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Autonomic influences on the production of cerebrospinal fluid

Material Information

Title:
Autonomic influences on the production of cerebrospinal fluid
Creator:
Haywood, Joseph Roscoe, 1949-
Publication Date:
Language:
English
Physical Description:
xii, 108 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Adrenergics ( jstor )
Agonists ( jstor )
Blood flow ( jstor )
Blood pressure ( jstor )
Blood vessels ( jstor )
Cerebrospinal fluid ( jstor )
Cholinergics ( jstor )
Choroid plexus ( jstor )
Dosage ( jstor )
Innervation ( jstor )
Autonomic Agents ( mesh )
Cerebrospinal Fluid ( mesh )
Dissertations, Academic -- Pharmacology -- UF ( mesh )
Pharmacology thesis Ph.D ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1976.
Bibliography:
Includes bibliographical references (leaves 99-107).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Joseph Roscoe Haywood.

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University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
024233686 ( ALEPH )
25764493 ( OCLC )
AEK7516 ( NOTIS )

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Full Text














AUTONOMIC INFLUENCES ON THE PRODUCTION
OF CEREBROSPINAL FLUID












By

JOSEPH ROSCOE HAYWOOD


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












UNIVERSITY OF FLORIDA
1976




AUTONOMIC INFLUENCES ON THE PRODUCTION
OF CEREBROSPINAL FLUID
By
JOSEPH ROSCOE HAYWOOD
A DISSERTATION PRESENTED TO THE
GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1976


To Carol
for her understanding, patience, and love
To Becca and Alyson
for giving me so much happiness


ACKNOWLEDGMENTS
I wish to express my sincere gratitude to Dr.
Betty P. Vogh for her guidance throughout this course of
study. She has been most helpful as a teacher, as a
researcher, and as an advisor, but especially as a friend
whose interest in her students extends beyond science.
I am also grateful to the other members of my
supervisory committee, Dr. C.Y. Chiou, Dr. Carl Feldherr,
Dr. David Silverman, and Dr. David Travis for their
contributions. The advice of Dr. Thomas Maren, Dr. Peter
Lalley, Dr. Sidney Cassin, and the faculty of the
Department of Pharmacology and Therapeutics is also
greatly appreciated.
I also thank Mr. Mark Loveland for his friendship,
sense of humor, and technical assistance and Ms. Joyce
Simon for her support and contribution in the preparation
of this manuscript.
My sincere thanks are also extended to my fellow
graduate students for their encouragement and friendship
during these years.
This research was supported by a traineeship from
the National Institutes of Health (GM-00760) and a research
grant from the National Institute of General Medical
Sciences (GM-16934).


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
LIST OF TABLES vi
LIST OF FIGURES viii
KEY TO ABBREVIATIONS ix
ABSTRACT x
INTRODUCTION 1
BACKGROUND 3
Autonomic Innervation and Function in
Cerebral Blood Vessels 3
Innervation of the Choroid Plexus 5
Blood Flow to the Choroid Plexus 8
Autonomic Nervous System Control of
Fluid Secretions 8
Rationale for Study 12
METHODS 13
Tests of Autonomic Nervous System Influence
on CSF Production 15
Tests of Autonomic Nervous System Influence
on Blood Flow 21
Evaluation of CSF Production Data 24
Statistical Analysis . 25
RESULTS 2 6
Ventriculocisternal Perfusion Studies
for Estimation of CSF Production Rate .... 26
IV


Changes in Blood Pressure and Heart Rate
during Ventriculocisternal Perfusion Studies 53
Effect of Autonomic Agonists on Blood Flow . 67
DISCUSSION 70
Modification of CSF Production by
Autonomic Influences 70
Mechanism of Changes in CSF Production
by Autonomic Influences 82
Practical Significance 91
APPENDICES
I Autonomic Innervation and Function of
Cerebral Blood Vessels 93
II Composition of Artificial Cerebrospinal
Fluid 98
REFERENCES 9 9
BIOGRAPHICAL SKETCH 108
v


2
3
4
5
6
7
8
9
10
11
12
13
LIST OF TABLES
Page
CONTROL PRODUCTION OF CEREBROSPINAL FLUID . 27
CARBACHOL DOSE-RESPONSE: CHANGES IN CSF
PRODUCTION 31
EFFECTS OF AUTONOMIC BLOCKING AGENTS ON CSF
PRODUCTION 37
PHENYLEPHRINE DOSE-RESPONSE: CHANGES IN
CSF PRODUCTION 38
THE EFFECT OF HEMICHOLINIUM-3 (HC-3) ON CSF
PRODUCTION DURING INTRAVENOUS INFUSION OF
PHENYLEPHRINE (Phe) 44
BETA ADRENERGIC AGONISTS DOSE-RESPONSE:
CHANGES IN CSF PRODUCTION 4 6
EFFECTS OF THEOPHYLLINE ON CSF PRODUCTION,
BLOOD PRESSURE, AND HEART RATE 51
EFFECTS OF CARBONIC ANHYDRASE INHIBITION
ALONE AND WITH AUTONOMIC BLOCKING AGENTS
ON CSF PRODUCTION 52
CARBACHOL DOSE-RESPONSE: CHANGES IN
BLOOD PRESSURE AND HEART RATE 54
EFFECTS OF AUTONOMIC BLOCKING AGENTS ON
BLOOD PRESSURE AND HEART RATE 58
PHENYLEPHRINE DOSE-RESPONSE: CHANGES IN
BLOOD PRESSURE AND HEART RATE 59
THE EFFECT OF HEMICHOLINIUM-3 (HC-3) ON BLOOD
PRESSURE AND HEART RATE DURING INTRAVENOUS
INFUSION OF PHENYLEPHRINE (Phe) 63
BETA ADRENERGIC AGONISTS DOSE-RESPONSE:
CHANGES IN BLOOD PRESSURE AND HEART RATE . 64
Vl


14 EFFECTS OF PHENYLEPHRINE, CARBACHOL, AND
SALBUTAMOL ON BLOOD FLOW TO THE CHOROID
PLEXI, BRAIN, SKELETAL MUSCLE, AND KIDNEY . 68
Vll


LIST OF FIGURES
FIGURE Page
1 Changes in the production of CSF with
time in control experiments 29
2 Carbachol dose-response for CSF production . 33
3 95% confidence interval on probit analysis
of the effect of carbachol on CSF
production 35
4 Phenylephrine dose-response for CSF
production 40
5 95% confidence interval on probit analysis
of the effect of phenylephrine on CSF
production 4 2
6 Salbutamol dose-response for CSF production. 48
7 95% confidence interval on probit analysis
of the effect of salbutamol on CSF
production 50
8 Carbachol dose-response for blood pressure . 56
9 Phenylephrine dose-response for blood
pressure 61
10 Salbutamol dose-response for blood pressure 66
11 Autonomic pathways to the choroid plexus I .73
12 Autonomic pathways to the choroid plexus II 77
13 Autonomic pathways to the choroid plexus III 80
viii


KEY TO ABBREVIATIONS
CBF
CSF
cpm
CAMP
cGMP
ED50
gm
HC-3
ITP
kg
yg
yi
mg
ml
mm
mM
msec
min
M
M.W.
sec
Phe
Cerebral Blood Flow
Cerebrospinal Fluid
Counts per minute
Adenosine 3',5' cyclic monophosphate
Guanosine 3', 5' cyclic monophosphate
Median effective dose (dose to
produce 50% effect)
Gram
Hemicholinium-3
l-isopropylamino-3(2 thiazoloxy)-
2 propanol HC1
Kilogram
Microgram
Microliter
Milligram
Milliliter
Millimeter
Millimolar
Millisecond
Minute
Molar
Molecular weight
Second
Phenylephrine
xx


Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fullment of the
Requirements for the Degree of Doctor of Philosophy
AUTONOMIC INFLUENCES ON THE PRODUCTION
OF CEREBROSPINAL FLUID
By
Joseph Roscoe Haywood
December, 1976
Chairperson: Betty P. Vogh
Major Department: Pharmacology and Therapeutics
For the first time, treatments which stimulate or
inhibit autonomic nervous system receptors were tested for
their effects on cerebrospinal fluid (CSF) production
using the dilution of blue dextran during ventriculo-
cisternal perfusion in cats. Initial control CSF formation
ranged from 15 to 25 yl/min and gradually decreased at the
rate of 0.015 yl/min each minute between 60 and 300 minutes.
The cholinergic agonist, carbachol, in single
intravenous injections caused a dose-dependent increase
in the production of new fluid. The maximum increase was
10.6 2.1 yl/min which was blocked by atropine.
Single intravenous injections of the alpha adrenergic
agonist, phenylephrine, also increased CSF production to
a maximum of 10.6 1.5 yl/min over control. This dose-
dependent effect was prevented by phentolamine and by
atropine. An increase in CSF formation during infusion
of phenylephrine was blocked by administration of hemi-
cholinium-3 through a reduction of endogenous
x


acetylcholine. The ability of atropine and hemicholinium
to inhibit the increase in CSF production by phenylephrine
demonstrates that the alpha adrenergic agonist probably
activates a cholinergic pathway. Atropine or phentolamine
alone reduced CSF formation 2.6 0.5 and 3.7 0.8 yl/min,
indicating that the cholinergic pathway exerts some control
over normal fluid production. Neither atropine nor phen
tolamine was able to add to the maximal reduction in CSF
formation caused by the carbonic anhydrase inhibitor
methazolamide.
The beta2 adrenergic agonist, salbutamol (albuterol),
also increased the rate of CSF formation, but it was only
half as effective as carbachol and phenylephrine. The
betag adrenergic agonist, l-isopropylamino-3(2 thiazoloxy)-
2 propanol HC1 (ITP), had no effect on CSF production.
Propranolol did not affect fluid formation but did reduce
the effect of salbutamol.
Bilateral stimulation of the cervical sympathetic
trunk reduced CSF production 3.0 0.6 yl/min. This
observation indicated that alpha adrenergic agonists
probably act at the choroid plexus to decrease CSF
formation; however, any direct action of circulating
alpha agonists is masked by activating the cholinergic
pathway.
Experiments were also performed to determine whether
autonomic agents influenced CSF production through hemo
dynamic changes or a direct action on the choroid plexus
epithelium. Blood pressure data for carbachol,
xi


phenylephrine, and salbutamol did not correlate with the
changes in CSF production. Blood flow studies showed
that none of the agonists caused statistically significant
changes in cerebral or choroid plexus blood flow. These
findings suggest that autonomic drugs probably act directly
on the choroid plexus epithelium to change the rate of CSF
formation.
Verapamil, an agent that blocks calcium channels, did
not alter a carbachol-induced increase in fluid production
indicating that the autonomic agents probably did not exert
their effect by influencing calcium fluxes. The phospho
diesterase inhibitor, theophylline, on the other hand,
increased the rate of CSF production. This effect
suggested that the autonomic treatments might alter fluid
formation by increasing or decreasing levels of the cyclic
nucleotides guanosine 3',5' cyclic monophosphate (cGMP) or
adenosine 3',5' cyclic monophosphate (cAMP).
Although the exact mechanisms remain to be elucidated,
it is clear that cholinergic and beta2 adrenergic receptors
can be stimulated to increase CSF formation significantly
and that the cholinergic pathway which predominates is
also activated by alpha adrenergic agonists.
xii


INTRODUCTION
Epithelia structurally similar to the choroid
plexus epithelium are found in the ciliary body of the
eye, the salivary gland, and the pancreas. Secretory
transport in these tissues is known to be controlled to
some extent by the autonomic nervous system. Therefore,
it is of interest to consider the influence of the
autonomic nervous system on the formation of cerebrospinal
fluid (CSF).
The columnar epithelial cells of the choroid plexus
are considered to be a primary site for the secretion of
CSF. This structure is an evagination of the walls of
the lateral and third ventricles of the brain and forms
the roof of the fourth ventricle. The choroid plexus
receives some innervation from the autonomic nervous
system. On the ventricular side, the outermost structure
of the choroid plexus is a single layer of epithelial
cells supported by connective tissue richly supplied with
blood vessels.
The autonomic nervous system is comprised of
sympathetic and parasympathetic components. The sympa
thetic system has short fibers from the central nervous
system to the sympathetic chain ganglia, then long
1


2
post-ganglionic fibers which innervate the tissues.
Sympathetic effects are classified into alpha, beta-p and
beta2 adrenergic actions and are mediated endogenously by
the adrenergic neurotransmitters norepinephrine and
epinephrine. Other agents can selectively stimulate alpha,
beta-^, or beta2 adrenergic activity if they have the
necessary structural modifications required for each
receptor. Phenylephrine is an alpha agonist; 1-isopropyl-
amino-3(2 thiazoloxy)-2 propanol HC1 (ITP) is a betag
agonist; and salbutamol is a beta2 agonist. The parasym
pathetic system has long fibers from the central nervous
system to ganglia close to the tissue being supplied,
then short post-ganglionic fibers which innervate the
tissue. The cholinergic neurotransmitter acetylcholine
mediates parasympathetic actions. Chemically related
agents such as carbachol can initiate cholinergic
activity when applied to the effector site.


BACKGROUND
In all systems in which fluid formation can be
altered by agents that affect the autonomic nervous system,
one must consider two basic means by which these effects
may be mediated. Alterations in fluid production may occur
in direct response to autonomic agents acting on receptors
located in the secretory cells. Also changes in fluid
production may possibly result from changes in hemodynamic
relationships increasing or decreasing pressure and flow
in the vessels of the choroid plexus. Functionally,
changes in hemodynamic relationships may interact with
direct changes in the secretory function of the epithelium
to alter fluid formation.
Autonomic Innervation and Function in Cerebral Blood Vessels
By following the nerve trunks passing into the
brain, anatomists identifed nerves supplying the large
pial vessels more than a century ago. Microscopists
subsequently described the presence of nerve fibers in
the finer cerebral vasculature, but classification of the
nerves as sympathetic and parasympathetic did not come
until the 1920's. Since the blood vessels of the choroid
3


4
plexus arise from cerebral arteries, a brief summary of
general cerebrovascular innervation and autonomic function
will be given before considering the choroid plexus
specifically. A more detailed presentation of the
autonomic innervation and responses of cerebral blood
vessels is given in Appendix I.
Sympathetic innervation of the large pial blood
vessels is supplied by the cervical sympathetic trunks.
This has been demonstrated by showing the disappearance
of adrenergic nerves after cervical sympathetic ganglionec-
tomy. However, smaller parenchymal vessels within the
brain receive adrenergic nerves from the brainstem. These
nerves survive bilateral cervical sympathectomy. Parasym
pathetic innervation of cerebral blood vessels originates
primarily through the facial nerve. Recent experiments
have shown that cholinergic nerves also come from sources
other than the cranial nerves.
A decrease in cerebral blood flow in dogs after
electrical stimulation of the cervical sympathetic trunk
or administration of norepinephrine has been demonstrated
by some investigators; however, others have been unable to
elicit these effects. On the other hand, stimulation of
the facial nerve or exogenous administration of acetyl
choline reproducibly and significantly increases cerebral
blood flow.
Cerebral blood vessels have shown more consistent
adrenergic responses in vitro than in the whole animal


5
experiments. Both electrical stimulation and alpha
adrenergic agents contract cerebral arteries in vitro,
but the maximum force of contraction is less than in
arteries from other tissues. Beta adrenergic responses
in cerebral vessels so far have been found only in the
cat. In this species, beta2 stimulation relaxes vessels
contracted with serotonin.
The significant observation from both the in vivo
and in vitro studies is that regardless of the nature of
the stimulation (i.e., electrical or pharmacological) the
adrenergic response of the cerebral vessel is small or
absent. In contrast, cholinergic stimulation has a strong
influence on cerebral blood vessels. In vitro acetyl
choline has been shown to relax contracted vessels.
Innervation of the Choroid Plexus
Since the choroid plexus is composed of an epithelium
resting on connective tissue containing blood vessels, it
is not feasible to discuss separately innervation of the
epithelium and innervation of the vessels with regard to
what is presently known about the anatomy of the tissue.
For this reason, evidence for nerves supplying both the
epithelia and the blood vessels of this secretory tissue
will be presented together.
Benedikt (1874) first observed nerve fibers around
blood vessels and near epithelial cells of the fourth


6
ventricle plexus. These fibers arose from a vagal nucleus
in the medulla which he called the XIII Cranial Nerve.
Later, innervation in the lateral plexus was described as
being mostly around the vessels, and it was suggested that
these nerves might be involved in vasomotor function
(Findlay, 1899). Stohr (1922) confirmed the observation
of Benedikt in the fourth ventricle plexus and also
demonstrated a nerve supply to the third ventricle plexus.
He suggested the nerve fibers were divided into "vascular
nerve fibers" entering with the arterial vessels and
"choroid plexus proper" fibers originating in a vagal
nucleus, in pontine nuclei, and in cerebral peduncles.
Junet (1926) also described nerves supplying the epithelial
cells of the plexus. Stohr's observation of separate
vascular and epithelial innervation to the fourth ventricle
plexus originating from the medulla was confirmed by Clark
(1928, 1934), and he extended the theory to include the
lateral plexus. Clark described the fibers as being
mostly in the connective tissue base of the choroid plexus
rather than around the epithelial cells. Some myelinated
nerves and a large number of unmyelinated nerves were
found in association with blood vessels and epithelial
cells without any of them ending directly on capillaries.
With electron microscopy, unmyelinated nerve fibers have
been identified near the choroid plexus epithelium
(Millen and Rogers, 1956) and around some blood vessels


7
supplying the plexus (Maxwell and Pease, 1956). Tennyson
(1975) has described a nerve process adjacent to an
epithelial cell. The cell membranes of both the nerve
and epithelium were thickened, indicating the presence of
a possible cell junction.
To determine the extent of adrenergic innervation,
two groups of investigators did cervical sympathetic
ganglionectomies. In the first study, a unilateral sympa
thectomy resulted in a patchy degeneration of nerve fibers
in the connective tissue of the ipsilateral and third
ventricle plexi of dogs and cats with most of the inner
vation remaining intact (Tsuker, 1947). The other study
demonstrated by fluorescence microscopy the presence of
an interconnected ground plexus of adrenergic nerves
between the vascular walls and the epithelial cells
(Edvinsson et al., 1974) These observations indicated
the lateral and third ventricle plexi had the highest
content of adrenergic innervation and the fourth ventricle
plexus content was much lower. After a bilateral sympa
thectomy, the fluorescence disappeared in all the choroid
plexus and the norepinephrine content of the plexus
decreased significantly (Edvinsson et al., 1972b). Cholin
ergic innervation of the choroid plexus epithelium and
blood vessels has also been described (Edvinsson et al.,
1973b). In cats and rabbits, cholinergic fibers were
observed for all the plexus in greater density than


8
adrenergic fibers. The network of cholinergic nerve
fibers remained intact after sympathetic ganglionectomy.
Blood Flow to the Choroid Plexus
Blood flow to the choroid plexus has been measured
in the intact whole animal in only one study. Aim and
Bill (1973), using radioactive microspheres, obtained a
control flow of 3.01 ml/gm min. Upon stimulation of the
cervical sympathetic chain, the blood flow to the plexus
did not change. Two groups have made in situ determin
ations of plexus blood flow. Welch (1963) while
measuring CSF production at the lateral choroid plexus in
the rabbit, observed a blood flow of approximately 2.86
ml/gm min. Pollay et al. (1972) perfused the choroid
plexus vessels of sheep and related blood flow to the
production of CSF. For a normal rate of formation they
concluded choroid plexus blood flow must be about 2.56
ml/gm min. Pharmacological agents affecting blood flow
were not used in any of these studies; however, Macri
et al. (1966) and Politoff and Macri (1966) showed that
serotonin and 1-norepinephrine reduced choroidal blood
flow in rabbits.
Autonomic Nervous System Control of Fluid Secretions
Fluid secretion occurs in several specialized
epithelial tissues. Sympathetic or parasympathetic


9
agonists have been shown to influence secretion in nearly
all of the secretory epithelia, but there has not been a
consistent pattern of effects. The salivary gland is
probably the most thoroughly studied of the tissues in
which there is autonomic control over fluid production.
In this system, both cholinergic and adrenergic agonists
increase secretion (Koelle, 1970). Cholinergic agonists
can produce approximately ten times as much fluid as beta
adrenergic agonists (Mangos etal., 1973). Petersen (1972)
has suggested that acetylcholine-activated secretion occurs
as a result of an uptake of extracellular calcium which
Douglas and Poisner (1963) showed was necessary for fluid
movement. The calcium, in turn, may cause an efflux of
potassium and an uptake of sodium which results in water
transfer.
The eye is another site in which fluid movement is
actively influenced by autonomic agonists. In this system
both production of fluid by the ciliary body and drainage
of aqueous humor through the trabecular meshwork are
under autonomic control (Chiou and Zimmerman, 1975). It
has been difficult to determine whether the cholinergic
system or the adrenergic system really predominates in
either mechanism, but it is certain that both are
influential. Paterson and Paterson (1972) have shown
that alpha adrenergic agonists cause a slight increase in
aqueous humor production and outflow facility. In contrast,


10
beta2 agonists (such as salbutamol) cause a decrease in
production and a large increase in flow through the
trabecular meshwork which can be blocked by propranolol
(Langham and Diggs, 1974 and Langham, 1976). The effects
of cholinergic agents on aqueous humor dynamics is more
complicated. The major effect in the whole animal is an
increased drainage of fluid (Ellis, 1971 and Macri and
Cevario, 1973). When acetylcholine and eserine, an anti
cholinesterase agent, are given intra-arterially in an
isolated perfused cat eye, the formation of aqueous humor
increases 10 to 15 yl/min (Macri and Cevario, 1973). The
action of acetylcholine can be blocked by atropine;
however, it can also be prevented with alpha adrenergic
receptor blocking agents or guanethidine, an adrenergic
neuron blocking agent, indicating the effect is probably
mediated by alpha receptors.
The acinar cells of the pancreas are also under
autonomic control (Thomas, 1967). Either vagal stimulaLion
or the administration of cholinomimetic drugs can elicit
moderate increases in fluid secretion and substantial
increases in the release of digestive enzymes. Norepi
nephrine and epinephrine decrease secretion possibly by
constricting the blood vessels which supply the tissue.
Fluid transfer in the toad bladder and frog skin
epithelia can be increased by beta adrenergic stimulation
(Foster, 1974). This presumably occurs through changes


11
in adenosine 3',5' cyclic monophosphate (cAMP) levels
since it can be mimicked by dibutyryl cAMP and potentiated
by phosphodiesterase inhibitors (Jard, 1974). On the
other hand, alpha adrenergic activation reduces water
movement stimulated by theophylline or antidiuretic
hormone (Foster, 1974 and Jard, 1974) but has no effect
in the absence of these agents. No observations have been
made concerning possible cholinergic actions.
Very little investigation has been made into the
role of autonomic agents in the production of CSF. Becht
and Gunnar (1921) set a needle in the cisterna magna of
cats and noted that epinephrine and pilocarpine increased
fluid outflow. They suggested that this was not true CSF
production since some of the collected fluid was drawn up
into the tube again after the action of the drug was
finished. Bhattacharya and Feldberg (1958) observed the
same effect during ventriculocisternal perfusion with
epinephrine, histamine, and tubocurare. In these exper
iments only volumes were measured, and it was suggested
that the increased flow during the perfusion of the
ventricles was due to a change in cerebral blood flow.
Vates et al. (1964) observed that norepinephrine
(2.5 X 10~6 M in the perfusate) caused a decrease in
production of about 24% during yentriculocisternal
perfusion.


