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ABSTRACT
ELECTRICAL ACTIVITY OF SINGLE CELLS IN THE

SEPTAL AREA OF RATS RESUDTING FROM CHANGES
IN PLASMA VOLUME AND OSMOLALITY

BY

John G. Bridge

The septal nucleus is an area of the brain that
has been implicated in a number of different activities.
Stimulation of the septal area in humans has been shown
to reduce awareness of pain, have a general calming effect,
and elicit sexual thoughts and/Or feelings. In some
species septal lesions interfere with performance in one
choice (go - no go) situations, but not with two choice
tasks, such as choosing the correct alley in a T—maze.
Some investigators have found septal lesions to interfere
with learning of reversal tasks; other report enhancement
of reversal performance. The "septal rage syndrome" is a
classical symptom of lesions of the rat or cat septum.

Involvement in water regulation is perhaps the
function of the septum most frequently investigated.

There is, for example, evidence that septal lesions

1

John G. Bridge

increase water ingestion and septal stimulation inhibits
ongoing drinking. Other evidence indicates that septal
cells reSpond to stimuli of one recognized component of
thirst (reduction of blood volume), but not to another (an
increase in blood osmotic pressure). To further explore
the role of the septum in water regulation, discharges of
single septal cells during alterations of blood volume and
osmotic pressure were observed.

In the first experiment firing rates of septal
cells in re3ponse to the hypovolemic component of dehydra—
tion were monitored. Rats on §g_libitum food and water
schedules were anesthetized and blood was extracted from
the femoral vein. During the extraction cells character-
istically increased discharge rates. After the extraction,
cells in rats that had large (5 cm3) extractions decreased
firing rates while cells of rats that received moderate
size (2—3.5 cm3) extractions displayed no typical discharge
pattern.

In experiment 2 septal activity in 8 water deprived
and 8 water satiated rats was monitored for 1 hour. After
a 10 minute base rate recording period the deprived rats
were stomach loaded with water; 20 minutes after the

2

John G. Bridge

stomach load they were injected subcutaneously with 1 cm3

16% NaCl. With this series of manipulations firing rates
of septal cells were recorded as the dehydrated rat became
relatively hydrated and then dehydrated again. Satiated
rats were injected with the saline solution at the end of
the 10 minute base rate recording period, and 20 minutes
later stomach loaded with water. With this procedure the
satiated rat became relatively dehydrated and then hydrated
again. Firing rates of cells in deprived rats and firing
rates of cells in satiated rats did not differ signifi—
cantly during the base rate period. However, 13 of the

16 cells discharged faster during the dehydrated period
than during the hydrated period (p < .02, sign test).

To observe responses of septal cells to changes in
blood osmotic pressure, solutions of hypertonic, isotonic,
or hypertonic saline were infused into the carotid artery.
Isotonic injections offered an index of the effect of the
injection E§£_§§, while deviations from this index during
hypotonic or hypertonic injections indicated changes in
firing rates coincidental with changes in osmotic pressure.
Injections characteristically increaSed firing rates; in—
creases after hypertonic injections were usually the

3

John G. Bridge

largest, while increases after hypotonic injections were
usually smallest.

There are septal cells that are re3ponsive to
plasma volume depletion (femoral vein extractions, exper—
iment 1), changes in osmotic pressure (carotid injections,
experiment 3), and combinations of these stimuli (stomach
loads of water and hypertonic injections, experiment 2).
In general, cells fired faster during hyperosmotic than
during hypoosmotic blood conditions. Cells also usually
discharged faster during blood hypervolemia than blood
hypovolemia. This combination of results was not antici-
pated--since hypovolemia and hypertonicity are both condi—
tions of the water deprived state, it was eXpected that

these conditions would both increase or both decrease

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firing rates of septal cells.

ELECTRICAL ACTIVITY OF SINGLE CELLS IN THE
SEPTAL AREA OF RATS RESULTING FROM CHANGES

IN PLASMA VOLUME AND OSMOLALITY

BY

John G. Bridge

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF ARTS

Department of Psychology

1972

ACKNOWLEDGEMENT

Thanks to my committee, Dr. Glenn I. Hatton
(chairman), Dr. John I. Johnson, and Dr. Lawrence I.
O'Kelly. And, thanks to: Keven Bridge (editing), Gary
Conners (electronics), Robert Contreras (microelectrodes),
and Emmy Haight (histology).

