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This is to certify that the
thesis entitled
ELECTRICAL AC”IVI‘TY OF SUPiiAO PTIC
NEUBONS I N HATER DEPRIVED RATS
DUhING SLO N I NTdAGAS'THI C IlQF‘USI ON ‘3
OF WATER
presented by

Charles Thomas Bennett

has been accepted towards fulfillment
of the requirements for

Ph. D . degree inPsycholoqy

@JW

Date Q/J! // 7/
/ 7

0-7639

 

 

 

 

.3
as
(\5

ABSTRACT
ELECTRICAL ACTIVITY OF SUPRAOPTIC
NEURONS IN WATER DEPRIVED RATS
DURING SLOW INTRAGASTRIC INFUSIONS
OF WATER

By
Charles Thomas Bennett

It is known that the supraOptic nucleus (SON) pro-
duces at least the humoral precursor of vaSOpressin, the
antidiuretic hormone (ADH). The nature of the relationship
between electrical activity of SON neurons to changes
in concentration of body fluids has not been clearly de-
fined. Therefore, to more precisely understand this relation-
ship, changes in activity of neurons in the SON and anterior
hypothalamus (AH) of rats were monitored during changes
in tonicity of body fluids. In EXperiment I, rats were
placed on a 23.5 hr water deprivation schedule for five
days. On the fifth day, before access to water, the rats
were anesthetized and prepared as if for unit recording.

They then received a 10 cm3 gastric water load at 1 cmB/min.

This rate is similar to the rate of water ingestion of

awake rats under similar dehydration conditions. At four min-
ute intervals for 2h min from the beginning of the water

load plasma osmolality (Poem)! plasma volume (Pv). and water

absorption were measured. It was found that by 11 min of ab-

sorption time Posm decreased significantly 3%, while 2.5 cm3

of water had been absorbed. Pv. on the other hand, did not

 

 

Charles Thomas Bennett
change until 18 min of absorption time.

In Experiment II, rats were treated Just as those were
in the previous study, but during the water load, unit activity
in the SON and AH were monitored. Following the stomach
load in some rats, a 1 cm3 16% NaCl injection was administer-
ed subcutaneously (SC). The following changes in unit
activity were observed:

SON unit activity was found to be significantly

faster in the water deprived rat, as compared to rats on an

 

ad libitum food and water schedule.

A transient, short latency increase in SON unit
activity was observed in response to initial filling of the
stomach with water. Following this, when Posm began to
significantly decrease, unit activity in the SON began to
also significantly decrease below baseline levels.

In response to a SC hypertonic saline injection, a
short latency, transient increase in SON unit activity was
seen. This increase occurred during a time period when awake
rats, treated in a similar fashion, exhibit a marked pain
reaction. By seven minutes post-injection, when Posm has
been shown to significantly increase, SON unit activmy became
significantly faster.

AH cells, treated in the same fashion, did not show
similar changes in unit activity in response to rapid
changes in Posm° This is true depsite the fact that fol-
lowing a period of water deprivation, unit activity in the AH

was significantly faster than in animals on an ad libitum

Charles Thomas Bennett

feeding and drinking schedule.

If (a) the initial short latency, transient changes
in SON unit activity following the water load or injection
can be attributed to peripheral stimuli, and, if (b)
the secondary changes in unit activity can be attributed

to shifts in P then this is apparently the first reported

OSIII'

evidence for convergence of stimuli that are potent releasers

of ADH.

 

ELECTRICAL ACTIVITY OF SUPRAOPTIC

NEURONS IN WATER DEPRIVED RATS

DURING SLOW INTRAGASTRIC INFUSIONS
OF WATER

By
Charles Thomas Bennett

 

A Thesis

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Psychology

1971

 

Acknowledgments

I want to express deep appreciation to Dr. Glenn
I. Hatton who guided my graduate training.

I wish to also acknowledge Dr. John I. Johnson,

Dr. Lawrence I. O'Kelly, and Dr. Ralph Pax who helped direct
this thesis.

Important to the completion of this thesis was the
assistance of the Department of Psychology and the lab-
oratory for Comparative Neurology, which supplied necessary
photographic and data analysis equipment.

I want to thank Mrs. John Haight for histological
assistance.

I want to express special thanks to Diane Bennett who
spent many late hours typing and doing the basic statistics.

This thesis was completed while the author was supported
by a predoctoral fellowship from the National Institute
of Mental Health, no. MH uau7u. The research was supported by
a grant from the National Institute of Neurological Dis-

eases and Stroke, no. NS O91h0 to Dr. Glenn I. Hatton.

11

 

 

 

 

TABLE OF CONTENTS

Page
ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . ii
LIST OF TABLES . . . . . . . . . . . . . . . . . . iv
LIST OF FIGURES . . . . . . . . . . . . . . . . . . v
LIST OF APPENDICES . . . . . . . . . . . . . . . . . ix
INTRODUCTION . . . . . . . . . . . . . . . . . . . i

fiperiment 1 O O O 0 O O O 0 O O O I O O O O 0 O O

 

MethOd o o o o o o c o o c o c o c o o o o o 9
Subjects . . . . . . . . . . . . . . . . . . . 9
Procedure . . . . . . . . . . . . . . . . . . 9
Results 0 O O O O O O O O 0 O O O O O O O O O 1 3
Discussion . . . . . . . . . . . . . . . . . . 13

Experiment 2 O O O O O I O O O O O O O O O O O O 0

Method .

Subjects . . . . . . . . . . . . . . . . . . . 25
Procedure . . . . . . . . . . . . . . . . . . 26
Results . . . . . . . . . . . . . . . . . . . 31
Discussion . . . . . . . . . . . . . . . . . . 31

RWERWCES O O O 9 O O O 0 O I O O O O O O O O O O 83
APPENDICES O O O O O O O O O O O O 0 O O O 0 O O O 88

iii

 

Table

1.

LIST OF TABLES

Page
Amount of water remaining in the stomachs
and small intestines of the animals in
fipel‘lment I. c c o o o c o o o o 114'
Description of treatment conditions. . . . . 29

iv

 

 

118T OF FIGURES

Figures

1.

Indicated are the mean (iS.E.) amount of
water absorbed as a function of minutes.

See text for an explanation of the

double abscissa. Nzh per point. . . . . .

Indicated are the mean (iS.E.) levels of
plasma osmolality for the different

groups as a function of minutes. See

text for an explanation of the double
abscissa. Ngh per point. . . . . . . .

Indicated are the mean (iS.E.) percent
changes in heart rate for the various

groups. Change in heart rate for each

rat was calculated from their respective

mean baseline rates. See text for
eXplanation of the double abscissa.

N=u per point. . . . . . . . . . .

Indicated are the mean (iS.E.) plasma

protein concentrations for each group as

a function of minutes. See text for an
explanation of the double abscissa.

Ngh per point. . . . . . . . . . .

Indicated are the mean (¢S.E.) percent
changes in heart rate for six animals
selected at random from the Load-In-

jected Group. During the stomach load,

change in heart rate for each rat was
calculated from their respective mean
baseline rates. During the injection,

the change was calculated from each rat's
mean minute rate for the last minute of

the load period. N=6. . . . . . . . .

Indicated are the mean (iS.E.) percent
changes in heart rate per ten seconds in

the first minute of the stomach load. The
animals are the same as those used in

Figure 5. N=6. . . . . . . . . . .

 

Page

16

19

21

2h

33

35

 

 

Figures

7. Indicated are the mean (iS.E.) percent
changes in heart rate per ten seconds
for the first three minutes following
the saline injection. The animals are
the same as those used in Figure 5. N=6. .

8. In panel A are indicated the mean (iS.E.)
spikes per second per minute for 12 SON
cells recorded from water deprived rats
(C)--()), and, 7 SON cells recorded from
rats on an ad libitum food and water sched-

ule (IF-CDL. . . . . . . . . . .

9. Indicated are the mean (iS.E.) spikes
per minute for five AH cells (H)
and two SON cells ( C)-<D). These
cells were recorded from rats that had
been water deprived. . . . . . . .

10. In panel A are indicated the mean (iS.E.)
spikes per second for 5 AH cells in the
Load Only Group as a function of min-
utes. The animals from which these cells
were recorded had been water deprived and
then received a 10 gm gastric water load
at the rate of 1 cm /min.

In panel B are indicated the mean (iS.E.)
spikes per second for 1+ SON cells. The
animals from which these cells were re-
corded were treated as those in panel A. .

11. In panel A are indicated the mean (iS.E.)
spikes per second for 17 AH cells as a
function of minutes. These cells were re-
corded from animals that had been water
depriged. As indicated, they received a
10 cm gastric load of water and a 1 cm3
16% NaCl subcutaneous injection. . . . .

