pS
InsP3-gated channel showed a similar increased Propen between
pH 8 and 11 from 0.07 ± 0.02 to 0.36 ± .14; a change of
approximately 5 fold (n=2) (Figure 6-1).
Appearance of Modal Patterns Differing in Gating Kinetics
In approximately ten percent of all patches recorded
(11 of 110), both the 30 (Figure 6-2) and 74 pS InsP3-gated
channel (data not shown) entered long duration open modes.
Channels left their most observed state (Propen = 0.33) and
entered one of three kinetically different modes. Modes
were defined based on cutoff open probability as follows;
Mode 1, Propen was > 0.90, Mode 2, the channel maintained a
sustained open state (Propen = 1.0) for a second or longer,
and Mode 3, Propen was < 0.15.
The different patterns of gating can be attributed to
the mean open and closed dwell-time in both the 30 and 74 pS
InsP3-gated channels (Figure 6-3). The sampling size of the
mean open (tQ) and closed time (tc) for channel modes varied
because not all 11 patches contained all three modal
patterns and because reliable estimates of tQ and tc could
not be made for modes having extremely low Propen or long
open durations. This small sampling size, prevented
statistical comparison of the mean open or closed duration
across modes and between the two conductance channels.

Figure 6-1. Effect of pH on the gating of inositol 1,4,5-
trisphosphate (InsP3)- activated ion channels in lobster
olfactory receptor neurons (ORNs).
(A) Unitary currents of the large-conductance (74 pS)
InsP3-gated channel at increasing pH values. Channel
activity was recorded by applying 2.4 x 1CT7 M InsP3 to the
inside face of cell-free patches of membrane voltage-clamped
at -60 mV. Pipette and bath solution was symmetrical =
control KA patch (see Table 1) . Open probability (Propen) ,
calculated from curve-fit amplitude histogram distributions
of these records, is reported on the right. C= closed
state, 0= open state.
(B) Line graph showing mean ± SEM Propen for the small (30
pS)-(--A--A--) (n=4) and large (74 pS) (n=2)
conductance InsP3-gated channel as a function of pH.

189

Figure 6-2. Unitary currents of the small-conductance
InsP3-gated channel in lobster ORNs shown expressing three
different kinetics: Mode 1 = long open duration, Propen >
0.90; Mode 2 = maintained open state, Propen = 1.0; Mode 3 =
long closed state with rapid flicker to open state, Propen =
0.15. The most observed kinetic state (Mt Obs) is shown for
comparison (Propen = 0.33) . Similar mode behavior is
observed in the large-conductance InsP3-gated channel (data
not shown) . C, 0, Propen, recording conditions and
configuration as in Figure 6-1.

N>
T3
>
í
í
o n
Mode 2
Most Observed State

Figure 6-3. Histogram of the mean open (tQ) and close (tc)
times of the small- (striped bar) and large- (solid bar)
conductance InsP3-gated channels as a function of mode.
Data were calculated based on single exponential fits of the
open and closed dwell-time histogram after separating into
Mode 1 (Propen > 0.90), Mode 2 (Propen = 1.0, and Mode 3 (Propen
< 0.15 with flickery transitions). The most observed state
(mt obs; Propen ~ 0.33) is shown for comparison. Error bar ±
SEM for sample size as noted. Note that values for Mode 2
were divided by 100 to fit to scale.

193
A
45-
40-
35-
o
0
CO
30-
E
25-
c
2
20-
15-
10-
5-
o
0
CO
E
_c
'o'
mt obs mode 1 mode 2 mode 3
mt obs mode 1 mode 2 mode 3

194
Qualitatively, exponential time constants governing the
rapid transitions from open to closed state are strikingly
different between the three modes and the most observed
state but not between the two InsP3-gated channels,
independent of kinetic state (Figure 6-3). Modes 1 and 2
had a mean open time constant 10 and 1000X greater than that
in the most observed state (Figure 6-3A) with mean closed
time constants as much as 3X less than that in the most
observed state (Figure 6-3B). The change in gating kinetics
of Mode 3 could be solely attributed to a 3X increase in tc
over that in the most observed state, since tQ was
unaffected. Unlike the most observed state, the kinetics of
the modal patterns showed variability from one patch to the
next; mean values for the 3 0 pS InsP3-gated channel were:
Mode 1 - tQ = 129.06 ± 59.18 msec, tc = 0.31 ± 0.03 msec,
Mode 2 - tQ = 2814.75 ± 1901.5 msec, tc = 0 msec, and
Mode 3 - tQ = 0.26 ± 0.05 msec, tc = 9.7 ± 4.21 msec. The
mean values for the 74 pS InsP3-gated channel were: Mode
1 - t0 = 52.91 ± 20.67 msec, tc = 0.40 ± 0.08 msec, Mode 2 -
tQ = 1620.62 ± 570.9 msec, tc = 0 msec, and Mode 3 - tQ =
0.28 ± 0.07 msec, tc = 9.91 ± 0.89 msec.
Once channels progressed into one of the three defined
modes (Figure 6-2), spontaneous transitions from one mode to
another were infrequent, with sojourns ranging 1-21 seconds
in each mode. One method to discern whether the modal
gating was a random or ordered process would be to perform a

195
runs analysis. Runs analysis of two 74 pS InsP3-gated
channels and one 30 pS InsP3-gated channel demonstrated that
the channels displayed modes of gating that were clustered;
a given mode of gating was maintained for an arbitrarily
defined time period before spontaneously changing to another
mode (Figure 6-4). Transitions between modes, however, were
random, i.e., independent of the previous history of gating,
with the exception of Mode 3, which always exited to the
most observed state. This is best exemplified in a
contingency table of InsP3 channel gating, in which the
number of time intervals that a particular mode is followed
by the same or different mode is tabulated (Figure 6-4).
Analysis of the diagonal (double line box) demonstrated that
the observed (italics) frequencies of repeats of the same
type of gating mode was significantly different than the
expected (bracketed) if the pattern of gating was purely
random (Chi-square, p < 0.005). Highly diagonalized
contingency tables were obtained for the other two InsP3-
gated channels analyzed in this manner (data not shown).
Secondly, the observed frequency of entering or exiting
Mode 3 from another state was always one or zero;
significantly different than the expected (Chi-square, p <
0.005). This was consistent with the non-random behavior of
the channel entering Mode 3 through Mode 2, and then finally
exiting Mode 3 to either a blank or the most observed state.
Frequency distributions were used to yield information about

Figure 6-4. Four x four contingency table of small-
conductance InsP3-channel mode gating. The gating pattern
of interval (K + 1) is shown in relation to gating of
interval K; interval length was arbitrarily set to 300 ms,
N= 326 intervals classified. The observed numbers of
intervals in each category are in italics; expected numbers
for random occurrence in brackets. Expected numbers were
calculated as the product of individual modal probabilities
times the total number of intervals. Chi-square
significance p < 0.005 (shaded boxes). Similar analysis
yielded comparable results for large-conductance InsP3-
channel mode gating (table not shown).

197
Blanks
Mode 1
Mode 2
Mode 3
Blanks
0
0
0
1
[< 1]
[< 1]
[< 1]
[1]
Mode 1
0
45
16
0
[<1]
[18]
[27]
[31]
Mode 2
0
18
131
1
[< 1]
[27]
[41]
[47]
Mode 3
1
0
0
109
[1]
[31]
[47]
[55]

Figure 6-5. Distribution histograms of run lengths for mode
gating in InsP3 channels, constructed by pooling data from
three patches (1 small, 2 large conductance). A run = A
series of intervals displaying channel activity in the same
mode. The duration of the sojourns in each mode was
calculated by multiplying the mean number of intervals
(exponential fit of the distribution) by 307.20 ms, the
defined, arbitrary interval duration. Calculated time
constants: Mode 1= 1.49 ± 0.09 s, Mode 2- 0.88 ± 0.03 s,
and 'Mode 3= 11.76 ± 6.7 s. * = Calculated average duration
of three runs.