This thesis was undertaken while the author was
on NASA grant number NSGT—SB and NDEA grant number 437-
002290. The research was supported in part by a grant
from the National Institute of Neurological Diseases and

Stroke (number NS 09140) to Dr. Glenn I. Hatton.

ii

LIST OF FIGURES. .

INTRODUCTION . . .
EXPERIMENT 1 . . .
Method . . . .
Subjects .
Procedure.
Results. .
EXPERIMENT 2 . . .
Method . . . .
Subjects .
Procedure.
Results. .
EXPERIMENT 3 . . .
Method . . . .
Subjects .
Procedure.

Results. .

TABLE

OF CONTENTS

iii

Page

10
12
25
25
25
26
28
38
38
38
39

42

TABLE OF CONTENTS (Cont.)

Page
DISCUSSION . . . . . . . . . . . . . . . . . . . . . 58
LIST OF REFERENCES 0 O O O O O O O O O O O O O O 0 O 71
APPENDICES O O O O O O O O O O 0 O O O O O O O O 0 O 74

iv

LIST OF FIGURES
Figure Page

1. Indicated are maximum, 0.5 mm lateral, and
1.0 mm lateral sagittal projections of the
rat septum. Below are confirmed recording
sites for each of three eXperiments in the
anterior—posterior dimension. Adapted
from de Groot, 1959 . . . . . . . . . . . . l3

2. Indicated are the mean (i S.E.) percent
changes from a five minute mean base rate.
N = 6 0 O O O O O O O O O O O O O O O O O O 15

3. Indicated are mean (: S.E.) percent changes
from base rate during and after a 5.0 cm3
extraction of blood from the femoral vein.
N = 6 O O O O O O O O O O O O O O 0‘ O O O O l7

4. Indicated are mean (i S.E.) percent changes
from base rate during and after a 2.0 cm
or 3.5 cm3 extraction of blood from the
femoral vein. N = 6 (two 3.5 cm3 and four
2.0 cm3 extractions). . . . . . . . . . . . l9

5. Indicated are discharge rates before, during,
and after a 5.0 cm3 extraction of blood
from the femoral vein. N = 6 . . . . . . . 23

6. Indicated are mean (t S.E.) percent of base
firing rate during a 10 minute stomach
load period (S.L.) in which 10.0 cm3 of
tap water were loaded at 1.0 cm3/minute,
a 20 minute post-stomach load period (Post—
S.L.), and a 20 minute post-injection
period (Post-inj.). The base (Dep base)

LIST OF FIGURES (Cont.)

Figure

7.

8.

9.

10.

Page

rate was recorded from 23.5 hour deprived

rats and the injection was 1.0 cm3 16%

NaCl administered subcutaneously in the

hip area. N = 8. . . . . . . . . . . . . . 29

Indicated are mean (i S.E.) percent of base

firing rate during a post—injection period
(post—inj) of 20 minutes, a 10 minute
stomach load period (post-S.E.) in which
10.0 cm3 of tap water were loaded at

1.0 cm3/minute and a 20 minute post-
injection period (post-inj). The base
(SAT base) rate was recorded from rats
after the 0.5 hour drink period of a 23.5
hour water deprivation schedule, and the
injection was 1.0 cm3 16%.NaC1 adminis-
tered in the hip area. N = 8 . . . . . . . 31

Indicated are mean (i S.E.) percent changes

in heart rate for six animals chosen at

random from the DEP group. Change in heart

rate for each rat was calculated frOm its
reapective mean baseline rates. N = 6. . . 34

Indicated are mean (t S.E.) percent changes

in heart rate for six animals chosen at

random from the SAT group. Changes in

heart rate for each rat was calculated

from its resPective mean baseline rates.

N = 6 . . . . . . . . . . . . . . . . . . . 36

Indicated are mean (t S.E.) discharge rates

during base rate periods, hypotonic, and
hypertonic NaCl injections into the left

carotid artery. Base means are six 10

second time bins; injection means are

three 10 second time bins . . . . . . . . . 43

vi

LIST OF FIGURES (Cont.)

Figure

11.

12.

13.

14.

15.

16.