12. Indicated are the median (Q and Q2) per-
cent changes of 9 of the cells shown in
Figure 11. These 9 cells showed a sig-
nificant decrease in firing rate during
the load period. Each cell's percent
change during the load was calculated
on the basis of each cell's respective
mean baseline rate. Percent change
following the injection was calculated

vi

Page

37

#0

“3

“6

49

 

Figures Page

from each cells mean firing rate
per minute during the last minute of
the load period. . . . . . . . . . . . . . . 52

13. Indicated are the mean (iS.E.) spikes
per second per ten seconds for the first
minute following the beginning of
the water load (panel A) and injection
(panel B). These are the same cells
that are depicted in Figure 11. . . . . . . . . 54

1h. Indicated are the mean (iS.E.) percent
changes in firing rate per ten seconds
for the first minute following the beginning
of the water load (panel A) and in-
jection (panel B). These are the same
cells depicted in Figure 13. . . . . . . . . 57

 

15. Indicated are portions of diagrams of
a rat brain at de Groot planes 6.6-8.2
AP. The black circles indicate location
of marking lesions which indicate that
the cell being recorded from was probably
a 30! neuron. The cpen circles indicate
marking lesions at the various other
recording sites. All animals from which
these cells had been recorded were
of the Ad Libitum Group. . . . . . . . . 59

16. Indicated are the location of marking
lesions in diagrams of a rat brain at
de Groot planes 7.0-8.2 AP. Black circles
indicate location of recordingsites
that are probably within the SON. Open
circles indicate other recording sites.
Animals from which these cells had
been recorded were in the Deprived
Group. . . . . . . . . 61

17. Indicated are diagrams of a rat brain
at de Groot planes 6.6-7.8 AP. Black
circles indicate marking lesions of
recording sites probably with in the
SON. Open circles indicate other re-
cording sites. All animals from which these
cells were recorded were in the Load
Only Group. . . . . . . . . 63

vii

 

 

 

Figures Page

18. Indicated are diagrams of a rat brain
out at de Groot planes 7.0-8.2 AP.
Black circles indicate probable SON
recording sites. Open circles indicate
neurons which were recorded from in
areas outside the SON. Animals from
which these cells were recorded were
in the Load Injected Group. . . . . . . . . 66

19. Indicated are the mean (iS.E.) percent
changes in firing rate for 13 minutes
following the beginning of the water
load. These are the same cells that
are depicted in Figure 11b. . . . . . . . . 71

20. Shown is a photograph of a frontal
cut of a rat brain embedded in cel-
loiden and stained in thionin. Indicat-
ed is the supraoptic nucleus (SO),
suprachiasmatic nucleus (SC), and the
Optic chiasm (0C). . . . . . . . . 98

 

21. A. Shown is a photograph of a frozen
section of a rat brain stained in
thionin and cut in the same plane as
Figure 20. Encroaching on the SO is
a marking lesion indicating a re-
cording site.

B. Shown is a photograph of a frozen

section of a rat brain stained in

thionin and cut in the same plane as

Figure 20. Encroaching on the SO is

another marking lesion indicating

a recording site. . . . . . . . . 100

22. A. Shown is a photograph of a frozen
section of a rat brain stained in
thionin and cut in the same plane
as the previous photographs. En-
croaching on the SO is a marking
lesion.

B. Shown is a photograph of a

frozen section of a rat brain

stained in thionin and cut in the

same plane as Figure 20. Seen is

a marking lesion located in the

Preoptic area. . . . . . . . . 102

viii

LIST OF APPENDICES

Page
APPENDIX A: Apparatus . . . . . . . . . . . . . . 88
APPMDIX B3 83" Data 0 e e o e e e e e e e e e e 90

 

ix

 

 

 

INTRODUCTION

Ingestion and excretion of water are two primary con-
trol mechanisms most mammals employ to insure a proper
water balance. When water is freely available, animals
usually have few problems maintaining normal hydration levels.
To do this, the volume of water lost must equal, for the
most part, the volume of water ingested. But, if ingestion is
restricted for any reason, the amount of water lost may
exceed the ingested volume (Chew, 1965). As a result, an
animal incurs a negative water balance, with a consequent
increase in the tonicity of body fluids, and a decrease in
their volume (Wolf, 1958).

To maintain life, it is imperative that an animal be
able to limit these changes. One way to do this is by the
conservation of body water. While there are several ways an
animal can conserve body water, one primary method is to
reduce renal water loss.

There are two hormonalsystems which can limit the
amount of water excreted by the kidney. However, these hor—
mones are released as the result of essentially different
stimuli. As renal blood pressure decreases (which can be a
consequence of the decrease in blood volume) aldosterone is

released. This hormone promotes the renal retention of sodium.

Then, as blood sodium levels increase, water tends to be
retained in the body (Pitts, 1968). But also, as a result

of excessive water loss, blood tonicity increases. This

can result in the increased circulating titers of vaSOpressin,
or the antidiuretic hormone (ADH). This hormone has the
effect of increasing the reabsorption of water in the prox-
imal tubules of the kidney (Pitts, 1968).

The control of the release of either aldosterone or
ADH is not completely understood. Of special interest here,
however, is the regulatory system controlling vascular ADH
titers. It is believed that there are two primary system
variables (blood volume and tonicity) that are potent stimu-
li for the release of ADH. Changes in the former are detect-
ed by baroreceptors in the carotid arteries and left
atrium (Share, 1968). As blood volume decreases these re-
ceptors mediate an increased release of ADH into the blood
(Bland, 1963). The exact neural pathways which subserve
this system have not been clearly established (Barker,
Crayton, and Nicoll, 1971).

It is believed that hyperosmolality of the blood will
result in increased release of ADH; and, that these changes
in blood tonicity are detected by osmosensitive neurons
located in the vascular bed of the carotid artery (Verney,
1947). There is some evidence to indicate that there may
also be hepatic osmosensitive cells important to the re-
lease of ADH (Haberich, 1965 and 1968; Lydtin, 1969). These

investigators have reported that as tonicity of the blood

 

perfusing the liver increases, there is an increased release
of ADH. How important the role of these latter receptors

are to the daily regulation of ADH secretion has not, however,
been established.

The importance of the hypothalamo-hypOphyseal system
to the elaboration of ADH had been established many years
ago (Fisher, Ingram, and Hanson, 1938; Harris, 19h7; Verney,
19h7). On the basis of data reported in these works, it
has generally been concluded that elements in the hypotha-
lamo-hypOphyseal system become activated as the result of
increases in effective osmotic pressure (EOP) of body
fluids. As this activation takes place, ADH (or, its humoral
precursor) is released into the blood.

Since that time, histochemical (Belislin, Bisset, and
Halder, 1967) and anatomical (Howe and Jewell, 1959) studies
have indicated that the supraoptico-hypophyseal system is
responsible for the production and release of ADH.

Early electrOphysiological investigations (Von Euler,
1953) indicated that the supraoptic nucleus (SON) might be
be detecting changes in EOP. His work indicated that there are
slow wave D.C. potential shifts following intacarotid (IC)
injections of hypertonic solutions. On the basis of these
"osmopotentials" he concluded that the SON might be a de-
tector component of the ADH release system.

A few years later, Cross and Green (1959) recorded from
single neurons in this area. They reported that cells in

the SON were osmosensitive (ie., the firing frequency of

 

certain cells changed with a short, reliable latency fol-
lowing an osmotic stimulus).
Since Cross and Green's now classic study, many in-

vestigators have substantiated the fact that at least some

 

of the cells in the SON are osmosensitive (Brooks, Koizumi,
and Zebalios, 1966:.Brookug Ushiyama, and Lange, 1962; Cross
and Silver, 1966; Dyball, 1969: Dyball and Koizumi, 1969:
Ishikawa, Koizumi, and Brooks, i966a; Ishikawa, Koizumi,

and Brooks, 1966B: Joynt, 196k; Koizumi, Ishikawa, and Brooks,

 

196“: Wang, 1969). However, not until recently (Dyball,
1969) has anyone attempted to correlate changes in SON
unit activity with the release of ADH. Dyball reported that
shortly after SON unit activity increased, there was an
elevation of ADH titers in jugular blood.

The fact remains, though, that none of the studies
cited above, in themselves, demonstrate that the SON
actually is a detector of osmotic pressure shifts. It could
be that activity changes in SON neurons, seen in response to
EOP shifts, are merely the result of influences impinging
on them from other neurons. If this is the case, the true
detectors cells are probably located within some subcortical
area. This notion has some support because isolated, de-
corticated brains seem capable of producing ADH in response
to EOP changes (Cross and Kitay, i967; Saito, Yoshida, and
Nakao, 1969: Suda, Koizumi, and Brooks, 1963; Sundsten and
Sawyer, 1961; Woods, Bard, and Bleir, 1966).

 

The problems of dennnstrating that the supraOptic nucle-
us is actually the detector component of this system
are enormous. Because the area of the hypothalamus is highly
vascularized (Haymaker, 1969), any attempt to surgically
isolate structures within it will disrupt the blood supply,

 

and increase the difficulty in interpreting the results.
As indicated above, it is debatable whether or not the
SON actually is an osmodetector. Despite this, and as in-

dicated earlier, the supraoptico-hypophyseal system seems

 

to be the effector organ of the ADH release system. However,
essentially all that is known about the electrOphysiological
preperties of this effector organ is that there are osmo-
sensitive neurons in it. This is true despite the fact that
there are at least three stimuli (blood volume, blood tonicity,
and pain, Pitts, 1968) which elicit the release of vasopres-
sin. Yet, if we are to better understand the neurophysiological
correlates of ADH release, we must know how cells in the
SON code the stimuli which impinge upon it: whether these
stimuli impinge directly on the neurons, or via nervous
pathways from other loci.

The purpose of this thesis, in part, is to determine
more precisely the manner in which SON neurons respond to
osmotic stimuli. To accomplish this, traditional methodol-
ogy could not be used. Apparently, without exception, in
every reported study dealing with the osmosensitivity of

SON neurons, osmotic stimuli (hypertonic solutions) have been

 

presented via intracortid (IC) cannulae. No one has ap-
parently even recorded from SON neurons in water deprived
animals. Further, no one has apparently monitored SON units
in water deprived animals as.they are being rehydrated by
means of oral water loads. And, apparently, no one has even
actually measured blood tonicity while concurrently recording
from cells in the hypothalamus.

If we are to understand the changes in SON unit activity
of awake animals, it would seem reasonable to monitor these
neurons under conditions which most closely approximate
those an animal might actually encounter. One of the pur-
poses of the thesis, then, is to determine changes in SON
unit activity of deprived animals, while they are being re-
hydrated with a gastric load of water.

It has been reported that, after a period of water dep-
rivation, rats, when given access to water, will drink at
the approximate rate of 2 cm3/min (Hatton and Bennett,

1970). This rate is, generally, maintained until almost 3 cm3
of water has been absorbed and plasma osmolality (Posm) has
decreased at least 1-2%. With this information, it is
possible to understand more clearly the relationship of SON
unit activity to changes in drinking behavior. For, if
changes in Posm and water absorption are being measured while
recording from SON units (as a water load is being given),
then the time relationship between the cessation of drinking

and changes in SON unit activity may be approximated.