C 20
1 6
Mode 3
03
C
3
cr
-Q
2
8
4
0
0 1
[El
2 3
, A-
5 6 7 891011121314151617
Pun Length (Number of Intervals)
53 1 9 78

200
the duration a channel remained in one modal pattern of
gating before switching to another (Figure 6-5). Histograms
of the length of a run of a single type of modal gating were
well fit by a single exponential for Mode 1 and Mode 2. The
run length for Mode 3 was not exponential, implying an exit
rate that did not follow a random or Markov process.
Effect of Ca2+ on InsP,-activated Channel Gating
Although neither InsP3-activated channel could be gated
directly by ImM [Ca2+] ± or 13 mM [Ca2+]0, as evidenced by the
lack of channel activity evoked in either Ca patch or PS
solution, the Propen as well as the unitary conductance was
dependent upon the Ca2+ concentration of the InsP3 solution.
The InsP3-gated channels did not represent Ca2+-activated
channels, such as those mediating calcium-induced calcium
release (CICR) , but rather Ca2+ modulated, InsP3-activated
channels. When the inside face of the patch was challenged
with 2.4 x 1CT7 M InsP3, three of four 30 pS channels (Figure
6-6A) and two of two 74 pS channels (Figure 6-6B) increased
Propen between [Ca2+] t of 0.001 and 1.0 mM. Further
increasing the [Ca2+] A from 1.0 mM to 3 0 mM reduced the slope
conductance of each channel type (Figure 6-6). The
reduction in slope conductance under high [Ca2+] ± was
generally observed across the entire recorded voltage range
(Figure 6-7A), but in two instances was due to a change from
inward rectification, and therefore only varied at
depolarizing potentials (Figure 6-7B).

Figure 6-6. Effect of Ca2+ on the gating of InsP3-gated ion
channels in lobster ORNs. Line graph of the open
probability (Propen) (--A--A--) and the slope conductance
(slope g)of a small conductance (A) or a large
conductance (B) InsP3-gated channel as a function of Ca2+
concentration. Recording conditions and configuration as in
Figure 6-1.

Slope g
Pr(open)
l0.2
CD
Slope g
>
-si
o
50
40
30
20
10
70
60
Pr(open)
202

Figure 6-7. Effect of Ca2* on the conductance of InsP3-gated
ion channels in lobster ORNs.
(A) Current (ordinate)-voltage (abscissa) relation of a
large conductance InsP,-gated channel recorded by applying
2.4 x 1CT7 M InsP3 in
1.0 mM Ca2+ (—▲ — ▲—) or 30 mM Ca2* (—• — •—) patch (see
Table 6-1) to the inside face of cell-free patches of
membrane.
(B) Current (ordinate)-voltage (abscissa) relation of a
small conductance InsP3-gated channel recorded by applying
2.4 x 10'7 M InsP3 in 0.001 mM Ca2+ (—#—#—), 0.01 mM Ca2+
(—o — o—), 0.1 mM Ca2* (—□ — □—), or 30 mM Ca2+ (—x — x—)
patch (see Table 6-1) to the inside face of cell-free
patches of membrane.
(A & B) Pipette solution =. control KA patch (see Table
6-1); Patches were voltage-clamped at -60 mV.

204

205
In a limited number of trials, it was possible to apply
InsP3 at five different [Ca2+] to the inside face of three
3 0 pS and one 74 pS InsP3-gated channel. Due to the
difficulty of patch integrity through all 5 solution
changes, the data from the 4 channels were pooled for the
analysis of the effect of Ca2+ on channel Propen. Chelation
with 11 mM EGTA reduced the Propen of the channels by 51 ± 13
percent (n=4) from that measured at 0.1 mM [Ca2+]i. Changing
the [Ca2+] i to 30 mM, increased the Propen to 157 ± 7 percent
(n=4) of that found in 0.1 mM [Ca2*]^ Propen remained near
that recorded in the 0.1 mM [Ca2+] t condition when 3 0 mM Ba2+
was substituted for Ca2+ (n=4)(Figure 6-8).
Pharmacology of Macroscopic Odor-evoked Current & InsP,-
qated Channels
To test the alternative hypothesis that InsP3 could
mobilize intracellular Ca2+, cells were preincubated with a
membrane permeant, Ca2+-ATPase pump inhibitor,
thapsiagargin. The magnitude of inward odor-evoked
macroscopic currents was not significantly different among
cells preincubated with 8 x 1CT7 M thapsiagargin and those
preincubated with the EtOH carrier alone (Figure 6-9,
Student's t-test).
Macroscopic inward odor-evoked currents that are
mediated by InsP3 (Chapter 5) were reversibly blocked by a
mixture of two transition metals that block voltage-
activated Ca2+ channels (5 mM Co/Cd) . The inward currents
were also partially and reversibly blocked by RR, a blocker

Figure 6-8. Histogram of the mean ± SEM open probability (Propen) of both the small-
and large-conductance InsP3-gated' channel (n=4) in lobster ORNs as a function of
internal Ca2* concentration (2.4 x 10‘7 M InsP3 in Ca Patches) . Configuration as in
Figure 6-1. +EGTA = 0.001 mM Ca Patch + 11 mM EGTA; Ba30 = 30 mM Ba Patch. Pipette
solution = control KA patch. Patches were voltage-clamped at -60 mV. See Table 6-1
for patch solution composition.

0.5
c
0
Q_
o
cl
0.4
0.3
0.2
0.1
0
-
gw
Wm
Hf
m
+ EGTA 0.001 0.01 0.1 30
Calcium in mM
Ba30
207

Figure 6-9. Histogram of the mean ± SEM macroscopic inward
odor-evoked current in lobster ORNs treated with 8 x 10'7 M
thapsiagargin (Thap) or with the EtOH carrier alone (EtOH)
two hours prior to recording. ORNs were voltage-clamped
at -60 mV. Odorants used to evoke the current include 10"3
M proline or a 1000X dilution of TET stock.

Response Magnitude (pA)
209

210
of InsP3-regulated Ca2+ channels (10 /xM RR) . Co/Cd
significantly reduced the peak current 84 ± 7.5 percent
(n=10) while RR reduced it 74 + 10.7 percent (n=20) of
initial control values (arcsin transformation of percentage
data followed by Student's t-test)(Figure 6-10A,B,D).
Application of 5 mM TEA, a blocker of all known K+ channels,
reduced the odor-evoked inward current by only 13.5 ± 9
percent, which was not significant nor contributable to all
cells tested (n=5)(Figure 6-10D). One micromolar TTX, an
exceptionally specific blocker of voltage-activated Na+
channels, had a heterogenous effect across 10 cells tested.
TTX delayed the termination of the odor-evoked inward
current (n=3), slowed the onset kinetics and reduced the
magnitude of the current (n=6), or completely blocked the
current (n=2)(Figure 6-10C,D).
The pharmacology of the InsP3-activated unitary
currents was consistent with that of the macroscopic odor-
evoked inward current, further supporting that InsP3-gated
channels underlie the excitatory current. Five millimolar
Co\Cd blocked 3 of 4 tested 30 pS channels and 3 of 4 tested
74 pS channels. Ten micromolar RR consistently blocked both
the 30 pS channel (n=3) and the 74 pS channel (n=2). One
micromolar TTX blocked 2 of 4 tested 30 pS channels and did
not block the 74 pS channel (n=4). TEA was not tested for
its effect on unitary currents. Ten millimolar Cs was not
tested for its effect on macroscopic currents but failed to

Figure 6-10. Pharmacological probes targeting the
macroscopic inward odor-evoked current in lobster ORNs.
(A) Whole-cell current evoked by application (arrow) of the
odorant TET (Odor Prepulse), TET plus 5 mM Co and 5 mM Cd
(Odor + 5 CoCd), and then the odorant TET (Odor Recovery).
(B) Whole-cell current evoked by application (arrow) of the
odorant TET (Odor Prepulse) , TET plus 10 /¿M ruthenium red
(Odor + 10 RR) .
(C) Whole-cell current evoked by application (arrow) of the
odorant TET (Odor Prepulse) , TET plus 1 /¿M tetrodotoxin
(Odor + 1 TTX). Top trace: TTX appears to affect
termination of the odor response (TTX-T). Middle trace:
TTX appears to affect the onset and magnitude of the odor
response (TTX-O). Bottom trace: TTX appears to affect the
termination of the odor response (TTX-T).
(D) Histogram plot of the mean ± SEM macroscopic inward
odor-evoked current of ORNs treated with various
pharmacological probes (solid bars). Odor Prepulse =
striped bar preceding each treatment, Odor Recovery =
striped bar following each treatment. TEA = 5 mM
tetraethylammonium. * = Significant difference, paired
t-test. Statistics not computed for heterogeneous effect of
TTX. TTX-T, TTX-O, TTX-B as in part C.
(A-D) ORNs voltage-clamped at a -60 mV holding potential.
Odorants used to evoke the current include 10~3 M proline,
taurine, glycine, betaine, or a 1000X dilution of TET stock.