Page

Indicated are mean (i S.E.) discharge rates
during base rate periods, hypertonic, and
hypotonic NaCl injections into the left
carotid artery. Base means are six 10
second time bins; injection means are
three 10 second time bins . . . . . . . . . 45

Indicated are mean (t S.E.) discharge rates
during base rate periods, hypertonic, and
hypotonic NaCl injections into the left
carotid artery. Base means are six 10
second time bins, injection means are
three 10 second time bins . . . . . . . . . 47

Indicated are mean (i S.E.) discharge rates
during base rate periods, isotonic, hyper-
tonic, and hypotonic NaCl injections into
the left carotid artery. Base means are
six 10 second time bins; injection means
are three 10 second time bins . . . . . . . 49

Indicated are mean (i S.E.) discharge rates
during base rate periods, isotonic, hyper—
tonic, and hypotonic NaCl injections into
the left carotid artery. Base means are
six 10 second time bins; injection means
are three 10 second time bins . . . . . . . 51

Indicated are mean (t S.E.) discharge rates
during base rate periods, hypertonic, and
hypotonic NaCl injections into the left
carotid artery. Base means are six 10
second time bins; injection means are
three 10 second time bins . . . . . . . . . 53

Indicated are mean (i S.E.) discharge rates
during base rate periods, isotonic, hypo-
tonic, and hypertonic NaCl injections into
the left carotid artery. Base means are
six 10 second time bins; injection means
are three 10 second time bins . . . . . . . 55

vii

INTRODUCT ION

During the past two decades, the role of the
lateral and medial septal nucleus in water regulation has
been the tOpic of numerous experiments. However, these
investigations have used ablation or stimulation tech-
niques; none used extracellular single unit recording.

A number of issues remain unresolved, apparently because
the eXperimental designs inherent in lesion and stimula—
tion studies prohibit a direct analysis of the role of
septal cells in water regulation.

The first and most basic issue is still in ques—
tion: Are septal cells involved in water regulation? In
more operationally definable terms this might be asked:
Do septal cells alter firing rates in response to changes
in blood volume or osmotic pressure? There is mounting
indirect evidence that they do, but some quite different
theories are tenable.

For example, the septum has been considered an
inhibitor of ongoing behavior; the septal lesioned animal

1

is unable to inhibit ongoing behavior (drinking as well
as other behavior). This theory has been supported by
experiments that demonstrate Passive Avoidance Response
(PAR) deficits in septal lesioned animals (Kaada, Rasmus-
sen, and Kveim, 1962; McCleary, 1961). But in 1965,
Harvey and Hunt found that septal lesioned rats could
maximize water rewards on Differential Reinforcement of
Law rates (DRL), and that differential water pre-loads
equalize bar pressing on Fixed Interval (FI) and Contin-
uous Reinforcement (CRF) schedules with water as a reward.
It was, perhaps, that study that turned attention to the
lateral and medial septum as areas directly involved in
water regulation. To date, evidence for this comes from
a number of perspectives: a) lesions in both lateral and
medial septal regions produce hyperdipsia (Blass and Han-
son, 1970: Harvey and Hunt, 1965); b) deprivation enhances
and satiation diminishes septal self-stimulation (Brady,
Boren, Conrad, and Sidman, 1957); c) cholinergic stimula-
tion of the lateral septum increases both food and water
intake (Fisher and Coury, 1962) and carbachol stimulation
of the medial septum increases drinking and decreases

eating (Grossman, 1964).

Hyperdipsia following septal lesions is a highly
consistent phenomenon, while increments in food ingestion
and the septal rage syndrome are not consistent results.
Lubar, Schaefer, and Wells (1969) found that hyperdipsic
effects are strongest with lesions in the middle and
posterior third of the septum (particularly in the anter-
ior two-thirds of the posterior third of the septum).
Effects of lesions in the anterior septum, they showed,
are much less than those of posterior lesions on water
intake, urine output, and urine osmolality. Injections
of pitressin tannate returned these measures to near
normal levels.

This literature that indicates septal cells prob—
ably are involved in water regulation contains a basis
for a hypothesis about the direction of the changes in
firing rate as an animal goes from one hydration state to
another. Since septal self-stimulation is enhanced by
deprivation and inhibited by satiation, and septal lesions
produce hyperdipsia, an intuitively attractive hypothesis
would be that the septum is a drinking inhibitory center,
and activity of septal cells decreases during deprivation

and increases during satiation.