 

 

Verney (1947) reported that an increase in diuresis
begins when concentration of body fluids decreases as little
as 1-2%. It would be expected, then, that similar changes
in P08m should result in both the cessation of drinking and
changes in unit activity of the SON. This hypothesis will

be tested in the present investigation.

Specifically, then, the purposes of this thesis are to
(a) establish whether there are consistent, correlated changes
in plasma osmolality and unit activity of the SON, and, (b)
determine the time relationship of these changes to the Posm
thresholds that are known to bring about the cessation of
drinking.

To accomplish this, in Experiment I animals were placed
on a water deprivation schedule. They were then anesthetized,
prepared as if for unit recording, and given a stomach
load of water at a rate similar to normal ingestion rates.
Changes in plasma osmolality, plasma volume (Pv), water
absorption, and heart rate were measured.

In Experiment II, SON units were monitored under these
same conditions. Some of these animals also received a
common thirst-producing stimulus, a subcutaneous hypertonic
saline injection.

These procedures (water load and saline injection)
bring about dramatic changes in blood tonicity. As a result,

only one cell was recorded from each animal. This procedure

has obvious limitations. But these are outweighed by the
fact that changes in unit activity could be compared to

changes in tonicity of body fluids that are similar to those
that might naturally occur.

 

EXPERIMENT I

INTRODUCTION

In this study, rats were placed on a water deprivation
schedule for five says. Before access to water on the
fifth day, they were anesthetized and prepared as if for
unit recording. They then received a gastric water load.
Changes in Posm’ on water absorption, and heart rate were
monitored.

With this data, some of the concommitant neural changes

were then examined in Experiment II.

METHOD

subjects

The animals used were 40 naive, male albino rats of
the Holtzman strain, 100-110 days old at the beginning of
the experimental treatments. They were housed in individual
cages under conditions of constant light and given Wayne
Mouse Breeder Blox ad libitum. While there was no humidity

control, temperature was maintained between 22—2500.

Procedure
After arrival into the laboratory, the rats were given

at least three days of free access to food and water in

 

 

 

10

their home cages. The animals were then placed on a 23.5 hr
water deprivation schedule. During their 0¢5hr access to
water, drinking bottles were placed on the rats' home
cages.

Before access to water on the fifth day, four rats were
randomly assigned to each of the following treatment con-
ditions:

Ten yin ngggg g §3222-~rats which were anesthetized
with Dial-urethane (Ciba). The anesthetic was given intra-
peritoneally (IP); the dosage being 0.7 cm3/kg plus a sup-
plementary dose of 0.02 cm3. In no case was any other sup-
plementary dose given.

Once anesthetized, a 2 cm3 heart blood sample was taken
from the left ventricle of their surgically exposed hearts.
Following this, stomachs, small intestines, caecum-colons
(taken as one) were clamped, removed, and stripped of excess
lipomal and mysenteric tissue. The organs were weighed wet,
then dried for approximately 2h hr at 100°C. This pro-
cedure has been shown to remove all tissue fluids (Bennett,
1969).

Immediately upon removal the blood was centrifuged at
3300rpm for h min. The plasma was then withdrawn, protein
concentration determined by refractometry, and the remaining
plasma sample was frozen in sealed glass vials for later

osmometric determinations in a freezing point osmometer.

.2 Min Group--rats which were anesthetized and then

 

11

prepared as if for unit recording. This preparation included
the following: (a) positioning of a PE 50 (Intramedic)

stomach tube, (b) positioning ~ sub-dermal cardiac electrodes,
the ground being attached to the stereotaxic apparatus, (c)
placing the rat in the stereotaxic instrument, (d) positioning

 

a rectal thermistor thermometer, (e) wrapping the animal with
a heating tape to maintain body temperature, (f) exposing
and cleaning the skull. Following these procedures, a ten

minute sample of heart rate was takenj To do this, cardiac

 

bioelectric activity was amplified through a low level pre-
amplifier, then displayed on an oscilloscope and recorded
on magnetic tape. (See Appendix A for a detailed description.)
When the baseline record of heart rate was taken, the animals
were treated as the 10 Min Pre-Load Group was.

.Thggg,4§, agd 9 Min Groups--rats which were treated
just like the 0 Min Group, except that 0.5 hr following the
surgical preparation for recording was completed, they re-
ceived stomach loads of 3, 6, and 9 cm3, respectively, of
tap water at room temperature. The load was delivered manually
at the rate of i cm3/min. Ten minutes prior to and then during
the load, heart rate was monitored and recorded on magnetic
tape. Body temperature was maintained throughout the pro-
cedures at 37i1°C° This was done through a regulated D.C.
power supply. Immediately following the load, the animals
were treated the same as the 10 Min Pre-Load Group.

Twelve, 15, ;§, 2;, and gngin Groups--rats which were

 

12

treated as the previous groups were, but received a 10 cm3
load of water delivered at the rate of 1 cm3/min. Then

12, 15, 18, 21, and 2h min, respectively, from the begin-
ning of the load, they were removed and treated like the
10 Min Pre-Load Group. Heart rate was also recorded 10 min

prior to and then during and after the water load.

 

Determination 22 water absorption. A detailed descrip-
tion is presented elsewhere (Bennett, 1969: Hatton and Bennett
1970). Essentially, the procedure was the following: Fluid

 

loss from the guts of the 10 Min Pre-Load Group provided

the amount of fluid in the tissues. Fluid loss from the guts

of animals which had been Stomach loaded provided the amount

of fluid in the tissues and lumen. Fluid loss from the guts

of animals which had been loaded, minus that from animals

that had not been loaded provided the amount of fluid in

the lumen of the intestinal tract. Therefore, the amount

of water absorbed is given by the following formula:
AJ=IJ-TFWJ

Where A=amount of water absorbed
I=amount of water loaded
TFN=amount of total water in the lumen of the small
intestine.
Determination 2; heart rate. Actual frequency of heart
rate was determined by an electronic counting system. (A

detailed description is presented in Appendix A.)

13

RESULTS AND DISCUSSION

.In Table 1 are shown the mean (iS.E.) amount of water
remaining in the stomachs and small intestines of the
various groups. Of interest here is that the maximum amount
of fluid remaining in the stomach does not occur until ap-
proximately the 9 min sampling.

Figure 1 shows the mean (¢S.E.) amount of water absorb-

ed as a function of time for the groups receiving the

 

stomach load. The double abscissa (in this, and the following
figures) is necessary because while a given animal was re-
moved at (for example) 9 min, and after 9 cm3 of water hat~
been loaded, approximately 2 min elapsed during which time
surgical procedures were performed. During these two minutes,
water that had been loaded could be absorbed and bring about
changes in blood tonicity.

The 3 Min Group had significantly (p(0.05) more water
in their intestinal tract than would be expected on the
basis of the 3 cm3 that had been loaded. These data in-
dicate that fluid had entered the intestinal lumen, either
from the peritoneal cavity or from the vascular system, or
both. By 6 min, the amount of water loaded equalled the amount
of water in the intestinal tract. This would mean that the
fluid that had entered the lumen previously had begun to
flux back into the blood and/or abdominal cavity.

Not until 9 min had a significant amount of water been

1h

Table 1

Amount of water remaining in the stomachs and small
intestines of the animals in Experiment I.

Group Amount in Stomach Amount in Small intestine
(cm3) (cm )

3 Min 3.910.17 1.7io.54

6 Min 3.310.33 1.810.69

9 Min 6.Qio.u8 1.910.68
12 Min u.3:o.68 2.610.36
15 Min 5.2io.8h 2.310.76
18 Min 6.2i0.82 2.410.50

21 Min 3.7io.72 2.1io.69
2n Min 3.610.82 1.7¢O.68

 

15

Figure 1.

Indicated are the mean (iS.E.) amount of water absorbed
as a function of minutes. See text for an explanation
of the double abscissa. N=h per point.

 

16

 

 

 

 

6..
5' —WATER LOAD——-l | 4)
/
o 4. (i)
g I I
a: 3‘
a)
2 2' l I
as " I g,
I
2 C o I
3 I
8 -|-I I
.1'2‘ (F
2
'3‘ l 1 1 :
3 6 9 I2 I5 |8 2| 24
MINUTES
5 8 II I4 I? 20 23 26

EFFECTIVE ABSORPTION TIME

 

 

17

absorbed. However, by the 18 min sampling, significantly
more water was in the intestinal tract than at either the
9 or 12 min sampling. The implication is that fluid again
began to flow back into the small intestine. Shortly after
this, as indicated by the 21 min sampling, water again began
to be absorbed into the blood.

Figure 2 shows the mean (iS.E.) P08m levels as a function
of time before, during, and after the stomach load of water.

Though there is a slight decrease in Posm by 5 min of ef-

 

fective absorption time, no significnat decrease in plasma
concentration occurred until 11 min. By that time, P08m had
decreased approximately 3%. There was then no statistically
significant change until 24 min following the beginning of

the load, when P again tended to decrease. The magnitude

cam
and time course of the first significant decrease observed,
here, is similar to that reported for awake, drinking rats
under similar hydration conditions (Hatton and Bennett, 1970).
Figure 3 shows the mean (iS.E.) change in heart rate as
a function of time during and after the load of water. There
is an initial small decrease in heart rate following the
beginning of the load. But, by the time the load is completed
(and, when plasma osmolality and stomach fluid volume de-
crease), heart rate returns to baseline levels.
Increases in heart rate during stomach loads have
been reported (O'Kelly, Hatton, Tucker, and Westall, 1965).

However, the loads in their study were given at a rather

rapid rate. But, despite this, 30 min following a 3% water

 

18

Figure 2.

Indicated are the mean (iS.E.) levels of plasma osmolality
for the different groups as a function of minutes. See
text for an explanation of the double abscissa. N=h per
point.