Percent Normalized Response
212
A
Odor + 5 CoCd
B
Odor + 10 RR
'C*
50 pA
1 S
Odor Prepulse
^ 1 TTX
20 pA Odor Prepulse
0 5 s
1 TTX
I

213
block either the 30 pS channel (n=5) nor the 74 pS channel
(n=3).
Ionic Selectivity of InsP,-gated Channels
The mean reversal potential for the odor-evoked inward
current was -46.5 ± 2.1 mV (n=26), over 80 mV negative of
the calculated Nernst potential for Ca 2+ (Eca = 34 mV)
(Figure 6-11), suggesting that the excitatory odor-evoked
inward current is a mixed current. The reversal potential
of twenty-one 30 pS InsP3-gated channels and six 74 pS
InsP3-gated channels were evaluated using two different ion
substitution paradigms. In the first paradigm, the
calculated Nernst potential for Cl', Na+, or K* was shifted
varying degrees from 0 mV and the Nernst potential for Ca2+
was set to 0 mV. Eleven of 15 30 pS channels and 3 of 4
74 pS channels reversed near 0 mV, under these conditions.
An example of one of the 30 pS channels is given in Figure
6-12. In symmetrical conditions the channel reversed at
0.11 mV (not shown) . When ECa = 0, ENa = -19, and EC1 = 30,
the channel reversed at 2.9 mV. When ECa = 0, ENa = -26, and
EC1 = 28, the channel reversed at 4.9 mV. An example of
one of 74 pS channels is given in Figure 6-13. In
symmetrical conditions the channel reversed at 4.5 mV (not
shown) . When ECa = 0, ENa = -19, and EC1 = 30, the channel
reversed at 8.8 mV. When ECa = 0, ENa = -26, and EC1 = 28,
the channel reversed at 10.7 mV. One of the three 74 pS
channels displayed extreme inward rectification at positive

Figure 6-11. Current reversal of macroscopic odor-evoked
inward currents in lobster ORNs.
(A) Odorant-evoked (TET) whole-cell currents in a lobster
ORN voltage-clamped at various membrane potentials.
(B) Histogram plot of the current reversal for 26 such ORN
recordings binned into 10 mV holding potential intervals (Vc
Range). Mean reversal potential = -46.5 ± 2.1 mV. Odorants
applied to evoke the current include (10“3 M) AMP, glycine,
proline, taurine, or betaine, or a 1000X dilution of TET
stock.

215
'20
-40
-50
-60
l -70
30 pA.
B
_C/3
q3
O
CD
.Q
E
D
[-70,-60] [-60,-50] [-50,-40] [-40,-30] [-30,-20]
Vc Range (mV)
500 msec

Figure 6-12. Current reversal of the small- (30 pS)
conductance InsP3-gated channel in a lobster ORN recorded in
ionically substituted solutions.
(A) Unitary currents evoked by 2.4 x 10'7 M InsP3 in NalOOCs
Bath (—□—□—) or Na Bath (—•—•—) when applied to the
inside-face of a cell-free patch of lobster ORN voltage-
clamped at various membrane potentials. C= closed state,
03= first open state, 02= second open state, 03= third open
state. See Table 6-1 for bath solution composition.
(B) Current (ordinate)-voltage (abscissa) relation of the
small-conductance InsP3-gated channel in A. Each data point
represents at least 12s of open channel events binned into a
point-by-point amplitude histogram, which was fitted by a
gaussian curve. The height' of the fitted distribution
determined the mean unitary current (in pA) and was plotted
as a function of voltage (in mV). The plotted relation was
fitted by linear regression (continuous line). Calculated
Nernst potentials for various ion species are denoted by the
arrows and specified in text. ECa was set to zero unless
noted otherwise. Symbols as in A.

217
A
(<=)
-30 mV r- Ql
t O2
(•)
c -80 mV
wrVru jydKl /1
-60
10 rvn\vwV^^^^^.w>»Vi*>r >
-50 i^V'V'
40
-30
1*J
- Twâ–  T
-10
0000

Figure 6-13. Current reversal of the large- (74 pS) conductance InsP3-gated channel
in a lobster ORN recorded in ionically substituted solutions.
(A) Unitary currents evoked by 2.4 x 10"7 M InsP3 in Na Bath (—• — •—) or NalOOCs
Bath (—o—o—) when applied to the inside-face of a cell-free patch of lobster ORN
voltage-clamped at various membrane potentials.
(B) Current (ordinate)-voltage (abscissa) relation of the large-conductance InsP3-
gated channel in A.
(A & B) Analysis and notation as in Figure 6-12.

219

Figure 6-14. Current reversal of the large- (74 pS)
conductance InsP3-gated channel in a lobster ORN recorded in
ionically substituted solutions.
(A) Unitary currents evoked by 2.4 x 1CT7 M InsP3 in Ca Bath
(—•—•—) when applied to the inside-face of a cell-free
patch of lobster ORN voltage-clamped at various membrane
potentials.
(B) Current (ordinate)-voltage (abscissa) relation of the
large-conductance InsP3-gated channel in A.
(A & B) Analysis and notation as in Figure 6-12.

221
A
-80 mV
C
o
-60
-40
-10
10
40
fflTYl
Na Ca Cl
J I J
12
60
50 ms
20 pA
80

222
holding potentials (Figure 6-14). In symmetrical
conditions, the channel reversed at 3.2 mV (not shown).
When ECa = 0, ENa = -2 6, and EC1 = 25, the channel reversed at
4.6 mV.
The remaining 4 30 pS channels and one 74 pS channel
that did not reverse near 0 mV, reversed independently of
any calculated Nernst potential and were inconclusive as to
selectivity (data not shown). For example, when ECa = 0 mV,
EC1 = 38.8 mV, ENa = 148.6 mV, and EK = 6.5 mV, the first of
these remaining 30 pS channels reversed at 23 mV. In the
second 30 pS channel, when ECa = 0 mV, EC1 = 45.3 mV,
ENa = 148.6 mV, and EK = 6.5 mV, the channel reversed at
-38 mV. In the third remaining 30 pS channel, when ECa = 0
mV, ENa = -72.3 mV, EC1 = 70.6 mV, and EK = 195.3 mV, the
channel reversed at -15.7 mV. In the fourth remaining 30 pS
channel, when ECa = 0 mV, ENa = -23 mV, EC1 = 2 5 mV, and
Ek = -195.3, the channel reversed at -14.5 mV. In the
remaining 74 pS channel, when ECa = 0 mV, ENa = 46.7 mV,
EC1 = -28 mV, and EK = -195 mV the channel reversed at
-43 mV.
In the second substitution paradigm, ECa was shifted
from 0 mV in order to discriminate between channels with
primarily Ca2+ permeability and non-selective cation
permeability. In conditions where ECa was other than 0 mV,
channels of both conductances either followed ECa,
at 0 mV, or were intermediate between ENa and ECa
remained

Figure 6-15. Current (ordinate)-voltage (abscissa) relation
of the small- (A-C) and large- (D-E) conductance InsP3-gated
channel when 2.4 x 10~7 M InsP3 was applied to the inside-
face of cell-free patches of lobster ORN under the following
ionic substitutions to force calculated ECa to be other than
0 mV. All other ions (Ex) at 0 mV unless shown otherwise.
(A) InsP3 in Ca Bath (—•—•—)
(B) InsP3 in Ca Patch (—•—•—) 0.01 mM
(C) InsP3 in Ca Patch (—•—•—) 1.0 mM, ( — □ — □—) 30 mM
(D) InsP3 in Ca Bath (—•—•—)
(E) InsP3 in Ca Patch (—A — A—) 1.0 mM, (
Analysis and notation as in Figure 6-12.
— •—•—) 30 mM

224

225
(Figure 6-15A-E). Three of six 30 pS channels reversed near
0 mV, implying non-selectivity. An example is given in
Figure 6-15A. In symmetrical conditions the channel
reversed at 10.1 mV (not shown) . When ECa = -83, EC1 = 88.6,
ENa = 148, and EK = 195, the channel reversed at -9.6 mV.
When ECa = 3 0 mV and the remaining ions were set to 0 mV,
another 30 pS channel reversed at 3.4 mV (Figure 6-15B).
The remaining three 30 pS channels reversed intermediate
between ENa and ECa such as the example in Figure 6-15C. In
symmetrical conditions the channel reversed at 8.4 mV (not
shown) . When ECa = -30, Ecl, ENa, and EK = 0, the current
reversed at -19.5 mV. When ECa was further shifted to
-74 mV, the current reversed at -28.7 mV.
Of the two 74 pS channels tested under conditions where
ECa was other than O mV, one channel reversed consistent
with ECa (Figure 6-15D) and the other channel reversed
intermediate between ENa and ECa (Figure 6-15E) . In the
example of the former, where ECa = -83, Ecl, ENa, and EK = 0,
the channel reversed at -55.8 mV with extreme rectification.
In the latter example, in symmetrical conditions the channel
reversed at 2.4 mV (not shown) . When ENa = 0 and ECa = 30,
the channel reversed at 25 mV. However, when ENa = 0 and
ECa = 60, the channel reversed at 20.3 mV, intermediate
between ECa and ENa.