12
Rationale for Study
The evidence presented satisfies two major criteria
for concluding that the autonomic nervous system may exert
an influence on CSF production. First, it has been shown
that the blood vessels and epithelia of the choroid plexus
receive both adrenergic and cholinergic innervation. The
adrenergic nerves extend from the cervical sympathetic
trunk whereas the cholinergic nerves, comprising most of
the tissue's innervation, originate from undefined
locations. Second, it is clear that both adrenergic and
cholinergic agents alter secretion in other epithelial
systems.
Therefore, it seemed reasonable to investigate CSF
production during pharmacological alteration of the adren
ergic and cholinergic nervous system with agonists and
antagonists and to examine the role of cerebral blood flow
as a possible mediator of effects of these agents on CSF
production. Such an investigation is the subject of this
dissertation.


METHODS
Cats have been used extensively for studies of
neurological and secretory functions in the brain and for
this reason were chosen for investigation of the role of
the autonomic nervous system on CSF production. Animals
weighing 2-4 kg were anesthetized with an intrahepatic
injection of 30 mg/kg sodium pentobarbital and further
maintenance doses were given intravenously as needed
throughout the experiment. Surgical procedures for the
insertion of a tracheal cannula and femoral arterial and
venous catheters were performed. Blood pressure and heart
rate were monitored with a Statham pressure transducer
connected to the femoral artery catheter and recorded on
a Grass polygraph. Body temperature was maintained at
37 C. Throughout each experiment, the animal was arti
ficially ventilated at a rate set to maintain arterial pH
at approximately 7.35, the normal control value r a cat.
By measuring arterial pH and adjusting the ventilatory
rate as needed, the correct rate was established and the
corresponding end-tidal CC>2 was recorded and subsequently
monitored to maintain pH at the desired level.
For ventriculocisternal perfusion, the cat's head
was placed in a standard stereotaxic apparatus. A midline
13


14
incision was made from the top of the head to the neck
and a burr hole was drilled at a point 2 mm lateral to
the saggital suture and 2 mm caudal to the coronal suture.
A 22-gauge spinal needle, attached through a Statham
pressure transducer to a Harvard infusion pump, was set in
the right lateral ventricle. Placement was determined by
a sharp drop in the pressure recording as the needle passed
beyond the brain parenchyma into the ventricle. This
needle was used to deliver perfusate and to monitor intra
ventricular pressure which was recorded throughout the
experiment. The tip of a 19-gauge needle was then placed
in the cisterna magna by separating the neck muscles at
the midline and puncturing the dura mater just below the
occipital condyle of the skull, and effluent was collected
through a short tube attached to the needle. An artificial
CSF (see Appendix II for the composition) was used for the
perfusion. pH of the perfusion fluid was adjusted to 7.4
by bubbling 5% CC>2 in C>2 through it until the desired pH
was achieved. The perfusion rate was 191 yl/min during
the first 15 minutes and 76.4 yl/min thereafter.
Following a 30 minute washout of the original CSF in the
ventricular system, four to eight control periods of 15
minutes each were collected prior to tests with drugs or
stimulation.
Production of CSF was calculated from the dilution
of blue dextran, M.W. 2 X 10^ daltons, (Pharmacia). The


15
collected effluent of each sampling period and the original
artificial perfusate were diluted (100 yl in a total
volume of 1.0 ml) for measurement of optical densities
(Gilford 2400 spectrophotometer). This dilution was
necessary to obtain a large enough volume to assay during
short sampling periods. In most experiments, perfusion
was continued six hours; none went beyond nine hours.
Fluid formation was calculated with the equation
developed by Heisey et al. (1962) for dilution of non-
diffusible substances:
('-in ~ ^out) (r)
Vf = ¡w
The difference between the concentration of blue dextran
in the infused buffer solution (Cj_n) and the effluent
(Cout) divided by the concentration of the effluent is the
dilution factor of the perfusate. The product of the
dilution factor and the rate of infusion (r) then provides
a value for the rate of formation of CSF.
Tests of Autonomic Nervous System
Influence on CSF Production
Stimulation of Cervical Sympathetic Trunks
Bilateral electrical stimulation of the cervical
sympathetic trunk was applied during ventriculocisternal
perfusion in some experiments. In addition to the surgical
procedure described for perfusion experiments, the cervical


16
sympathetic ganglion was exposed and the two preganglionic
sympathetic fibers were isolated from the vagi. The
preganglionic fibers were then passed through a platinum
electrode which was connected to a stimulator. The nerve
fibers were stimulated with 10 volts at a frequency of 10
shocks per second; each shock lasted for 0.5 msec. Nicti
tating membrane responses were also monitored during these
experiments to insure that adequate stimulation was
bilaterally applied. This was done by gently pulling each
membrane away from the eye, placing a suture in it and
attaching the suture to a Myograph-B (Narco Biosystems)
transducer which permitted the recording of membrane
responses. The experimental sampling period was 15
minutes in duration, with stimulation continuing throughout
the period. Control samples were collected during two
hours preceding and two hours following stimulation.
Modification of CSF Production by Application of
Pharmacological Agents
Drugs tested to determine a possible autonomic
influence on CSF production were carbamylcholine chloride
(carbachol) (Sigma), phenylephrine HC1 (Sigma), salbutamol
(albuterol in the U.S.) (Schering), atropine methyl
nitrate (Sigma), phentolamine mesylate (Ciba-Geigy),
propranolol HC1 (Ayerst), l-isopropylamino-3(2 thiazoloxy)-
2 propanol HC1 (ITP) (Syntex), aminophylline (theophylline)
(Parke-Davis), verapamil (Knoll Pharmaceutical), and
methazolamide (Lederle). All autonomic agonists were


17
administered in volumes less than 1 ml; all other drugs
were given in volumes less than 3 ml. These volumes
were considered small enough not to alter significantly
the total volume of blood.
At least one and one-half hours elapsed between
administrations of autonomic agonists when the drugs were
given to test the individual effects. No agonist was
tested during the first 30 minutes after an autonomic
blocking agent was given so as to test the effect of the
blocking agent on CSF formation. Subsequently, agonists
were injected at approximately 30 minute intervals.
The doses of the autonomic agonists used in these
experiments were selected on the basis of the blood
pressure dose-response curves. Doses on either side of
the ED5Q for blood pressure and the dose which gave the
maximal blood pressure response were tested for their
effect on CSF production. Salbutamol, however, required
a dose higher than that giving maximal blood pressure
effect to elicit a maximal effect on CSF formation. Doses
of the autonomic antagonists used were chosen for their
effectiveness in blocking peripheral actions of the
agonists.
Cholinergic agonist and antagonist. Carbachol was
chosen as the cholinergic agonist for its specificity at
the cholinergic post-ganglionic effector site and prolonged
half-life in the whole animal compared to acetylcholine.


18
It was dissolved in normal saline in concentrations up
to 10 yg/ml and administered intravenously in doses of
0.03, 0.1, 0.3, or 3.0 yg/kg body weight. As in all
experiments involving agonists, three 3-minute effluent
samples were collected beginning immediately after
injection of the drug.
Atropine (0.15 mg/kg) was given to test for a direct
effect on CSF formation. During atropine treatment,
carbachol was given in doses of 1.0, 3.0, and 30.0 yg/kg
to test for blockade of cholinergic effects on blood
pressure and CSF production. Phenylephrine (30 yg/kg)
and salbutamol (3 yg/kg) were also administered during
atropine treatment.
Alpha adrenergic agonist and antagonist. Phenyl
ephrine was chosen as the agonist for these experiments
because of its specificity for alpha adrenergic receptors.
It was dissolved in normal saline in concentrations up
to 300 yg/ml and administered intravenously in single
doses of 3.0, 10.0, 30.0, and 100.0 yg/kg body weight
or infused over a nine minute period (30-35 yg/kg min)
intravenously. Single injections were followed by three
3-minute samples. During infusions 3-minute samples were
also collected. Phentolamine was chosen as the alpha
receptor-blocking agent for its specificity and reversi
bility. The direct action of phentolamine on CSF formation
was tested at the dose of 2 mg/kg body weight. During


19
alpha adrenergic blockade, single injections of 30 yg/kg
phenylephrine, 3 yg/kg salbutamol, and 3 yg/kg carbachol
were tested. To further examine the mechanism of action
of phenylephrine, another group of experiments was done
in which the nine minute phenylephrine infusion was
preceded three minutes earlier by an intravenous injection
of 50 yg/kg of hemicholinium-3.
Beta adrenergic agonists and antagonist. To
ascertain specificity of beta adrenergic function both a
betai anc^ a heta2 agonist were tested to determine their
effect on CSF production. The beta^ agent selected was
l-isopropylamino-3(2 thiazoloxy)-2 propanol HC1 (ITP).
This compound has been shown to be specific for cardio-
acceleration and stimulation of lipolysis without affecting
beta2 actions (Lockwood and Lum, 1974). The drug was
dissolved in normal saline less than five minutes before
use to be certain that the drug was still active, and doses
of 100 and 300 yg/kg body weight were administered intra
venously. Beta2 activity was determined with salbutamol.
Salbutamol has been shown to have bronchodilator activity
and to increase blood flow to the dog hindlimb more
effectively than isoproterenol, and these effects are
reduced in the presence of propranolol (Cullum et al.,
1969). This drug was dissolved in normal saline in
concentrations up to 200 yg/ml and was given intravenously
in doses of 0.1, 1.0, 3.0, 10.0, and 30.0 yg/kg body
weight. For both agents, cisternal effluent was collected


20
over three 3-minute periods after drug administration.
The general beta blocking agent propranolol (2.0 mg/kg)
was tested to determine its effect on CSF production and
was given to antagonize beta2 adrenergic effects. Phenyl
ephrine and carbachol were also tested in the presence of
propranolol.
Non-autonomic agents. Three non-autonomic drugs
were studied with regard to their influence on CSF
production: theophylline, verapamil, and methazolamide.
Theophylline has been shown to stimulate the respiratory
rate, increase urine flow, and stimulate the rate and force
of contraction of the heart. Some of these actions of
theophylline are thought to result from the ability of the
agent to inhibit phosphodiesterase, the enzyme responsible
for cyclic nucleotide hydrolysis (Amer and Kreighbaum,
1975). To test whether autonomic agents might be acting
through changes in cyclic nucleotide levels to affect
CSF production, theophylline was administered intravenously
in the form of aminophylline in doses equivalent to 10 and
20 mg/kg theophylline. Again, CSF perfusate was collected
over three 3-minute periods following drug injection.
Verapamil has been demonstrated to block inward calcium
channels in cardiac muscle (Kohlhardt et al., 1972).
Since calcium influx is important in acetylcholine-
stimulated salivary secretion, verapamil was given via
the perfusate at a concentration of 10"
M to test its


21
effect on carbachol activity; three 3-minute sampling
periods followed the administration of carbachol.
Carbonic anhydrase inhibitors, acetazolamide and benzol-
amide, have been shown to reduce CSF formation between 50
and 75% (Davson and Segal, 1970; Oppelt et al., 1964).
The carbonic anhydrase inhibitor, methazolamide was chosen
for its high lipid solubility providing ready access to
the secretory tissues. In these experiments methazolamide
(30 mg/kg) was given intravenously over a period of 30
minutes. CSF production in methazolamide treated cats was
also studied in the presence of atropine (0.15 mg/kg) or
phentolamine (2.0 mg/kg). Samples were collected in ten
minute periods for at least two hours after both drugs
had been administered.
Tests of Autonomic Nervous System
Influence on Blood Flow
Radioactive microspheres were used to measure blood
flow to the choroid plexus, brain, kidney, and biceps
brachii muscle. The kidney and muscle flows were used as
reference data. Briefly, the measurement depends on the
immediate lodging of spheres in capillary beds propor
tional to the capillary flow of the tissue. After delivery
of the spheres to the left side of the heart, the spheres
will have the same distribution as the blood leaving the
heart. Simultaneously withdrawing blood and microspheres


22
into a syringe at a known rate then provides the basis for
estimation of flow in tissues (Wagner et al. 1969). Both
femoral arteries and a femoral vein were catheterized, and
a tracheal cannula was inserted. One of the arterial
catheters was connected to a pump set to withdraw blood
at 2.72 ml/min. The chest was opened at the midline and
the pericardium incised. A catheter was placed in the
left atrium and secured. The animal was then given 3500
units of sodium heparin to prevent clotting of blood. End-
tidal CO2 was carefully monitored during the experiment to
insure the same pC02 during each injection of microspheres
since brain blood flow is highly sensitive to pCC>2 changes.
The microspheres were suspended by the 3 M Company
in 20% dextran in Tween 80 for injection. Microspheres
15 5 microns in diameter were chosen because this size
has been shown to be optimal for cerebral blood flow
measurements, providing high reproducibility and less than
2% shunting of spheres past the capillary beds (Marcus et
al., 1976). Buckberg et al. (1971) showed a minimum of
400 spheres must be present to measure reliably the blood
flow of a tissue. Therefore, the number of counts per
minute (cpm) per microsphere was determined to estimate
a dose that would satisfy this requirement. In all exper
iments the cpm in each tissue represented more than the
minimum required number of spheres, thus validating the
calculation of flow from the radioactivity in the tissue.
O C
In each experiment injection of
Strontium spheres ten


23
seconds after beginning blood withdrawal gave control
, i4i
blood flows, and injection of Cerium spheres later gave
blood flows during the peak influence of 3 yg/kg carbachol,
30 yg/kg phenylephrine, or 3 yg/kg salbutamol on CSF
production. The drugs were given about five seconds
before the blood withdrawal began. Blood withdrawal was
continued 90 seconds to guarantee at least one circulation
time. The animal was sacrificed and the choroid plexus
of the lateral and fourth ventricles was removed and
weighed. The brain, kidney, and muscle were also taken,
weighed, and ashed. The radioactivity was counted in a
Beckman Biogamma Scintillation Counter.
Blood flow to each of the organs was calculated
from the relation
Q = L_
d d'
whence,
A (F) (d)
U (d-)
where Q is the blood flow (ml/min), F is the rate at
which the arterial blood is sampled, d is the cpm of the
tissue being determined, and d' is the cpm of the isotope
in the arterial blood sample. To standardize the values,
the blood flows are expressed as ml/gm min considering
tissue weight.


24
Evaluation of CSF Production Data
The changes in CSF production caused by the various
treatments were calculated in the following manner: For
agonists, a least squares linear regression was applied
to the control CSF productions of each experiment. This
regression was based on CSF formation during one or more
control periods before any treatment and at least three
post-treatment control periods as determined by the return
of blood pressure and heart rate to pre-drug levels. No
values for post-treatment controls were taken sooner than
30 minutes after administration of short-acting agonists
such as carbachol, phenylephrine, and salbutamol. This
line was used to determine the theoretical control CSF
production at any given time over a two or three hour
span of the experiment. The production rate measured over
a three minute period after the agonist was given was
compared with the theoretical control for that time, and
the difference taken as the change in production caused
by the drug. Since the autonomic blocking agents and
methazolamide have relatively long active half-lives,
control periods were not availble after administration of
these drugs. For these experiments, four or more periods
prior to the time the agent was given were used to determine
a least squares regression line and calculate theoretical
control CSF productions. Changes in production were
determined in these experiments as for the agonists.


Statistical Analysis
All the changes in CSF production are expressed as
mean standard error of the mean (SE). Statistical
significance of changes in production caused by several
treatments was established by pairing theoretical control
and experimental data for each experiment and using the
paired one-tailed t-test (Goldstein, 1964). Significance
of changes in CSF production before and after treatment
with blocking agents or methazolamide was determined by
using the unpaired two-tailed t-test (Goldstein, 1964).
To ascertain whether increasing doses of autonomic
agonists yielded a dose-response relationship, the indi
vidual responses for each experiment were converted to
probits, a measure of the per cent of maximum response
obtained. A linear regression was then performed and
a plot of log dose vs. probit was produced with a 95%
confidence interval on the regression line. Significance
of the line was established by testing its slope against
a null hypothesis (Snedecor and Cochran, 1967). In all
cases, significance was assumed when the t-value exceeded
the minimum value for p<.05. Non-significance is denoted
by N.S. in the data.


RESULTS
Ventriculocisternal Perfusion Studies
for Estimation of CSF Production Rate
Control Experiments
Control data were collected in four experiments in
which no drugs were given. Control data from 24 exper
iments in which only short-acting autonomic agonists such
as carbachol, phenylephrine, and salbutamol were given
were added to these, avoiding data collected within 30
minutes after drug. The two groups of data were combined
since the slopes of the 4 untreated (-0.042 yl/min each
minute) and 24 treated animals (-0.015 yl/min each minute)
were not statistically significant from each other or
different from zero when tested against a null hypothesis.
These combined data, shown in Table 1 and Figure 1,
indicate a steep increase in the optical density of the
CSF perfusate reflected as a decrease in CSF production
during the first 60 minutes of the experiment according
to a regression line with a slope of -0.143 yl/min per
minute. This initial apparent decline in fluid formation
was probably due to an incomplete washout of the original
CSF in the ventricular system. A more gradual decline
follows ranging from 19.7 1.0 to 15.8 0.7 yl/min over
26


27
TABLE 1
CONTROL PRODUCTION OF CEREBROSPINAL FLUID
Time (min)a
n
Production
(yl/min) SE
30
24
24.0
+
1.5
45
21
20.7
+
1.3
60
27
19.7
+
1.0
75
10
17.5
+
1.2
90
8
19.7
+
1.2
105
6
16.9
+
1.6
120
17
17.0
+
0.8
135
22
16.2
+
0.7
150
27
16.8
+
0.7
165
24
16.5
+
0.7
180
28
16.7
+
0.6
195
11
17.2
+
0.7
210
7
15.6
+
1.0
225
14
15.1
+
0.9
240
24
15.9
+
0.7
255
20
15.1
+
0.7
270
25
14.9
+
0.6
285
22
15.6
+
0.5
300
19
15.8
+
0.7
Regression line for 0 to 60 minutes
y = -0.143 x + 27.9
r = -0.96
Regression line for 60 to 300 minutes
y = -0.015 x + 19.3
r = -0.81
aDuration of perfusion after washout of original CSF.


Figure 1. Changes in the production of CSF with time in
control experiments. The ordinate is the mean
CSF production value SE for at least 6
experiments at each point. The abcissa is
time in minutes. The regression lines for
0 to 60 minutes and 60 to 300 minutes are
represented by a solid line.


28
26
24
22
20
18
16
14
12
10
8
6
4
2
A i A 1 1 * -L
30 60 90 120 150 180 210 240 270 300
Time Cmin)
N)
O


30
the next 240 minutes in which the slope of the regression
line is -0.015 yl/min each minute. These data compare
favorably with the accepted control CSF production value
of approximately 20 yl/min (Davson, 1967) and with the
slope of -0.025 yl/min per minute for a regression line
on CSF formation in the monkey (Martins et al., 1974).
Cervical Sympathetic Stimulation
Only those experiments in which successful bilateral
stimulation occurred were included in the study. This was
determined by recording the contraction of the nictitating
membrane. After an initial decrease in the response of the
membrane during stimulation, the tension caused by the
contraction remained steady, and no fatigue was observed
during the experiment. Preganglionic stimulation of 10
volts at a frequency of 10 shocks/sec each with a duration
of 0.5 msec decreased CSF production 3.0 0.6 yl/min in
five animals (p<.05 in paired t-test) .
Cholinergic Agonist and Antagonist
Carbachol significantly increased the rate of CSF '
formation to 10.6 2.1 yl/min over control (Table 2,
Figure 2). Over a dosage range of 0.3 yg/kg to 3.0 yg/kg
probit analysis (Figure 3) yielded a significant slope of
1.52 (p<.05). These relationships among the CSF
production data were taken to indicate a carbachol dose-
response. The ED50 for carbachol taken from the probit
plot was 0.12 yg/kg. High doses of this cholinergic
agonist occasionally produced slight increases in intra
ventricular pressure.


TABLE 2
CARBACHOL DOSE-RESPONSE: CHANGES IN CSF PRODUCTION
Dose (yg/kg) Control CSF Production Change in CSF Production3
(yl/min) SE(yl/min) SE (n)
0.03
19.5
+
2.2
+ 2.0
+
0.6
(6)
0.10
28.9
+
5.7
+ 4.6
+
1.0
(5)
0.30
18.4
+
1.1
+ 7.8
+
1.6
(5)
3.0
16.8
+
1.8
+ 10.6

2.1
(6)
i.ob
15.8
+
2.3
+ 0.6

0.4
(3)
3.0b
14.1
+
1.5
-0.3

1.4
(3) d
rQ
o
o
ro
9.9
+
2.1
+ 2.7

1.0
(5)
3.0G
12.3
+
0.6
+ 15.3

6.5
(4)e
Change in CSF production calculated for each
experiment as the difference between the control
and the peak effect of the drug
Given within 120 min after 0.15 mg/kg atropine
c R
10 M verapamil in CSF perfusate
dp < .05 when compared with 3 yg/kg carbachol alone
0
N.S. when compared with 3 yg/kg carbachol alone


Figure 2. Carbachol dose-response for CSF production. The
ordinate is the mean increase in CSF production
over the theoretical control value SE plotted
on a linear scale. The abcissa is the dose of
carbachol in pg/kg body weight plotted on a
logarithmic scale. (# ) represents
carbachol alone. (O) is carbachol during
0.15 mg/kg atropine.


0 03 0 10 0 30
Carbachoi C^ig/ kg)
ee


Figure 3.
95% confidence interval on probit analysis of
the effect of carbachol on CSF production. The
ordinate is probits. The abcissa is the dose
of carbachol in pg/kg body weight plotted on a
logarithmic scale. The slope of the probit
regression line is 1.52 which is significant
(p<.05). The dose of carbachol producing an
EDcjq response is 0.12 pg/kg.