19

 

§

302 $\(

I D
297 \ I

292

 

 

287

N
(n
N

PLASMA OSMOLALI TY (wOsu/KG)
__C)\
/O
f"
70’
O\_
O.—

 

 

-IO 0 3 6 9 I2 l5 l8 2| 24
MINUTES
5 8 ll I4 I7 20 23 26
EFFECTIVE ABSORPTION TIME

20

Figure 3.

Indicated are the mean (iS.E.) percent changes in
heart rate for the various groups. Change in heart rate for
each rat was calculated from their respective mean baseline
rates. See text for explanation of the double abscissa.

N=h per point.

PERCENT CHANGE IN HEART RATE

21

 

 

 

 

 

 

 

 

 

 

 

 

   

4 WATER LOAD‘——-{
3
2 o
| MCREASE] / \O
o ....-.....".::_—_- ————:===.=-_-:=_-: .__ _—:_—.=- :
' [Because] 0\ /
2 ' | O/Q
3 9‘0
I

4 \I
5 T
6

3 6 9 I2 I5 I8 2| 24

MINUTES
5 8 II I4 I7 20 23 26

EFFECTIVE ABSORPTION TIME

22

load (comparable to the 10 cm3 load given here), heart rate
had returned to near baseline levels, as it did in this study.

Mean Pv changes (18.E.) as reflected by plasma protein
concentration are shown in Figure 4 during and after the
stomach load. The magnitude of change in plasma volume are
greater than those reported for awake, drinking rats under
similar dehydration conditions (Hatton and Bennett, 1970).
In addition, the absolute level of plasma protein concentra-
tion is lower than that reported for normal, deprived rats.
This difference in hemodilution might be attributed to the
effect of being under anesthesia for approximatley 45 min
Ibefore experimental treatments had begun.

Also, in the present study, Pv decreases occurred
severalminutes after the first significant change reported
by Hatton and Bennett. This could be caused by the initial

hemodilution that was observed in these animals.

 

 

 

23

Figure A.

Indicated are the mean (iS.E.) plasma protein concentra-
tions for each group as a function of minutes. See text for
an explanation of the double abscissa. N=b per point.

PLASMA PROTEIN (g/IOOmI)

6.5

6.0

5.5

2’4

 

l I l

 

6 9 l2 l5 I8 2| 24
MINUTES
8 II I4 I? 20 23 26

EFFECTIVE ABSORPTION TIME

 

 

25
EXPERIMENT II

INTRODUCTION

In the previous experiment, changes in Posm: Pv,
water absorption and heart rate were established in response
to a water load. This was done in rats prepared for unit
recording. In the present study, animals were treated as
those in Experiment I, but during the water loads, unit

activity in the SON was actually recorded.

 

Twenty-five minutes after the water load was begun, a
1 cm3 16% subcutaneous (SC) NaCl injection was administered
to some rats. Awake rats, under similar hydration con-
ditions, will drink in approximately 6 min following such an
injection, and, when Posm has increased 2-h% (Hatton and Almli,
1969).

Following experimental manipulations, changes in unit
activity examined in relation to the decreases in Posm
reported in the previous study, and the increases in Posm

following the NaCl injection reported by Hatton and Almli.

METHOD

Subjects

Animals used were 8h male albino rats of the Holtzman

strain, 100-110 days old when the eXperimental treatments

26

began. The rats were maintained under conditions similar to
those in the previous study. During experimental treatments,
15 animals died, and are not included in the data.

General recording procedure . These procedures were
generally the same for each treatment condition.

The animals were anesthetized as in the previous study.
Once recording procedures were begun, no supplementary doses
were given.

The animals were then prepared for unit recording.

This preparation included: (a) the positioning of a PE 50
(Intramedic) stomach tube: (b) implanting a sub-dermal cardiac
electrode (the ground was attached to the stereotaxic ap-
paratus): (c) positioning a rectal thermister thermometer for
recording body temperature: (d) wrapping the animal in heating
tape to maintain body temperature: (e) the placement of the
animal in a stereotaxic instrument: (f) the exposing of the
dura through a trephine hole in the calvarium: (g) the re-
moval of the dura and covering the exposed neural tissue

with mineral 011. Body temperature was maintained as in the
previous study.

Once an animal was surgically prepared, a glass insu-
lated microelectrodeIexposed tip, 15-40;: widest shaft
diameter, #0-6QAO was lowered at DeGroot coordinates 9.8 AP,
.11.“ Lat, and 8.5 DV. The skull was positioned horizontally
in the apparatus.

Once the electrode was lowered into the brain, neural

 

 

 

27

activity was amplified by a low level pre-amplifier. It

was then visually displayed on a dual beam oscilloscOpe

(as was bioelectric activity of the heart). When experimental
treatments began, unit and cardiazelectrical activity

was recorded on magnetic tape. (See Appendix A for a de-
tailed description of the amplification and recording system)

After the microelectrode was lowered to at least 8.5 mm
from the tOp of the cortex, isolation of a single unit was
attempted. This procedure was the following: The electrode
was slowly lowered manually. Once the extracellularly re-
corded action potentials of a given cell were at least SQwv
above baseline noise (which usually was 25-50pv) the low-
ering of the electrode was stepped. If the amplitude of the
action potential was sufficient to reliably trigger the
sweep of the oscilloscOpe, the cell was considered to be
isolated.

Following this, the various eXperimental treatments
were begun.

Immediately following recording, a small D.C. cathodal
(ZQAB for 5 sec) marking lesion was made through the tip of
the recording electrode. Following lesioning, the animals
were perfused with physiological saline and then a 10%
formo-saline solution. The brains were removed and stored in
the latter solution. Prior to frozen sectioning, some brains
were placed for 2-3 days in sucro-formaline solution. Once

the brains were sectioned, they were stained in cresyl violet

 

28

or thionin. The recording sites were then verified.

Treatment conditions. Once the rats arrived in the lab-
oratory, they received at least three days of free access to
food and water. At this time they were assigned to one of
the two basic treatment conditions: Ad Libitum or Deprived.
(See Table 2.)

Following the three day adaptation period, the rats in
the Ad Libitum Group received at least five more days of

free access to food and water. These animals were then anes-

 

thetized and prepared for unit recording. The electrode was
lowered to the predicted location of the SON. Once a cell
was isolated, ten minutes of activity was recorded before a
marking lesion was made. Two to four cells were recorded
from each animal of this group.

The animals in the Deprived Condition were placed on
a 23.5 hr water deprivation schedule similar to the one
used in Experiment I. Then on Day 5 of the deprivation
schedule, prior to access to water, the animals were anesthe-
tized and prepared for unit recording. Prior to actual re-
cording, the rats in the Deprived Condition were further sub-
divided into one of the following treatment conditions:

Deprived Qpppp--once a cell was electrically isolated,
49 min of activity was recorded on magnetic tape. A small
marking lesion was then made.

‘Lpgg’gply Qgppp--once a cell was isolated, in the ap-

proximate area of the SON, 10 min of baseline activity was

 

 

 

29

Table 2
Treatment conditions
Group Treatment
Ad Libitum Group Five days of free
access to food and
water before unit
recording.

Deprivation conditions

 

Deprived Group Five days on 23.5 hr
water deprivation
schedule before unit
recording.

Load Only Group Deprivatio period,
plus 10 cm gastric
water load during
unit recording.

Load-Injected Group Deprivation period
plus water load, plus
1 cm3 16% NaCl sc in-
jection.

 

 

 

 

30

recorded. Immediately following this, a 10 cm3 stomach load
of water at room temperature was manually delivered at
1 cm3/min. For 39 min from the beginning of the load, unit
activity was then recorded. A marking lesion was then made.
Lppg-Injected Epppp--animals which were treated as the
Load Only Group were, but, 2% min from the beginning of the
load a 1 cm3 16% NaCl subcutaneous (SC) injection was given
in the lower back. Fifteen more minutes of activity were then
recorded. A small marking lesion was then made.
Frequency analysi . Determination of frequency of
unit activity and heart rate was determined by electronic
counters. (See Appendix A for a detailed description of

this data analysis system.)

31

RESULTS AND DISCUSSION

19222 2.93:.

Figure 5 shows the mean percent changes (18.E.) for
heart rate during and after the water load, and following the
NaCl injection for six animals randomly sampled from the
Load-Injected Group. A random sample was used here merely
for convenience sake. The changes in heart rate seen in these
animals are comparable to those seen in the animals in
Experiment I (cf. Figure 3). There is an initial significant
(p<0.05) bradycardia which is maintained until 10 min, at
which time heart rate returns to near baseline levels.

In the first min following the injection, there is a
significant (p<0.05) tachycardia. Heart rate then continues
to increase until approximately 10 min following the injection.

Figure 6 shows the mean (iS.E.) percent changes in
heart rate for the first minute following the beginning of the
load. Immediately following the beginning of the water load,
a'ixlradycardia (p<0.05) is seen.

In Figure 7 are shown the mean (iS.E.) changes in heart
rate per ten seconds immediately following the injection.
In contrast to the bradycardia seen in the early portions
of the stomach load, the tachycardia observed following the
injection develops apparently more gradually.

It is well known that there are gastric stretch receptors
that contribute to the vagal afferents (Paintal, 195b, 1963).

 

 

 

 

 

 

32

Figure 5.

Indicated are the mean (iS.E.) percent changes in heart
rate for six animals selected at random from the Load-In-
jected Group. During the stomach load, change in heart rate
for each rat was calculated from their respective mean
baseline rates. During the injection, the change was cal-
culated from each rat's mean minute rate for the last minute
of the load period. N=6.

PERCENT CHANGE IN HEART RATE

6

I?)

a:

O)

N

 

33

 

LOAD

INCREASE

..-"'..'.‘ I

 

 

 

 

 

 

 

I 7

 

 

 

 

DECREASE

l3 l9
MINUTES

 

INJECTION

I 7

 

-"-----""..- I

 

 

I3

 

 

 

3U

Figure 6.