226
Discussion
Gating Properties
While pH sensitivity has not been investigated for
cerebellar InsP3-gated channels, pH modulates the kinetics
of other ion channels, such as the ICa conductance of L-type
Ca2+ channels (Prod'hom, Pietrobon, and Hess, 1987, 1989).
Alkaline pH is known to increase binding affinity of the
InsP3 ligand to its receptor in isolated catfish olfactory
ciliary membranes (Kalinoski et al., 1992). Under alkaline
conditions, the 5 to 12 fold increase in Propen for the
small- and large-conductance InsP3-gated channel in lobster
is not unlike the 6 fold increases in InsP3 binding affinity
observed in catfish olfactory cilia (Kalinoski et al.,
1992). Therefore, physiological changes in pH could modify
the gating of the InsP3-activated channels in lobster ORNs
by altering the affinity of InsP3 binding.
The ability of Ca2+ to modulate both the small- and
large-conductance InsP3-gated channel in a concentration
dependent manner is similar to the InsP3-triggered Ca2+ flux
in the cerebellum, which is stimulated at low [Ca2+] and
inhibited at higher [Ca2+] (Ferris, Huganir, and Snyder,
1990; lino and Endo, 1992; Mignery, Johnson, and Siidhof,
1992; Luttrell, 1993). Yet oddly for InsP3-gated channels
in lobster ORNs, an increase in Propen of the channels in
response to increased [Ca2+] ± is coupled with a decrease in
channel conductance. These data suggest that Ca2+ could

227
increase channel open frequency throughout the tested
concentration range and concurrently, but independently,
affect channel permeation at high Ca2+, possibly by
targeting the pore of the protein.
Cerebellar InsP3 receptors are dependent on the Ca2+
content of the endoplasmic reticulum (Worley, Baraban,
Colvin, and Snyder, 1987) and it has been suggested that
calsequestrin, calmedin, or calreticulin may serve as the
Ca2+ sensing unit, independent of the channel protein
(Ferris et al., 1990; Berridge, 1993). Although the latter
is conceivable for olfactory InsP3 receptors, the former is
irrelevant for lobster InsP3-gated channels in situ (Ache,
Hatt, Breer, Zufall, in press), which are located on outer
dendritic processes of the ORN; an area devoid of organelles
(Grünert and Ache, 1988). The fact that cultured lobster
ORNs incubated with thapsiagargin demonstrated no change in
odor-evoked inward currents, shown previously to be mediated
by InsP3 (Fadool and Ache, 1992a) and shown in this study to
be carried partially by Ca2+, suggests that the source of
the Ca2+ providing feedback regulation of the InsP3 receptor
is provided extracellularly via Ca2+ influx across the
plasma membrane. Rather than the detection of internal Ca2+
stores by the receptor or an associated protein, Ca2+ influx
through the InsP3 channel itself may modulate its gating
throughout a large Ca2+ concentration range. Secondarily
permeation is affected at high Ca2+ concentrations, blocking

228
channel conductance, perhaps as a type of negative feedback
loop. Ca2+ ions have been reported to inactivate the very
channel they pass through (Yue, Backx, and Imredy, 1991),
necessitating that the conducting ions that accumulate near
the pore exert a negative feedback on Ca2+ flux as modeled
by DeFelice (1993) .
The long open modes observed in both the small- and
large-conductance InsP3-gated channels of lobster ORNs has
never been reported for cerebellar InsP3-gated channels, nor
is the function of modal kinetic schemes in general well
understood. Modal gating is inferred when a channel changes
its kinetic behavior suddenly and maintains it for several
seconds in the absence of solution changes or changes in
membrane potential (Delcour, Lipscombe, and Tsien, 1992) .
Long open modes have been observed in a number of systems
utilizing Ba2+ as a current carrier (Hess, Lansman, and
Tsien, 1984; Nowycky, Fox, and Tsien, 1985; Pietrobon and
Hess, 1990; Mazzanti, Galli, and Ferroni, 1992) to last for
hundreds of milliseconds with only brief closures, much like
Mode 1 or Mode 2 InsP3-gated channels in our study. Three
different modes of gating occur in N-type Ca2+ channels in
bullfrog sympathetic neurons (Delcour et al., 1993), with
low, medium, and high Propen comparable to the Propen of 0.15,
0.33, and 0.91-1.00 of the InsP3-gated channels in our
system. Spontaneous changes in the pattern of gating could
influence the total contribution of the InsP3-gated channels

229
to Ca2+ or cation entry. For example a transition from
normal Propen gating (0.33) to high Propen gating (0.91-1.00,
Mode 1-2) could greatly increase the amount of current flow
across the plasma membrane. Concurrently, a transition from
normal Propen gating (0.33) to low Propen gating (0.15, Mode 3)
could downregulate the total ion entry or current produced.
As suggested by Delcour et al. (1993), a mode might be the
expression of a modulatory state of a channel, superimposed
on the set of conformational states occupied during normal
gating. Because we observed the long open modes
infrequently, in at most 10% of the channels recorded, one
possibility is that the long openings could be attributed to
spontaneous lapses in Ca2+ block of the conducting channel
(see section above; DeFelice, 1993). Another possibility is
that local ligand concentration, phosphorylation state, or
GTP-protein binding could favor mode transition in the
InsP3-gated channels (Bean, 1989; Elmslie, Zhou, Jones,
1990; Yue, Herzig, and Marban, 1990).
Ionic Selectivity and Pharmacology
The failure of the mean reversal potential of the
macroscopic odor-evoked inward current to coincide with the
calculated equilibrium potential for any ion species,
implies that the whole-cell excitatory current is a mixed
current. A large percentage of the current (74-84%) may be
mediated by an underlying Ca2+ channel, since it can be
blocked by Co/Cd and RR. This is consistent with the

230
pharmacological response of the underlying InsP3-gated
channels that are also both blocked by these probes.
Discrepancy arises in the pharmacology of InsP3-activated
currents in olfactory tissue, so far reported in catfish
(Restrepo et al., 1990), moth (Stengl, 1993), and lobster
(this chapter). Insect ORNs are TTX insensitive, blocked by
TEA, and insensitive to externally applied Ca2+ channel
blockers (Stengl, 1993). This is clearly at odds with the
heterogeneous TTX pharmacology and TEA insensitivity of the
odor-evoked inward currents in lobster, the TTX blockability
of the small-conductance InsP3-gated channel, and the RR and
Co/Cd sensitivity of both the large- and small-conductance
InsP3-gated channels. InsP3-gated channels in catfish and
lobster ORNs, are sensitive to Ca2+ channel blockers, yet
InsP3-evoked currents in catfish and moth ORNs are abolished
by removal of external Ca2+ or Ca2+ chelation. The evidence
that both the small- and large-conductance InsP3-gated
channel can be recorded in solutions where Ca2+ is the
predominant ion and also in EGTA-chelated conditions,
suggests that the current carried by each channel can also
be carried by Na+ in the absence of a Ca2+-requiring
mechanism.
In the lobster, ionic substitution demonstrates that
half of the small-conductance InsP3-gated channels are non-
selective to cations, while the remaining half are
intermediary between Na+ and Ca2+. Although based upon a

231
more limited data sample, the large-conductance InsP3 may be
more selective for Ca2+ over other cations. In regards to
Ca2+ permeability (not selectivity), the olfactory InsP3-
gated channels (Restrepo et al., 1990; Stengl, 1993; this
chapter) are not unlike those that occur in the cerebellum
(Maeda et al., 1991). Only the catfish olfactory
InsP3-gated channel is highly Ca2+ selective and shows more
than one conductance state, being most like cerebellar IP3-
gated channels. In lobster, at least two different channels
are gated by InsP3 (Chapter 5), not a single channel with
multiple conductive states as is true of InsP3-gated
channels in the cerebellum (Watras et al., 1991) .
When one considers that the lobster InsP3-gated
channels are recognized by an antibody targeting a mammalian
cerebellar InsP3 receptor (Ca2+ channel)(Chapter 5), are
sensitive to drugs targeting Ca2+ channels, and have either
a non-selective cation, Na+ and Ca2+ selective, or Ca2+
selective permeability, it is tempting to speculate that the
InsP3-gated channels in lobster represent evolutionary or
developmental intermediaries between Na+ and Ca2+ channels.
Such a suggestion has been made for Na+ currents in
jellyfish that are blocked by pharmacological probes
targeting Ca2+ channels (Anderson, 1987). Subsequent
molecular sequencing confirmed the jellyfish Na+ channel to
be more closely homologous to Na+ channels than Ca2+, and