Prob its
3
2
1
0 03 0 10
Carbachol (|jg/ kg )
030
U>
On


36
Atropine caused a decrease of 2.6 0.5 yl/min in
CSF production which was statistically significant
(Table 3). The cholinergic blocking agent also prevented
the increase stimulated by 3 yg/kg carbachol (Table 2).
The 30 yg/kg carbachol dose increased CSF production
during atropine blockade by 2.7 1.0 yl/min. The 3 yg/kg
dose of carbachol was administered during phentolamine
and propranolol blockade of adrenergic receptors in one
or two experiments. No alteration in the effect of
carbachol was evident. When verapamil (10-^ M) was
present in the artificial CSF perfusate, there was also
no change in the response to this dose of carbachol
(Table 2). Verapamil alone did not alter normal CSF
production.
Alpha Adrenergic Agonist and Antagonist
Phenylephrine caused a dose-dependent increase in
CSF production (Table 4, Figure 4). The change in response
with increasing amounts of drug was subjected to probit
analysis (Figure 5). A slope of 3.41 was obtained from
the plot indicating significance (p<.05). The ED^g for
phenylephrine was 12 yg/kg. A slight increase in intra
ventricular pressure sometimes occurred with high doses
of phenylephrine.
Phentolamine decreased production 3.7 0.8 yl/min
(p<.05) (Table 3). This alpha adrenergic blocking agent
also significantly reduced the effect of 30 yg/kg phenyl
ephrine, i.e., from 10.0 1.6 yl/min to -0.8 0.7 yl/min


TABLE 3
EFFECTS OF AUTONOMIC BLOCKING AGENTS ON CSF PRODUCTION
Drug
Dose (mg/kg)
Control CSF Production
(pl/min) SE
Change in CSF Production'
(yl/min) SE (n)
Atropine
0.15
17.1 1.3
-2.6 0.5 (9) b
Phentolamine
2.0
15.4 1.0
-3.7 0.8 (6)b
Propranolol
2.0
16.9 1.8
-0.7 0.6 (4)C
aChange in CSF production calculated for
each experiment as the difference between
the control and the peak effect of the drug
bp < .05 with paired t-test
c
N.S. with paired t-test
u>


TABLE 4
PHENYLEPHRINE DOSE-RESPONSE: CHANGES IN CSF PRODUCTION
Dose (yg/kg) Control CSF Production Change in CSF Production3
(yl/min) SE (yl/min) SE (n)
3
16.2
+
1.0
+ 0.3
+
1.2
(5)
10
22.6
+
3.9
+ 2.6
+
0.6
(5)
30
20.0
+
2.8
+ 10.0
+
1.6
(7)
100
10.9
+
2.0
+ 10.6

1.5
(7)
30b
15.1

1.2
-0.8

0.7
(6) d
30C
15.8

1.6
+ 0.4

1.2
(5) d
aChange in CSF production calculated for each
experiment as the difference between the control
and the peak effect of the drug
bGiven within 120 min after 2.0 mg/kg phentolamine
c ,
Given within 120 min after 0.15 mg/kg atropine
p < .01 when compared with 30 yg/kg phenylephrine alone


Figure 4.
Phenylephrine dose-response for CSF production.
The ordinate is the mean increase in CSF pro
duction over the theoretical control value
SE plotted on a linear scale. The abcissa is
the dose of phenylephrine in yg/kg body weight
plotted on a logarithmic scale. ( )
represents phenylephrine alone. (O) is
30 yg/kg phenylephrine during 2.0 mg/kg phen-
tolamine. (*) is 30 yg/kg phenylephrine
during 0.15 mg/kg atropine.


40
->
3 10
Phenylephrine (^jg/kg)
x
30
100


Figure 5. 95% confidence interval on probit analysis of
the effect of phenylephrine on CSF production.
The ordinate is probits. The abcissa is the dose
of phenylephrine in yg/kg body weight plotted on
a logarithmic scale. The slope of the probit
regression line is 3.41 which is significant
(p<.05). The dose of phenylephrine producing an
EDj-q response is 12 yg/kg.


42


43
(p<.01) (Table 4, Figure 4). Atropine, the cholinergic
blocking agent, also effectively prevented the increase
in CSF production caused by phenylephrine (Table 4,
Figure 4). In the presence of atropine, 30 yg/kg
phenylephrine increased CSF formation only 0.4 1.2
yl/min which differed significantly (p<.01) from the
increase due to the alpha agonist alone. Propranolol
did not influence the effect of phenylephrine in the only
experiment in which it was tested.
Phenylephrine was also administered in an intravenous
infusion (approximately 30 yg/kg min) over nine minutes.
In the first three minutes, CSF production increased
14.1 4.0 yl/min, 24.1 4.5 yl/min in the next three
minutes, and 18.6 2.0 yl/min in the final three minutes
(Table 5). Administration of 50 yg/kg hemicholinium-3
to the animal three minutes prior to the phenylephrine
infusion significantly reduced the increase in production
in four of six cats. In these four animals, the increase
in production due to phenylephrine was limited to 2.2
1.0 yl/min (p<.05 compared with phenylephrine alone) in
the first period, 2.5 1.6 yl/min (p<.01) in the next
three minutes, and 0.8 1.8 yl/min (p<.01) in the final
three minutes (Table 5). When the two non-responsive
animals are included, the effect of hemicholinium
pretreatment appears to cause no significant change in
the effect of phenylephrine (Table 5). Hemicholinium
alone did not influence the rate of CSF production.


TABLE 5
THE EFFECT OF HEMICHOLINIUM-3 (HC-3) ON CSF PRODUCTION
DURING INTRAVENOUS INFUSION OF PHENYLEPHRINE (Phe)
3 minutes
6 minutes
9 minutes
Change in CSF Production3
(yl/min) SE during Phe*3
alone in four cats
+14.1 4.0
+24.1 4.5
+18.6 2.
Change in CSF Production
(yl/min) SE during Phe
after HC-3 pretreatmentc
in four cats
+2.2 1.0d
+2.5 1.6e
+0.8 1.
Change in CSF Production
(yl/min) SE during Phe
after HC-3 pretreatmentc
in six cats
+7.0 3.lf
+ 9.3 4.4f
+
CO
o
1+
aChange in CSF production calculated for each experiment as the difference
between the control and the peak effect of the drug
h
Phenylephrine infused intravenously for 9 minutes, approximately 30 yg/kg min
c .
Hemicholinium-3 (50 yg/kg) given 3 minutes prior to phenylephrine infusion
p < .05 when compared with phenylephrine infusion alone
0
p < .01 when compared with phenylephrine infusion alone
^N.S. when compared with phenylephrine infusion alone


45
Beta Adrenergic Agonists and Antagonist
The beta2 agonist, salbutamol, increased CSF
production about half as much as carbachol or phenylephrine
(Table 6, Figure 6). A dose-response effect was demon
strated by a probit analysis of the changes in CSF
formation (Figure 7). The regression line had a signif
icant slope of 0.96 (p<.05). The ED^q for salbutamol
was 1.5 yg/kg.
Beta blockade with propranolol did not change CSF
production (Table 3). Propranolol did appear to reduce
the increase observed with 3.0 yg/kg salbutamol, but the
difference was not significant (Table 6, Figure 6).
Atropine (four experiments) and phentolamine (one exper
iment) did not appear to alter the salbutamol response.
The beta-]_ agonist, ITP, did not significantly change the
production of CSF in either 100 or 300 yg/kg doses.
Theophylline
Theophylline significantly increased CSF formation
(Table 7). Doses of 10 and 20 mg/kg increased production
3.3 0.8 yl/min and 9.2 1.4 yl/min, respectively. The
difference (p<.01) in the increases with the two doses
suggest the effect was probably dose-dependent.
Carbonic Anhydrase Inhibition
The carbonic anhydrase inhibitor, methazolamide,
significantly reduced the rate of CSF formation (Table 8).
The maximal decrease was 11.2 1.4 yl/min; over several


TABLE 6
BETA ADRENERGIC AGONISTS DOSE-RESPONSE: CHANGES IN CSF PRODUCTION
Dose (yg/kg) Control CSF Production Change in CSF Production3
(yl/min) SE(yl/min) SE (n)
SALBUTAMOL
0.1
21.6
1.8
+ 0.5
+
0.5
(5)
1.0
22.0
2.0
+ 3.3
+
1.3
(4)
3.0
18.3
1.8
+ 3.8
+
1.6
(5)
10.0
17.4
2.3
+ 4.0

1.2
(7)
30.0
16.3
1.0
+ 5.7

1.4
(5)
3.0b
13.0
2.7
+ 0.7

0.2
(4>S
3.0C
10.1
2.6
+2.8

2.0
(4)S
ITPd
100
16.0
1.1
-0.8

1.0
(5)
300
19.5
3.4
+ 0.1

1.6
(4)
aChange in CSF production calculated for each exper
iment as the difference between the control and the
peak effect of the drug
Given within 120 min after 2.0 mg/kg propranolol
Q
Given within 120 min after 0.15 mg/kg atropine
l-isopropylamino-3(2 thiazoloxy)-2 propanol HC1
0
N.S. when compared with 3.0 yg/kg salbutamol alone


Figure 6. Salbutamol dose-response for CSF production. The
ordinate is the mean increase in CSF production
over the theoretical control value SE plotted
on a linear scale. The abcissa is the dose of
salbutamol in yg/kg body weight plotted on a
logarithmic scale. ( - ) represents
salbutamol alone. (O) is 3.0 yg/kg salbutamol
during 2.0 mg/kg propranolol. (a) is 3.0 yg/kg
salbutamol during 0.15 mg/kg atropine.


SalbutamoI ( jjg/ kg )
Increase in CSF Production (jjl/min)
i
N9 O N> ^ O)
' l 1 i
8 V
oo


Figure 7. 95% confidence interval on probit analysis of
the effect of salbutamol on CSF production.
The ordinate is probits. The abcissa is the
dose of salbutamol in yg/kg body weight plotted
on a logarithmic scale. The slope of the probit
regression line is 0.96 which is significant
(p<.05). The dose of salbutamol producing an
ED50 response is 1.5 yg/kg.


o
*0


TABLE 7
EFFECTS OF THEOPHYLLINE ON CSF PRODUCTION, BLOOD PRESSURE, AND HEART RATE
Dose
(mg/kg)
Control CSF
Production
(yl/min) SE
Change in CSF
Production3 (yl/
min) SE (n)
Change in Mean
Pressure (mm
SE (n)
Blood
Hg)
Change in Heart
Rate (beats/min)
SE (n)
10
15.8 2.1
+3.3 0.8b (5)
-37 6 (5)
+ 25 5
(5)
20
16.9 1.4
+9.2 1.4b'c (4)
-54 7 (4)
+ 35 3
(4)
Change in CSF production
calculated for
each
experiment as the difference between the control
and the peak effect of the drug
<
.01 with paired t-test
.01 when compared with 10 mg/kg theophylline


TABLE 8
EFFECTS OF CARBONIC ANHYDRASE INHIBITION ALONE AND WITH
AUTONOMIC BLOCKING AGENTS ON CSF PRODUCTION
Treatment
Control CSF Production
(yl/min) SE
Maximum Change in
CSF Production3
(yl/min) SE (n)
Mean Change in
CSF Production*3
(yl/min) SE (n)
Q
Methazolamide
19.0
1.. 7
-11.2 1.4 (6)
-9.4 1.2 (6)
Methazolamide +
Phentolamine1^
19.1
2.4
-13.6 1.8 (5)f
-12.5 1.6 (5)f
Methazolamide +
Atropinee
16.0
2.2
-10.6 0.9 (5)f
-8.8 1.0 (5)f
Calculated for each experiment as the difference
between the control and the peak effect of the drug
Calculated for each experiment as the difference
between the control and the effect of the drug over
two or more periods
Q
30 mg/kg methazolamide given over 30 minutes
ci
2.0 mg/kg phentolamine
0
0.15 mg/kg atropine
^N.S. when compared with methazolamide alone


53
collection periods the mean drop in production was
9.4 1.2 yl/min. The CSF formation rate during metha-
zolamide treatment was approximately 7 to 9 yl/min. Since
atropine and phentolamine successfully decreased fluid
formation, these drugs were tested in combination with
methazolamide for possible additive effects. From Table 8
it is evident that the autonomic blocking agents caused no
further significant decrease in production. It was also
noted in several experiments that carbachol and phenyl
ephrine partially override the carbonic anhydrase inhi
bition .
Changes in Blood Pressure and Heart Rate
during Ventriculocisternal Perfusion Studies
Cervical Sympathetic Stimulation
Bilateral preganglionic cervical sympathetic
stimulation (10 volts, 10 shocks/sec, each shock lasting
0.5 msec) produced an increase in mean arterial blood
pressure of 42 27 mm Hg. Heart rate did not change
significantly.
Cholinergic Agonist and Antagonist
Doses of carbachol ranging from 0.03 to 3.0 yg/kg
were administered. From Table 9 and Figure 8 it is clear
that the drug had a dose-response effect in decreasing
blood pressure (p<.01 by probit analysis). The heart rate
also decreased beginning at the 3 yg/kg dose (Table 9).
Atropine caused an initial drop of 43 5 mm Hg in mean


TABLE 9
CARBACHOL DOSE-RESPONSE: CHANGES IN BLOOD PRESSURE AND HEART RATE
Dose (yg/kg)
Change in Mean Blood Pressure Change in Heart Rate
(mm Hg) SE (n) (beats/min) SE (n)
0.
,03
-28

4
(7)
-1

1
(7)
0.
,10
-38

6
(6)
0

2
(6)
0.
. 30
-57

5
(6)
3

2
(6)
3.
, 0
-81

10
(6)
-63

8
(5)
1.
,oa
-6

3
(4)
-1

3
(4)
3.
,0a
-9

6
(2) C
0

0
(2)
30.
.0a
+ 12

13
(5)
2

2
(5)
3.
. ob
-50

6
(4) d
-38

15
(4
Given within 120 min after 0.15 mg/kg atropine
d10 verapamil in CSF perfusate
Q
p < .01 when compared with 3.0 yg/kg alone
dN.S. when compared with 3.0 yg/kg carbachol alone


Figure 8. Carbachol dose-response for blood pressure. The
ordinate is the change in mean blood pressure
(mm Hg) SE plotted on a linear scale. The
abcissa is the dose of carbachol in yg/kg body
weight plotted on a logarithmic scale. Carbachol
alone is represented by (# ) .
Carbachol during 0.15 mg/kg atropine is represented
by (O : O) .


0 03 0 10 0 30
Carbachol Cpg/kg)
Change in
i
S
T
Mean Blood Pressure (mm/Hg)
i i i *1
S S S
> T I
95


57
arterial blood pressure and a decrease in heart rate of
17 3 beats/min (Table 10). Since both of these
parameters had returned to pre-drug levels within 30
minutes after atropine administration, these actions
were thought to be unrelated to the anti-cholinergic
action of the drug. The atropine successfully blocked the
fall in blood pressure seen with both 1 and 3 yg/kg
carbachol and the decrease in heart rate seen with 3 yg/kg
carbachol (Table 9). After atropine, 30 yg/kg carbachol
produced a slight, but insignificant, increase in blood
pressure, possibly through nicotinic stimulation of
sympathetic ganglia. No change in heart rate was observed
with this dose of carbachol during atropine treatment.
The cardiovascular effects of 3 yg/kg were not affected
by the presence of verapamil in the CSF perfusate (Table 9).
Alpha Adrenergic Agonist and Antagonist
Phenylephrine, given in doses between 3 and 100 yg/kg
body weight, increased blood pressure to a peak of 133 11
mm Hg (Table 11 and Figure 9). Probit analysis yielded a
statistically significant slope (p<.01) and an ED5q of 17
yg/kg. Although the lower doses of phenylephrine (3 and
10 yg/kg) caused a significant decrease in heart rate,
the larger amounts of drug caused no significant change
(Table 11). Phentolamine caused a decrease in mean blood
pressure of 40 7 mm Hg and an increase in heart rate
of 24 5 beats/min (Table 10). Both blood pressure and
heart rate returned to control values within 30 minutes.


TABLE 10
EFFECTS OF AUTONOMIC BLOCKING AGENTS ON BLOOD PRESSURE AND HEART RATE
Drug
Dose (mg/kg)
Change in Mean
(mm Hg)
Blood Pressure3
SE (n)
Change in Heart Rate3
(beats/min) SE (n)
Atropine
0.15
-43 5
(17)
-17
3 (16)
Phentolamine
2.0
-40 7
(ID
+ 24
5 (11)
Propranolol
2.0
-15 2
(4)
-41
11 (4)
^Measured


TABLE 11
PHENYLEPHRINE DOSE-RESPONSE: CHANGES IN BLOOD PRESSURE AND HEART RATE
Dose (yg/kg) Change in Mean Blood Pressure Change in Heart Rate
(mm Hg)
+
SE
(n)
(beats/min)
+
SE (n)
3
+26
+
4
(9)
-9
+
4
(8)
10
+ 47
+
6
(14)
-11
+
3
(12)
30
+ 8 5
+
9
(15)
+ 2
+
5
(13)
100
+ 133

11
(14)
-17
12
(13)
30a
+ 21
+
4
(6)c
-6
+
4
(6) d
30b
+ 73
+
5
(7)d
-13
+
4
<7)d
aGiven within 120 min after 2.0 mg/kg phentolamine
Given within 120 min after 0.15 mg/kg atropine
c
p < .05 when compared with 30 yg/kg phenylephrine alone
cl
N.S. when compared with 30 yg/kg phenylephrine alone


Figure 9. Phenylephrine dose-response for blood pressure.
The ordinate is the change in mean blood
pressure (mm Hg) SE plotted on a linear
scale. The abcissa is the dose of phenylephrine
in yg/kg body weight plotted on a logarithmic
scale. Phenylephrine alone is represented by
( ) ( O) is 30 yg/kg phenylephrine
during 2.0 mg/kg phentolamine. ( a. ) is 30 yg/kg
phenylephrine during 0.15 mg/kg atropine.


140
120
100
80
60
40
20
3 10 30 100
Phenylephrine Cpg/kg)
O


62
This blocking agent significantly reduced the pressor
action of 30 yg/kg phenylephrine (Table 11). In the
presence of atropine the phenylephrine-induced changes
in blood pressure and heart rate were not altered (Table
11). Phenylephrine administered in an infusion (30 yg/kg
min) over nine minutes increased mean arterial pressure
by about 66 mm Hg (Table 12). Heart rate decreased
significantly only in the first three minute period.
Pretreatment with hemicholinium-3 did not significantly
affect the increase in blood pressure or the change in
heart rate due to phenylephrine (Table 12).
Beta Adrenergic Agonists and Antagonist
Salbutamol was given in doses between 0.1 and
30.0 yg/kg body weight. The blood pressure decreased
significantly in response to all doses tested; however,
the maximum fall occurred after 1.0 yg/kg (Table 13 and
Figure 10). A dose-response increase in heart rate
occurred as determined by probit analysis. Propranolol
dropped the mean arterial blood pressure slightly and
significantly decreased the heart rate immediately after
administration (Table 10). Unlike the other autonomic
blocking agents, these effects of propranolol persisted
for two or more hours. Propranolol successfully blocked
the cardiovascular responses to 3 yg/kg salbutamol
(Table 13). Atropine did not significantly alter the
changes in mean blood pressure and heart rate caused by


TABLE 12
THE EFFECT OF HEMICHOLINIUM-3 (HC-3) ON BLOOD PRESSURE AND HEART RATE
DURING INTRAVENOUS INFUSION OF PHENYLEPHRINE (Phe)
3
Phea
minutes
HC-3 + Pheb
6
Phe
minutes
HC-3 + Phe
9 minutes
Phe HC-3 + Phe
Change in Mean
+ 69 3
+63 10C
+ 70 6
+69 9C
+ 60 7
+63 11
Blood Pressure (mm Hg)
SE (n)
(4)
(6)
(4)
(6)
(4)
(6)
Change in Heart
-32 13
-30 15C
+ 6 23
-16 19C
-10 19
-7 20C
Rate (beats/min)
SE (n)
(4)
(6)
(4)
(6)
(4)
(6)
aPhenylephrine infused intravenously for 9 minutes,
approximately 30 yg/kg min
b
Hemicholinium-3 (50 yg/kg) given 3 minutes prior to
phenylephrine infusion
c
N.S. when compared with phenylephrine infusion alone


TABLE 13
BETA ADRENERGIC AGONISTS DOSE-RESPONSE: CHANGES IN BLOOD PRESSURE AND HEART RATE
Dose (yg/kg) Change in Mean Blood Pressure Change in Heart Rate
(mm Hg) SE (n) (beats/min) SE (n)
SALBUTAMOL
0.1
-27

5
(6)
+ 3
+
3
(6)
1.0
-40

8
(9)
+ 17
+
4
(8)
3.0
-43

7
(9)
+ 33

5
(9)
10.0
-45

5
(9)
+ 39

6
(9)
30.0
-45

5
(7)
+ 58

8
(7)
3.0a
-10

2
<4)d
+ 1

1
(4)d
3.0b
-19

13
(4)e
+ 18

5
(4)e
ITPC
100
-11
+
6
(5)
+ 21

9
(5)
300
-17
+
5
(4)
+ 20
11
(4)
aGiven within 120 min after 2.0 mg/kg propranolol
Given within 120 min after 0.15 mg/kg atropine
c
l-isopropylammo-3 (2 thiazoloxy)-2 propanol HC1
p < .05 when compared with 3.0 yg/kg salbutamol alone
0
N.S. when compared with salbutamol alone
Oh


Figure 10. Salbutamol dose-response for blood pressure.
The ordinate is the change in mean blood
pressure (mm Hg) SE plotted on a linear
scale. The abcissa is the dose of salbutamol
in yg/kg body weight plotted on. a logarithmic
scale. Salbutamol alone is represented by
( #) (O) is 3.0 yg/kg salbutamol
during 2.0 mg/kg propranolol. (a) is 3.0
yg/kg salbutamol during 0.15 mg/kg atropine.


Salbutamol Cpg/kg)
Change in Mean Blood Pressure Cmm Hg)
99


67
salbutamol. The betag agonist, ITP, increased heart
rate at 100 and 300 yg/kg doses (Table 13), A small
decrease in mean arterial blood pressure was also
observed.
Non-autonomic Drugs
Theophylline given intravenously significantly
decreased mean arterial blood pressure and increased the
heart rate (Table 7). In both cases the effects appeared
to be dose-related. The dose of 10 mg/kg decreased blood
pressure 37 6 mm Hg and increased heart rate 25 5
beats/min, whereas 20 mg/kg decreased blood pressure
54 7 mm Hg and increased heart rate 35 3 beats/min.
Verapamil in the perfusate did not influence peripheral
blood pressure or heart rate. Methazolamide also had
no significant peripheral cardiovascular effects.
Effect of Autonomic Agonists on Blood Flow
One group of three cats served as an internal
control; i.e., each animal received two control injections
of microspheres sequentially. In these animals, no
changes in blood flow were detected in any of the four
tissues (Table 14). Other groups of cats received
30 yg/kg phenylephrine, 3.0 yg/kg carbachol, or 3.0 yg/kg
salbutamol as the experimental treatment during the second
injection of microspheres. The control blood flows
(ml/gm min) for these animals were 2.26 .28 for the


TABLE 14
EFFECT OF PHENYLEPHRINE, CARBACHOL, AND SALBUTAMOL ON BLOOD FLOW TO
THE CHOROID PLEXI, BRAIN, SKELETAL MUSCLE, AND KIDNEY
Treatment
Choroid Plexi
Change
Control After
Flowa Treatment
Brain
Skeletal
Muscle
Kidney
Control
Flow
Change
After
Treatment
Control
Flow
Change
After
Treatment
Control
Flow
Change
After
Treatment
Control
X
1.94
-.06
. 42
+ .02
. 028
-.001
1.63
-.14
SE
. 46
.18
.07
. 06
.006
.004
. 37
.19
Carbachol
X
2.33
+ .81
.37
-.06
.041
+ .018
1.94
-.58
3 ug/kg
SE
. 79
.93
.02
.04
.012
.011
.23
. 11
Phenylephrine
X
2.51
+ .77
.63
-.09
.070
+ .013
1.24
-.58
30 yg/kg
SE
.70
. 86
.09
.16
. 025
.019
. 43
.36
Salbutamol
X
2.16
+ .35
.40
+ .01
.049
+ .024
1.74
-.45
3 yg/kg
SE
.33
.50
.09
.05
.011
.028
.49
.26
Mean Control
X
2.26
. 46
.048
1.64
Flow
SE
.28
. 04
. 008
.19
Blood flow expressed as ml/gm min wt tissue weight


69
choroid plexus, 0.46 .04 for the brain, .048 .008 for
the muscle, and 1.64 .19 for the kidney. None of the
drugs caused a significant change in blood flow to any of
the tissues examined (Table 14) .