Another line of reasoning leads to the Opposite
hypothesis, that septal cell firing decreases during sa—
tiation and increases during deprivation. Cross and Green
(1959), Dyball and Koizumi (1968), and Suda, Koizumi, and
Brooks (1963) have established that the cells of the supra—
Optic nucleus increase activity and ADH release increases
in response to increased blood tonicity. Hayward and Smith
(1963) have demonstrated that septal stimulation (and stim-
ulation of other limbic structures) causes pituitary re—
lease of ADH. With this line of reasoning one can account
for polydipsia in septal lesioned rats: without excitatory
stimulation from the septal nucleus, supraoptic activity
diminishes, ADH release decreases, and diuresis leads to
additional water ingestion. From this perSpective it ap-
pears that septal cells should be hyperactive during de-
privation and hypoactive during satiation. With these two
arguments leading to different conclusions about the rela—
tive discharge rate of septal cells during deprivation and
satiation, a direct measurement of this phenomenon would
be helpful.

While these first two questions involve (a) whether
or not septal cells are involved in water regulation, and

(b) if so, whether the cells are more active during

deprivation or satiation, a different area of research has
indicated that the septum may be sensitive to vascular
volume shifts, but not to osmotic preSsure changes. Blass
and Hanson (1970) injected septal lesioned rats and normal
rats with either polyethylene glycol (PG) to induce hypo—
volemia, hypertonic saline to cause blood hypersmolality,
or isotonic saline (preceded by 6 hrs. food and water
deprivation), and then measured intakes at 15, 30, 60, 90,
and 120 minutes post injection. Lesions damaged both
lateral and medial septal areas extensively. The PG septal
rats drank more than the PG normal rats in the first 15
minutes, but intakes were identical from then on. In the
hypertonic saline group, septals drank less than normals
in the first 15 minutes, but more during the 30, 60, 90,
and 120 minute periods; however, none of these differences
appears significant. Finally, in the deprivation and iso-
tonic saline group, septals drank more than controls at
each interval. Blass and Hanson interpreted these data as
evidence that hyperdipsia in septal rats is in response to
hypovolemia, but not to hypersmolality. They also measured
hematocrit and serum sodium, and concluded that normal and
septal rats differ on these measures neither in ad lib nor

in PG injected states. To Blass and Hanson this indicated

that septal hyperdipsia is not a result of a shift in body
fluid balance; that is, the septal rats are drinking more
than normal rats in re3ponse to similar blood volume and
osmotic conditions.

A few procedural anomalies and some more recent
experiments cast doubt on the results and interpretations
of the Blass and Hanson study. Almli (1970) noted that
hypertonicity accompanies hypovolemia when the sample is
taken as the animal initiates drinking, even when a "pure"
volume manipulation such as hemorrhage is made. Perhaps
rats in Blass and Hanson's hypovolemia group were, in fact,
reSponding to hypertonic cues that follow a hypovolemic
manipulation. One disappointment in the Blass and Hanson
article is that they used very small N's and rarely re-
ported significance levels. Finally, Wishart and Mogenson
(1970) referenced an unpublished study that fails to con-
firm that septal rats over-respond to hypovolemia. For
these and other minor reasons, the Blass and Hanson article
has been discredited to the extent that the hypothesis that
septal cells are sensitive to vascular volume changes but
not to osmotic pressure changes should be carefully exam—

ined.

The present study is designed to investigate the

questions raised above. These are:

(a) How do septal cells respond to changes in the

hydration state of the animal?

(b) How does hemorrhagic hypovolemia alter the firing

rates of septal cells?

(c) What is the response of septal cells to carotid
injections of hypo-, iso-, and hypertonic saline

injections?

The firing rates of septal cells as a function of
different hydration states were determined by two methods.
The first compared the firing rates of septal cells under
ad lib food and water conditions, 23.5 hour water depriva-
tion, and satiation (immediately after the 0.5 hour drink
period). In the second method, firing rates before, during,
and after both stomach loads of water and subcutaneous
injections of hypertonic saline were examined. To analyze
the Blass and Hanson prOposal that septal cells respond to
vascular volume shifts but not to osmotic pressure changes,
units were recorded before, during, and after either injec-

tions of saline solutions into the common carotid artery

or extractions of blood from the femoral vein. Heart rates
were monitored during all exPeriments. Histological slides
of the recording sites were made with either 40 micron
frozen sections and cresyl violet stain, or 25 micron

celloidin sections and Heidenhain, Weil, or Thionin stain.