232
revealed a different selectivity filter than that reported
in higher animals (Anderson, Holman, and Greenberg, 1993).
Data support a subtle distinction between the two
InsP3-gated channels in lobster ORNs. Although separated by
conductance, kinetics, and voltage dependence (Chapter 5),
the channels cannot be distinguished based upon modulation
by increased pH or Ca2+, modal gating patterns,
pharmacology, nor ionic selectivity. Although the latter
properties fail to provide functional distinction between
the two InsP3-gated channels, they may help define the novel
properties of olfactory, plasma membrane InsP3-gated
channels.
Modulation of output is typically shaped by the
presence of more than one channel type in most neurons. The
apparent redundancy of having more than one channel gated by
the same second messenger, however, is unclear. Diversity
in olfactory receptor neurons is classically attributed to
differential expression of odor receptor proteins across a
population of cells as well as to differential tuning of a
single expressed receptor to different odor qualities. The
excitatory transduction mechanism in lobster ORNs has the
capacity to recruit at least 3 and possibly 4 types of
second messenger-gated ion channels: a 30 pS InsP3-gated
non-selective cation channel, a 74 pS InsP3-gated non-
selective cation channel, a 193 pS IP4-gated Ca2+ selective
channel (Chapter 7), and possibly a 74 pS InsP3-gated Ca2+

233
selective channel. The distribution and type of second
messenger-gated ion channel ultimately and perhaps equally
contributes to ORN diversity.

CHAPTER 7
IP4-GATED ION CHANNELS IN NEURONS
Introduction
Stimulus-induced turnover of phosphoinositide is a
major intracellular signalling system that mediates the
action of neurotransmitters, hormones, and growth factors
(Figure 7-1; Irvine, 1990; Neher, 1992a; Putney, 1992,
Berridge, 1993). Inositol 1,3,4,5-tetrakisphosphate
[InsP4(1,3,4,5) ] , either alone (Higashida and Brown, 1986;
Hill et al., 1988; Ely et al., 1990; Gawler et al., 1990;
Parker and Ivorra, 1991; De Waard et al., 1992; Guse et al.,
1992; Lückhoff and Clapham, 1993) or in combination (Irvine
and Moor, 1986, 1987; Morris et al., 1987; Changya et al.,
1989a, 1989b; DeLisle et al., 1992) with inositol 1,4,5-
trisphosphate [InsP3 (1,4,5) ] , enhances Ca2+ entry into cells,
sequesters or mobilizes Ca2+, or triggers a change in
membrane potential. Whether InsP4(1,3,4,5) directly
activates channels in neurons and the extent to which these
receptors interact with InsP3(l,4,5) receptors in excitable
cells is unknown. We report that lobster olfactory receptor
neurons (ORNs) express an InsP4(1,3,4,5) receptor that is a
NOTE: This chapter will be submitted for publication in the
journal Nature and conforms to its style requirements.
234

Figure 7-1. A non-threatening view of the inositol
phospholipid metabolic pathway. The shell of the turtle
represents the inositol ring, the solid circle the phosphat
moiety, and the head & tail of the turtle, the chair
conformation of the molecule. The shaded molecules are the
inositol phosphates that were tested to determine whether
they could evoke channel activity in inside-out cell-free
patches of lobster ORN plasma membrane.
Turtles representing the inositol moiety were first
presented by Dr. B. Agranoff and are used with his
permission.

236
myo-inositol

237
functional channel differing in conductance, kinetics and
voltage sensitivity from two previously reported plasma
membrane InsP3 (1,4,5)-gated channels (Chapters 5-6).
InsP4 (1,3,4,5)-gated channels interacted with the
InsP3 (1,4,5)-gated channels by altering the Propen of the
channels in what may be a novel mechanism for regulating
Ca2* entry. InsP4 (1,3,4,5)-gated channels may be considered
one arm of a dualistic mechanism by which inositol
phospholipids (IP) regulate olfactory signal detection in
these neurons.
Results and Discussion
InsP4 (1,3,4,5) (6.4 x 1CT6 M) activated unitary
currents in 25 of 42 cell-free patches of plasma membrane
taken from cultured lobster ORN somata (Chapters 2-3; Figure
7-2) . The average slope conductance of this channel (193 ±
13.0 pS, n=19) was 3-6 times that of two InsP3-activated
channels (30 pS, 74 pS) present in these neurons (Chapter
5) . The average open probability (Propen) for the 193 pS
channel at -60 mV was 0.11 ± 0.02 (n=14) (Figure 7-3A). The
mean open time (tQ) for the channel was 5.03 ± 0.97 msec
(n=ll) (Figure 7-3B) . The Propen of the channel was voltage
dependent; the voltage function described a bell-shaped
curve that decreased at hyperpolarizing and depolarizing
extremes (Figure 7-4). Together with conductance, these
gating properties distinguished the 193 pS channel from the
two InsP3 (1,4,5)-gated channels, which have two open states

Figure 7-2 (A) Unitary currents activated by applying 6.4 x 10'6 M inositol 1,3,4,5-
tetrakisphosphate [InsP4 (1,3,4,5) ] to the inside face of a patch of membrane pulled
from the soma of a lobster olfactory receptor neuron (ORN). The patch was voltage-
clamped at various holding potentials. C= closed state, 0X = one open channel, 02 =
two open channels. (B) X-Y plot of the amplitude of the unitary currents shown in A
as a function of holding potential. Each data point represents the mean unitary
current of at least 12s of open channel events determined based on a gaussian
distribution fit of a point-by-point amplitude histogram at each holding potential.
The data points were fit by locally weighted regression (continuous line). The mean
conductance, determined from the slope of a linear regression at negative holding
potentials for 19 channels, was 193 ± 13.0 pS.
METHODS. Distinct clusters of ORNs were dissected from the olfactory organs (lateral
antennular filaments) of adult specimens of the Caribbean spiny lobster, Panulirus
argus, enzymatically dissociated, and sustained in primary culture as described
previously (Chapters 2-3). The cultured cells were viewed at 40X magnification with
Hoffman optics for patch-clamp recording. Patch electrodes, fabricated from 1.8 mm
O.D. borosilicate glass and fire polished to a tip diameter of approximately 1.0 ¡ím
(bubble number 4.8), produced seal resistances between 8 and 14 GO. Selected
phospholipids were "spritzed" on the inner face of cell-free patches utilizing a
previously described delivery system (Chapter 2). Unitary currents were recorded in
symmetrical solutions, amplified, and processed as in an earlier study (Chapter 5).
Briefly, signals were amplified with an integrating patch amplifier (Dagan 3900).
The analog records were filtered at 2 kHz and recorded on videotape. On playback,
the records were sampled every 100 /¿sec for processing on an IBM-compatible computer,
using pCLAMP software (Axon Instruments). Patch solution (in mM):
30 NaCl, 11 EGTA, 10 HEPES, 1 CaCl2, 180 K-acetate, and 696 glucose, pH 7.0.

i v » O t’
AUJ
oz
—r*
C)
l)(
I)
0
a
V slu 0l7
Tpmr
r~
U9
01?
)9~ 00 1-
^yrrwfijníY-rnfí^

Figure 7-3 (A) Histogram of the point-by-point amplitude
of a digitized record containing three 193 pS channels
activated by 6.4 x 10"6 M InsP4 (1,3,4,5) in a cell-free patch
from a lobster ORN. 36432 open channel events were fit by
gaussian distributions (continuous line). C= closed state,
03 = one open channel, 02 = two open channels, 03 = three
open channels. The mean open probability (Propen) ,
calculated from the area under the distributions, for 14
such ORNs at -60 mV was 0.11 ± 0.02. Inset shows a portion
of the record used to generate the histogram. The density
of the 193 pS channel was relatively high: patches
containing 3 to 9 channels were not uncommon. Given an
average surface area of 380.13 /xm2 for the soma of cultured
ORNs (Chapter 3), the calculated average density of the
193 pS channel was 0.60 ± 10.80 /xm2. (B) Histogram of the
open dwell time (tQ) of 2052 openings of a single 193 pS
channel activated by 6.4 x 10'6 M InsP4 (1,3,4,5) in a cell-
free patch from a lobster ORN. The distribution was best
fit by a single exponential (continuous line). Mean tQ =
5.03 ± 0.97 msec (n=ll).
METHODS. To calculate the lower and upper channel density
of the InsP4(1,3,4,5) channel the following were taken into
consideration to calculate between 228 and 4105 channels per
neuron: average surface area = 380.13 /xm2, percentage of
patches containing the 193 pS channel = 60%, electrode tip
diameter = 0.5 - 1.0 /xm, and number of channels per patch =
1 - 9. For patches containing more than one channel the
open probability was calculated as
Propen = 0_lPol,_±_l__LPi) + 2(P2) + 3 (P3I_.±, • ....
N
where N = number of channels, P0 = closed state probability,
P3 = probability of one channel open, P2 = probability of
two channels open, etc.