Indicated are the mean (iS.E.) percent changes in heart
rate per ten seconds in the first minute of the stomach
load. The animals are the same as those used in Figure 5.

 

 

 

35

 

 

 

 

INCREASE

DECREASE

30 4O 50 60

SECONDS

20

 

1.
Ike

IO

 

 

 

 

_ _ _ _ q

 

 

wk<m .529; z. woz<Io szomwa

 

 

36

Figure 7.

Indicated are the mean (iS.E.) percent changes in heart
rate per ten seconds for the first three minutes following
the saline injection. The animals are the same as those
used in Figure 5. N=6.

PERCENT CHANGE IN HEART RATE

 

37

 

INCREASE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DECREASE

O :. =’_ .. v _ - — — "'.."I'.’...'."."-'I'."""‘

 

 

 

IO 30 50 70 90 IIO I30 I50 l70
SECONDS

 

38

The changes in heart rate that are seen following the
beginning of the load could be the result of increased
activity in such mechanoreceptors. However, in addition to
these vagal afferents, it is known that abdominal sympa-
thetic afferents may influence heart rate (Sarnoff and Yamada,
1959). But because of the lack of evidence, it is unclear
whether the bradycardia and the tachycardia seen in the
present study are reflexively mediated through either one, or
both, of the abdominal afferents.
Neural activity

In Figure 8a are shown the mean(¢S.E.) spikes per
second for a 10 min sample of activity of SON cells re-
corded from deprived rats, and, rats on an ad libitum
food and water regimen. The overall mean from the deprived
animals is 11.910.2 spikes per second: and, the mean from the
ad libitum animalais 2.4:0.1 Spikes per second. The
difference is statistically significant (p<0.05). This is
clear evidence that SON unit activity is faster after a
period of water deprivation.

In Figure 8b is the mean minute (13.E.) spikes per sec-
ond for cells located in the anterior hypothalamus (AH),
but outside of the SON. The upper line is the mean minute
activity of cells recorded from animals that had been
on a water deprivation schedule (overall mean is 14.3:0.2
spikes/second). The lower line indicates the rate of firing
of.AH cells in rats that were on an ad libitum feeding and

 

 

39

Figure 8.

In panel A are indicated the mean (iS.E.) spikes per
second per minute for 12 SON cells recorded from water deprived
rats (C)--() ), and, 7 SON cells recorded from rats on
an ad libitum food and water schedule (H ).

In panel B are indicated the mean (is. E. ) spikes per
second per minute for 17 AH cells recorded from water deprived
rats ( CP-()) , and, 8 AH cells recorded from rats on an
ad libitum food and water schedule ( H ).

SPIKES PER SECOND

1&0

 

DEP
SON

AL

I —'.VEV-‘

AL

 

 

 

AH B

I23456789I0
MINUTES

 

 

 

 

hi

drinking schedule (overall mean is 8.9iO.2 spikes per
second). The mean difference between those two groups is
significant (p<0.05).

As is indicated in panel 8a, SON cells are apparently
differentially affected by the water deprivation procedure.
This might be expected on the basis of their presumed role
in the elaboration of ADH in the dehydrated state, but
more about this point later.

Because of the rather heterogeneous population of cells
that are represented in panel 8b, it would be hazardous to
make any statements about the effect of water deprivation on
unit activity of any specific cell in the AH. But, generally,
water deprivation apparently does result in significant in-
creases in AH cellular activity.

Figure 9 shows the mean (iS.E.) activity in spikes per
second for #9 min of cells in the AH and SON. The mean
minute activity of the two SON cells recorded here is slightly
slower than that reported for deprived rats in Figure 8a.
However, the mean minute activity of the SON cells in Figure
9 is significantly faster than the cells recorded from in
animals of the Ad Libitum Condition (cf. Figure 8a). But,
most importantly, there are no dramatic shifts in activity
of these cells during the 49 min recording session. Then,
any changes that are seen following other experimental
treatments, might be attributed to the effects of those

manipulations.

#2

Figure 9.

Indicated are the mean (iS.E.) Spikes per second per
minute for five AH cells ( H ) and two SON cells
( C)--() ). These cells were recorded from rats that had been
water deprived.

-__-.. .-._._‘ .. ,. .- —"'"-_ "" ""'_"

“3

 

6

AH

 

 

 

 

c7:

SON

(D

7

5.0... .'e 0......'e

IIIIulIISIIII I

I 7 I3 IS 25 3| 37 43 49
MINUTES

SPIKES PER SECOND

 

nu

Figure 10a shows the mean (iS.E.) activity in spikes
per second of 5 AH cells of rats that were subjected to
the water load, then recorded from for 29 more minutes.

The mean minute activity of these cells during baseline is
11.1:0.“ spikes per second. The mean minute activity of these
cells during the water load perkd is 10.1iO.1 spikes per
second. Taking into account the variability about these
means, the activity during baseline is not different than
that during the load period. However, each mean firing rate
per minute during the load is below the mean rate per minute
during the baseline. The probability of this occuring on
the basis of chance is very small (p<0.01). Exactly why this
occurs is unclear. It seems reasonable, however, that it is
related to water entering the stomach.

In experiment I, it was reported that Posm significantly
decreases by the 11th minute of effective absorption time.

No change in AH mean minute firing rate is seen during that
period following the water load. This is interpreted to
mean that the rapid Posm changes that occurred following
the stomach load of water did not effect the firing rate of
these AH cells.

In Figure 10b is indicated the mean (iS.E.) minute firing
rate in spikes per second for three cells in the SON treated
the same as those cells in Figure 10a. The mean minute
firing rate of these cells during baseline is 10.9iO.6 spikes

per second. Mean minute firing rate of these cells increases

“5

Figure 10.

In panel A are indicated the mean (iS.E.) spikes
per second for 15 AH cells in the Load Only Group as
a function of minutes. The animals from which these cells
were regarded had been water deprived and then Seceived
a 10 cm gastric water load at the rate of 1 cm /min.

In panel B are indicated the mean (iS.E.) spikes per
second for h SON cells. The animasl from which these cells
were recorded were treated as those in panel A.

SPIKES PER SECOND

I6

I4

LI6

 

 

BASELINE

I 7

 

AH

 

SON

LOAD

I 7 I3
MINUTES

I9 25 3|

 

37

A?

slightly in the early portions of the load. By the thirteenth
minute following the beginning of the load, mean minute firing
rate is below that seen during baseline. For the remainder
of the recording session, mean activity per minute of these
cells never becomes as fast as it was during baseline. It
is interesting that activity of these cells decreases
below baseline by the 13th minute (which corresponds favorably
with the time when Posm decreases occur).

In Figure 11a is the mean (iS.E.) minute activity in
spikes per second of 17 AH cells. Twelve minutes following
the completion of the water load, a 1 cm3 16% NaCl 80
injection was administered. As Was the case with the AH
cells of the Load Only Group, the mean minute activity of
these cells, during the load period, is below that seen during
baseline. Because this decrease occurs within the first
minute, it seems reasonable that this depression in frequency
is the result of water entering the stomach. Most important
here, however, is the fact no significant change in mean
activity which might be attributed to changes in Posmo occurred
either during the load, or following the injection.

In contrast to this is the mean frequency per minute
of 12 SON cells plotted in panel 11b. These cells were
treated as those in panel 11a were. There is an initial
increase in activity which gradually decreases throughout
the remainder of the load procedure. The animals from which

these cells were recorded were given (as those in panel 11a

48

Figure 11.

In panel A are indicated the mean (iS.E.) spikes per
second for 17 AH cells as a function of minutes. These cells
were recorded from animals that had been water deprived.

As indicated, they received a 10cm gastric load of water
and a 1 cm3 16% NaCl SC injection.

A In panel B are indicated the mean (iS.E.) spikes per
second for 12 SON cells. The animals from which these cells
were recorded were treated as those in panel A.

’49

 

 

BASELINE

SPIKES PER SECOND

 

AH

LOAD

SON

I 7 I3
MINUTES

W

INJECTION

 

 

IS I 7 I3

50

were) a hypertonic saline injection. Immediately following

the injection, there was a significant increase in mean

minute firing rate of these cells. By four minutes, this
increase had been attenuated to some degree. But, by seven
minutes, the cells' activity had again increased significantly.

Figure 12 indicates the median (Q1 and Q3) percent change
from baseline for the SON cells depicted in 11b, which
showed a significant change in mean minute firing rate. All
but two of these cells showed a median percent increase in
the first minute. This trend continued until minute seven,
by which time activity of the cells was actually decreasing.
By 13 min, all but one of the cells was firing below baseline.
On the basis of random distribution, the changes in firing
rates seen at 13 min would be expected only 5% of the time.
Throughout the remaining portion of the session, the
activity of the cells tended to decrease. It is interesting,
of course, that by thirteen minutes Posm had been shown to
have significantly decreased.

In the right had side of Figure 12 are the median
percent changes that occur following the injection: (a) the
initial increase immediately following the injection, (b) the
decrease by four minutes, and, then, (c) the dramatic
increase by seven minutes. As pointed out earlier, it has
been shown that there is a significant increase in Posm
following such an injection.

In Figure 13 are indicated the mean (iS.E.) firing

51

Figure 12.

Indicated are the median (Q and Q ) percent changes of
9 of the cells shown in Figure 1i. Thes 9 cells showed a
significant decrease in firing rate during the load period.
Each cell's percent change during the load was calculated on
the basis of each cell's respective mean baseline firing
rate. Percent change following the injection was calculated
from each cells mean minute rate during the last minute
of the load period.

52

 

 

 

 

 

 

 

 

 

 

I60 20
STOMACH LOAD
I20 I60
{“9
80
Z
4 I20
I
0 40 8
I-
z 0 - ........'.'.
8 4o -
E ' .
IL 40 e L
. e o -0--. ------....
80 INJECTION

 

 

I47IOI3I6|922 I47IOI3
MINUTES

53

Figure 13.