EXPERIMENT 1

Pilot data indicated that in determining the re-
sponse of septal cells to the volume component of dehydra-
tion stimuli a femoral vein extract was superior to the
tail cut method; in addition to resulting in greater blood
flow than tail cuts, femoral vein extracts allowed the

eXperimenter to extract blood at a constant predetermined

rate.
Method
Subjects

Subjects were 18 male albino rats, 105—125 days
old and 350-425 grams on the recording date, supplied by
Holtzman Company of Madison, Wisconsin. They were housed
in single cages with constant light and ad lib food and

water.

10
Procedure

Prior to surgery all subjects received 0.7 g/kg
Dial-urethane anesthetic; occasionally, supplementary doses
of 0.03—0.07 g were necessary to anesthetize the animal
completely. Each rat was shaved on the crown, chest, and
abdomen with electric clippers. The femoral vein was ex-
posed and a 0.025 o.d. x 0.012 i.d. 22 cm. catheter was
inserted approximately six to seven cm. toward the heart
by passing it through the vein wall in a 17 gauge needle
and tying the vein on the proximal Side of the insertion,
and the needle then removed. The catheter was filled with
sodium heparin and the outer end joined to a heparinized
syringe by a 26 ga. stainless steel needle. (Once the sur—
gery was complete, the wound was closed and held with wound
clips. A needle electrode served as a heart lead and was
subcutaneously onto the chest; both the heart lead and the
catheter were prevented from slipping by affixing them to
the skin with masking tape. Next, ear pins were inserted,
and the rat stereotaxically positioned. Body temperature
was maintained at 36-38°C. Each subject was wrapped in a
variable temperature heating coil and both the rat and the
coil were wrapped in a towel; temperature was monitored

with a rectal thermister inserted to approximately 7—8 cm.

11

After the skull was surgically exposed, a 0.4 cm.
hole was trephined, dura mater removed, and a tungsten elec-
trode with glass insulation (except for a 5—8 micron exposed
tip) was lowered to the septum. Leads from the electrode
and the heart probe went to a junction box with off-lesion-
record Options. In the lesion position, current could be
supplied to the electrode tip for a marking lesion (20 ”A
anodal current for five seconds); in the recording position,
impulses from the electrode and the heart lead went to chan-
nel one and channel two, resPectively, of a dual beam os-
cillosc0pe and to a stereo tape recorder, via a low level
preamplifier set to amplify times 1000. Impulses were mon-
itored auditorily with a loudspeaker, and an on—line counter
was available to record the rates of impulses whose ampli-
tude exceeded the threshold of the oscillosc0pe trigger.

Once a cell was sufficiently isolated (the signal—
to—noise ratio allowed reliable recording of the unit dis-
charge), magnetic tape records were taken during the fol-
lowing procedure:

1. Five minutes of base rate.
2. A two to five minute extract period in which

2-5 cm3 of blood were extracted at a rate of

approximately 1.0 cm3 per minute.

3. A 50 minute post-extract period.

12
Results

Distribution of recording sites on the anterior-
posterior dimension is shown in Figure l; for recording
sites of individual cells, see Appendix B. The cytoarchi—
tectural demarcation of the lateral and medial septum
(shown by the DeGroot atlas used in this appendix) was
particularly vivid with Heidenhain and Weil fiber staining
techniques, revealing the medial area to be a much more
fiberous portion of the septum than is the lateral region.
The results of experiment 1, expressed in percent of base
rate, are summarized in Figures 2-4. The control group
was comprised of three normal control rats and three sur-
gery controls that had the femoral vein exposed and a
catheter inserted but no blood drawn. Because the normal
controls and surgery controls were similar in firing pat-
terns throughout the one hour recording session, they were
combined to form the control group of N = 6 in Figure 2.
As the graph indicates, at no point did the mean discharge
rate differ reliably from its original five minute base
rate. But there was a tendency for these cells to increase
in rate over the entire time of the recording session.

Statistically significant differences in rates may have

13

Figure l.--Indicated are maximum, 0.5 mm lateral, and
1.0 mm lateral sagittal projections of the
rat septum. Below are confirmed recording
sites for each of three exPeriments in the
anterior-posterior dimension. Adapted
from de Groot, 1959.

14

$2. 2.3 93 «an. o» «0.554

 

op od 06
o t.
m _ m ... m .
a . < n. «a
5.559.. ”.58.: 5.554

 

 

 

 

 

n_<mm._.<.._ 22 o._u u . .
...ZMPXM 232322 a ll

<mm< 44.5mm n_O

ZOCbMpOmn. n_<._:_._o<m

0 ID

ID

 

3Nl'l 'IVHHVHHINI W083 WW

15

Figure 2.-—Indicated are the mean (i S.E.) percent
changes from a five minute mean base rate.
N = 6.