DISCUSSION
Modification of CSF Production by Autonomic Influences
The data show significant increases in CSF production
as a result of stimulation of the cholinergic system by
pharmacological agents that influence the autonomic nervous
system. The cholinergic agonist, carbachol, caused a dose-
dependent increase in the rate of CSF formation up to
10.6 2.1 yl/min that was blocked by atropine. Further
evidence for cholinergic control of secretion is found in
the phenylephrine data. This alpha adrenergic agonist
also increased CSF formation by 10.6 1.5 yl/min; this
increase was blocked by both phentolamine and atropine.
These data suggested that the alpha adrenergic agonist
probably was stimulating a cholinergic pathway to release
acetylcholine to increase CSF production. Since hemi-
cholinium blocks the uptake of choline for the synthesis
of acetylcholine (Gardiner, 1957), hemicholinium was given
prior to an infusion of phenylephrine. The infusion of
phenylephrine without hemicholinium increased the
production rate to more than twice that following a
single injection of the drug. However, when the animal
was pretreated with the hemicholinium, the infusion of
phenylephrine was not able to increase fluid production
70


71
in four of six animals. These experiments confirmed that
the increase in CSF formation caused by phenylephrine
depends on the synthesis and release of acetylcholine.
This relationship is depicted in Figure 11 which shows,
alpha adrenergic receptors that may or may not be supplied
by a neuron. The failure of hemicholinium in the other
two animals might be explained by larger stores of acetyl
choline or particularly high levels of choline. An
inadequate concentration of hemicholinium is probably not
a cause for this lack of effect.
A marked similarity exists among several secretory
systems with respect to cholinergic function and parasym
pathetic innervation. Cholinergic stimulation of the
salivary glands causes a profuse secretion (Koelle, 1970).
Parasympathetic innervation for these glands originates
from the inferior and superior salivary nuclei of the
brainstem (Mitchell, 1953). Secretion by the pancreas
and by the stomach can also be enhanced by cholinergic
agonists (Koelle, 1970). Both of these tissues derive
their parasympathetic innervation from the dorsal vagal
nucleus which is located immediately adjacent to the
salivary nuclei in the brainstem (Mitchell, 1953). The
production of aqueous humor by the ciliary body is also
stimulated by cholinergic agents (Macri, 1971). The
source of the cholinergic innervation in this tissue is
not defined. Increase in aqueous humor formation is


Figure 11. Autonomic pathways to the choroid plexus I.
The effector site has a cholinergic receptor.
(++) indicates the relative effectiveness
of the activated receptor to increase CSF
production. The cholinergic nerve supplying
the choroid plexus, in turn, can be stimulated
by alpha adrenergic agonists. The location of
the alpha adrenergic receptor remains undefined.


EFFECTOR SITE
+ +
CHOLINERGIC
RECEPTORS
ALPHA
ADRENERGIC
RECEPTOR


74
apparently mediated by a preganglionic cholinergic
stimulation of an adrenergic effector (Maori, 1971).
The origin of the cholinergic nerve supply to the
choroid plexus is unknown. Early anatomical descriptions
of nerves in the choroid plexus suggested that the inner
vation might originate in the dorsal vagal nucleus of the
brainstem (Benedikt, 1874; Stohr, 1922; Clark, 1932).
Although there are no data confirming that these are
cholinergic nerves, they arise in the same area responsible
for parasympathetic-stimulated pancreatic and gastric
secretion. This area is immediately adjacent to the
superior and inferior salivary nuclei (Mitchell, 1953).
In spite of the proximity of origin to other secretory
nuclei, one major observation thus far marks the cholin-
ergic-stimulated production of CSF as unique among cholin
ergic secretory systems: the activation of cholinergic
pathways by alpha adrenergic agonists.
As in most secretory systems, for the choroid plexus
it is unknown whether the cholinergic effector site is
vascular or epithelial. Cerebral blood vessels receive
some cholinergic innervation from the facial, glosso
pharyngeal, and vagal cranial nerves (Mitchell, 1953)
and from pathways arising within the brain (Edvinsson,
1975). Innervation from within the central nervous
system has also been shown for the blood vessels and the
epithelium of the choroid plexus (Clark, 1928 and 1932).


75
The role of the sympathetic nervous system in the
production of CSF is complex. The action of the alpha
adrenergic agonist, phenylephrine, in stimulating fluid
formation through a cholinergic pathway (Figure 11) has
already been discussed. The beta2 agonist, salbutamol,
also caused a dose-dependent increase in CSF formation;
however, the maximum effect was an increment of 5.7 1.4
yl/min which is approximately half of the cholinergic
response. The action of salbutamol appeared to result from
a direct stimulation of beta receptors at the effector
site (Figure 12) since neither atropine nor phentolamine
influenced its effect, whereas the general beta blocking
agent, propranolol, did reduce the salbutamol effect.
Beta-j_ receptors are apparently absent in the choroid plexus,
for ITP did not influence production at all.
Since norepinephrine is present in the choroid plexus
and disappears after cervical sympathectomy (Edvinsson
et al., 1972b; Edvinsson et al., 1974), alpha adrenergic
agonists may act at the choroid plexus to influence CSF
production directly. There are two observations that
suggest alpha adrenergic agonists may decrease CSF
production. In this study, bilateral cervical sympathetic
stimulation reduced the rate of CSF formation 3.0 0.6
yl/min. In addition, when norepinephrine is in the
perfusate during ventriculocisternal perfusion, CSF
production is decreased approximately 24% (Vates et al.,


Figure 12. Autonomic pathways to the choroid plexus II.
In addition to the cholinergic pathway, the
effector site also has a beta2 adrenergic
receptor. (+) indicates that activation of
these receptors also increases CSF production,
but to a lesser extent than cholinergic
stimulation.


77
EFFECTOR SITE
+ +
4-
CHOLINERGIC
RECEPTOR
BETA2
ADRENERGIC
RECEPTOR
ALPHA
ADRENERGIC
RECEPTOR


78
1964) A proposed alpha adrenergic pathway, completing
the autonomic innervation of the choroid plexus, is
depicted in Figure 13. However, any direct decrease an
alpha adrenergic agonist might cause at the choroid plexus
would probably be masked by the simultaneous stimulation
of production by the cholinergic pathway as described
above. Since atropine itself affects the rate of CSF
formation, no attempt was made to block the cholinergic
receptors and then study the alpha adrenergic system.
Although the sympathetic nervous system has a
greater effect on fluid production in other secretory
systems than it has on CSF formation, the actions are
variable depending on the tissue. Alpha adrenergic
agonists increase aqueous humor production by the ciliary
body through an increase in arterial blood pressure (Maori
et al., 1974). Beta2 agonists, on the other hand,
decrease aqueous humor formation (Langham, 1976). In the
salivary gland, both alpha and beta adrenergic agonists
increase fluid production (Emmelin, 1967). However, the
two systems influence secretion in different ways:
alpha adrenergic stimulation causes a secretion that is
watery and contains high potassium, whereas beta stimu
lation causes secretion that is viscous and contains
digestive enzymes. In the pancreas both norepinephrine
and epinephrine decrease secretion; specific beta agonists
such as isoproterenol have not been tested (Thomas, 1967).


Figure 13. Autonomic pathways to the choroid plexus III.
An alpha adrenergic receptor may also be at
the effector site in addition to the
cholinergic receptor and beta2 adrenergic
receptor. (-) indicates that this pathway
decreases the production of CSF.


80
EFFECTOR SITE
+ +
CHOLINERGIC
RECEPTOR
V
beta2
ADRENERGIC
RECEPTOR
ALPHA
ADRENERGIC
RECEPTOR


81
These secretory tissues derive their alpha adrenergic
innervation through the ganglia of the sympathetic chain
as does the choroid plexus.
The relatively minor physiological role of the
autonomic nervous system in the normal production of CSF
is best demonstrated by observing the effects of the
autonomic blocking agents. Atropine, the cholinergic
blocking agent, decreased the rate of CSF formation
2.6 0.5 yl/min indicating the cholinergic system may
continually stimulate fluid secretion but only to the
extent of about 15 to 20% of the fluid produced. Phentol-
amine also reduced CSF formation. This decrease may be
occurring through alpha adrenergic blockade of the
cholinergic pathway since the change in CSF production
is approximately the same as that caused by atropine;
however, this is not confirmed. The beta adrenergic
system may have little direct function in the normal
secretory processes of the choroid plexus since propranolol
did not influence CSF production. Nevertheless, it is
possible that in unanesthetized animals the autonomic
nervous system may exert a greater influence on normal
fluid production than is observed here or may cause
temporary fluctuations in the rate of fluid formation.


82
Mechanism of Changes in CSF Production
by Autonomic Influences
In two previous investigations, epinephrine and
pilocarpine were shown to cause an increase in the flow of
CSF from the cisterna magna followed shortly by a reduction
in CSF outflow (Becht and Gunnar, 1921; Bhattacharya and
Feldberg, 1958). These authors suggested that epinephrine
and pilocarpine increased cerebral blood flow and the
resulting increase in brain volume forced subarachnoid
CSF out through the cisternal cannula without affecting
true CSF production. Increased volumes were also observed
in response to the autonomic agonists used in the study
presented in this dissertation. The increased rate of
outflow during drug treatment averaged approximately
35 yl/min over control for a three minute collection
period. If pressure on the subarachnoid fluid was a
partial cause of this phenomenon, the additional fluid
collected would have come first from the cisterna magna
and basal cisterns which contained the dye marker at the
same dilution as the control perfusate. Hence, any error
in the calculation of production changes as a result of
this would cause an underestimate of the dilution of the
inflowing perfusate and an underestimate of the actual
increase in fluid production. By measuring only volumes,
the early investigators could not reliably show whether
the agents actually increased fluid formation. The


83
increases in CSF production calculated from dye dilution
in this study indicate that autonomic agents do alter
fluid formation.
The increase in CSF production caused by cholinergic
and adrenergic agonists can be ascribed to two possible
mechanisms of action, depending on the effector site.
Either a change in the hemodynamic relationships or a
direct action on the secretory cells might result in
alterations in CSF formation by these drugs. An increase
in the supply of blood to the brain or choroid plexus
would provide more fluid to produce an ultrafiltrate of
plasma in the CSF. Such a transfer of plasma water would
increase CSF volume.
Peripheral blood pressure changes do not correlate
well with CSF production changes. Carbachol and salbutamol
decrease blood pressure; this could decrease the supply of
blood to the secretory tissue. Phenylephrine, on the
other hand, increases peripheral blood pressure which
might serve to increase blood flow to the brain and
choroid plexus and thus to increase the rate of CSF.
formation. However, this alpha adrenergic agonist also
increases blood pressure during treatment with atropine
and hemicholinium which block the increased CSF production
by phenylephrine.
Since blood pressure changes were measured periph
erally, choroid plexus and cerebral blood flow data were
considered necessary to determine possible vascular actions


84
of these agents at the site of CSF formation. The control
blood flow to the choroid plexus measured by microspheres
was 2.26 0.28 ml/gm min. This flow rate is comparable
to 3.01 ml/gm min observed by Aim and Bill (1973) for the
choroid plexus using the same technique. It is also
similar to the estimates of choroid plexus blood flow by
Welch (1963) and Pollay et al. (1972). The flow to the
choroid plexus represents one of the greatest amounts of
blood per tissue weight delivered to any tissue. Carbachol
and phenylephrine increased mean plexus blood flow 0.81
and 0.77 ml/gm min, respectively. Salbutamol increased
blood flow to the choroid plexus 0.35 ml/gm min. The
effects of these drugs on blood flow are in about the
same proportions as their effects on CSF production.
However, none of these changes in choroid plexus blood
flow differ statistically from control flows because of
the wide variation in the changes. These data, along with
the data of Aim and Bill (1973) showing no effect on
plexus blood flow by sympathetic stimulation, seem to
indicate that the autonomic nervous system does not
significantly alter the blood flow to the choroid plexus.
Cerebral blood flow in the control experiments was
0.46 .04 ml/gm min. This value is approximately the
same as the flows measured for the dog (Marcus et al.,
1976) and sheep (Hales, 1973). There was no alteration
in brain blood flow after carbachol, phenylephrine, or


85
salbutamol. When Salanga and Waltz (1973) stimulated the
facial nerve, vasodilation occurred only if the nerve was
cut where it left the brainstem. This suggests that
sensory nerve fibers detecting cerebral vasodilation might
pass back into the central nervous system through the
facial nerve causing a reflex vasoconstriction. If such a
pathway does exist, it would also explain the ineffec
tiveness of salbutamol and carbachol to increase cerebral
blood flow. The inability of phenylephrine to alter
cerebral blood flow is probably due to its low efficacy on
alpha adrenergic receptors in cerebral blood vessels
(Duckies and Bevan, 1976). Blood pressure and blood flow
data seem to indicate that the autonomic treatments
performed in this study produce minimal effects on the
production of CSF by way of changes in blood flow. Also,
it is clear that any increase in the rate of outflow seen
during drug treatment was not mediated by a generalized
increase in blood flow to the brain. An increase in
choroid plexus blood flow could have forced additional
perfusate from the ventricles.
Verapamil and theophylline were used to investigate
two possible ways in which autonomic agents could influence
CSF production by direct action on secretory cells. The
presence of calcium is a requirement for cholinergic
stimulation of salivary secretion (Douglas and Poisner,
1963). If calcium influx has a role in increasing CSF


86
production stimulated by carba.ch.ol, then verapamil, which
blocks calcium channels (Kohlhardt et al., 1972), should
prevent the increase in fluid formation caused by the
cholinergic agonist. Verapamil has been shown to be
effective when administered on either side of the epithe
lium and was given intraventricularly (Bentley, 1974).
Verapamil did not affect either normal CSF formation or
fluid production stimulated by carbachol; therefore,
fluid secretion probably occurs through a mechanism other
than a calcium-induced activation.
Since the effects of both cholinergic and adrenergic
neurotransmitters may be mediated by changes in cyclic
nucleotide levels (Greengard, 1976), an increase in
guanosine 3',5' cyclic monophosphate (cGMP) or adenosine
3',5' cyclic monophosphate (cAMP) might be the means by
which carbachol, phenylephrine, and salbutamol increase
the production of CSF. Theophylline, which inhibits the
destruction of the nucleotides by phosphodiesterase among
other actions, was used to enhance the levels of the
cyclic nucleotides. These experiments showed that
theophylline caused a significant dose-dependent increase
in CSF formation. The higher dose increased fluid
production 9.2 1.4 yl/min which is approximately equal
to the maximum effect of the autonomic agonists. Unfor
tunately, theophylline is not totally specific for either
cGMP or cAMP phosphodiesterases (Amer and Kreighbaum,


87
1975), so it is not possible to show whether the effects
mediated by cyclic nucleotide action are cholinergic,
beta2 adrenergic, or both. Whereas the theophylline
experiments suggest that increases in cyclic nucleotide
levels are responsible for the increases in fluid
production elicited by carbachol, phenylephrine, and
salbuatmol administration, they do not provide conclusive
evidence that enhancement of the cyclic nucleotide
concentrations takes place in choroid plexus epithelia.
However, the relatively large blood flow to the choroid
plexus does not significantly change during treatment
with any of the autonomic agents that increase CSF
production. Apparently, the blood flow only provides
fluid and electrolyte substrate for the secretory
processes of the choroid plexus epithelium. The data
strongly imply that autonomic agents influence CSF
production by causing changes primarily at the choroid
plexus epithelium.
The changes in CSF production mediated by the
epithelial cells are probably a result of alterations in
the transport of electrolytes. Three pathways have been
demonstrated for the secretory transfer of electrolytes
in the CSF: the catalysis of bicarbonate formation by
carbonic anhydrase, the transport of cations by the
ouabain-sensitive sodium-potassium ATPase, and the
probable transport of chloride by an ethacrynic acid-
sensitive pump. Inhibition of any of the three mechanisms


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FILES


AUTONOMIC INFLUENCES ON THE PRODUCTION
OF CEREBROSPINAL FLUID
By
JOSEPH ROSCOE HAYWOOD
A DISSERTATION PRESENTED TO THE
GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1976

To Carol
for her understanding, patience, and love
To Becca and Alyson
for giving me so much happiness

ACKNOWLEDGMENTS
I wish to express my sincere gratitude to Dr.
Betty P. Vogh for her guidance throughout this course of
study. She has been most helpful as a teacher, as a
researcher, and as an advisor, but especially as a friend
whose interest in her students extends beyond science.
I am also grateful to the other members of my
supervisory committee, Dr. C.Y. Chiou, Dr. Carl Feldherr,
Dr. David Silverman, and Dr. David Travis for their
contributions. The advice of Dr. Thomas Maren, Dr. Peter
Lalley, Dr. Sidney Cassin, and the faculty of the
Department of Pharmacology and Therapeutics is also
greatly appreciated.
I also thank Mr. Mark Loveland for his friendship,
sense of humor, and technical assistance and Ms. Joyce
Simon for her support and contribution in the preparation
of this manuscript.
My sincere thanks are also extended to my fellow
graduate students for their encouragement and friendship
during these years.
This research was supported by a traineeship from
the National Institutes of Health (GM-00760) and a research
grant from the National Institute of General Medical
Sciences (GM-16934).

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
LIST OF TABLES vi
LIST OF FIGURES viii
KEY TO ABBREVIATIONS ix
ABSTRACT x
INTRODUCTION * 1
BACKGROUND 3
Autonomic Innervation and Function in
Cerebral Blood Vessels 3
Innervation of the Choroid Plexus 5
Blood Flow to the Choroid Plexus 8
Autonomic Nervous System Control of
Fluid Secretions 8
Rationale for Study 12
METHODS 13
Tests of Autonomic Nervous System Influence
on CSF Production 15
Tests of Autonomic Nervous System Influence
on Blood Flow 21
Evaluation of CSF Production Data 24
Statistical Analysis . . . 25
RESULTS 2 6
Ventriculocisternal Perfusion Studies
for Estimation of CSF Production Rate .... 26
IV

Changes in Blood Pressure and Heart Rate
during Ventriculocisternal Perfusion Studies . 53
Effect of Autonomic Agonists on Blood Flow . . 67
DISCUSSION 70
Modification of CSF Production by
Autonomic Influences 70
Mechanism of Changes in CSF Production
by Autonomic Influences 82
Practical Significance 91
APPENDICES
I Autonomic Innervation and Function of
Cerebral Blood Vessels 93
II Composition of Artificial Cerebrospinal
Fluid 98
REFERENCES 9 9
BIOGRAPHICAL SKETCH 108
v

2
3
4
5
6
7
8
9
10
11
12
13
LIST OF TABLES
Page
CONTROL PRODUCTION OF CEREBROSPINAL FLUID . . 27
CARBACHOL DOSE-RESPONSE: CHANGES IN CSF
PRODUCTION 31
EFFECTS OF AUTONOMIC BLOCKING AGENTS ON CSF
PRODUCTION 37
PHENYLEPHRINE DOSE-RESPONSE: CHANGES IN
CSF PRODUCTION 38
THE EFFECT OF HEMICHOLINIUM-3 (HC-3) ON CSF
PRODUCTION DURING INTRAVENOUS INFUSION OF
PHENYLEPHRINE (Phe) 44
BETA ADRENERGIC AGONISTS DOSE-RESPONSE:
CHANGES IN CSF PRODUCTION 4 6
EFFECTS OF THEOPHYLLINE ON CSF PRODUCTION,
BLOOD PRESSURE, AND HEART RATE 51
EFFECTS OF CARBONIC ANHYDRASE INHIBITION
ALONE AND WITH AUTONOMIC BLOCKING AGENTS
ON CSF PRODUCTION 52
CARBACHOL DOSE-RESPONSE: CHANGES IN
BLOOD PRESSURE AND HEART RATE 54
EFFECTS OF AUTONOMIC BLOCKING AGENTS ON
BLOOD PRESSURE AND HEART RATE 58
PHENYLEPHRINE DOSE-RESPONSE: CHANGES IN
BLOOD PRESSURE AND HEART RATE 59
THE EFFECT OF HEMICHOLINIUM-3 (HC-3) ON BLOOD
PRESSURE AND HEART RATE DURING INTRAVENOUS
INFUSION OF PHENYLEPHRINE (Phe) 63
BETA ADRENERGIC AGONISTS DOSE-RESPONSE:
CHANGES IN BLOOD PRESSURE AND HEART RATE . . 64
Vl

14 EFFECTS OF PHENYLEPHRINE, CARBACHOL, AND
SALBUTAMOL ON BLOOD FLOW TO THE CHOROID
PLEXI, BRAIN, SKELETAL MUSCLE, AND KIDNEY . . 68
Vll

LIST OF FIGURES
FIGURE Page
1 Changes in the production of CSF with
time in control experiments 29
2 Carbachol dose-response for CSF production . . 33
3 95% confidence interval on probit analysis
of the effect of carbachol on CSF
production 35
4 Phenylephrine dose-response for CSF
production 40
5 95% confidence interval on probit analysis
of the effect of phenylephrine on CSF
production 4 2
6 Salbutamol dose-response for CSF production. . 48
7 95% confidence interval on probit analysis
of the effect of salbutamol on CSF
production 50
8 Carbachol dose-response for blood pressure . . 56
9 Phenylephrine dose-response for blood
pressure 61
10 Salbutamol dose-response for blood pressure . 66
11 Autonomic pathways to the choroid plexus I . .73
12 Autonomic pathways to the choroid plexus II . 77
13 Autonomic pathways to the choroid plexus III . 80
viii

KEY TO ABBREVIATIONS
CBF
CSF
cpm
CAMP
cGMP
ED50
gm
HC-3
ITP
kg
yg
yi
mg
ml
mm
mM
msec
min
M
M.W.
sec
Phe
Cerebral Blood Flow
Cerebrospinal Fluid
Counts per minute
Adenosine 3',5' cyclic monophosphate
Guanosine 3', 5' cyclic monophosphate
Median effective dose (dose to
produce 50% effect) ‘
Gram
Hemicholinium-3
l-isopropylamino-3(2 thiazoloxy)-
2 propanol HC1
Kilogram
Microgram
Microliter
Milligram
Milliliter
Millimeter
Millimolar
Millisecond
Minute
Molar
Molecular weight
Second
Phenylephrine
xx

Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fullment of the
Requirements for the Degree of Doctor of Philosophy
AUTONOMIC INFLUENCES ON THE PRODUCTION
OF CEREBROSPINAL FLUID
By
Joseph Roscoe Haywood
December, 1976
Chairperson: Betty P. Vogh
Major Department: Pharmacology and Therapeutics
For the first time, treatments which stimulate or
inhibit autonomic nervous system receptors were tested for
their effects on cerebrospinal fluid (CSF) production
using the dilution of blue dextran during ventriculo-
cisternal perfusion in cats. Initial control CSF formation
ranged from 15 to 25 yl/min and gradually decreased at the
rate of 0.015 yl/min each minute between 60 and 300 minutes.
The cholinergic agonist, carbachol, in single
intravenous injections caused a dose-dependent increase
in the production of new fluid. The maximum increase was
10.6 ± 2.1 yl/min which was blocked by atropine.
Single intravenous injections of the alpha adrenergic
agonist, phenylephrine, also increased CSF production to
a maximum of 10.6 ± 1.5 yl/min over control. This dose-
dependent effect was prevented by phentolamine and by
atropine. An increase in CSF formation during infusion
of phenylephrine was blocked by administration of hemi-
cholinium-3 through a reduction of endogenous
x

acetylcholine. The ability of atropine and hemicholinium
to inhibit the increase in CSF production by phenylephrine
demonstrates that the alpha adrenergic agonist probably
activates a cholinergic pathway. Atropine or phentolamine
alone reduced CSF formation 2.6 ± 0.5 and 3.7 ± 0.8 yl/min,
indicating that the cholinergic pathway exerts some control
over normal fluid production. Neither atropine nor phen¬
tolamine was able to add to the maximal reduction in CSF
formation caused by the carbonic anhydrase inhibitor
methazolamide.
The beta2 adrenergic agonist, salbutamol (albuterol),
also increased the rate of CSF formation, but it was only
half as effective as carbachol and phenylephrine. The
betag adrenergic agonist, l-isopropylamino-3(2 thiazoloxy)-
2 propanol HC1 (ITP), had no effect on CSF production.
Propranolol did not affect fluid formation but did reduce
the effect of salbutamol.
Bilateral stimulation of the cervical sympathetic
trunk reduced CSF production 3.0 ± 0.6 yl/min. This
observation indicated that alpha adrenergic agonists
probably act at the choroid plexus to decrease CSF
formation; however, any direct action of circulating
alpha agonists is masked by activating the cholinergic
pathway.
Experiments were also performed to determine whether
autonomic agents influenced CSF production through hemo¬
dynamic changes or a direct action on the choroid plexus
epithelium. Blood pressure data for carbachol,
xi

phenylephrine, and salbutamol did not correlate with the
changes in CSF production. Blood flow studies showed
that none of the agonists caused statistically significant
changes in cerebral or choroid plexus blood flow. These
findings suggest that autonomic drugs probably act directly
on the choroid plexus epithelium to change the rate of CSF
formation.
Verapamil, an agent that blocks calcium channels, did
not alter a carbachol-induced increase in fluid production
indicating that the autonomic agents probably did not exert
their effect by influencing calcium fluxes. The phospho¬
diesterase inhibitor, theophylline, on the other hand,
increased the rate of CSF production. This effect
suggested that the autonomic treatments might alter fluid
formation by increasing or decreasing levels of the cyclic
nucleotides guanosine 3',5' cyclic monophosphate (cGMP) or
adenosine 3',5' cyclic monophosphate (cAMP).
Although the exact mechanisms remain to be elucidated,
it is clear that cholinergic and beta2 adrenergic receptors
can be stimulated to increase CSF formation significantly
and that the cholinergic pathway which predominates is
also activated by alpha adrenergic agonists.
Xll

INTRODUCTION
Epithelia structurally similar to the choroid
plexus epithelium are found in the ciliary body of the
eye, the salivary gland, and the pancreas. Secretory
transport in these tissues is known to be controlled to
some extent by the autonomic nervous system. Therefore,
it is of interest to consider the influence of the
autonomic nervous system on the formation of cerebrospinal
fluid (CSF).
The columnar epithelial cells of the choroid plexus
are considered to be a primary site for the secretion of
CSF. This structure is an evagination of the walls of
the lateral and third ventricles of the brain and forms
the roof of the fourth ventricle. The choroid plexus
receives some innervation from the autonomic nervous
system. On the ventricular side, the outermost structure
of the choroid plexus is a single layer of epithelial
cells supported by connective tissue richly supplied with
blood vessels.
The autonomic nervous system is comprised of
sympathetic and parasympathetic components. The sympa¬
thetic system has short fibers from the central nervous
system to the sympathetic chain ganglia, then long
1

2
post-ganglionic fibers which innervate the tissues.
Sympathetic effects are classified into alpha, beta^, and
beta2 adrenergic actions and are mediated endogenously by
the adrenergic neurotransmitters norepinephrine and
epinephrine. Other agents can selectively stimulate alpha,
beta-^, or beta2 adrenergic activity if they have the
necessary structural modifications required for each
receptor. Phenylephrine is an alpha agonist; 1-isopropyl-
amino-3(2 thiazoloxy)-2 propanol HC1 (ITP) is a betag
agonist; and salbutamol is a beta2 agonist. The parasym¬
pathetic system has long fibers from the central nervous
system to ganglia close to the tissue being supplied,
then short post-ganglionic fibers which innervate the
tissue. The cholinergic neurotransmitter acetylcholine
mediates parasympathetic actions. Chemically related
agents such as carbachol can initiate cholinergic
activity when applied to the effector site.

BACKGROUND
In all systems in which fluid formation can be
altered by agents that affect the autonomic nervous system,
one must consider two basic means by which these effects
may be mediated. Alterations in fluid production may occur
in direct response to autonomic agents acting on receptors
located in the secretory cells. Also changes in fluid
production may possibly result from changes in hemodynamic
relationships increasing or decreasing pressure and flow
in the vessels of the choroid plexus. Functionally,
changes in hemodynamic relationships may interact with
direct changes in the secretory function of the epithelium
to alter fluid formation.
Autonomic Innervation and Function in Cerebral Blood Vessels
By following the nerve trunks passing into the
brain, anatomists identifed nerves supplying the large
pial vessels more than a century ago. Microscopists
subsequently described the presence of nerve fibers in
the finer cerebral vasculature, but classification of the
nerves as sympathetic and parasympathetic did not come
until the 1920's. Since the blood vessels of the choroid
3

4
plexus arise from cerebral arteries, a brief summary of
general cerebrovascular innervation and autonomic function
will be given before considering the choroid plexus
specifically. A more detailed presentation of the
autonomic innervation and responses of cerebral blood
vessels is given in Appendix I.
Sympathetic innervation of the large pial blood
vessels is supplied by the cervical sympathetic trunks.
This has been demonstrated by showing the disappearance
of adrenergic nerves after cervical sympathetic ganglionec-
tomy. However, smaller parenchymal vessels within the
brain receive adrenergic nerves from the brainstem. These
nerves survive bilateral cervical sympathectomy. Parasym¬
pathetic innervation of cerebral blood vessels originates
primarily through the facial nerve. Recent experiments
have shown that cholinergic nerves also come from sources
other than the cranial nerves.
A decrease in cerebral blood flow in dogs after
electrical stimulation of the cervical sympathetic trunk
or administration of norepinephrine has been demonstrated
by some investigators; however, others have been unable to
elicit these effects. On the other hand, stimulation of
the facial nerve or exogenous administration of acetyl¬
choline reproducibly and significantly increases cerebral
blood flow.
Cerebral blood vessels have shown more consistent
adrenergic responses in vitro than in the whole animal

5
experiments. Both electrical stimulation and alpha
adrenergic agents contract cerebral arteries in vitro,
but the maximum force of contraction is less than in
arteries from other tissues. Beta adrenergic responses
in cerebral vessels so far have been found only in the
cat. In this species, beta2 stimulation relaxes vessels
contracted with serotonin.
The significant observation from both the in vivo
and in vitro studies is that regardless of the nature of
the stimulation (i.e., electrical or pharmacological) the
adrenergic response of the cerebral vessel is small or
absent. In contrast, cholinergic stimulation has a strong
influence on cerebral blood vessels. In vitro acetyl¬
choline has been shown to relax contracted vessels.
Innervation of the Choroid Plexus
Since the choroid plexus is composed of an epithelium
resting on connective tissue containing blood vessels, it
is not feasible to discuss separately innervation of the
epithelium and innervation of the vessels with regard to
what is presently known about the anatomy of the tissue.
For this reason, evidence for nerves supplying both the
epithelia and the blood vessels of this secretory tissue
will be presented together.
Benedikt (1874) first observed nerve fibers around
blood vessels and near epithelial cells of the fourth

6
ventricle plexus. These fibers arose from a vagal nucleus
in the medulla which he called the XIII Cranial Nerve.
Later, innervation in the lateral plexus was described as
being mostly around the vessels, and it was suggested that
these nerves might be involved in vasomotor function
(Findlay, 1899). Stohr (1922) confirmed the observation
of Benedikt in the fourth ventricle plexus and also
demonstrated a nerve supply to the third ventricle plexus.
He suggested the nerve fibers were divided into "vascular
nerve fibers" entering with the arterial vessels and
"choroid plexus proper" fibers originating in a vagal
nucleus, in pontine nuclei, and in cerebral peduncles.
Junet (1926) also described nerves supplying the epithelial
cells of the plexus. Stohr's observation of separate
vascular and epithelial innervation to the fourth ventricle
plexus originating from the medulla was confirmed by Clark
(1928, 1934), and he extended the theory to include the
lateral plexus. Clark described the fibers as being
mostly in the connective tissue base of the choroid plexus
rather than around the epithelial cells. Some myelinated
nerves and a large number of unmyelinated nerves were
found in association with blood vessels and epithelial
cells without any of them ending directly on capillaries.
With electron microscopy, unmyelinated nerve fibers have
been identified near the choroid plexus epithelium
(Millen and Rogers, 1956) and around some blood vessels

7
supplying the plexus (Maxwell and Pease, 1956). Tennyson
(1975) has described a nerve process adjacent to an
epithelial cell. The cell membranes of both the nerve
and epithelium were thickened, indicating the presence of
a possible cell junction.
To determine the extent of adrenergic innervation,
two groups of investigators did cervical sympathetic
ganglionectomies. In the first study, a unilateral sympa¬
thectomy resulted in a patchy degeneration of nerve fibers
in the connective tissue of the ipsilateral and third
ventricle plexi of dogs and cats with most of the inner¬
vation remaining intact (Tsuker, 1947). The other study
demonstrated by fluorescence microscopy the presence of
an interconnected ground plexus of adrenergic nerves
between the vascular walls and the epithelial cells
(Edvinsson et al., 1974) . These observations indicated
the lateral and third ventricle plexi had the highest
content of adrenergic innervation and the fourth ventricle
plexus content was much lower. After a bilateral sympa¬
thectomy, the fluorescence disappeared in all the choroid
plexus and the norepinephrine content of the plexus
decreased significantly (Edvinsson et al., 1972b). Cholin¬
ergic innervation of the choroid plexus epithelium and
blood vessels has also been described (Edvinsson et al.,
1973b). In cats and rabbits, cholinergic fibers were
observed for all the plexus in greater density than

8
adrenergic fibers. The network of cholinergic nerve
fibers remained intact after sympathetic ganglionectomy.
Blood Flow to the Choroid Plexus
Blood flow to the choroid plexus has been measured
in the intact whole animal in only one study. Aim and
Bill (1973), using radioactive microspheres, obtained a
control flow of 3.01 ml/gm min. Upon stimulation of the
cervical sympathetic chain, the blood flow to the plexus
did not change. Two groups have made in situ determin¬
ations of plexus blood flow. Welch (1963) , while
measuring CSF production at the lateral choroid plexus in
the rabbit, observed a blood flow of approximately 2.86
ml/gm min. Pollay et al. (1972) perfused the choroid
plexus vessels of sheep and related blood flow to the
production of CSF. For a normal rate of formation they
concluded choroid plexus blood flow must be about 2.56
ml/gm min. Pharmacological agents affecting blood flow
were not used in any of these studies; however, Macri
et al. (1966) and Politoff and Macri (1966) showed that
serotonin and 1-norepinephrine reduced choroidal blood
flow in rabbits.
Autonomic Nervous System Control of Fluid Secretions
Fluid secretion occurs in several specialized
epithelial tissues. Sympathetic or parasympathetic

9
agonists have been shown to influence secretion in nearly
all of the secretory epithelia, but there has not been a
consistent pattern of effects. The salivary gland is
probably the most thoroughly studied of the tissues in
which there is autonomic control over fluid production.
In this system, both cholinergic and adrenergic agonists
increase secretion (Koelle, 1970). Cholinergic agonists
can produce approximately ten times as much fluid as beta
adrenergic agonists (Mangos etal., 1973). Petersen (1972)
has suggested that acetylcholine-activated secretion occurs
as a result of an uptake of extracellular calcium which
Douglas and Poisner (1963) showed was necessary for fluid
movement. The calcium, in turn, may cause an efflux of
potassium and an uptake of sodium which results in water
transfer.
The eye is another site in which fluid movement is
actively influenced by autonomic agonists. In this system
both production of fluid by the ciliary body and drainage
of aqueous humor through the trabecular meshwork are
under autonomic control (Chiou and Zimmerman, 1975). It
has been difficult to determine whether the cholinergic
system or the adrenergic system really predominates in
either mechanism, but it is certain that both are
influential. Paterson and Paterson (1972) have shown
that alpha adrenergic agonists cause a slight increase in
aqueous humor production and outflow facility. In contrast,

10
beta2 agonists (such as salbutamol) cause a decrease in
production and a large increase in flow through the
trabecular meshwork which can be blocked by propranolol
(Langham and Diggs, 1974 and Langham, 1976). The effects
of cholinergic agents on aqueous humor dynamics is more
complicated. The major effect in the whole animal is an
increased drainage of fluid (Ellis, 1971 and Macri and
Cevario, 1973). When acetylcholine and eserine, an anti¬
cholinesterase agent, are given intra-arterially in an
isolated perfused cat eye, the formation of aqueous humor
increases 10 to 15 yl/min (Macri and Cevario, 1973). The
action of acetylcholine can be blocked by atropine;
however, it can also be prevented with alpha adrenergic
receptor blocking agents or guanethidine, an adrenergic
neuron blocking agent, indicating the effect is probably
mediated by alpha receptors.
The acinar cells of the pancreas are also under
autonomic control (Thomas, 1967). Either vagal sti - > ; !ation
or the administration of cholinomimetic drugs can elicit
moderate increases in fluid secretion and substantial
increases in the release of digestive enzymes. Norepi¬
nephrine and epinephrine decrease secretion possibly by
constricting the blood vessels which supply the tissue.
Fluid transfer in the toad bladder and frog skin
epithelia can be increased by beta adrenergic stimulation
(Foster, 1974). This presumably occurs through changes

11
in adenosine 3',5' cyclic monophosphate (cAMP) levels
since it can be mimicked by dibutyryl cAMP and potentiated
by phosphodiesterase inhibitors (Jard, 1974). On the
other hand, alpha adrenergic activation reduces water
movement stimulated by theophylline or antidiuretic
hormone (Foster, 1974 and Jard, 1974) but has no effect
in the absence of these agents. No observations have been
made concerning possible cholinergic actions.
Very little investigation has been made into the
role of autonomic agents in the production of CSF. Becht
and Gunnar (1921) set a needle in the cisterna magna of
cats and noted that epinephrine and pilocarpine increased
fluid outflow. They suggested that this was not true CSF
production since some of the collected fluid was drawn up
into the tube again after the action of the drug was
finished. Bhattacharya and Feldberg (1958) observed the
same effect during ventriculocisternal perfusion with
epinephrine, histamine, and tubocurare. In these exper¬
iments only volumes were measured, and it was suggested
that the increased flow during the perfusion of the
ventricles was due to a change in cerebral blood flow.
Vates et al. (1964) observed that norepinephrine
(2.5 X 10"6 M in the perfusate) caused a decrease in
production of about 24% during yentriculocisternal
perfusion.

12
Rationale for Study
The evidence presented satisfies two major criteria
for concluding that the autonomic nervous system may exert
an influence on CSF production. First, it has been shown
that the blood vessels and epithelia of the choroid plexus
receive both adrenergic and cholinergic innervation. The
adrenergic nerves extend from the cervical sympathetic
trunk whereas the cholinergic nerves, comprising most of
the tissue's innervation, originate from undefined
locations. Second, it is clear that both adrenergic and
cholinergic agents alter secretion in other epithelial
systems.
Therefore, it seemed reasonable to investigate CSF
production during pharmacological alteration of the adren¬
ergic and cholinergic nervous system with agonists and
antagonists and to examine the role of cerebral blood flow
as a possible mediator of effects of these agents on CSF
production. Such an investigation is the subject of this
dissertation.

METHODS
Cats have been used extensively for studies of
neurological and secretory functions in the brain and for
this reason were chosen for investigation of the role of
the autonomic nervous system on CSF production. Animals
weighing 2-4 kg were anesthetized with an intrahepatic
injection of 30 mg/kg sodium pentobarbital and further
maintenance doses were given intravenously as needed
throughout the experiment. Surgical procedures for the
insertion of a tracheal cannula and femoral arterial and
venous catheters were performed. Blood pressure and heart
rate were monitored with a Statham pressure transducer
connected to the femoral artery catheter and recorded on
a Grass polygraph. Body temperature was maintained at
37° C. Throughout each experiment, the animal was arti¬
ficially ventilated at a rate set to maintain arterial pH
at approximately 7.35, the normal control value fcr a cat.
By measuring arterial pH and adjusting the ventilatory
rate as needed, the correct rate was established and the
corresponding end-tidal CC>2 was recorded and subsequently
monitored to maintain pH at the desired level.
For ventriculocisternal perfusion, the cat's head
was placed in a standard stereotaxic apparatus. A midline
13

14
incision was made from the top of the head to the neck
and a burr hole was drilled at a point 2 mm lateral to
the saggital suture and 2 mm caudal to the coronal suture.
A 22-gauge spinal needle, attached through a Statham
pressure transducer to a Harvard infusion pump, was set in
the right lateral ventricle. Placement was determined by
a sharp drop in the pressure recording as the needle passed
beyond the brain parenchyma into the ventricle. This
needle was used to deliver perfusate and to monitor intra¬
ventricular pressure which was recorded throughout the
experiment. The tip of a 19-gauge needle was then placed
in the cisterna magna by separating the neck muscles at
the midline and puncturing the dura mater just below the
occipital condyle of the skull, and effluent was collected
through a short tube attached to the needle. An artificial
CSF (see Appendix II for the composition) was used for the
perfusion. pH of the perfusion fluid was adjusted to 7.4
by bubbling 5% CC>2 in C>2 through it until the desired pH
was achieved. The perfusion rate was 191 yl/min during
the first 15 minutes and 76.4 yl/min thereafter.
Following a 30 minute washout of the original CSF in the
ventricular system, four to eight control periods of 15
minutes each were collected prior to tests with drugs or
stimulation.
Production of CSF was calculated from the dilution
of blue dextran, M.W. 2 X 10^ daltons, (Pharmacia). The

15
collected effluent of each sampling period and the original
artificial perfusate were diluted (100 yl in a total
volume of 1.0 ml) for measurement of optical densities
(Gilford 2400 spectrophotometer). This dilution was
necessary to obtain a large enough volume to assay during
short sampling periods. In most experiments, perfusion
was continued six hours; none went beyond nine hours.
Fluid formation was calculated with the equation
developed by Heisey et al. (1962) for dilution of non-
diffusible substances:
('-in ~ ^out) (r)
Vf = ¡w
The difference between the concentration of blue dextran
in the infused buffer solution (C-¡_n) and the effluent
(Cout) divided by the concentration of the effluent is the
dilution factor of the perfusate. The product of the
dilution factor and the rate of infusion (r) then provides
a value for the rate of formation of CSF.
Tests of Autonomic Nervous System
Influence on CSF Production
Stimulation of Cervical Sympathetic Trunks
Bilateral electrical stimulation of the cervical
sympathetic trunk was applied during ventriculocisternal
perfusion in some experiments. In addition to the surgical
procedure described for perfusion experiments, the cervical

16
sympathetic ganglion was exposed and the two preganglionic
sympathetic fibers were isolated from the vagi. The
preganglionic fibers were then passed through a platinum
electrode which was connected to a stimulator. The nerve
fibers were stimulated with 10 volts at a frequency of 10
shocks per second; each shock lasted for 0.5 msec. Nicti¬
tating membrane responses were also monitored during these
experiments to insure that adequate stimulation was
bilaterally applied. This was done by gently pulling each
membrane away from the eye, placing a suture in it and
attaching the suture to a Myograph-B (Narco Biosystems)
transducer which permitted the recording of membrane
responses. The experimental sampling period was 15
minutes in duration, with stimulation continuing throughout
the period. Control samples were collected during two
hours preceding and two hours following stimulation.
Modification of CSF Production by Application of
Pharmacological Agents
Drugs tested to determine a possible autonomic
influence on CSF production were carbamylcholine chloride
(carbachol) (Sigma), phenylephrine HC1 (Sigma), salbutamol
(albuterol in the U.S.) (Schering), atropine methyl
nitrate (Sigma), phentolamine mesylate (Ciba-Geigy),
propranolol HC1 (Ayerst), l-isopropylamino-3(2 thiazoloxy)-
2 propanol HC1 (ITP) (Syntex), aminophylline (theophylline)
(Parke-Davis), verapamil (Knoll Pharmaceutical), and
methazolamide (Lederle). All autonomic agonists were

17
administered in volumes less than 1 ml; all other drugs
were given in volumes less than 3 ml. These volumes
were considered small enough not to alter significantly
the total volume of blood.
At least one and one-half hours elapsed between
administrations of autonomic agonists when the drugs were
given to test the individual effects. No agonist was
tested during the first 30 minutes after an autonomic
blocking agent was given so as to test the effect of the
blocking agent on CSF formation. Subsequently, agonists
were injected at approximately 30 minute intervals.
The doses of the autonomic agonists used in these
experiments were selected on the basis of the blood
pressure dose-response curves. Doses on either side of
the ED5Q for blood pressure and the dose which gave the
maximal blood pressure response were tested for their
effect on CSF production. Salbutamol, however, required
a dose higher than that giving maximal blood pressure
effect to elicit a maximal effect on CSF formation. Doses
of the autonomic antagonists used were chosen for their
effectiveness in blocking peripheral actions of the
agonists.
Cholinergic agonist and antagonist. Carbachol was
chosen as the cholinergic agonist for its specificity at
the cholinergic post-ganglionic effector site and prolonged
half-life in the whole animal compared to acetylcholine.