241
CM
I
o
c
>
LJ
0)
_Q
0
5
10
Time (ms)
15

Figure 7-4. Plot of the open probability (Propen ) of a 193 pS channel activated by
InsP4 (1,3,4,5) in a cell-free patch from a lobster ORN as a function of holding
potential. The data were fit by a locally weighted regression (continuous line).
Values at and bordering the reversal potential (-10 to +10 mV) could not provide a
meaningful Propen calculation and were excluded from the fit.

Propen
0.4 -
100 -80 -60 -40 -20 0 20 40 60 80 100
Holding Potential (mV)
243

244
and either decrease their Propen throughout the depolarizing
range or are voltage independent (Chapter 5).
The 193 pS channel was selective for InsP4 (1,3,4,5) in
25 (86%) of 29 patches (Table 7-1). In the remaining four
(14%) patches, InsP4(1,3,4,5) activated unitary currents
with average slope conductances similar to those of the
InsP3 (1,4,5)-activated channels (29.9 ± 4.8 pS, n=3;
90.5 pS, n=l). The 30 and 74 pS channels were selective for
InsP3(l,4,5) in 104 (95%) of 110 patches. Of seven other
metabolites in the inositol phospholipid pathway,
InsP3(l,4,5) (2.4 x 10“7 M) activated the 193 pS channel, in
only 6 (5%) of 110 patches as did inositol hexaphosphate
(InsP6) (10‘5 M) in one patch. None of the other metabolites
gated channel activity. The inactivity of inositol 1,3,4-
trisphosphate [InsP3 (1,3,4) ] excluded the possibility that
the 193 pS channel was activated by membrane associated 3-
phosphatases secondarily generating InsP3(l,3,4) from
InsP4(1,3,4,5) (Irvine, 1991).
As the solutions tested free of contaminating inositol
phospholipid metabolites, we presume that the relatively low
incidence at which InsP3(l,4,5) activated the 193 pS channel
and InsP4(1,3,4,5) activated the 30 and 74 pS channels
reflects low affinity cross receptor binding. Other
inositol metabolites bind InsP4(1,3,4,5) and InsP3(l,4,5)
receptors in mammalian brain only at high concentrations and
with low affinity (Enyedi and Williams, 1988; Ferris et al.,

245
Table 7-1. Selectivity of various metabolites of the inositol phospholipid pathway for three different types of channels in cell-free
patches of cultured lobster ORNs. InsP3(l,4,5) and InsPaO .3,4,5) were commercial lots tested by NMR and hplc as 99 and 95%
pure, respectively. Analysis did not detect contaminants as products of phospholipid metabolism.
Mean Channel Conductance
Phospholipid
Concentration (M)
No. of
Patches
No. of Patches 30 pS
Containing Channels
74 pS
193 pS
Inositol
10°
7
0
0
0
0
Inositol 1-monophosphate
10'5
8
0
0
0
0
Inositol 1,2-cyclic monophosphate
10'5
9
0
0
0
0
Inositol 1,4-biphosphate
3xl0'4
7
0
0
0
0
Inositol 1,3.4-tnsphosphate
I0‘5
9
0
0
0
0
Inositol 1,4,5-tnsphosphate
2.4xl0'7
184
110
61
43
6
Inositol 1,3,4,5-tetrakisphosphate
6.4xl0'6
42
29
3
1
25
Inositol hexaphosphate
10'5
13
2
0
1
1

246
1989; Theibert et al., 1991, 1992). The apparent
selectivity of the InsP3 (1,4,5)-gated channels in the
lobster, is greater than that reported for the catfish
olfactory InsP3(l,4,5) receptor, which binds InsP3(l,4,5)
and InsP4 (1,3,4,5) with equal affinity (Kalinoski et al.,
1992) .
The InsP4(1,3,4,5)- and InsP3(1,4,5)-gated channels
interacted when they occurred in the same patch of membrane.
Interaction altered the Propen but not the conductance of the
channels (Figure 7-5A), suggesting an altered gating
mechanism without interference of the pore region. In
patches containing both types of channels, the Propen of the
InsP4(1,3,4,5)-activated channel significantly increased
when the two ligands were co-presented, compared to that
under InsP4 (1,3,4,5) stimulation alone (n=16, paired t-test,
Figure 7-5B) . Interaction required that the InsP3 (1,4,5) -
activated channel be gated and not simply present. The
Propen of the InsP4 (1,3,4,5)-activated channel was not
significantly different in patches that contained both IP-
activated channels and patches that only contained
InsP4 (1,3,4,5)-activated channels (n=8, Student's t-test,
Figure 7-5B) . The InsP4 (1,3,4,5)-activated channel,
decreased the Propen of both InsP3 (1,4,5)-act ivated channels.
In this direction, interaction only required the
InsP4 (1,3,4,5)-activated channel be present, not gated. Co¬
presenting both ligands had no effect on the Propen
of the

Figure 7-5. InsP3- and InsP4-gated channels interact to alter the open probability
(Propen) but not the conductance state of the respective channels in cultured lobster
ORNs.
A) Representative records of unitary currents activated by applying 6.4 x 10'6 M
InsP4 (1,3,4,5) and 2.4 x 10'7 M InsP3(l,4,5) to the inner face of cell-free patches of
lobster ORNs. InsP4(1,3,4,5) (trace 1) and InsP3(l,4,5) (trace 4) activated the
193 pS and the 74 pS channel, respectively, in the same patch. Presenting both
ligands simultaneously to the same patch as in traces 1 and 4 increased the Propen of
the InsP4 (1,3,4,5)-activated channel (trace 2 vs trace 1), but not the InsP3 (1,4,5) -
activated channel (trace 5 vs trace 4). Both InsP4(1,3,4,5)- (*) and InsP3 (1,4,5) -
( + ) activated channel activity can be distinguished in trace 5. The Propen of the
InsP4 (1,3,4,5)-activated channel in a patch that contained only this type of channel
(trace 3) did not differ from that when both types of channels were present (trace
1) . The Propen of the InsP3 (1,4,5)-activated channel, however, was greater in the
absence of an InsP4-activated channel (trace 6 vs trace 4). Propen, C, 01( and 02 as
in Figure 7-2.
B) Bar graph comparing the normalized open probability (Propen) for all channels
tested as in A. Solid bar = Propen for that channel when both channels were contained
in the same patch in response to its own ligand. Striped bar = Propen for that
channel when then co-presented with both ligands. * = Significant difference, p s
0.05; Arcsin transformation of percentage data followed by paired t-test. Open bar =
Propen for that channel activated by its own ligand in patches known to only contain
that type of channel. * = Significant difference, p s 0.05; Arcsin transformation of
percentage data followed by Student's t-test.