16

 

 

 

 

 

30
MINUTES

 

 

P

h—
0 O O O
O K) o IO
N

1|.th ONIUIJ BSVG :IO 3'6

50

4O

20

IO

17

Figure 3.--Indicated are mean (t S.E.) percent chan es
from base rate during and after a 5.0 cm
extraction of blood from the femoral vein.
N = 6.

18

m

m¥004m ”3.32.2 m Pedmhxw ...mon.
m . _ ...xw um<m

- q d d u 1 4

 

 

.00

00.

On.

CON

BIVH 9N|Hl=l BSVB :IO %

19

Figure 4.-—Indicated are mean (t S.E.) percent changes
from base rate during and after a 2.0 cm3
or 3.5 cm3 extraction of blood from the
femoral vein. N = 6 (two 3.5 cm3 and four
2.0 cm3 extractions).

20

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d

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m _ ...xm mm<m

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21

been observed if the sessions had been extended for longer
periods. During the extraction of blood producing hypo-
volemia, the 5 cm3 extract group (Figure 3) showed a
tendency to increase, and this was followed by a decrease
during the post-extract period. The 5 cm3 extract group
did not differ from the controls during the extract, but
each post—extract mean was significantly (p < .05) lower
than the control group mean. Extracts of intermediate
sizes (2.0 cm3, N = 4, and 3.5 cm3, N = 2) showed mean
reSponses intermediate to those of the control and the
5.0 cm3 groups (Figure 4). However, individual records
of the intermediate size extracts reveal that rather than
discharging in an intermediate pattern, these cells char—
acteristically reSpond in a pattern similar to either the
control group or the 5.0 cm3 group. Of the four cells
with 2.0 cm3 extractions of blood, two were similar to the
control group and two were similar to the 5.0 cm3 group;
likewise, the 3.5 cm3 group was evenly divided. This
accounts for the large amount of variance in the inter-
mediate group.

While a measure of percent of base rate offers a
good index of the effects of the manipulation on single

units, it may be misleading in assessing the overall

22

changes in septal activity, since a percent change measure
would give slower firing cells a diSproportionately greater
weight than faster firing units. To obtain an index of
overall discharge rate changes (and to reveal how closely
the percent change measure reflects the overall change
measure), the absolute number of discharges per second
minus the mean base discharge rate was calculated for each
cell during the extract and post-extract periods, and this
mean absolute change for the 5.0 cm3 group is shown in
Figure 5.

Confirmation of the recording sites revealed that
in the control group 4 of the 6 cells were in the lateral
septal area. Of the 6 cells in the 5 cm3 extract group
all were in the medial septal region, and in the 2 3.5 cm3
extract group 3 of 6 cells were in the medial septum. No
differential effects between the lateral and medial septal
nuclei were observed, nor were any differential effects
apparent along anterior—posterior, dorsal-ventral, or

medial-lateral dimensions. For anatomical Sites of indi-

vidual cells, see Appendix B.

23

Figure 5.--Indicated are discharge rates before, during,
and after a 5.0 cm3 extraction of blood from
the femoral vein. N = 6.

24

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9.00.5 3.32.2 n bedmhxm ...mom
0 _

...xw wm<m

 

 

 

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dat

EXPERIMENT 2

This experiment determined single unit firing
rates during water deprivation and satiation, stomach
loads of water, and subcutaneous injections of hypertonic
saline solutions. With this design, two persPectives were
available: a) discharge rates of septal cells during
naturally occurring states of hydration and dehydration
were measured when the animals were recorded under deprived
and satiated conditions, and b) unit discharges in reSponse
to relative changes in hydration were measured under ex-
perimentally controlled conditions during and after the

stomach load and hypertonic injection recording periods.

Method

Subjects

Sixteen naive, male albino rats of the Holtzman
strain 350-425 g and 101-110 days old on the recording

date were housed in individual cages under constant light.

25

26

For a minimum of five days preceding the recording date,
each subject was adapted to a 23.5 hour water deprivation

schedule; food (to Appendix) was available 2g libitum.