18
It was dissolved in normal saline in concentrations up
to 10 yg/ml and administered intravenously in doses of
0.03, 0.1, 0.3, or 3.0 yg/kg body weight. As in all
experiments involving agonists, three 3-minute effluent
samples were collected beginning immediately after
injection of the drug.
Atropine (0.15 mg/kg) was given to test for a direct
effect on CSF formation. During atropine treatment,
carbachol was given in doses of 1.0, 3.0, and 30.0 yg/kg
to test for blockade of cholinergic effects on blood
pressure and CSF production. Phenylephrine (30 yg/kg)
and salbutamol (3 yg/kg) were also administered during
atropine treatment.
Alpha adrenergic agonist and antagonist. Phenyl¬
ephrine was chosen as the agonist for these experiments
because of its specificity for alpha adrenergic receptors.
It was dissolved in normal saline in concentrations up
to 300 yg/ml and administered intravenously in single
doses of 3.0, 10.0, 30.0, and 100.0 yg/kg body weight
or infused over a nine minute period (30-35 yg/kg min)
intravenously. Single injections were followed by three
3-minute samples. During infusions 3-minute samples were
also collected. Phentolamine was chosen as the alpha
receptor-blocking agent for its specificity and reversi¬
bility. The direct action of phentolamine on CSF formation
was tested at the dose of 2 mg/kg body weight. During

19
alpha adrenergic blockade, single injections of 30 yg/kg
phenylephrine, 3 yg/kg salbutamol, and 3 yg/kg carbachol
were tested. To further examine the mechanism of action
of phenylephrine, another group of experiments was done
in which the nine minute phenylephrine infusion was
preceded three minutes earlier by an intravenous injection
of 50 yg/kg of hemicholinium-3.
Beta adrenergic agonists and antagonist. To
ascertain specificity of beta adrenergic function both a
betai anc^ a heta2 agonist were tested to determine their
effect on CSF production. The beta^ agent selected was
l-isopropylamino-3(2 thiazoloxy)-2 propanol HC1 (ITP).
This compound has been shown to be specific for cardio-
acceleration and stimulation of lipolysis without affecting
beta2 actions (Lockwood and Lum, 1974). The drug was
dissolved in normal saline less than five minutes before
use to be certain that the drug was still active, and doses
of 100 and 300 yg/kg body weight were administered intra¬
venously. Beta2 activity was determined with salbutamol.
Salbutamol has been shown to have bronchodilator activity
and to increase blood flow to the dog hindlimb more
effectively than isoproterenol, and these effects are
reduced in the presence of propranolol (Cullum et al.,
1969). This drug was dissolved in normal saline in
concentrations up to 200 yg/ml and was given intravenously
in doses of 0.1, 1.0, 3.0, 10.0, and 30.0 yg/kg body
weight. For both agents, cisternal effluent was collected

20
over three 3-minute periods after drug administration.
The general beta blocking agent propranolol (2.0 mg/kg)
was tested to determine its effect on CSF production and
was given to antagonize beta2 adrenergic effects. Phenyl¬
ephrine and carbachol were also tested in the presence of
propranolol.
Non-autonomic agents. Three non-autonomic drugs
were studied with regard to their influence on CSF
production: theophylline, verapamil, and methazolamide.
Theophylline has been shown to stimulate the respiratory
rate, increase urine flow, and stimulate the rate and force
of contraction of the heart. Some of these actions of
theophylline are thought to result from the ability of the
agent to inhibit phosphodiesterase, the enzyme responsible
for cyclic nucleotide hydrolysis (Amer and Kreighbaum,
1975). To test whether autonomic agents might be acting
through changes in cyclic nucleotide levels to affect
CSF production, theophylline was administered intravenously
in the form of aminophylline in doses equivalent to 10 and
20 mg/kg theophylline. Again, CSF perfusate was collected
over three 3-minute periods following drug injection.
Verapamil has been demonstrated to block inward calcium
channels in cardiac muscle (Kohlhardt et al., 1972).
Since calcium influx is important in acetylcholine-
stimulated salivary secretion, verapamil was given via
the perfusate at a concentration of 10”^
M to test its

21
effect on carbachol activity? three 3-minute sampling
periods followed the administration of carbachol.
Carbonic anhydrase inhibitors, acetazolamide and benzol-
amide, have been shown to reduce CSF formation between 50
and 75% (Davson and Segal, 1970; Oppelt et al., 1964).
The carbonic anhydrase inhibitor, methazolamide was chosen
for its high lipid solubility providing ready access to
the secretory tissues. In these experiments methazolamide
(30 mg/kg) was given intravenously over a period of 30
minutes. CSF production in methazolamide treated cats was
also studied in the presence of atropine (0.15 mg/kg) or
phentolamine (2.0 mg/kg). Samples were collected in ten
minute periods for at least two hours after both drugs
had been administered.
Tests of Autonomic Nervous System
Influence on Blood Flow
Radioactive microspheres were used to measure blood
flow to the choroid plexus, brain, kidney, and biceps
brachii muscle. The kidney and muscle flows were used as
reference data. Briefly, the measurement depends on the
immediate lodging of spheres in capillary beds propor¬
tional to the capillary flow of the tissue. After delivery
of the spheres to the left side of the heart, the spheres
will have the same distribution as the blood leaving the
heart. Simultaneously withdrawing blood and microspheres

22
into a syringe at a known rate then provides the basis for
estimation of flow in tissues (Wagner et al. , 1969). Both
femoral arteries and a femoral vein were catheterized, and
a tracheal cannula was inserted. One of the arterial
catheters was connected to a pump set to withdraw blood
at 2.72 ml/min. The chest was opened at the midline and
the pericardium incised. A catheter was placed in the
left atrium and secured. The animal was then given 3500
units of sodium heparin to prevent clotting of blood. End-
tidal CO2 was carefully monitored during the experiment to
insure the same pC02 during each injection of microspheres
since brain blood flow is highly sensitive to pCC>2 changes.
The microspheres were suspended by the 3 M Company
(S)
in 20% dextran in Tween 80^ for injection. Microspheres
15 ± 5 microns in diameter were chosen because this size
has been shown to be optimal for cerebral blood flow
measurements, providing high reproducibility and less than
2% shunting of spheres past the capillary beds (Marcus et
al., 1976). Buckberg et al. (1971) showed a minimum of
400 spheres must be present to measure reliably the blood
flow of a tissue. Therefore, the number of counts per
minute (cpm) per microsphere was determined to estimate
a dose that would satisfy this requirement. In all exper¬
iments the cpm in each tissue represented more than the
minimum required number of spheres, thus validating the
calculation of flow from the radioactivity in the tissue.
O C
In each experiment injection of
Strontium spheres ten

23
seconds after beginning blood withdrawal gave control
141
blood flows, and injection of Cerium spheres later gave
blood flows during the peak influence of 3 yg/kg carbachol,
30 yg/kg phenylephrine, or 3 yg/kg salbutamol on CSF
production. The drugs were given about five seconds
before the blood withdrawal began. Blood withdrawal was
continued 90 seconds to guarantee at least one circulation
time. The animal was sacrificed and the choroid plexus
of the lateral and fourth ventricles was removed and
weighed. The brain, kidney, and muscle were also taken,
weighed, and ashed. The radioactivity was counted in a
Beckman Biogamma Scintillation Counter.
Blood flow to each of the organs was calculated
from the relation
Q = L_
d d'
whence,
A _ (F) (d)
U (d-)
where Q is the blood flow (ml/min), F is the rate at
which the arterial blood is sampled, d is the cpm of the
tissue being determined, and d' is the cpm of the isotope
in the arterial blood sample. To standardize the values,
the blood flows are expressed as ml/gm min considering
tissue weight.

24
Evaluation of CSF Production Data
The changes in CSF production caused by the various
treatments were calculated in the following manner: For
agonists, a least squares linear regression was applied
to the control CSF productions of each experiment. This
regression was based on CSF formation during one or more
control periods before any treatment and at least three
post-treatment control periods as determined by the return
of blood pressure and heart rate to pre-drug levels. No
values for post-treatment controls were taken sooner than
30 minutes after administration of short-acting agonists
such as carbachol, phenylephrine, and salbutamol. This
line was used to determine the theoretical control CSF
production at any given time over a two or three hour
span of the experiment. The production rate measured over
a three minute period after the agonist was given was
compared with the theoretical control for that time, and
the difference taken as the change in production caused
by the drug. Since the autonomic blocking agents and
methazolamide have relatively long active half-lives,
control periods were not availble after administration of
these drugs. For these experiments, four or more periods
prior to the time the agent was given were used to determine
a least squares regression line and calculate theoretical
control CSF productions. Changes in production were
determined in these experiments as for the agonists.

Statistical Analysis
All the changes in CSF production are expressed as
mean ± standard error of the mean (SE). Statistical
significance of changes in production caused by several
treatments was established by pairing theoretical control
and experimental data for each experiment and using the
paired one-tailed t-test (Goldstein, 1964). Significance
of changes in CSF production before and after treatment
with blocking agents or methazolamide was determined by
using the unpaired two-tailed t-test (Goldstein, 1964).
To ascertain whether increasing doses of autonomic
agonists yielded a dose-response relationship, the indi¬
vidual responses for each experiment were converted to
probits, a measure of the per cent of maximum response
obtained. A linear regression was then performed and
a plot of log dose vs. probit was produced with a 95%
confidence interval on the regression line. Significance
of the line was established by testing its slope against
a null hypothesis (Snedecor and Cochran, 1967). In all
cases, significance was assumed when the t-value exceeded
the minimum value for p<.05. Non-significance is denoted
by N.S. in the data.

RESULTS
Ventriculocisternal Perfusion Studies
for Estimation of CSF Production Rate
Control Experiments
Control data were collected in four experiments in
which no drugs were given. Control data from 24 exper¬
iments in which only short-acting autonomic agonists such
as carbachol, phenylephrine, and salbutamol were given
were added to these, avoiding data collected within 30
minutes after drug. The two groups of data were combined
since the slopes of the 4 untreated (-0.042 yl/min each
minute) and 24 treated animals (-0.015 yl/min each minute)
were not statistically significant from each other or
different from zero when tested against a null hypothesis.
These combined data, shown in Table 1 and Figure 1,
indicate a steep increase in the optical density of the
CSF perfusate reflected as a decrease in CSF production
during the first 60 minutes of the experiment according
to a regression line with a slope of -0.143 yl/min per
minute. This initial apparent decline in fluid formation
was probably due to an incomplete washout of the original
CSF in the ventricular system. A more gradual decline
follows ranging from 19.7 ± 1.0 to 15.8 ± 0.7 yl/min over
26

27
TABLE 1
CONTROL PRODUCTION OF CEREBROSPINAL FLUID
Time (min)a
n
Production
(yl/min) ± SE
30
24
24.0
+
1.5
45
21
20.7
+
1.3
60
27
19.7
+
1.0
75
10
17.5
+
1.2
90
8
19.7
+
1.2
105
6
16.9
+
1.6
120
17
17.0
+
0.8
135
22
16.2
+
0.7
150
27
16.8
+
0.7
165
24
16.5
+
0.7
180
28
16.7
+
0.6
195
11
17.2
+
0.7
210
7
15.6
+
1.0
225
14
15.1
+
0.9
240
24
15.9
+
0.7
255
20
15.1
+
0.7
270
25
14.9
+
0.6
285
22
15.6
+
0.5
300
19
15.8
+
0.7
Regression line for 0 to 60 minutes
y = -0.143 x + 27.9
r = -0.96
Regression line for 60 to 300 minutes
y = -0.015 x + 19.3
r = -0.81
aDuration of perfusion after washout of original CSF.

Figure 1. Changes in the production of CSF with time in
control experiments. The ordinate is the mean
CSF production value ± SE for at least 6
experiments at each point. The abcissa is
time in minutes. The regression lines for
0 to 60 minutes and 60 to 300 minutes are
represented by a solid line.

28
26
24
22
20
18
16
14
12
10
8
6
4
2
30 60 90 120 150 180
Time (min)
210
240
270
300
N)

30
the next 240 minutes in which the slope of the regression
line is -0.015 yl/min each minute. These data compare
favorably with the accepted control CSF production value
of approximately 20 yl/min (Davson, 1967) and with the
slope of -0.025 yl/min per minute for a regression line
on CSF formation in the monkey (Martins et al., 1974).
Cervical Sympathetic Stimulation
Only those experiments in which successful bilateral
stimulation occurred were included in the study. This was
determined by recording the contraction of the nictitating
membrane. After an initial decrease in the response of the
membrane during stimulation, the tension caused by the
contraction remained steady, and no fatigue was observed
during the experiment. Preganglionic stimulation of 10
volts at a frequency of 10 shocks/sec each with a duration
of 0.5 msec decreased CSF production 3.0 ± 0.6 yl/min in
five animals (p<.05 in paired t-test).
Cholinergic Agonist and Antagonist
Carbachol significantly increased the rate of CSF '
formation to 10.6 ± 2.1 yl/min over control (Table 2,
Figure 2). Over a dosage range of 0.3 yg/kg to 3.0 yg/kg
probit analysis (Figure 3) yielded a significant slope of
1.52 (p<.05). These relationships among the CSF
production data were taken to indicate a carbachol dose-
response. The ED50 for carbachol taken from the probit
plot was 0.12 yg/kg. High doses of this cholinergic
agonist occasionally produced slight increases in intra¬
ventricular pressure.

TABLE 2
CARBACHOL DOSE-RESPONSE: CHANGES IN CSF PRODUCTION
Dose (yg/kg) Control CSF Production Change in CSF Production3
(yl/min) ± SE(yl/min) ± SE (n)
0.03
19.5
+
2.2
+ 2.0
+
0.6
(6)
0.10
28.9
+
5.7
+ 4.6
+
1.0
(5)
0.30
18.4
+
1.1
+ 7.8
+
1.6
(5)
3.0
16.8
+
1.8
+ 10.6
±
2.1
(6)
i.ob
15.8
+
2.3
+ 0.6
±
0.4
(3)
3.0b
14.1
+
1.5
-0.3
±
1.4
(3) d
rQ
o
o
ro
9.9
+
2.1
+ 2.7
±
1.0
(5)
3.0G
12.3
+
0.6
+ 15.3
±
6.5
(4)e
Change in CSF production calculated for each
experiment as the difference between the control
and the peak effect of the drug
Given within 120 min after 0.15 mg/kg atropine
c — R
10 M verapamil in CSF perfusate
dp < .05 when compared with 3 yg/kg carbachol alone
0
N.S. when compared with 3 yg/kg carbachol alone

Figure 2. Carbachol dose-response for CSF production. The
ordinate is the mean increase in CSF production
over the theoretical control value ± SE plotted
on a linear scale. The abcissa is the dose of
carbachol in pg/kg body weight plotted on a
logarithmic scale. (• —• ) represents
carbachol alone. (O) is carbachol during
0.15 mg/kg atropine.

0 03 0 10 0 30
Carbachoi Cpg/ kg)
ee

Figure 3.
95% confidence interval on probit analysis of
the effect of carbachol on CSF production. The
ordinate is probits. The abcissa is the dose
of carbachol in pg/kg body weight plotted on a
logarithmic scale. The slope of the probit
regression line is 1.52 which is significant
(p<.05). The dose of carbachol producing an
ED^q response is 0.12 pg/kg.

Prob its
3
2
1
0 03 0 10
Carbachol (|jg/ kg )
030
u>
On

36
Atropine caused a decrease of 2.6 ± 0.5 yl/min in
CSF production which was statistically significant
(Table 3). The cholinergic blocking agent also prevented
the increase stimulated by 3 yg/kg carbachol (Table 2).
The 30 yg/kg carbachol dose increased CSF production
during atropine blockade by 2.7 ± 1.0 yl/min. The 3 yg/kg
dose of carbachol was administered during phentolamine
and propranolol blockade of adrenergic receptors in one
or two experiments. No alteration in the effect of
carbachol was evident. When verapamil (10-^ M) was
present in the artificial CSF perfusate, there was also
no change in the response to this dose of carbachol
(Table 2). Verapamil alone did not alter normal CSF
production.
Alpha Adrenergic Agonist and Antagonist
Phenylephrine caused a dose-dependent increase in
CSF production (Table 4, Figure 4). The change in response
with increasing amounts of drug was subjected to probit
analysis (Figure 5). A slope of 3.41 was obtained from
the plot indicating significance (p<.05). The ED^g for
phenylephrine was 12 yg/kg. A slight increase in intra¬
ventricular pressure sometimes occurred with high doses
of phenylephrine.
Phentolamine decreased production 3.7 ± 0.8 yl/min
(p<.05) (Table 3). This alpha adrenergic blocking agent
also significantly reduced the effect of 30 yg/kg phenyl¬
ephrine, i.e., from 10.0 ± 1.6 yl/min to -0.8 ± 0.7 yl/min

TABLE 3
EFFECTS OF AUTONOMIC BLOCKING AGENTS ON CSF PRODUCTION
Drug
Dose (mg/kg)
Control CSF Production
(pl/min) ± SE
Change in CSF Production'
(yl/min) ± SE (n)
Atropine
0.15
17.1 ± 1.3
-2.6 ± 0.5 (9) b
Phentolamine
2.0
15.4 ± 1.0
-3.7 ± 0.8 (6)b
Propranolol
2.0
16.9 ± 1.8
-0.7 ± 0.6 (4)C
aChange in CSF production calculated for
each experiment as the difference between
the control and the peak effect of the drug
bp < .05 with paired t-test
c
N.S. with paired t-test
u>

TABLE 4
PHENYLEPHRINE DOSE-RESPONSE: CHANGES IN CSF PRODUCTION
Dose (yg/kg) Control CSF Production Change in CSF Production3
(yl/min) ± SE (yl/min) ± SE (n)
3
16.2
+
1.0
+ 0.3
+
1.2
(5)
10
22.6
+
3.9
+ 2.6
+
0.6
(5)
30
20.0
+
2.8
+ 10.0
+
1.6
(7)
100
10.9
+
2.0
+ 10.6
±
1.5
(7)
30b
15.1
±
1.2
-0.8
±
0.7
(6) d
30C
15.8
±
1.6
+ 0.4
±
1.2
(5) d
aChange in CSF production calculated for each
experiment as the difference between the control
and the peak effect of the drug
bGiven within 120 min after 2.0 mg/kg phentolamine
c ,
Given within 120 min after 0.15 mg/kg atropine
p < .01 when compared with 30 yg/kg phenylephrine alone

Figure 4.
Phenylephrine dose-response for CSF production.
The ordinate is the mean increase in CSF pro¬
duction over the theoretical control value ±
SE plotted on a linear scale. The abcissa is
the dose of phenylephrine in yg/kg body weight
plotted on a logarithmic scale. (• • )
represents phenylephrine alone. (O) is
30 yg/kg phenylephrine during 2.0 mg/kg phen-
tolamine. (*) is 30 yg/kg phenylephrine
during 0.15 mg/kg atropine.

40
-1- 1
3 10
Phenylephrine (^jg/kg)
x
30
100

Figure 5. 95% confidence interval on probit analysis of
the effect of phenylephrine on CSF production.
The ordinate is probits. The abcissa is the dose
of phenylephrine in yg/kg body weight plotted on
a logarithmic scale. The slope of the probit
regression line is 3.41 which is significant
(p<.05). The dose of phenylephrine producing an
EDj-q response is 12 yg/kg.

42

43
(p<.01) (Table 4, Figure 4). Atropine, the cholinergic
blocking agent, also effectively prevented the increase
in CSF production caused by phenylephrine (Table 4,
Figure 4). In the presence of atropine, 30 yg/kg
phenylephrine increased CSF formation only 0.4 ± 1.2
yl/min which differed significantly (p<.01) from the
increase due to the alpha agonist alone. Propranolol
did not influence the effect of phenylephrine in the only
experiment in which it was tested.
Phenylephrine was also administered in an intravenous
infusion (approximately 30 yg/kg min) over nine minutes.
In the first three minutes, CSF production increased
14.1 ± 4.0 yl/min, 24.1 ± 4.5 yl/min in the next three
minutes, and 18.6 ± 2.0 yl/min in the final three minutes
(Table 5). Administration of 50 yg/kg hemicholinium-3
to the animal three minutes prior to the phenylephrine
infusion significantly reduced the increase in production
in four of six cats. In these four animals, the increase
in production due to phenylephrine was limited to 2.2 ±
1.0 yl/min (p<.05 compared with phenylephrine alone) in
the first period, 2.5 ± 1.6 yl/min (p<.01) in the next
three minutes, and 0.8 ± 1.8 yl/min (p<.01) in the final
three minutes (Table 5). When the two non-responsive
animals are included, the effect of hemicholinium
pretreatment appears to cause no significant change in
the effect of phenylephrine (Table 5). Hemicholinium
alone did not influence the rate of CSF production.

TABLE 5
THE EFFECT OF HEMICHOLINIUM-3 (HC-3) ON CSF PRODUCTION
DURING INTRAVENOUS INFUSION OF PHENYLEPHRINE (Phe)
3 minutes
6 minutes
9 minutes
Change in CSF Production3
(yl/min) ± SE during Phe*3
alone in four cats
+14.1 ± 4.0
+24.1 ± 4.5
+18.6 ± 2.
Change in CSF Production
(yl/min) ± SE during Phe
after HC-3 pretreatmentc
in four cats
+2.2 ± 1.0d
+2.5 ± 1.6e
+0.8 ± 1.
Change in CSF Production
(yl/min) ± SE during Phe
after HC-3 pretreatmentc
in six cats
+7.0 ± 3.lf
+ 9.3 ± 4.4f
+
CO
o
1+
aChange in CSF production calculated for each experiment as the difference
between the control and the peak effect of the drug
h
Phenylephrine infused intravenously for 9 minutes, approximately 30 yg/kg min
c .
Hemicholinium-3 (50 yg/kg) given 3 minutes prior to phenylephrine infusion
p < .05 when compared with phenylephrine infusion alone
0
p < .01 when compared with phenylephrine infusion alone
^N.S. when compared with phenylephrine infusion alone

45
Beta Adrenergic Agonists and Antagonist
The beta2 agonist, salbutamol, increased CSF
production about half as much as carbachol or phenylephrine
(Table 6, Figure 6). A dose-response effect was demon¬
strated by a probit analysis of the changes in CSF
formation (Figure 7). The regression line had a signif¬
icant slope of 0.96 (p<.05). The ED^q for salbutamol
was 1.5 yg/kg.
Beta blockade with propranolol did not change CSF
production (Table 3). Propranolol did appear to reduce
the increase observed with 3.0 yg/kg salbutamol, but the
difference was not significant (Table 6, Figure 6).
Atropine (four experiments) and phentolamine (one exper¬
iment) did not appear to alter the salbutamol response.
The beta-]_ agonist, ITP, did not significantly change the
production of CSF in either 100 or 300 yg/kg doses.
Theophylline
Theophylline significantly increased CSF formation
(Table 7). Doses of 10 and 20 mg/kg increased production
3.3 ± 0.8 yl/min and 9.2 ± 1.4 yl/min, respectively. The
difference (p<.01) in the increases with the two doses
suggest the effect was probably dose-dependent.
Carbonic Anhydrase Inhibition
The carbonic anhydrase inhibitor, methazolamide,
significantly reduced the rate of CSF formation (Table 8).
The maximal decrease was 11.2 ± 1.4 yl/min; over several

TABLE 6
BETA ADRENERGIC AGONISTS DOSE-RESPONSE: CHANGES IN CSF PRODUCTION
Dose (yg/kg) Control CSF Production Change in CSF Production3
(yl/min) ± SE(yl/min) ± SE (n)
SALBUTAMOL
0.1
21.6 ±
1.8
+ 0.5
+
0.5
(5)
1.0
22.0 ±
2.0
+ 3.3
+
1.3
(4)
3.0
18.3 ±
1.8
+ 3.8
+
1.6
(5)
10.0
17.4 ±
2.3
+ 4.0
±
1.2
(7)
30.0
16.3 ±
1.0
+ 5.7
±
1.4
(5)
3.0b
13.0 ±
2.7
+ 0.7
±
0.2
(4>S
3.0C
10.1 ±
2.6
+2.8
±
2.0
(4)S
ITPd
100
16.0 ±
1.1
-0.8
±
1.0
(5)
300
19.5 ±
3.4
+ 0.1
±
1.6
(4)
aChange in CSF production calculated for each exper¬
iment as the difference between the control and the
peak effect of the drug
Given within 120 min after 2.0 mg/kg propranolol
Q
Given within 120 min after 0.15 mg/kg atropine
l-isopropylamino-3(2 thiazoloxy)-2 propanol HC1
0
N.S. when compared with 3.0 yg/kg salbutamol alone

Figure 6. Salbutamol dose-response for CSF production. The
ordinate is the mean increase in CSF production
over the theoretical control value ± SE plotted
on a linear scale. The abcissa is the dose of
salbutamol in yg/kg body weight plotted on a
logarithmic scale. (• -• ) represents
salbutamol alone. (O) is 3.0 yg/kg salbutamol
during 2.0 mg/kg propranolol. (a) is 3.0 yg/kg
salbutamol during 0.15 mg/kg atropine.