Indicated are the mean (iS.E.) spikes per second per
ten second time bins of the first minute following the be-
ginning of the water load (panel A) and injection (panel B).
These are the same cells that are depicted in Figure 11.

SPIKES PER SECOND

 

51+

 

 

 

 

 

 

WATER LOAD

 

SALI NE INJECTION

I0 20 I0 40 50
SECONDS

 

60

 

 

fiifimm-h-‘fm'fl _

55

rates of SON cells immediately following the beginning of
the stomach load (panel a) and the injection (panel b).
In Figure in are the mean (iS.E.) percent changes during these
same periods. The change in firing rate following the
beginning of the load is more rapid, and transient, than
that following the injection. The changes in SON unit ac-
tivity that occur during these periods will be discussed
in more detail later.
Histology

Figure 15 shows portions of diagrams of a rat brain
at de Groot planes 6.6-8.3 AP. The black circles indicate
location of marking lesions which indicate that the cell
being recorded from was probably a SON neuron. The cpen
circles indicate marking lesions at the various other
recording sites. All animals from which these cells had been
recorded from were of the Ad Libitum Group.

Figure 16 indicates location of marking lesions in
diagrams of a rat brain at de Groot planes 7.0-8.2 AP.
Black circles again indicate location of recording sites
that are probably within the SON. Open circles indicate
other recording sites. Animals from which these cells had
been recorded from were in the Deprived Group.

Figure 1? indicates diagrams of a rat brain at de Groot
planes 6.6-7.8 AP. Black circles indicate marking lesions
of recroding sites probably within the SON. Open circles
indicate other recording sites. All animals from which

these cells were recorded from were in the Load Only Group.

56

Figure 1b.

Indicated are the mean (iS.E.) percent changes in firing
rate per ten seconds for the first minute following the
beginning of the water load (panel A) and injection (panel B).
These are the same cells depicted in Figure 13.

 

 

PERCENT CHANGE

57

 

0
0|

0)
0|

.fi
0|

 

N
0|

/

5 WATER LOAD

 

N
N
0'
O

 

I25

25 e SALINE INJECTION

I0 20 30 40
SECONDS

50

 

B
60

 

58

Figure 15.

Indicated are portions Of diagrams of a rat brain
at de Groot planes 6.6-8.2 AP. The black circles indicate
location Of marking lesions which indicate that the cell
being recorded from was probably within the SON. The
Open circles indicate marking lesions at various other
recording sites. All animals from which these cells had
been recorded were of the Ad Libitum Group.

 

59

 

 

 

__L_

,-.L. _.
U

 

 

 

 

 

 

60

Figure 16.

Indicated are the location Of marking lesions in
diagrams Of a rat brain at de Groot planes t.0-8.2 AP.
Black circles indicate location Of recording sites that
are probably within the SON. Open circles indicate other
recording sites. Animals from which these cells had been
recorded were in the Deprived Group.

61

 

 

 

 

 

62

Figure 17.

Indicated are diagrams of a rat brain at de Goot planes
6.6-7.8 AP. Black circles indicate marking lesions of
recording sites probably within the SON. Open circles in-
dicate other recording sites. All animals from which these
cells were recorded were in the Load Only Group.

 

63

 

 

 

 

 

 

 

 

 

 

 

 

 

 

a." TV

6A

In the bottom section is indicated a recording site pro-
bably within the tuberal portion of the SON. Interesting
is that this cell responded as did the anterior SON cells.
Figure 19 indicates diagrams of a rat brain out at
de Groot planes 7.0-8.2 AP. Black circles indicate probable
SON recording sites. Open circles indicate neurons which
were recorded from in areas outside the SON. Animals from
which these cells were recorded from were in the Lead In-
jected Group.
In Appendix A are photomicrographs of some of these

recording sites.

65

Figure 18.

Indicated are diagrams of a rat brain out at
de Groot planes 7.0-8.2 AP. Black circles indicate probable
SON recording sites. Open circles indicate neurons which
were recorded from in areas outside the SON. Animals from
which these cells were recorded from were in the Load
Injected Group.

 

66

 

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e s e I e

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It-
0

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I
.

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67

GENERAL DISCUSSION

In Experiment I, changes in Posmv Pv' water absorption,
and heart rate were measured in water deprived animals which
had received a stomach load Of water. In addition, these
animals were anesthetized and prepared for unit recording.
As was pointed out, the time course and magnitude of changes
that occur, during and after the water load, in Posm and
water absorption are comparable to those that occur in awake,
drinking rats under similar deprivation conditions. In
Experiment II, unit activity in the anterior hypothalamus
Of rats (under conditions similar to the first study) was

recorded.

‘ngp activity changes pp response pp 33335 lpggp

§QN activity. Mean minute firing rates Of SON cells are
significantly faster in a deprived animal than in one given
free access to food and water. This was also true wifiiother
AH cells. Cells within the SON are extremely homogeneous in
respect to their cellular morphology (Rechardt, 1969). On
the basis Of this, it seems reasonable that the difference in
firing rates is not the result Of a sampling bias, but
actually reflects the fact that increases in tonicity of
body fluids result in faster firing rates Of SON cells.

It has been well documented that the production of an

action potential by a neuron requires metabolic energy (Ochs,

68

1965). The notion that SON cells increase their metabolic
activity during periods Of water stress receives some

support from histochemical (Watt, 1970) and anatomical studies
(Hatton, Johnson, and Malatesta, 1971). And the increases in
metabolic activity of SON cells, indicated by these studies
might be the result, in part, Of the increased firing rate

of these cells. The increased protein synthesis indicated by
the studies cited above might also reflect the increased
production of ADH which is a polypeptide hormone.

The initial increases in unit activity following the
beginning of the stomach load were uneXpected. Because of
the short latency with which the increase in firing rates
occur, it is likely that this response is mediated through
gastric stretch receptor stimulation. Activity Of chemo-
receptors in the stomach should not be ruled out. However,
apparently no evidence has been gathered for such receptors
responding to water (Paintal, 1963).

As pointed out earlier, there is abundant evidence that
baroreceptors in the stomach respond to passive distention.
There is, however, apparently no evidence on the relationship
Of these baroreceptors to SON unit activity. It has been
shown, however, that palpation. of the surgically exposed
stomach will result in dramatic increases in ADH titers in
the blood (Moran and Zimmermann, 1969). In any case, whether
this effect might be mediated through vagal or sympathetic

afferents has apparently not been determined.

69

Because gastric mechanoreceptors have different
thresholds (Paintal, 1963), it would not be unlikely that
they are mediating (in part, at least) the early changes in
SON activity. In any case, their influence on the SON is
probably rather transitory. By 7 min, following the beginning
of the stomach load, the frequency Of firing of SON units
, has decreased to near baseline levels (See Figure 20), but,
complete gastric filling had not yet taken place (cf. Table 1).

It should be appreciated, though, that the initial
increase in firing rates Of SON cells, following the begin-
ning Of the load, occurred when only 0.25 cm3 Of water had
entered the stomach.

Following the first phasic increase in unit activity
following the beginning Of the water load, there is no
significant change in mean rate Of firing Of SON cells
until 12 minutes. By this time most cells are firing below
baseline. Then generally, as seen in Figure 11b, the activity
of these cells tends to continue to decrease. Interesting,

Of course, is the fact that by 11 min of effective absorption
time there is a significant 3% decrease in Posm- It would
seem reasonable that the decrease in P08m is at least con-
tributing to the decrease in SON activity.

Pv on the other hand, as indicated by protein con-
centration, does not shift until approximately 18 min, 80,
it seems unlikely that Pv, pp; pp, in bringing about the
changes in activity that are seen at 12 min.

70

Figure 19.

Indicated are the mean (iS.E.) percent changes in firing
rate for 13 minutes following the beginning of the water

load. These are the same cells that are depicted in Figure
11b.

 

71

 

OI
IO

  

INCREASE

NGE
s s

A
N
'0

I0

 

       

 

    

."'..'..'....' "I 'I'."'-

 

PERCENT CH
IO

   

DECREASE

 

 

 
  

WATER LOAD

 

  

 

   

I23456789IO|II2|3

MINUTES

 

72

The difficulty in attributing the decrease in SON
activity to Posm decreases, or to changes in stomach fluid
volume should be appreciated. Because changes in P081:! and
gastric fluid volume are occurring at approximately the
same time, the role that each plays in the decrease Of SON
activity is unclear. But, because unit activity in the SON
returns to baseline levels by 7 min after the load starts,
it seems reasonable that the change in rate seen at 12 min
is the result Of Posm decreases. If, then, the decrease in
SON activity below baseline rates can be attributed tO de-
creases in Poem changes, then the magnitude of the decrease

in P (3%) during this period might be considered to be

osm
a plasma osmotic pressure threshold.

.AH activity. Following five days on a water deprivation
schedule, mean firing rates per minute of AH cells were sig-
nificantly faster than that seen in the Ad Libitum Group.

But, in contrast to this, and as indicated in Figures 10 and

11, AH cells generally show no dramatic changes in mean

firing rates per minute as the result of fairly rapid alter-
ations in the tonicity Of the plasma. There were, however, three
cells in the Preoptic Area (POA) which did show a change in
firing in response to the water load. Once the load began,

there was a slight significant decrease in the rate of these
cells,. then a gradual increase. However, no other cellular

activity changes were recorded. In contrast to this, other

investigators have found decreases in POA activity in response

73

to either gastric (Thorton, 1969) or IC water loads (Brooks,
Koizumi, and Zebalios, 1966: Ishikawa, Koizumi, and Brooks,
1966b: Joynt, 196A).