Procedure

 

Recording procedure was the same as described in
experiment one. Subjects were assigned to either a de-
prived (DEP) group or a satiated (SAT) group; in the DEP
group subjects were removed from their home cages at the
end of the 23.5 hour water deprivation period, weighed,
and injected with 1.0 g/kg Dial-urethane (mixed in our
laboratory). When the rat was sufficiently anesthetized,
a polyethylene tube was inserted into the stomach. A
heart lead was affixed and the animal was stereotaxically
positioned as in the hypovolemia experiment. Once a cell
was isolated, a ten minute deprived base was recorded.
Then, a 10.0 cm3 stomach load of room temperature tap
water was injected via the polyethylene tube at approxi—
mately 0.25 cm3/15 seconds (10.0 cm3 in 10.0 minutes) with
a 20 cm3 plastic syringe. A post-stomach load period
(P-SL) of 20 minutes followed in which no manipulation

was made. (The stomach load tube was left in the animal

27

throughout the experiment.) At the end of the P—SL period--
40 minutes into the recording session—~a 1.0 cm3 16%»NaC1
solution was injected subcutaneously into the hip area.
This injection always took less than 10 seconds, and the
lO-second time bin in which it occurred was not included
in the data analysis. Finally, a post-injection period
(P-INJ) of 20 minutes was recorded. Following the record-
ing session a blood sample was extracted by heart puncture,
centrifuged at 3300 rpm, the plasma analyzed for protein
with a refractometer and frozen for later osmometry.
Subjects in the SAT group were removed from the
home cage immediately after the 0.5 hour drink period of
the 23.5 hour deprivation schedule. As with the DEP group,
once the animal was anesthetized a stomach tube was in—
serted, a heart lead was affixed to the chest, and the rat
was stereotaxically positioned. The order of manipulations
in the SAT group differed from that for the DEP group in
that the hypertonic saline injection preceded the stomach
load of water in the SAT animals. After a lO—minute base
rate recording, the SAT animals were injected with 1.0 cm3
of NaCl, and a 20-minute P-INJ period was recorded. The

recording session continued through a 10-minute stomach

load period (10.0 cm3 at 0.25 cm3/15 seconds) and a 20 minute

28

P-SL period. Blood sampling and perfusion procedures were

identical to those described for the DEP group.

Results

By comparing the 10 minute base rates of the DEP
and SAT groups it was determined that, under these Opera-
tionally defined conditions of water deprivation and sat-
iation, firing rates of septal cells in dehydrated rats
do not differ significantly from firing rates of cells in
relatively hydrated rats (DEP X': S.E. = 0.594 t 0.067;

SAT X't S.E. = 0.0501 t 0.255). The firing rates of septal
cells in response to DEP and SAT group manipulations are
summarized in Figures 6 and 7. In general, cells tended
to increase discharge rates in response to hypertonic in-
jections and decrease discharge rates in reSponse to stomach
loads of water. When the mean firing rate during the
stomach load and P-SL periOd was compared to the mean
firing rate of the P-INJ period, 13 of the 16 units (8 DEP
and 8 SAT) discharged faster in response to the hypertonic
injection than to the stomach loads (p < .02). Osmotic
pressure of the DEP group was higher than that of the SAT

group (306.2 f 1.7 and 297.3 i 3.8 mOs, p < .01), but the

29

Figure 6.—-Indicated are mean (i S.E.) percent of base
firing rate during a 10 minute stomach load
period (S.L.) in which 10.0 cm3 of tap water
were loaded at 1.0 cm3/minute, a 20 minute
post-stomach load period (Post—S.L.), and a
20 minute post-injection period (Post-inj.).
The base (Dep base) rate was recorded from
23.5 hour deprived rats and the injection
was 1.0 cm3 16% NaCl administered subcuta-
neously in the hip area. N = 8.

30

...z. ...—.mon.

‘

m

Ix}

 

 

 

 

9.00.5 MPDZE m
....m -...mon.

‘ fl

0

wmdm
.5 duo

1'0

1

 

‘36

00.

O
8
sum omens asve so

31

Figure 7.-—Indicated are mean (i S.E.) percent of base
firing rate during a post-injection period
(post-inj) of 20 minutes, a 10 minute stomach
load period (post-S.E.) in which 10.0 cm3 of
tap water were loaded at 1.0 cm3/minute and
a 20 minute post-injection period (post-inj).
The base (SAT base) rate was recorded from
rats after the 0.5 hour drink period of a
23.5 hour water deprivation schedule, and
the injection was 1.0 cm3 16%.NaC1 adminis-
tered in the hip area. N = 8.