A
lnsP4
lnsP4 + lnsP3
Propen dnsP4)
lnsP4
I n s P 3
Propen (lnsP3)
0.01
I11SP4 + lnsP3
T 0.009
. . I
* * *
lnsP3
B
T 6*
28.5
10
Channel
248

249
InsP3 (1,4,5)-activated channels (n=10, paired t-test, Figure
7-5B) , but the Propen of the InsP3 (1,4,5)-activated channel
was significantly increased in patches that only contained
InsP3 (1,4,5)-activated channels as opposed to patches that
contained both IP-activated channels (n=6, Student's t-test,
Figure 7-5B). Interaction between InsP4- and InsP3-
activated receptors in other, non-excitable cells has been
ascribed to a variety of mechanisms (Irvine, 1990, 1991).
The ER InsP3 receptor is reputedly locked into an inactive
conformation by being bound to a plasma membrane InsP4
receptor at the InsP3 binding site. InsP4 binding
dissociates the coupled receptors to allow Ca2+ entry and
free access of InsP3 to its receptor. Such a model may not
generalize to our system where both IP-activated channels
are plasma membrane bound. Receptors in suitably close
proximity in the same membrane might also be expected to
block cytoplasmic binding sites; the presence of InsP4-
activated channels in lobster ORNs decreased the open
channel activity of the InsP3 receptor. Contrary to the
model, however, the InsP3-activated channel was stimulated
in the absence of InsP4 and in patches that did not contain
InsP4-activated channels.
We suspect that the 193 pS channel characterized in the
soma functions in olfactory transduction. Cultured lobster
ORN somata express transduction elements that are otherwise
confined to the outer dendrite, the presumed site of odor

250
transduction in situ (Chapters 2-6). In preliminary-
experiments (Ache et al., in press), a channel activated by
InsP4(1,3,4,5) , and two activated by InsP3(1,4,5) , all with
conductances within picosiemens of those reported in culture
(Chapters 5 & 7), were recorded in the outer dendrite.
Odors rapidly and transiently stimulate the production of
InsP3(l,4,5) (Ache et al., in press), the metabolic
precursor of InsP4 (1,3,4,5) , in the outer dendrite. Odor-
stimulated production of InsP3(l,4,5) would increase Ca2+
influx through the InsP3 (1,4,5)-activated channels (Chapter
6), allowing activation of a Ca-dependent 3-kinase to
produce InsP4 (1,3,4,5) to gate the 193 pS channel. Such a
large conductance channel could serve to enhance detection
of the odor signal by rapidly increasing the onset of the
receptor potential.
If, as in other systems, the InsP4(1,3,4,5)-activated
channel in lobster ORNs is calcium permeable, as preliminary
experiments suggest (n=2, data not shown), then multiple
channels would mediate Ca2+ entry into the cell. Dual
receptors mediating Ca2+ flux have been implicated in
controlling fertilization (Galione et al., 1993; Lee et al.,
1993) and two Ca2+ releasing channels have been colocalized
immunocytochemically in brain (Sharp et al., 1993) . Having
more than one Ca2+ mobilizing channel may be a fundamental
property of excitable cells such as olfactory receptor

neurons and interaction among the channels may provide f
control of neuronal output.

CHAPTER 8
SUMMARY
Sensory transduction pertains to all the mechanisms by
which a specific type of stimulus energy is transformed into
an electrical response by a specialized sensory cell:
detection, amplification, encoding & discrimination,
adaptation & termination, sensory channel gating, electrical
response, and finally transmission to the brain (Shepherd,
1991). A variety of transductory elements - from putative
G-protein coupled receptors to second-messenger gated ion
channels - have been uncovered in the past four years,
rapidly changing our view of olfactory signalling and
detection. The data presented in my dissertation focused
primarily on one specific stage of transduction, sensory
channel gating (Chapters 5-7), and to a lesser extent on
detection, discrimination (Chapter 3), and amplification
(Chapter 4).
At the onset of my research, I developed appropriate in
vitro conditions to sustain lobster olfactory receptor cells
in primary cell culture (Chapter 2). Neurite outgrowth and
cell viability were strongly affected by choice of adherent
substratum, presence of serum, and length of animal
captivity. Neither nerve growth factor, HEPES, nor
preconditioned media from the target organ, the olfactory
252

253
lobe, had any observable effect on either longevity or
neurite outgrowth. The diversity of known growth factors
(i.e. insulin, EGF, PDGF, etc.) in other systems has greatly
expanded since my initial experiments and it would be worth
while to pursue these as potential neurotrophic factors for
lobster ORNs. Whether or not a required trophic factor or
trace element could have sustained the neurons beyond the
average 23 days remains unanswered. Nonetheless, a cell
line was not required for successful physiological recording
or, presumably, receptor protein expression. The defined
culture conditions for these peripheral neurons can probably
be modified to sustain other marine invertebrate neurons,
including lobster CNS neurons, in vitro.
The finding that cultured ORNs maintained respon¬
siveness to odors (Chapter 2) yet were morphologically more
compact than their counterparts in situ lead to the prospect
of using these dissociated cultured neurons to study the
transduction elements that were normally confined to the
relatively inaccessible outer dendrites. The cultured ORNs
had to be surveyed for their degree of odor sensitivity and
selectivity to discern whether they were an appropriate
model (Chapter 3). The nature of the adequate stimuli, the
degree of tuning (response spectra) of the cells, the
threshold of sensitivity, the resting biophysical
properties, and the dual polarity of the odor-evoked
currents were all consistent with chemosensitivity in the

254
cultured cells being olfactory, mimicking that of the cells
in situ.
The ability of odors to evoke currents in cultured ORNs
that lacked processes suggested that lobster ORNs could be
induced in vitro to insert all elements of the transduction
cascade in the soma, including those that normally may have
been confined to processes. This greatly facilitated
analysis of transduction in these cells as Chapters 4-7
attest. Most recently, inositol 1,4,5-trisphosphate- (IP3)
and inositol 1,3,4,5- (IP4) gated ion channels were recorded
in a vesiculated preparation of lobster outer dendrites,
suggesting by localization that these second messenger-gated
channels were transductory in nature (Ache and Hatt, in
preparation). Riveting was the finding that these ion
channels in the outer dendrite had estimated conductances
and heparin & voltage sensitivity identical to those
channels characterized in culture (Chapters 5-7); further
substantiating the use of cultured neurons.
The finding that GTP-binding proteins linked odorant
stimulation to the generation of both outward (inhibitory)
and inward (excitatory) currents in lobster ORNs (Chapter 4)
was not unexpected given the rich evidence for G protein
mediation of olfactory signalling in other species, and the
discovery of a multigene family of putative olfactory
receptors coupled to G proteins. What G protein subtype
mediated these transductory currents was unclear, especially

255
as other olfactory related G proteins demonstrated
ribosylation and the lobster ORNs could not be functionally
blocked by the bacterial toxins. It must be concluded that
at least one of the transductory G proteins is resistant to
ribosylation as even injection of an active subunit does not
alter odor-evoked current magnitude. The lobster ORNs
probably contain a G protein of the o;q family, all of which
are PTX insensitive. A second G protein, more similar to G0
of the oii family, is likely coupled to excitatory odor
transduction in lobster ORNs. One or the other or both
G proteins, coupled to one or several cell-surface
receptors, may create a heterogeneous expression pattern
across a population of ORNs to give each receptor its own
blueprint of excitatory receptor cell output. As the odor-
evoked outward (inhibitory) currents are clearly G protein
dependent, and the nature of this protein subtype remains
undetermined, I could easily envision another, yet defined,
class of G proteins mediating this transduction cascade.
The o;q family was only discovered three years ago and its
enzymatic effector clearly defined two years ago. I feel
that this multigene family will be a target of intensive
research as it is so highly conserved across eukaryotes.
Which G protein subtypes functionally couple to what enzyme
or ion channel effector is an unexplored avenue in olfactory
transduction.

256
The finding outlined in Chapter 5 that IP3 directly
gates an ion channel in the plasma membrane was the first
evidence for such a channel in neurons. The IP3-activated
channels had kinetic properties of odor-activated channels
in the ORNs and pharmacological properties of intracellular
IP3-activated channels in other systems. An antibody
directed against an intracellular, cerebellar IP3 receptor
recognized a protein in the lobster ORNs of similar
molecular weight to the mammalian receptor. Interestingly,
the antibody selectively increased the magnitude of odor-
evoked inward currents and the open probability of IP3-
activated unitary currents. The ability to insert a patch
of membrane containing an IP3-gated channel into a recipient
cell, which could then be stimulated by an odorant to evoke
channel activity, provided further evidence for IP3 as an
olfactory second messenger mediating excitation in lobster
ORNs .
The discovery that two channels - differing in
conductance, voltage dependence, and dwell-time kinetics -
were gated by IP3 (Chapter 5) lead to the investigation of
the channels' intricate gating properties, ionic
selectivity, pharmacology, and ligand specificity (Chapters
6-7). Although these distinctions did not provide strong
evidence that the channels were functionally dissimilar to
each other, they provided a body of knowledge about the
novel biophysical properties of IP3-gated channels in

257
olfactory neurons, in comparison to those located
intracellularly, as in the cerebellum. The finding that
elevated pH or [Ca2+] ± increased the open probability,
whereas the latter simultaneously decreased the conductance
of both channels, implied that modulation of channel gating
could affect opening and closing of the channel in addition
to ion permeation. Both IP3-gated channels displayed
spontaneous transitions in gating kinetics across three
distinct patterns or modes. While the presence of mode
behavior is not well understood in channel biology, one
could envision these long open modes the result of many
molecular mechanisms, from transient lapses in open channel
block, to changes in phosphorylation state of the channel
protein.
Both the small- and large-conductance IP3-gated channel
mimicked the pharmacology of the macroscopic odor-evoked
inward current. Although each were reversibly blocked by
typical calcium channel blockers, the experiments to measure
the channel reversal potential using ionically substituted
conditions were inconclusive. Further permeation
experiments are required to determine the extent to which
these channels are Ca2+ selective.
What can be inferred from the current reversal data of
Chapter 6, is that the excitatory current is clearly a mixed
current, most probably comprised of several channel species.
As direct odorant gating of receptors does not appear to be