SalbutamoI ( jjg/ kg )
Increase in CSF Production (jjl/min)
i
N9 O N> ^ O)
' i l 1 â–  i
8 V
oo

Figure 7. 95% confidence interval on probit analysis of
the effect of salbutamol on CSF production.
The ordinate is probits. The abcissa is the
dose of salbutamol in yg/kg body weight plotted
on a logarithmic scale. The slope of the probit
regression line is 0.96 which is significant
(p<.05). The dose of salbutamol producing an
ED50 response is 1.5 yg/kg.

o
«o
s)'qo.

TABLE 7
EFFECTS OF THEOPHYLLINE ON CSF PRODUCTION, BLOOD PRESSURE, AND HEART RATE
Dose
(mg/kg)
Control CSF
Production
(yl/min) ± SE
Change in CSF
Production3 (yl/
min) ± SE (n)
Change in Mean
Pressure (mm
± SE (n)
Blood
Hg)
Change in Heart
Rate (beats/min)
± SE (n)
10
15.8 ± 2.1
+3.3 ± 0.8b (5)
-37 ± 6 (5)
+ 25 ± 5
(5)
20
16.9 ± 1.4
+9.2 ± 1.4b'c (4)
-54 ± 7 (4)
+ 35 ± 3
(4)
Change in CSF production
calculated for
each
experiment as the difference between the control
and the peak effect of the drug
<
.01 with paired t-test
.01 when compared with 10 mg/kg theophylline

TABLE 8
EFFECTS OF CARBONIC ANHYDRASE INHIBITION ALONE AND WITH
AUTONOMIC BLOCKING AGENTS ON CSF PRODUCTION
Treatment
Control CSF Production
(yl/min) ± SE
Maximum Change in
CSF Production3
(yl/min) ± SE (n)
Mean Change in
CSF Production*3
(yl/min) ± SE (n)
Q
Methazolamide
19.0 ±
1.. 7
-11.2 ± 1.4 (6)
-9.4 ± 1.2 (6)
Methazolamide +
Phentolamine1^
19.1 ±
2.4
-13.6 ± 1.8 (5)f
-12.5 ± 1.6 (5)f
Methazolamide +
Atropinee
16.0 ±
2.2
-10.6 ± 0.9 (5)f
-8.8 ± 1.0 (5)f
Calculated for each experiment as the difference
between the control and the peak effect of the drug
Calculated for each experiment as the difference
between the control and the effect of the drug over
two or more periods
30 mg/kg methazolamide given over 30 minutes
ci
2.0 mg/kg phentolamine
0
0.15 mg/kg atropine
^N.S. when compared with methazolamide alone

53
collection periods the mean drop in production was
9.4 ± 1.2 yl/min. The CSF formation rate during metha-
zolamide treatment was approximately 7 to 9 yl/min. Since
atropine and phentolamine successfully decreased fluid
formation, these drugs were tested in combination with
methazolamide for possible additive effects. From Table 8
it is evident that the autonomic blocking agents caused no
further significant decrease in production. It was also
noted in several experiments that carbachol and phenyl¬
ephrine partially override the carbonic anhydrase inhi¬
bition .
Changes in Blood Pressure and Heart Rate
during Ventriculocisternal Perfusion Studies
Cervical Sympathetic Stimulation
Bilateral preganglionic cervical sympathetic
stimulation (10 volts, 10 shocks/sec, each shock lasting
0.5 msec) produced an increase in mean arterial blood
pressure of 42 ± 27 mm Hg. Heart rate did not change
significantly.
Cholinergic Agonist and Antagonist
Doses of carbachol ranging from 0.03 to 3.0 yg/kg
were administered. From Table 9 and Figure 8 it is clear
that the drug had a dose-response effect in decreasing
blood pressure (p<.01 by probit analysis). The heart rate
also decreased beginning at the 3 yg/kg dose (Table 9).
Atropine caused an initial drop of 43 ± 5 mm Hg in mean

TABLE 9
CARBACHOL DOSE-RESPONSE: CHANGES IN BLOOD PRESSURE AND HEART RATE
Dose (yg/kg)
Change in Mean Blood Pressure Change in Heart Rate
(mm Hg) ± SE (n) (beats/min) ± SE (n)
0.
,03
-28
±
4
(7)
-1
±
1
(7)
0.
,10
-38
±
6
(6)
0
±
2
(6)
0.
. 30
-57
±
5
(6)
3
±
2
(6)
3.
, 0
-81
±
10
(6)
-63
±
8
(5)
1.
,oa
-6
±
3
(4)
-1
±
3
(4)
3.
,0a
-9
±
6
(2) C
0
±
0
(2)
30.
.0a
+ 12
±
13
(5)
2
±
2
(5)
3.
. ob
-50
±
6
(4) d
-38
±
15
(4
Given within 120 min after 0.15 mg/kg atropine
d10 verapamil in CSF perfusate
Q
p < .01 when compared with 3.0 yg/kg alone
dN.S. when compared with 3.0 yg/kg carbachol alone

Figure 8. Carbachol dose-response for blood pressure. The
ordinate is the change in mean blood pressure
(mm Hg) ± SE plotted on a linear scale. The
abcissa is the dose of carbachol in yg/kg body
weight plotted on a logarithmic scale. Carbachol
alone is represented by (#——— •) .
Carbachol during 0.15 mg/kg atropine is represented
by (O : O) .

0 03 0 10 0 30
Carbachol Cpg/kg)
Change in
i
S
T
Mean Blood Pressure (mm/Hg)
i i i »L
é S S S
» i —T »
99

57
arterial blood pressure and a decrease in heart rate of
17 ± 3 beats/min (Table 10). Since both of these
parameters had returned to pre-drug levels within 30
minutes after atropine administration, these actions
were thought to be unrelated to the anti-cholinergic
action of the drug. The atropine successfully blocked the
fall in blood pressure seen with both 1 and 3 yg/kg
carbachol and the decrease in heart rate seen with 3 yg/kg
carbachol (Table 9). After atropine, 30 yg/kg carbachol
produced a slight, but insignificant, increase in blood
pressure, possibly through nicotinic stimulation of
sympathetic ganglia. No change in heart rate was observed
with this dose of carbachol during atropine treatment.
The cardiovascular effects of 3 yg/kg were not affected
by the presence of verapamil in the CSF perfusate (Table 9).
Alpha Adrenergic Agonist and Antagonist
Phenylephrine, given in doses between 3 and 100 yg/kg
body weight, increased blood pressure to a peak of 133 ± 11
mm Hg (Table 11 and Figure 9). Probit analysis yielded a
statistically significant slope (p<.01) and an ED5q of 17
yg/kg. Although the lower doses of phenylephrine (3 and
10 yg/kg) caused a significant decrease in heart rate,
the larger amounts of drug caused no significant change
(Table 11). Phentolamine caused a decrease in mean blood
pressure of 40 ± 7 mm Hg and an increase in heart rate
of 24 ± 5 beats/min (Table 10). Both blood pressure and
heart rate returned to control values within 30 minutes.

TABLE 10
EFFECTS OF AUTONOMIC BLOCKING AGENTS ON BLOOD PRESSURE AND HEART RATE
Drug
Dose (mg/kg)
Change in Mean
(mm Hg) ±
Blood Pressure3
SE (n)
Change in Heart Rate3
(beats/min) ± SE (n)
Atropine
0.15
-43 ± 5
(17)
-17
± 3 (16)
Phentolamine
2.0
-40 ± 7
(ID
+ 24
± 5 (11)
Propranolol
2.0
-15 ± 2
(4)
-41
± 11 (4)
^Measured

TABLE 11
PHENYLEPHRINE DOSE-RESPONSE: CHANGES IN BLOOD PRESSURE AND HEART RATE
Dose (yg/kg) Change in Mean Blood Pressure Change in Heart Rate
(mm Hg)
+
SE
(n)
(beats/min)
+
SE (n)
3
+26
+
4
(9)
-9
+
4
(8)
10
+ 47
+
6
(14)
-11
+
3
(12)
30
+ 8 5
+
9
(15)
+ 2
+
5
(13)
100
+ 133
±
11
(14)
-17
± 12
(13)
30a
+ 21
+
4
(6)c
-6
+
4
(6) d
30b
+ 73
+
5
(7)d
-13
+
4
(7)d
aGiven within 120 min after 2.0 mg/kg phentolamine
bGiven within 120 min after 0.15 mg/kg atropine
c
p < .05 when compared with 30 yg/kg phenylephrine alone
cl
N.S. when compared with 30 yg/kg phenylephrine alone

Figure 9. Phenylephrine dose-response for blood pressure.
The ordinate is the change in mean blood
pressure (mm Hg) ± SE plotted on a linear
scale. The abcissa is the dose of phenylephrine
in yg/kg body weight plotted on a logarithmic
scale. Phenylephrine alone is represented by
(• ©) . ( O) is 30 yg/kg phenylephrine
during 2.0 mg/kg phentolamine. ( a. ) is 30 yg/kg
phenylephrine during 0.15 mg/kg atropine.

140
120
100
80
60
40
20
3 10 30 100
Phenylephrine Cpg/kg)

62
This blocking agent significantly reduced the pressor
action of 30 yg/kg phenylephrine (Table 11). In the
presence of atropine the phenylephrine-induced changes
in blood pressure and heart rate were not altered (Table
11). Phenylephrine administered in an infusion (30 yg/kg
min) over nine minutes increased mean arterial pressure
by about 66 mm Hg (Table 12). Heart rate decreased
significantly only in the first three minute period.
Pretreatment with hemicholinium-3 did not significantly
affect the increase in blood pressure or the change in
heart rate due to phenylephrine (Table 12).
Beta Adrenergic Agonists and Antagonist
Salbutamol was given in doses between 0.1 and
30.0 yg/kg body weight. The blood pressure decreased
significantly in response to all doses tested; however,
the maximum fall occurred after 1.0 yg/kg (Table 13 and
Figure 10). A dose-response increase in heart rate
occurred as determined by probit analysis. Propranolol
dropped the mean arterial blood pressure slightly and
significantly decreased the heart rate immediately after
administration (Table 10). Unlike the other autonomic
blocking agents, these effects of propranolol persisted
for two or more hours. Propranolol successfully blocked
the cardiovascular responses to 3 yg/kg salbutamol
(Table 13). Atropine did not significantly alter the
changes in mean blood pressure and heart rate caused by

TABLE 12
THE EFFECT OF HEMICHOLINIUM-3 (HC-3) ON BLOOD PRESSURE AND HEART RATE
DURING INTRAVENOUS INFUSION OF PHENYLEPHRINE (Phe)
3
Phea
minutes
HC-3 + Pheb
6
Phe
minutes
HC-3 + Phe
9 minutes
Phe HC-3 + Phe
Change in Mean
+ 69 ± 3
+63 ± 10C
+ 70 ± 6
+69 ± 9C
+ 60 ± 7
+63 ± 11°
Blood Pressure (mm Hg)
± SE (n)
(4)
(6)
(4)
(6)
(4)
(6)
Change in Heart
-32 ± 13
-30 ± 15C
+ 6 ± 23
-16 ± 19C
-10 ± 19
-7 ± 20C
Rate (beats/min)
± SE (n)
(4)
(6)
(4)
(6)
(4)
(6)
aPhenylephrine infused intravenously for 9 minutes,
approximately 30 yg/kg min
Id
Hemicholinium-3 (50 yg/kg) given 3 minutes prior to
phenylephrine infusion
c
N.S. when compared with phenylephrine infusion alone

TABLE 13
BETA ADRENERGIC AGONISTS DOSE-RESPONSE: CHANGES IN BLOOD PRESSURE AND HEART RATE
Dose (yg/kg) Change in Mean Blood Pressure Change in Heart Rate
(mm Hg) ± SE (n) (beats/min) ± SE (n)
SALBUTAMOL
0.1
-27
+
5
(6)
+ 3
+
3
(6)
1.0
-40
±
8
(9)
+ 17
+
4
(8)
3.0
-43
±
7
(9)
+ 33
±
5
(9)
10.0
-45
±
5
(9)
+ 39
±
6
(9)
30.0
-45
±
5
(7)
+ 58
±
8
(7)
3.0a
-10
±
2
<4)d
+ 1
±
1
(4)d
3.0b
-19
±
13
(4)e
+ 18
±
5
(4)e
ITPC
100
-11
+
6
(5)
+ 21
±
9
(5)
300
-17
+
5
(4)
+ 20
±11
(4)
aGiven within 120 min after 2.0 mg/kg propranolol
Given within 120 min after 0.15 mg/kg atropine
c
l-isopropylammo-3 (2 thiazoloxy)-2 propanol HC1
p < .05 when compared with 3.0 yg/kg salbutamol alone
0
N.S. when compared with salbutamol alone
Oh

Figure 10. Salbutamol dose-response for blood pressure.
The ordinate is the change in mean blood
pressure (mm Hg) ± SE plotted on a linear
scale. The abcissa is the dose of salbutamol
in yg/kg body weight plotted on. a logarithmic
scale. Salbutamol alone is represented by
(• ©) . (O) is 3.0 yg/kg salbutamol
during 2.0 mg/kg propranolol. ( a) is 3.0
yg/kg salbutamol during 0.15 mg/kg atropine.

Salbutamol Cpg/kg)
99

67
salbutamol. The betag agonist, ITP, increased heart
rate at 100 and 300 yg/kg doses (Table 13), A small
decrease in mean arterial blood pressure was also
observed.
Non-autonomic Drugs
Theophylline given intravenously significantly
decreased mean arterial blood pressure and increased the
heart rate (Table 7). In both cases the effects appeared
to be dose-related. The dose of 10 mg/kg decreased blood
pressure 37 ± 6 mm Hg and increased heart rate 25 ± 5
beats/min, whereas 20 mg/kg decreased blood pressure
54 ± 7 mm Hg and increased heart rate 35 ± 3 beats/min.
Verapamil in the perfusate did not influence peripheral
blood pressure or heart rate. Methazolamide also had
no significant peripheral cardiovascular effects.
Effect of Autonomic Agonists on Blood Flow
One group of three cats served as an internal
control; i.e., each animal received two control injections
of microspheres sequentially. In these animals, no
changes in blood flow were detected in any of the four
tissues (Table 14). Other groups of cats received
30 yg/kg phenylephrine, 3.0 yg/kg carbachol, or 3.0 yg/kg
salbutamol as the experimental treatment during the second
injection of microspheres. The control blood flows
(ml/gm min) for these animals were 2.26 ± .28 for the

TABLE 14
EFFECT OF PHENYLEPHRINE, CARBACHOL, AND SALBUTAMOL ON BLOOD FLOW TO
THE CHOROID PLEXI, BRAIN, SKELETAL MUSCLE, AND KIDNEY
Treatment
Choroid Plexi
Change
Control After
Flowa Treatment
Brain
Skeletal
Muscle
Kidney
Control
Flow
Change
After
Treatment
Control
Flow
Change
After
Treatment
Control
Flow
Change
After
Treatment
Control
X
1.94
-.06
. 42
+ .02
. 028
-.001
1.63
-.14
SE
. 46
.18
.07
. 06
.006
.004
. 37
.19
Carbachol
X
2.33
+ .81
.37
-.06
.041
+ .018
1.94
-.58
3 yg/kg
SE
. 79
.93
.02
.04
.012
.011
.23
. 11
Phenylephrine
X
2.51
+ .77
.63
-.09
.070
+ .013
1.24
-.58
30 yg/kg
SE
.70
. 86
.09
.16
.025
.019
. 43
.36
Salbutamol
X
2.16
+ .35
.40
+ .01
.049
+ .024
1.74
-.45
3 yg/kg
SE
.33
.50
.09
.05
.011
.028
.49
.26
Mean Control
X
2.26
. 46
.048
1.64
Flow
SE
.28
. 04
. 008
.19
Blood flow expressed as ml/gm min wt tissue weight

69
choroid plexus, 0.46 ± ,04 for the brain, .048 ± .008 for
the muscle, and 1.64 ± .19 for the kidney. None of the
drugs caused a significant change in blood flow to any of
the tissues examined (Table 14) .

DISCUSSION
Modification of CSF Production by Autonomic Influences
The data show significant increases in CSF production
as a result of stimulation of the cholinergic system by
pharmacological agents that influence the autonomic nervous
system. The cholinergic agonist, carbachol, caused a dose-
dependent increase in the rate of CSF formation up to
10.6 ± 2.1 yl/min that was blocked by atropine. Further
evidence for cholinergic control of secretion is found in
the phenylephrine data. This alpha adrenergic agonist
also increased CSF formation by 10.6 ± 1.5 yl/min; this
increase was blocked by both phentolamine and atropine.
These data suggested that the alpha adrenergic agonist
probably was stimulating a cholinergic pathway to release
acetylcholine to increase CSF production. Since hemi-
cholinium blocks the uptake of choline for the synthesis
of acetylcholine (Gardiner, 1957), hemicholinium was given
prior to an infusion of phenylephrine. The infusion of
phenylephrine without hemicholinium increased the
production rate to more than twice that following a
single injection of the drug. However, when the animal
was pretreated with the hemicholinium, the infusion of
phenylephrine was not able to increase fluid production
70

71
in four of six animals. These experiments confirmed that
the increase in CSF formation caused by phenylephrine
depends on the synthesis and release of acetylcholine.
This relationship is depicted in Figure 11 which shows,
alpha adrenergic receptors that may or may not be supplied
by a neuron. The failure of hemicholinium in the other
two animals might be explained by larger stores of acetyl¬
choline or particularly high levels of choline. An
inadequate concentration of hemicholinium is probably not
a cause for this lack of effect.
A marked similarity exists among several secretory
systems with respect to cholinergic function and parasym¬
pathetic innervation. Cholinergic stimulation of the
salivary glands causes a profuse secretion (Koelle, 1970).
Parasympathetic innervation for these glands originates
from the inferior and superior salivary nuclei of the
brainstem (Mitchell, 1953). Secretion by the pancreas
and by the stomach can also be enhanced by cholinergic
agonists (Koelle, 1970). Both of these tissues derive
their parasympathetic innervation from the dorsal vagal
nucleus which is located immediately adjacent to the
salivary nuclei in the brainstem (Mitchell, 1953). The
production of aqueous humor by the ciliary body is also
stimulated by cholinergic agents (Macri, 1971). The
source of the cholinergic innervation in this tissue is
not defined. Increase in aqueous humor formation is

Figure 11. Autonomic pathways to the choroid plexus I.
The effector site has a cholinergic receptor.
(++) indicates the relative effectiveness
of the activated receptor to increase CSF
production. The cholinergic nerve supplying
the choroid plexus, in turn, can be stimulated
by alpha adrenergic agonists. The location of
the alpha adrenergic receptor remains undefined.

EFFECTOR SITE
+ +
CHOLINERGIC
RECEPTORS
ALPHA
ADRENERGIC
RECEPTOR

74
apparently mediated by a preganglionic cholinergic
stimulation of an adrenergic effector (Maori, 1971).
The origin of the cholinergic nerve supply to the
choroid plexus is unknown. Early anatomical descriptions
of nerves in the choroid plexus suggested that the inner¬
vation might originate in the dorsal vagal nucleus of the
brainstem (Benedikt, 1874; Stohr, 1922; Clark, 1932).
Although there are no data confirming that these are
cholinergic nerves, they arise in the same area responsible
for parasympathetic-stimulated pancreatic and gastric
secretion. This area is immediately adjacent to the
superior and inferior salivary nuclei (Mitchell, 1953).
In spite of the proximity of origin to other secretory
nuclei, one major observation thus far marks the cholin-
ergic-stimulated production of CSF as unique among cholin¬
ergic secretory systems: the activation of cholinergic
pathways by alpha adrenergic agonists.
As in most secretory systems, for the choroid plexus
it is unknown whether the cholinergic effector site is
vascular or epithelial. Cerebral blood vessels receive
some cholinergic innervation from the facial, glosso¬
pharyngeal, and vagal cranial nerves (Mitchell, 1953)
and from pathways arising within the brain (Edvinsson,
1975). Innervation from within the central nervous
system has also been shown for the blood vessels and the
epithelium of the choroid plexus (Clark, 1928 and 1932).

75
The role of the sympathetic nervous system in the
production of CSF is complex. The action of the alpha
adrenergic agonist, phenylephrine, in stimulating fluid
formation through a cholinergic pathway (Figure 11) has
already been discussed. The beta2 agonist, salbutamol,
also caused a dose-dependent increase in CSF formation;
however, the maximum effect was an increment of 5.7 ± 1.4
yl/min which is approximately half of the cholinergic
response. The action of salbutamol appeared to result from
a direct stimulation of beta receptors at the effector
site (Figure 12) since neither atropine nor phentolamine
influenced its effect, whereas the general beta blocking
agent, propranolol, did reduce the salbutamol effect.
Beta-j_ receptors are apparently absent in the choroid plexus,
for ITP did not influence production at all.
Since norepinephrine is present in the choroid plexus
and disappears after cervical sympathectomy (Edvinsson
et al., 1972b; Edvinsson et al., 1974), alpha adrenergic
agonists may act at the choroid plexus to influence CSF
production directly. There are two observations that
suggest alpha adrenergic agonists may decrease CSF
production. In this study, bilateral cervical sympathetic
stimulation reduced the rate of CSF formation 3.0 ± 0.6
yl/min. In addition, when norepinephrine is in the
perfusate during ventriculocisternal perfusion, CSF
production is decreased approximately 24% (Vates et al.,

Figure 12. Autonomic pathways to the choroid plexus II.
In addition to the cholinergic pathway, the
effector site also has a beta2 adrenergic
receptor. (+) indicates that activation of
these receptors also increases CSF production,
but to a lesser extent than cholinergic
stimulation.

77
EFFECTOR SITE
+ +
4*
CHOLINERGIC
RECEPTOR
BETA2
ADRENERGIC
RECEPTOR
ALPHA
ADRENERGIC
RECEPTOR

78
1964) . A proposed alpha adrenergic pathway, completing
the autonomic innervation of the choroid plexus, is
depicted in Figure 13. However, any direct decrease an
alpha adrenergic agonist might cause at the choroid plexus
would probably be masked by the simultaneous stimulation
of production by the cholinergic pathway as described
above. Since atropine itself affects the rate of CSF
formation, no attempt was made to block the cholinergic
receptors and then study the alpha adrenergic system.
Although the sympathetic nervous system has a
greater effect on fluid production in other secretory
systems than it has on CSF formation, the actions are
variable depending on the tissue. Alpha adrenergic
agonists increase aqueous humor production by the ciliary
body through an increase in arterial blood pressure (Maori
et al., 1974). Beta2 agonists, on the other hand,
decrease aqueous humor formation (Langham, 1976). In the
salivary gland, both alpha and beta adrenergic agonists
increase fluid production (Emmelin, 1967). However, the
two systems influence secretion in different ways:
alpha adrenergic stimulation causes a secretion that is
watery and contains high potassium, whereas beta stimu¬
lation causes secretion that is viscous and contains
digestive enzymes. In the pancreas both norepinephrine
and epinephrine decrease secretion; specific beta agonists
such as isoproterenol have not been tested (Thomas, 1967).