The basis for the discrepancy between this study and
those which have reported such findings is unclear. Most
investigators in the area do not report their exact record-
ing sites. SO any comparison Of exact recording sites and
changes in cellular activity is impossible. The studies that
have reported histological findings have used goats and cats.
But, because of the Species differences, anatomical comparisons
between these is made difficult. In addition, these inves-
tigators did not monitor the osmotic state of their animals.
As a result, it is difficult to make any quantitative state-
ments about the change in blood tonicity which their manip-
ulations produced.

The lack Of evidence of AH cells responding to decreases
in tonicity of the blood in the present study should not be
taken to mean that no such cells exist. Because of the rel-
atively few number of cells investigated in the present
study, it would not be at all suprising if some Of these
cells were not isolated.

There is also a significant procedural difference
between other recording studies investigating neural osmo-
sensitivity and the present study. Only one other study
apparently has employed the procedure of recording during

and after a stomach load of water (Thornton, 1969). In this

 

71:

study, 3 cells in the POA were found to decrease frequency
to the load. It is not clear, however, whether Or not these

responses were to decreases in tonicity Of the body fluids.

Changes Ap‘pplp activity lpyrespgnse pp pypertonic injections

SEN activity. Cells in the SON showed a clear and
consistent reaction to the hypertonic injection. In the
first minute following a similar subcutaneous injection,
most awake rats exhibit a marked pain response (ie., squeal—
ing, biting at the injection site, etc.). During this time
period, SON cells showed a marked significant increase in
firing rate. During the time period when the pain reaction
is subsiding in awake animals (usually within a minute or
so) the activity Of these cells has markedly decreased.
Interesting is that nocioceptive stimuli have been shown to
result in a marked antidiuresis and release of ADH into the
blood (Verney, 19h7: Tata and Buzalkov, 1966: Moran and
Zimmermann, 1969). Because Of the short latency and transi-
tory nature Of the changes in firing rates Of these cells
(following the injection), it seems reasonable to assume that
these changes are the result of the saline injection acting
upon peripheral sensory receptors.

The central pathways which might mediate this response
are quite Obscure. Mills and Wang (1964) have shown, though,
that ADH is released in response to peripheral pain: and,
that this reSponse is probably not mediated through the

 

75

medial lemniscal system. Their evidence further indicates
that nocioceptive stimuli (radial nerve stimulation) result-
ing in ADH release probably pass by way of the extralemniscal
pathway to the centre median nucleus Of the thalamus. How-
ever, the pathways that might act upon the SON directly

have not been established.

The transitory increase in firing rates Of these
cells after the injection is followed by a significant
and maintained (throughout the recording session) increase
in activity (Figure 12). In awake rats under similar hy-
dration conditions, it has been shown that there are usually
significant 2-3% increases in Posm by 6 min following such
an injection (Hatton and Almli, 1969). Coincident with the
increase in Posm- the rats initiate drinking.

It seems reasonable to assume the increase in SON
activity seen by the 7th minute is the result of shifts in
osmotic pressure Of body fluids. Because the changes in firing
rates are also temporally coincident with a 2-u% increase in
Posmv it might be assumed that this change represents a
threshold for firing rates of these cells.

pp pp;p_activity. As indicated earlier, no mean minute
changes in activity Of AH cells was Observed following the
SC injection of saline. However, other investigators have
found cells in these general areas that have responded (in—
creased frequency) following intracarotid (10) injections of

hypertonic solutions. Here, again, there is a difficulty in

76

determining the apparent discrepancy. There is apparently

only one other study which reported the use Of SC NaCl in-
jections while recording from the hypothalamus (Hatton and
Almli, 1970). These investigators reported one cell in the
posterior hypothalamus that increased its firing rate follow-
ing the injection. Despite this, no clear statements should

be made on the basis Of the present data, with regard to the
presence (or absence) of AH cells sensitive to rapid increases

in tonicity Of body fluids.

Review of changes in unit activity in response to water
loads and salinei injections

In general, the following changes were Observed in
the animals studied: (a) As the load began, there was a
short latency hypothalamic and cardiasreSponse: the former
being an increase in SON unit activity, the latter being
a bradycardia. This response is probably mediated via gastric
stretch receptors. As pointed out, if this is true, these
receptors are reaponding to 0.25 cm3 entering the stomach.
As the water load proceeds, the stomach continues to fill
until approximately 11 min Of effective absorption time.
Until this time, a clear bradycardia is seen. As the contents
in the stomach diminish, heart rate returns to near baseline
levels. On the other hand, firing rate Of SON cells returns
to near baseline levels by 7 min.

(b) By 12 min post-load, these SON cells decreased their

firing rate significantly below baseline. This period also

77

correSponds to the first significant decrease in Posm'
Because the initial neural response to the load had subsided
by 7 min, the decrease at 12 min might be the result Of the
change in Posm' Under these conditions, it would not be
expected that the change in SON activity were due to Pv
changes because no significant change in Pv was seen until
approximately 18 min Of effective absorption time.

If the decrease in unit activity below baseline was
the result Of Posm shifts, then, the 3% decrease in Posm seen
at this time could be considered a threshold for decreased
firing frequency.

(c) Approximately 25 min after the beginning Of the load,
a 1 cm3 16% NaCl injection was given to some animals. Fol-
lowing the injections, there was a tachycardia and a transient
increase in rate of firing Of SON cells. By h min, this
increase had attenuated. But, by 7 min, the cells' activity
tended to again increase, coincident with a 2-h% increase
in Posm' Also during this period, heart rate again tended to
increase.

It seems reasonable that this two phase change in
unit activity is the result : (1) of peripheral stimuli
(resulting from the injection) causing the initial transient
change in firing rate, and, (2) of a change in tonicity Of
body fluids resulting in the second more sustained rise
in firing rate.

While it seems reasonable to attribute some of the changes

78

in activity Of SON cells to changes in tonicity of the body
fluids, it is difficult, on the basis of the present data,
to determine the influence on unit activity of 'peripheral
stimuli versus that Of Posm'

Also on the basis Of the data, it is impossible
to determine the actual nature Of the osmotic influence on
activity Of the SON cells. It could be that changes in EOP
Of the extracellular fluid are directly affecting unit ac-
tivity in the SON. On the other hand, EOP changes could be
detected by neural elements located in other areas of the
central or peripheral nervous system. There is no sound
evidence that any given area in the CNS affects SON unit
activity (in response to osmotic pressure changes). There
is, however, evidence that osmoreceptors in the hepatic
circulation can alter SON unit activity (Metveeva and Osi-
povich, 1970). And, as has been pointed out earlier, changes
in concentration Of the hepatic circulation can bring about
changes in diuresis.

Of course, because blood pressure was not recorded
in the present studies, its possible influence on SON activity
cannot be completely assessed.

Relationship 2: S93 activity pp drinkipg behavior.

Hatton and Bennett (1970) reported that significant
decreases in P08m may result in the cessation Of drinking.
These investigators also reported that water deprived rats,
when given access to water will drink for approximately

10 min, by which time 3 cm3 of water has been absorbed and

79

P08m has decreased 1-2%. In the present study, similar changes
in P08m and water absorption were reported. Also, during
this time period SON unit activity decreases.

The temporal relationship between SON unit activity
and the cessation Of drinking is interesting. This cor-
relation, Of course, should not necessarily be taken to
mean that the SON plays a role in bringing about the ces-
sation Of drinking.

It is well documented that interruption of the supra-
OpticO-hypOphyseal system results in a syndrome called dia-
betes insipidus (Fisher, Ingram, and Hanson, 1938): when,
following such lesions, the Observed polydipsia is secondary
to the resulting polyuria. However, in some cases the poly-
dipsia is thought to be primary. This is true because (a)
in some cases the polydipsia actually proceeds the polyuria
(Smith and McCann, 1962 a and b), and, (b) the polydipsia
occurs in nephrectomized animals (Rolls, 1970). If the SON
plays no role in the initiation or cessation of drinking,
then drinking thresholds following SON lesions should not
be disrupted. However, this has not apparently been measured.

As stated earlier (p. 7), it was expected that SON
unit activity would be closely correlated with changes in
drinking behavior. This was expected not because the SON
necessarily plays a role in drinking behavior, but because
similar changes in Posm result in changes in both the release

Of ADH and drinking.

8O

Stimulus convergence

In addition to changes in Posm and blood volume, it
has been shown that peripheral stimuli (like gastric pal-
pation and pain) will result in the increased release of ADH.
There has been, however, apparently no reported evidence
that these different stimuli act on the same SON neurons.

In the present study, the data indicated that changes
in gastric filling, stimuli that can elicit pain, and changes
in Posm can apparently alter the firing rates of single SON
neurons. If this data is supported, it is, then, the first
reported evidence that the stimuli which are potent re-

leasers Of ADH converge on single SON neurons.

M2152

There was a short latency bradycardia following the
beginning of the water load in both experiments. This
bradycardia is maintained as the stomach fills, but, then,
gradually diminishes. This presumably occurs as the result of
gastric stretch receptors initially being activated, adapting,
and then diminishing in activity as the amount of fluid in
the lumen Of the stomach decreases. It should be pointed
out that if activation of gastric mechanoreceptors mediates
the bradycardia, they do so, initially, in response to 0.25
cm3 Of water entering the stomach.

The tachycardia seen following the injection is a little

more difficult to interpret. It develops rather gradually

 

 

81

over the first 3 min, by which time it stabilizes. But, by
7 min, it begins to increase and then plateaus at a higher
level by 10 min (cf. Figure 5 and 7). There are apparently
no comparable data on which to assume whether these two
phases in the changes in heart rate are the result Of two
processes or not.

It has been well documented that intravascular infusions
Of hypertonic (or isotonic) solutions will result in a tachy-
cardia (Bard, 1968). The second increase in heart rate is, how—
ever, coincident with an increase in Posmv presumably the

result Of the hypertonic saline injection.

Summary
SON unit activity was found to be significantly faster

in the water deprived rat, as compared to rats on an
ad libitum food and water schedule.