258
a likely transduction mechanism (unpublished data), it would
benefit to use the cultured neurons for the investigation of
other second messengers mediating the excitatory current,
such as Ca2+, G-protein gated ion channels, nitric oxide,
other inositol phospholipid metabolites, or members of the
eicosanoid pathway.
Finally with the knowledge that other phospholipids are
suspected to serve as intracellular signals, a survey of IP3
metabolites (Chapter 7) revealed that IP4 directly activated
an ion channel in the plasma membrane of the ORNs that
differed in conductance, kinetics, density, and voltage
sensitivity from the IP3-gated channels. Most intriguing, I
found that the IP4-activated channel mutually interacted
with the IP3-activated channels to alter the open
probability but not the conductance of the channel. In¬
activated channels appear to be one arm of a dualistic,
interactive mechanism by which inositol phospholipids
regulate signal detection in these neurons.
My results suggest that our current model of olfactory
transduction in lobster ORNs (Figure 8-1) is overly
simplistic and will need to account for additional putative
second messengers (e.g. IP4) , modulatory factors (e.g. Ca2+
or phosphorylation), or even cross-talk (regulatory
interaction between cascades). Most importantly, the model
must reflect potential alternative ways in which the
transduction elements can be packaged within single cells.

Figure 8-1. Model of the dual, opposing second messenger cascades hypothesized to
mediate olfactory transduction in lobster ORNs. Some odor molecules (triangle) bind
to receptor proteins linked to an as yet undefined G-protein. This G-protein
mediates adenylate cyclase conversion of ATP to cAMP, which directly gates a
potassium channel to hyperpolarize the cell. Other odor molecules (square) bind to
receptor proteins linked to a G0- and/or Gq-like G-protein. These G-proteins mediate
phospholipase C conversion of PIP2 to IP3, which directly gates a non-selective
cation channel and possibly a Ca2*-dependent channel to depolarize the cell. The
output of the cell to the CNS depends on the net level of depolarization resulting
from co-activation of the two transduction cascades.
Taken with permission from Fadool and Ache, In press.

odor molecules INHIBITORY PATHWAY
260

261
Distribution of opposing cascades (excitatory/inhibitory)
and construction of cascades with different G proteins and
second messenger-gated ion channel types has the potential
to create diversity in receptor cell output. For example, a
G0-like protein coupled to IP3 production, which secondarily
gates a large conductance channel in a cell lacking an
inhibitory cascade would produce a different signal than a
cell containing Gq-like and G0-like proteins simultaneously
coupled to IP3 production, which gates a small conductance
channel in a cell that also contains the inhibitory cascade.
An emerging theory in the chemoreceptive field--"One
receptor protein, one olfactory receptor cell"--is
dominating meeting symposia and shaping predictive models in
the literature (e.g. Lancet et al., 1993). In the absence
of functional evidence, this theory remains just that.
Electrophysiological and biochemical cross-receptor binding
studies in the lobster and catfish are not consistent with
such an exclusion model, although at this time, they are
unable to rule it out entirely (Caprio and Byrd, 1984; Bruch
and Rulli, 1988; Caprio et al., 1989; McClintock et al.,
1989b, Michel et al., 1991, 1992b; Fadool et al., 1993). So
the BIG question for lobster chemoreception remains, whether
or not a single ORN can bind both excitatory and inhibitory
odorant compounds to a single cell-surface receptor type
that is linked to two or three different G proteins
subtypes, which individually mediate at least the activation

262
of effector enzymes in the production of second messenger
molecules, which in turn clearly gate more than one type of
ion channel. The ORN could finely tune its transmission to
the brain by utilizing machinery from both transduction
cascades inside a single cell (Ache et al., in press) but it
remains to be understood whether this is accomplished with a
single class of olfactory receptor proteins, or whether ORNs
express multiple receptor proteins coupled individually to
each cascade (Figure 8-1). Secondarily, it is not
understood whether all cells express both transduction
cascades, or only a subset possess multiple mechanisms.
Independent of receptor protein expression or cascade
distribution across cells, lobster olfactory transduction
clearly utilizes more than one pathway. My dissertation has
not changed this proposition (as hypothesized in Chapter 1),
but rather has provided details of the mechanisms. It has
instigated a different perspective of olfactory signalling:
the detection of odor qualities by an ORN is not only
dependent upon the type and tuning of its olfactory receptor
protein, but the type, distribution, modulation, and
interaction of its second messenger-gated ion channels.

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BIOGRAPHICAL SKETCH
Debra Ann Fadool was born to Elizabeth Lynn Lewis and
Robert Servetus Frey on 15 November 1962 in Highland Park,
Illinois. When she was a year old, she and her family,
including her sister Elizabeth Lynn, moved to Cincinnati
Ohio, where she resided and attended grade school at Kilgore
Elementary. When she was six, her father, her sister, and
she moved to Louisville, Kentucky, where she completed grade
school at Norton Elementary and Belknap Elementary. She
attended middle school at Highland Jr. High School. She
attended high school at Atherton High and Louisville
Central, graduating in 1981. It was during these years that
she became disciplined in athletics, competing nationally in
synchronized swimming and qualifying for the All State cross
country team. It was also during this period that she
acquired a self-taught interest in science, which developed
out of boredom for something constructive in a school system
which was undergoing long awaited desegregation. She
directed her high school science education by participating
in state and regional math and science fairs, which gave her
the incentive to pursue biology at Albion College, Michigan.
There she fostered her childhood interest in teaching
through the example of several excellent professors, and a
289

290
love of marine biology through an off-campus semester in the
U.S.V.I., St. Croix. She graduated magna cum laude from
Albion College--MVP and all MIAA, NCAAIII cross country &
track; biology and english majors, May of 1985. She taught
at Newfoundland Harbor Marine Institute, Big Pine Key,
Florida, the summer before going to Kingston, Rhode Island,
where she earned a Master of Science jointly under the
supervision of Dr. Phyllis Brown, an analytical chemist, and
Gabriel Kass-Simon, a physiologist. She returned to
Louisville and married James Michael Fadool at St. Paul's
United Methodist Church. They resided in East Lansing,
Michigan, where she completed additional classwork, taught
biology lectures, and was a research associate for Michigan
State University. She had her first son, James Calvin, the
summer of 1989, shortly afterwhich the family moved to St.
Augustine, Florida, to allow her to commence her doctorate
research at the Whitney Laboratory under the direction of
Barry Ache, a chemosensory physiologist. She studied
neurobiology at Marine Biological Laboratory, Woods Hole,
Massachusetts, the summer of 1991. She gave birth to her
second son the spring of 1993, traveled to Sapporo Japan to
participate in an international smell and taste meeting, and
returned to St. Augustine to complete her requirements for
the degree of Doctor of Philosophy in zoology through the
University of Florida, December 1993. She looks forward to
studying as a postdoctoral scientist under the direction of

291
Irwin Levitan, a membrane biophysicist at Brandéis
University, Waltham, Massachusetts, while her husband
studies under the direction of John Dowling, a developmental
biologist at Harvard University, Boston, Massachusetts.

I certify that I have read this study and that in my
opinion it conforms to accej
presentation and is fully/adequate', ii
iards~-'Of scholarly
and quality, as
igree of Docpor
of \P
'h^Llosophy.
:
/ /
l^/
Barry W. Acl
Professor of Zoology
and Neuroscience
I certify that I have read this study and that in my
opinion it conforms to acceptable.standards of scholarly
presentation and is fully 'adequate, in scope and quality, as
a dissertation for the degrefe Doctor of Philosophy.
Peter A.V.' Anderson
Professor of Neuroscience
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Barbara-Anne Battelle
Associate Professor of Neuroscience
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
//
STf.
William E.S.Carr
Professor of Zoology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree/of Doctor o£. Philosophy.
fichele Wheatly
Associate Professor of Zoold?
This dissertation was submitted to the Graduate Faculty
of the Department of Zoology in the College of Liberal Arts
and Sciences and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of
Doctor of Philosophy.
December 1993
Dean, Graduate School

UNIVERSITY OF FLORIDA
3 1262 08553 9384




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