Excitatory signal transduction mediated by inositol phospholipid metabolism in lobster (Panulirus argus) olfactory recep...

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Title:
Excitatory signal transduction mediated by inositol phospholipid metabolism in lobster (Panulirus argus) olfactory receptor neurons
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xii, 291 leaves : ill., photos ; 29 cm.
Language:
English
Creator:
Fadool, Debra Ann, 1962-
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Subjects / Keywords:
Zoology thesis Ph.D
Dissertations, Academic -- Zoology -- UF
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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
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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

































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)












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S


CSN
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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.


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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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Hid
~V1O


0)
E

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asuodslu pez!pJiepuelS UBe\l


'i
S I0"
0









outward


current.


Thresholds


were


at least


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-


*
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rA


I