A transient, short latency increase in SON unit activity
was Observed in response to initial filling of the stomach
with water. Following this, when Posm began to significantly
decrease, unit activity in the SON also decreased significantly
below baseline levels.

In response to a SC hypertonic saline injection, a
short latency, transient increase in SON unit activity was
seen. This increase ocurred during a time period when many
awake rats, treated in a similar fashion, exhibit a marked

pain reaction. By seven minutes post-injection, when Posm

82

has been shown to significantly increase. SON unit activity
became significnatly faster.

AH cells, treated in the same fashion, did not show
similar changes in unit activity in response to rapid
changes in Posm° This is true despite the fact that fol-
lowing a period Of water deprivation, unit activity in the
AH was significantly faster than in animals on an ad lib-
itum feeding and drinking schedule.

If (a) the initial short latency, transient changes in
SON unit activity, following the water load or injection,
can be attributed to peripheral stimuli, and, if (b) the second-
ary changes in unit activity can be attributed to shifts in
Posm: then this is apparently the first reported evidence for
convergence on SON sells of stimuli that elicit the release
Of ADH.
Addendum

In the Load Only and Load Injection Groups, 16 SON cells
were recorded from. Of these, 12 showed transient increases
in firing rates during the first 10 seconds of the water load.
The other four cells showed no increases in firing rate until
approximately the second minute of the load.

It is unclear whether the increase in firing rate of the
12 SON cells was mediated neurally or by changes in blood
pressure. But, on the basis of their reaction to water initially
entering the stomach, there seems to be two pOpulations of SON
cells, one set that shows a rather rapid change, and, one

a much slower.

LI ST OF REFERENCES

83

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neurohypOphysis in response to hemorrhage and infusion
Of hypertonic saline in dogs. Endocrinology, 1969, 85:72-78.

Sarnoff, D. and Yamada, K., Evidence for reflex control of
arterial pressure from abdominal receptors with special
references to the pancrease, Circulation Research,

1959. 7:325-335-

Share, L., Control Of plasma ADH titer in hemorrhage: Role Of
atrial and artrial receptors, American Journal 23 Ppysiology,
1968, 215:1384-1389.

Suda, 1., Koizumi, H., and Brooks, McC., A study Of unitary
activity in the supraOptic nucleus of the hypothalamus,
Japanese Journal 2; Papiology, 1963, 13:374-385.

Sundsten, J.W. and Sawyer, C.R., Osmotic activation of neuro-
hypOphyseal homone release in rabbits with hypothalamic
islands, Experimental NeuroloSY. 1961, 4:545-561.

Tata, P.S. and Buzalkov, D., vaSOpressin studies in the rat.
III. Inability Of ethanol anesthesia to prevent ADH secretion
dug to pain and hemorrhage, Pflugers Archiv, 1966, 290:

29 -297 e

Thornton, L.W., Effects Of repeated infusions Of hypertonic
saline solutions on the electrical activity of hypothalamic
neural units in goats, Ph.D. Thesis, Michigan State University,

1969.

Von Euler, G.A., A preliminary note on slow hypothalamic
osmOpotentials, Acta Physiologica Scandanavia, 1953, 29:

133-136.

Verney, E.Bt,‘The antidiuretichormone and factors which

determine its release. Proceedings 23 the Royal Society,
London, Series B, 1947, 135: 5-10 .

Wang, M.B., The distribution and control of osmosensitive
cells within the hypothalamus of the Opossum, Didelphis
virginiana, Neuroendocrinology, 1969, 21:51-67.

 

 

87

Watt, R.M., Metabolic activity in single supraoptic neurones
and its relation to osmotic stimulation, Brain Research,
1970, 21:443-447.

Wolf, A.V., Thirst, Springfield: C.C. Thomas, 1958.

Woods, J.W., Bard, P., and Bleir, D., Functional capacity
of the de-afferented hypothalamus: Water balance and re-
sponses to osmotic stimuli in the decerebrate cat and rat,

Journalqu NeurOphysiOlogy, 1966, 29:751-767.

APPENDIX A

Apparatus

 

 

88

Eguipment and materials used 3p making microelectrodes.

stainless steel tubing, 26 gauge, Superior Tube CO.
Tungsten wire, 0.005", Sylvania.

Glass tubing, Corning Glass.

Hydrofluoric acid, Merck, Inc.

Micrometer eyepiece, x8, Zeiss.

Stereozoom dissecting microscOpe, Nikon.

Equipment used lp blood analysis.

1.
2.
3.

Centrifuge, International Equipment CO.
Refractometer, American Optical CO., Instrument Div.

Freezing point osmometer, Precision Instrument Corp.

Eguipment and materials used yp bioelectric data recording

and analysi .

1.
2.
3.
4.
5.
6.
7.
8

9.
10.
11.
12.
13.
14.

Stereotaxic instrument, KOpf.

Preamplifiers, 122, Textronix.

Power supply, 125, Textronix.

OscilloscOpe, 502A, Textronix.

Tape recorder, 1028, Magnecord.

Electronic counter, 5512A, Hewlett Packard.
Printout counter, Digitron.

Audiomonitor, AM8, Grass Instruments.
Audiomonitor, AM5-B, Grass Instruments.
Thermistor thermometer, Yellow Springs Instruments.
Micromanipulator, Narishige.

Magnetic tape, Scotch, 1.5 mil acetate, %"x2500'.
Stimulator, SB-Cr, Grass Instruments.

Stimulus isolation unit, SIU 4678, Grass Instruments.

 

89

15. Technical Measurement Corp., Mneumotron Div.,
Amplitude discriminator 605
Peak detector 607
Amplitude to time discriminator 606
Computer Of averaged transients 400B

16. Oscilloscope, 565, Textronix

17. Lesion maker, G. Connors.

APPENDIX B
Raw Data

 

90
EIperiment I

Plasma Osmolality, mOSmZkg

Presurgery 0 Min 3 Min
300 311 292
307 291 290
303 302 299
299 303 300

9 Min 12 Min 15 Min
291 290 294
287 287 293
296 287 297
295‘ 293 290
21 Min 24 Min

287 279

289 290

293 292

301 286

Protein concentration, gliOOml
Presurgery 0 Min 3 Min
6.3 6.3 6.3
6.3 5.9 6.3
6.3 6.2 6.3
6.3 6.3 6.2

9 Min 12 Min 15 Min
6.4 6.1 6.3
6.0 6.4 6.1
6.0 509 509
6.1 5.7 5.7
21 Min 24 Min

5.8 5.4

6.2 5.9

5.6 5.8

5.9 5.1

6 Min

298
304
301
301

18 Min

289
288

295
296

Water absorbed, ml

 

Experiment I

3 Min 6R1n 9 Min 12 Min
-1.93 1.23 -1.63 2.53
-3.03 -1.03 0.83 2.13
-2.33 1.33 2.93 4.83
-3.93 -2.73 1.43 2.83
15 Min 18 Min 21 Min 24 Min
6.13 0.83 5.23 3.73
1e23 ‘1037 5014’3 6.73
2.23 -1.37 4.43 4.53
0.63 3063 2013 3.73
Percent change pp heart rate
3 Min 6 Min 9 Min 12 Min
2.9 2.1 7.2 10.4
2.0 2.8 2.6 10.1
3.2 4.9 7.2 1.0
3.4 2.5 1.8 7.0
15 Min 18 Min 21 Min 24 Min
12.8 1.7 1.0 7.7
7.9 2.0 14.7 6.8
1.0 1.7 12.7 1.1
5.4 2.6 8.8 1.0

 

Group, §pikes/second

92
Experiment II

c OEIIS .-

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potha

Minute
2

 

Unit activit
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Cell

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777766555555 USCN 7765555 UA CN44444

 

 

34 37 40

Minute
31

Cell
NO.

93
EXperiment II

 

Unit activitz, animals lg the Deprived Group, fipikes/second
cells

Supraoptic Nucleus

19 22 25 28

46 ug

16
Load-Injected group, Spikes/

13
seconq,.Anterior glpothalamic cells

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46? 781346789
1.11. 112222222

94
EXperiment II

 

 

 

 

 

 

 

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S S 1 1
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nm 32 2 11 n 1 2 31. 1
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mm 1 42 111 MR 21 211 31 1
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h‘ 7 312976509.56 h—O 1 828618223655
t 131 2 ts 11 111 511
n_ 47263428488 Ju 455572401899
1r 0 o O o O o o o O o 18 O 00000000 O o o o
o 4 48870.601036 1 1 977445473383
Mn 32 1 11 km 1 1 1 21 1
mm 94191789156 MN 38.9562975501
O O O O O O O O O O O ..... O C O 0
1M 1 90230251039 10 7 106359351293
w 1132 2 11 “mm 21 1 1 21 1
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95
cont.

18

EXperiment II

animals in the Load-In acted Gr
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Unit activit ,
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No. 19 22

 

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Experiment II

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Minute
1
Minute
19

activitz,

second

 

Cell

No.
Cell

No.

 

Unit

9?

Figure 20.

Shown is a photograph of a frontal cut of a rat brain
embedded in celloiden and stained in thionin. Indicated
is the supraoptic nucleus (SO), suprachiasmatic nucleus
(SC), and the optic chiasm (0C).

 

98

 

 

 

99

Figure 21.

A. Shown is a photograph of a frozen section of a rat
brain stained in thionin and cut in the same plane as
Figure 20. Encroaching on the SO is a marking lesion in-
dicating a recording site.

B. Shown is a photograph of a frozen sectiau of a rat
brain stained in thionin and cut in the same plane as
Figure 20. Encroaching on the SO is another marking lesion
indicating a recording site.

100

 

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101

Figure 22.

A. Shown is a photograph of a frozen section of a rat
brain stained in thionin and cut in the same plane as the
previous photographs. Encroaching on the SC is a marking
lesion.

B. Shown is a photograph of a frozen section of a rat
brain stained in thionin and cut in the same plane as
Figure 20. Seen is a marking lesion located in the
preOptic area.

102

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