Figure 13. Autonomic pathways to the choroid plexus III.
An alpha adrenergic receptor may also be at
the effector site in addition to the
cholinergic receptor and beta2 adrenergic
receptor. (-) indicates that this pathway
decreases the production of CSF.

80
EFFECTOR SITE
+ +
CHOLINERGIC
RECEPTOR
V
beta2
ADRENERGIC
RECEPTOR
ALPHA
ADRENERGIC
RECEPTOR

81
These secretory tissues derive their alpha adrenergic
innervation through the ganglia of the sympathetic chain
as does the choroid plexus.
The relatively minor physiological role of the
autonomic nervous system in the normal production of CSF
is best demonstrated by observing the effects of the
autonomic blocking agents. Atropine, the cholinergic
blocking agent, decreased the rate of CSF formation
2.6 ± 0.5 yl/min indicating the cholinergic system may
continually stimulate fluid secretion but only to the
extent of about 15 to 20% of the fluid produced. Phentol-
amine also reduced CSF formation. This decrease may be
occurring through alpha adrenergic blockade of the
cholinergic pathway since the change in CSF production
is approximately the same as that caused by atropine;
however, this is not confirmed. The beta adrenergic
system may have little direct function in the normal
secretory processes of the choroid plexus since propranolol
did not influence CSF production. Nevertheless, it is
possible that in unanesthetized animals the autonomic
nervous system may exert a greater influence on normal
fluid production than is observed here or may cause
temporary fluctuations in the rate of fluid formation.

82
Mechanism of Changes in CSF Production
by Autonomic Influences
In two previous investigations, epinephrine and
pilocarpine were shown to cause an increase in the flow of
CSF from the cisterna magna followed shortly by a reduction
in CSF outflow (Becht and Gunnar, 1921; Bhattacharya and
Feldberg, 1958). These authors suggested that epinephrine
and pilocarpine increased cerebral blood flow and the
resulting increase in brain volume forced subarachnoid
CSF out through the cisternal cannula without affecting
true CSF production. Increased volumes were also observed
in response to the autonomic agonists used in the study
presented in this dissertation. The increased rate of
outflow during drug treatment averaged approximately
35 yl/min over control for a three minute collection
period. If pressure on the subarachnoid fluid was a
partial cause of this phenomenon, the additional fluid
collected would have come first from the cisterna magna
and basal cisterns which contained the dye marker at the
same dilution as the control perfusate. Hence, any error
in the calculation of production changes as a result of
this would cause an underestimate of the dilution of the
inflowing perfusate and an underestimate of the actual
increase in fluid production. By measuring only volumes,
the early investigators could not reliably show whether
the agents actually increased fluid formation. The

83
increases in CSF production calculated from dye dilution
in this study indicate that autonomic agents do alter
fluid formation.
The increase in CSF production caused by cholinergic
and adrenergic agonists can be ascribed to two possible
mechanisms of action, depending on the effector site.
Either a change in the hemodynamic relationships or a
direct action on the secretory cells might result in
alterations in CSF formation by these drugs. An increase
in the supply of blood to the brain or choroid plexus
would provide more fluid to produce an ultrafiltrate of
plasma in the CSF. Such a transfer of plasma water would
increase CSF volume.
Peripheral blood pressure changes do not correlate
well with CSF production changes. Carbachol and salbutamol
decrease blood pressure; this could decrease the supply of
blood to the secretory tissue. Phenylephrine, on the
other hand, increases peripheral blood pressure which
might serve to increase blood flow to the brain and
choroid plexus and thus to increase the rate of CSF.
formation. However, this alpha adrenergic agonist also
increases blood pressure during treatment with atropine
and hemicholinium which block the increased CSF production
by phenylephrine.
Since blood pressure changes were measured periph¬
erally, choroid plexus and cerebral blood flow data were
considered necessary to determine possible vascular actions

84
of these agents at the site of CSF formation. The control
blood flow to the choroid plexus measured by microspheres
was 2.26 ± 0.28 ml/gm min. This flow rate is comparable
to 3.01 ml/gm min observed by Aim and Bill (1973) for the
choroid plexus using the same technique. It is also
similar to the estimates of choroid plexus blood flow by
Welch (1963) and Pollay et al. (1972). The flow to the
choroid plexus represents one of the greatest amounts of
blood per tissue weight delivered to any tissue. Carbachol
and phenylephrine increased mean plexus blood flow 0.81
and 0.77 ml/gm min, respectively. Salbutamol increased
blood flow to the choroid plexus 0.35 ml/gm min. The
effects of these drugs on blood flow are in about the
same proportions as their effects on CSF production.
However, none of these changes in choroid plexus blood
flow differ statistically from control flows because of
the wide variation in the changes. These data, along with
the data of Aim and Bill (1973) showing no effect on
plexus blood flow by sympathetic stimulation, seem to
indicate that the autonomic nervous system does not
significantly alter the blood flow to the choroid plexus.
Cerebral blood flow in the control experiments was
0.46 ± .04 ml/gm min. This value is approximately the
same as the flows measured for the dog (Marcus et al.,
1976) and sheep (Hales, 1973). There was no alteration
in brain blood flow after carbachol, phenylephrine, or

85
salbutamol. When Salanga and Waltz (1973) stimulated the
facial nerve, vasodilation occurred only if the nerve was
cut where it left the brainstem. This suggests that
sensory nerve fibers detecting cerebral vasodilation might
pass back into the central nervous system through the
facial nerve causing a reflex vasoconstriction. If such a
pathway does exist, it would also explain the ineffec¬
tiveness of salbutamol and carbachol to increase cerebral
blood flow. The inability of phenylephrine to alter
cerebral blood flow is probably due to its low efficacy on
alpha adrenergic receptors in cerebral blood vessels
(Duckies and Bevan, 1976). Blood pressure and blood flow
data seem to indicate that the autonomic treatments
performed in this study produce minimal effects on the
production of CSF by way of changes in blood flow. Also,
it is clear that any increase in the rate of outflow seen
during drug treatment was not mediated by a generalized
increase in blood flow to the brain. An increase in
choroid plexus blood flow could have forced additional
perfusate from the ventricles.
Verapamil and theophylline were used to investigate
two possible ways in which autonomic agents could influence
CSF production by direct action on secretory cells. The
presence of calcium is a requirement for cholinergic
stimulation of salivary secretion (Douglas and Poisner,
1963). If calcium influx has a role in increasing CSF

86
production stimulated by carba.ch.ol, then verapamil, which
blocks calcium channels (Kohlhardt et al., 1972), should
prevent the increase in fluid formation caused by the
cholinergic agonist. Verapamil has been shown to be
effective when administered on either side of the epithe¬
lium and was given intraventricularly (Bentley, 1974).
Verapamil did not affect either normal CSF formation or
fluid production stimulated by carbachol; therefore,
fluid secretion probably occurs through a mechanism other
than a calcium-induced activation.
Since the effects of both cholinergic and adrenergic
neurotransmitters may be mediated by changes in cyclic
nucleotide levels (Greengard, 1976), an increase in
guanosine 3',5' cyclic monophosphate (cGMP) or adenosine
3',5' cyclic monophosphate (cAMP) might be the means by
which carbachol, phenylephrine, and salbutamol increase
the production of CSF. Theophylline, which inhibits the
destruction of the nucleotides by phosphodiesterase among
other actions, was used to enhance the levels of the
cyclic nucleotides. These experiments showed that
theophylline caused a significant dose-dependent increase
in CSF formation. The higher dose increased fluid
production 9.2 ± 1.4 yl/min which is approximately equal
to the maximum effect of the autonomic agonists. Unfor¬
tunately, theophylline is not totally specific for either
cGMP or cAMP phosphodiesterases (Amer and Kreighbaum,

87
1975), so it is not possible to show whether the effects
mediated by cyclic nucleotide action are cholinergic,
beta2 adrenergic, or both. Whereas the theophylline
experiments suggest that increases in cyclic nucleotide
levels are responsible for the increases in fluid
production elicited by carbachol, phenylephrine, and
salbuatmol administration, they do not provide conclusive
evidence that enhancement of the cyclic nucleotide
concentrations takes place in choroid plexus epithelia.
However, the relatively large blood flow to the choroid
plexus does not significantly change during treatment
with any of the autonomic agents that increase CSF
production. Apparently, the blood flow only provides
fluid and electrolyte substrate for the secretory
processes of the choroid plexus epithelium. The data
strongly imply that autonomic agents influence CSF
production by causing changes primarily at the choroid
plexus epithelium.
The changes in CSF production mediated by the
epithelial cells are probably a result of alterations in
the transport of electrolytes. Three pathways have been
demonstrated for the secretory transfer of electrolytes
in the CSF: the catalysis of bicarbonate formation by
carbonic anhydrase, the transport of cations by the
ouabain-sensitive sodium-potassium ATPase, and the
probable transport of chloride by an ethacrynic acid-
sensitive pump. Inhibition of any of the three mechanisms

88
has been shown to decrease the rate of CSF formation.
Inhibition of carbonic anhydrase by acetazolamide,
benzolamide, or methazolamide reduces fluid production
approximately 50 to 70% (Pollay and Davson, 1963; Davson
and Segal, 1970; Broder and Oppelt, 1969; and this study).
That this effect involves the movement of bicarbonate ions
into CSF was first demonstrated by Maren and Broder (1970)
and further quantified by Vogh and Maren (1975) . Ouabain
decreases the production of CSF 50 to 60% by its inhibition
of sodium-potassium ATPase (Vates et al., 1964; Davson and
Segal, 1970; Garg and Mathur, 1975). A 50 to 60% reduction
in CSF formation is seen with ethacrynic acid (Domer,
1969; Miner and Reed, 1971) and furosemide (McCarthy and
Reed, 1974). Active transfer .of chloride ions inhibited
by ethacrynic acid and furosemide has been described for
the ascending limb of Henle's loop in the kidney (Burg,
1974), and thus may be presumed to occur in CSF secretion.
The possibility of an active chloride pump in the choroid
plexus is supported by the findings that the chloride
concentration in the CSF can be maintained during reduced
plasma levels of the ion (Abbott et al., 1971; Bourke
et al., 1970).
Assuming that changes in cGMP are associated with
cholinergic responses as in other tissues (Greengard,
1976), levels of cGMP may be increased at the critical
site for CSF production by cholinergic agonists or by
alpha adrenergic stimulation of the cholinergic pathway

89
and decreased by inhibition of the cholinergic pathway.
Concentrations of cAMP, on the other hand, generally
change in response to agents that affect the adrenergic
receptors. Therefore cAMP may be increased by beta2
stimulation and decreased by direct activation of the
alpha adrenergic receptors. The cholinergic pathway
appears to predominate over the direct adrenergic pathway
since activation of the cholinergic receptors causes a
greater increase in CSF production, and inhibition of
cholinergic receptors, but not beta2 adrenergic receptors,
causes a decrease in CSF formation. Any increase in
concentration of either cyclic nucleotide is presumed to
activate a protein kinase system which stimulates the
secretion of electrolytes and water through one of the
possible transport pathways.
The transport mechanism that the autonomic nervous
system affects through the cyclic nucleotides is still
unknown. The cyclic nucleotides may be activating one
or more of the transport systems described or systems
not yet discerned. However, some data on this matter
were collected in this study for the carbonic anhydrase
system. During carbonic anhydrase inhibition, carbachol
and phenylephrine increased CSF production approximately
the same amount as when the drugs were given alone. This
would support the possibility that autonomic agents acted
through a separate mechanism. On the other hand, although

90
atropine and phentolamine decreased CSF formation when
given alone, neither caused a further reduction in
combination with methazolamide. This would seem to
indicate that the pathway for carbonic anhydrase inhibitors
and inhibitors of the cholinergic pathway are the same or
overlapping. However, it should be noted that the dose of
methazolamide given decreased the rate of CSF production to
about 8 yl/min. This is probably the maximal reduction
possible since none of the agents that have been tested
can decrease the rate of formation below 6 to 9 yl/min
in cats (Vates et al., 1964; Garg and Mathur, 1975). This
baseline production may be accounted for by diffusion
across the ventricular ependyma (Milhorat, 1972). If
submaximal doses of a carbonic anhydrase inhibitor a.re
combined with an inhibitor of another transport system,
additive effects in reducing CSF formation might be seen
down to 6 to 9 yl/min (Garg and Mathur, 1975).
Regardless of which transport system(s) the
autonomic nervous system control(s) through cyclic
nucleotide levels, the role of the nucleotides is probably
to serve as activators for the enhancement of fluid
secretion above normal. The reduction in CSF production
by atropine, phentolamine, or cervical sympathetic
stimulation was only 15 to 20% of control, indicating a
relatively minor role in the normal function of the
transport system. This is especially small when compared
to the ability of specific transport inhibitors to decrease

91
the rate of CSF formation by 50 to 70%. On the other
hand, the autonomic agonists and theophylline were shown
to increase CSF production from 15 to 20 yl/min to 25 to
30 yl/min, an increment of 50 to 70% of control.
Practical Significance
These experiments have suggested that the changes
mediated by the autonomic agonists probably result from a
direct activation of the choroid plexus epithelium. They
have also disclosed a unique system in which alpha adren¬
ergic agents stimulate a cholinergic pathway to increase
fluid formation. This study does not resolve the question
of how CSF is secreted or even exactly how the autonomic
agents influence fluid formation; however, the data do
contribute to the understanding of a very complex
secretory system.
The physiological significance of autonomic function
in the production of CSF remains uncertain. The basal
rate of CSF formation may be partially dependent upon
autonomic nervous activity. Receptors for autonomic
agonists may play an important role in bringing about
fluctuation in CSF production; however, the value of
such fluctuations is not clear at this time.
One of the original goals of this study was to seek
an agent that acted through the autonomic nervous sytem
to decrease the production of CSF. Such an agent might

92
then be used alone or in addition to carbonic anhydrase
inhibitors in the therapy of hydrocephalus caused by an
increased secretion of CSF or as an adjunct to the
treatment of obstructive hydrocephalus. Failure to
achieve additive effects from methazolamide and blockade
of the cholinergic pathway does not necessarily mean that
autonomic agents have no role in the treatment of hydro¬
cephalus. In this pathologic situation, reduction of
cholinergic stimulation to production may be highly
beneficial if this stimulation is excessive. Furthermore,
if plasma catecholamines are elevated during increased
intraventricular volume as may be suggested by the obser¬
vation that hypertensive cerebrovascular disease sometimes
accompanies normal pressure hydrocephalus (Earnest et al.,
1974), an alpha adrenergic receptor blocking agent or
perhaps atropine may be useful in reducing new CSF
formation.
Although the physiological and clinical significance
of the data presented in this dissertation is uncertain,
this study does provide important basic information for
the design of future experiments on anatomical organi¬
zation of nerve pathways, on secretory mechanisms, and on
practical application in human disease.

APPENDIX I
Autonomic Innervation and Function
of Cerebral Blood Vessels
The innervation of cerebral blood vessels has been
identified according to sympathetic and parasympathetic
function. Forbes and Wolf (1928) demonstrated a sympa¬
thetic influence on cerebral blood vessels by stimulating
the cervical sympathetic trunk and observing a constriction
of pial vessels. More recently, investigators have used
fluorescence microscopy to confirm the presence of
adrenergic fibers to several pial vessels including the
circle of Willis, the anterior cerebral artery, and the
middle of cerebral artery (Falck et al., 1968; Nielsen and
Owman, 1967). Fluorescence disappears from these vessels
after a cervical sympathetic ganglionectomy. Adrenergic
endings on pial blood vessels have also been shown with
electron microscopy in which the sympathetic terminals
can be identified as containing small granular vesicles
(Iwayama et al., 1970; Nielsen et al., 1971). The small
parenchymal vessels within the brain, however, have an
alternative source of adrenergic innervation which
originates in the brainstem rather than the cervical
sympathetic chain. After a bilateral sympathectomy,

94
Hartman (1973) detected the continuing prescence of
dopamine-B-hydroxylase, the enzyme responsible for
norepinephrine synthesis, in the deep vessels of the brain
by immunofluorescence, and Edvinsson et al. (1973a) found
persistent fluorescence (indicating norepinephrine) in
the vessels supplying the inner structures. Raichle et
al. (1975) have suggested that these nerves arise from
the locus coelruleus since carbachol applied to the
structure bilaterally causes a significant decrease in
cerebral blood flow.
The parasympathetic innervation of the cerebral
vasculature is also well established. Forbes and Wolf
(1928) stimulated the vagus and observed cerebral vaso¬
dilatation. However, this was shown to be due to a
secondary activation of the facial cranial nerve in the
medulla (Chorobski and Penfield, 1932). The presence
of cholinergic fibers in pial vessels was confirmed by
acetylcholinesterase staining (Edvinsson et al., 1972a).
Electron microscopists have observed nerve terminals in
pial vessels containing small agranular vesicles and a
few large granular vesicles which are characteristic of
cholinergic fibers (Edvinsson et al., 1972a; Iwayama et al.,
1970; Motavkin and Osipova, 1973; Nielsen et al., 1971).
Edvinsson (1975) has also performed experiments in which
the.intracranial parts of the cranial nerves were cut,
and found no loss of acetylcholinesterase staining in

95
blood vessels occurring within two weeks. This seems to
indicate a source of cholinergic innervation other than
the facial nerve.
Some investigators have been able to show changes in
cerebral blood flow (CBF) by electrical stimulation of the
nerves supplying the blood vessels or by agents that affect
autonomic receptors. Most of the work investigating
sympathetic control of cerebral blood vessels has involved
stimulation of the cervical sympathetic trunk. Forbes
and Wolf (1928) first showed that sympathetic stimulation
constricted pial blood vessels. Later it was demonstrated
that the constriction could be abolished with cocaine, an
agent that prevents norepinephrine reuptake, or ergotamine,
an alpha adrenergic blocking agent (Forbes and Cobb, 1938).
More recently, an electromagnetic flowmeter at the internal
maxillary artery was used to measure a decrease in CBF
during sympathetic stimulation; this was blocked by
phentolamine (Lluch et al., 1975). D'Alecy and Feigl
(1972) also demonstrated a reduction in flow using a
flowmeter. On the other hand, Raper et al. (1972) could
not show any vasoconstriction with sympathetic stimulation,
and no change in CBF was observed with the microsphere
technique during cervical sympathetic stimulation (Aim
and Bill, 1973). Two studies have been found in which
norepinephrine was given. Meyer and Welch (1972) observed
a constriction of the cerebral vessels whereas Raper et al.
C1972) did not detect any change in vessel diameter.

96
Investigation of parasympathetic control of cerebral
blood vessels has been less extensive, but the investi¬
gators seem to be in agreement that vasodilation does
result from parasympathetic stimulation. After Chorobski
and Penfield (1932) demonstrated dilation of cerebral
vessels by stimulating the facial nerve at the geniculate
ganglion, it was discovered that atropine could block the
action (Forbes and Cobb, 1938). More recently, Salanga
and Waltz (1973) confirmed the increase in CBF with
stimulation of the facial nerve after the nerve had been
severed from the brainstem. Acetylcholine, administered
intravenously or intra-arterially, also increased cerebral
blood vessel diameter (Carpi, 1972).
In vitro studies have been useful in characterizing
autonomic cerebrovascular responses. Bevan and Bevan
(1973) found that the maximal contraction of the rabbit
basilar artery with electrical stimulation in vitro was
about 25% of the maximum for the ear artery. Alpha
adrenergic agonists have also been studied in isolated
vessels. Several investigators have demonstrated that
norepinephrine contracts cerebral vessels in vitro:
however, the response of large cerebral vessels to
norepinephrine is less than can be observed with other
agents such as serotonin (Nielsen and Owman, 1971; Toda
and Fujita, 1973; Dalske et al., 1974; Duckies and Bevan,
1976). Duckies and Bevan (1976) also noted that the

97
rabbit basilar artery showed a biphasic dose-response
constriction following alpha adrenergic agonists with
both phases having sigmoidal log dose-response curves.
One curve ranged from 10"^ to 10“^ M and reached a
plateau, then the other curve started at about 10~^ M
and reached a maximum at 10 M. In both sensitive
portions of the dose-response curve, phenylephrine showed
only one-tenth the potency of 1-norepinephrine. The
authors suggest that the response to the lower doses
represents the physiological response while the response
to higher doses represents the activation of a non-
adrenergic receptor that can be non-specifically elicited
by high concentrations of an adrenergic agonist. This
interpretation may also draw support from the observations
of Nielsen and Owman (1971) and Duckies and Bevan (1976)
that high concentrations of isoproterenol can contract
the vascular smooth muscle. The existence of beta
adrenergic receptors in the cerebral vasculature seems to
vary with species. So far the rabbit and the cat have
been tested with isoproterenol, and only the cat showed
a vasodilator response. When cat cerebral vessels were
contracted in vitro with serotonin, both isoproterenol
and tertbutaline relaxed the vessels, and the effect was
blocked by propranolol (Edvinsson and Owman, 1974).
_Q _ r
Acetylcholine (10 to 10 M) also relaxed cerebral
vessels contracted in vitro (Edvinsson, 1975).

APPENDIX II
Composition of Artificial Cerebrospinal Fluid
[modified from Vates et al . , (1964)]
Salt
mM
NaCl
130.0
KC1
2.5
MgS04
1.0
NaH2P04
0.5
Na2HP04
0.5
CaCl2
2.5
NaHC03
20.0
2.5 mg/ml of blue dextran was added immediately
prior to each experiment
98

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BIOGRAPHICAL SKETCH
Joseph Roscoe Haywood was born on May 29, 1949 in
Columbus, Ohio. He attended the Wilmington, Ohio City
School System and graduated from Wilmington Senior High
School in 1967. He continued his education at the Univer
sity of Kentucky in Lexington, Kentucky where he received
a Bachelor of Science degree in zoology in 1971. That
same year he started graduate work in the Thomas Hunt
Morgan School of Biological Sciences at the University
of Kentucky and obtained a Master of Science degree in
zoology in 1972. The author continued his graduate study
leading to the degree of Doctor of Philosophy in the
Department of Pharmacology and Therapeutics at the
University of Florida. He is married to the former Carol
Louise Kouka of Western Springs, Illinois, and they have
two children, Rebecca Laurice and Alyson Michelle.
108

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality
as a dissertation for the degree of Doctor of Philosophy.
Betty P. Vogh, Chairperson
Associate Professor of Pharmacology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality
as a dissertation for the degree of Doctor of Philosophy.
JO
^ILl
too
C.Y. Chiou
Associate Professor of Pharmacology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality
as a dissertation for the degree of Doctor of Philosophy.
Carl Feldherr
Associate Professor of Anatomy

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality
as a dissertation for the degree of Doctor of Philosophy.
V3^
David Silverman
Associate Professor of Pharmacology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality
as a dissertation for the degree of Doctor of Philosophy.
David M. Travis
Professor of Pharmacology
This dissertation was submitted to the Graduate Faculty of
the College of Medicine and to the Graduate Council, and
was accepted as partial fulfillment of the requirements
for the degree of Doctor of Philosophy.
October, 1976
Dean, College of Medicine
Dean, Graduate School

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