3.2

..muhmoa

9.00.5 3.32.: 0
4m 52. ....mon.

 

 

 

 

 

 

 

00.

O
8
any 9mm: asve .40 x

33

protein concentrations of the DEP group (5.1 + 0.2 g/100 ml)
and the SAT group (5.1 i 0.1 g/100 ml) were nearly iden—
tical. Heart rates of the DEP and SAT groups are analyzed
in Figures 8 and 9.

In both the SAT and DEP groups 7 of 8 cells were

in the lateral septum. For individual recording sites see

Appendix B.

34

Figure 8.—-Indicated are mean (i S.E.) percent changes
in heart rate for six animals chosen at
random from the DEP group. Change in heart
rate for each rat was calculated from its
reSpective mean baseline rates. N = 6.

...z. -.rmon.
0

9.00.5 3.32:2 m

...m ......won.
0

..m

n

wm<m
duo.

 

S d I

35

d

 

 

 

q

 

C CD
31.th IBVBH asvs W083 3'9NVHO “x.

CD

36

Figure 9.—-Indicated are mean (t S.E.) percent changes
in heart rate for six animals chosen at
random from the SAT group. Changes in
heart rate for each rat was calculated
from its reSpective mean baseline rates.

N = 6.

37

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d d

mxoonfi 5.32.2 m
..m ...2.-._.mon.

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Elva lHVBH asva woas BSNVHO x

 

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I

O

EXPERIMENT 3

To determine the effects of changes of blood toni-
city on firing rates of septal cells, solutions of hypo—
tonic, isotonic, and hypertonic saline were injected into
the left common carotid artery. Since no injection is
without a volume component, the isotonic injections were
necessary to reflect the effects of the volume increase.
Firing rates during and after injections of hypertonic and
hypotonic solutions are then compared to both the original
base firing rate and the firing rate of the isotonic in-
jections to reflect changes due to the alterations in

osmotic pressure.

Method

Subjects

Five naive, male albino rats of the Holtzman
strain, 350-425 g and 101-116 days old on the recording

date were housed in individual cages under constant light.

38

39

For a minimum of five days preceding the recording date,

food and water was available §g_libitum.

Procedure

 

Subjects were anesthetized as were those in the
first two eXperiments, although supplementary doses were
more frequently needed during the surgery for the present
eXperiment. Once the rat was anesthetized, the left common
carotid artery was exposed by cutting the skin along the
throat, separating the left sternomastoideus muscle from
the sternothyroideus muscle, cutting the tissue away from
the artery with a scalpal, lifting the artery with a pair
of small tweezers, and slipping a piece of plastic tubing
behind it. A 26 ga needle was slipped through the left
branch of the sternothyroideus muscle and into the carotid
artery. Prior to the eXperiment a small (0.03 cm3) chamber
made by a shortened neck of a 1.0 cm3 plastic syringe had
been inserted into the needle head and sealed with dental
acrylic. From this chamber two 25 cm lengths of plastic
tubing (0.012 I.D. x 0.025 0.D.) led to two 1.0 cm3 plastic
syringes that held the solutions of varying tonicity to be

injected into the carotid artery.

40

Once the needle was in the artery, the plastic tube
behind the artery was removed and the incision closed with
wound clips. With the wound clips placed closely together
so that only the two plastic tubes could emerge from the
skin (and not the bulbous dental acrylic portion), the
apparatus was held in place at two points: the Skin and
the left sternomastoideus muscle. So that the needle would
not slip out of the artery while the rat was prepared for
recording, a small towel was rolled from each side toward
the middle and taped along the abdomen of the rat from
head to tail; when the rat was turned over to be stereo—
taxically positioned, the injection apparatus protruded
between the two rolls of the towel, and these rolls sup—
ported the rat. Skull surgery and isolation of a unit was
as described in experiment one, except that the heating
coil was folded along the back of the rat rather than
wrapped around it.

Prior to the eXperiment the syringes were loaded
with solutions of the desired tonicity. When an injection
of one tonicity was followed by one of a different toni—
city, it was necessary to flush the chamber between the
two injections so that the chamber would contain a solution

whose salinity approximated that of the next injection.

41

Pilot data indicated that this was best done by a small
(0.05 cm3) flush injection, depressing the plunger of the
syringe containing the solution to be injected next. When
three different solutions were to be injected during the
same session, it was necessary to replace one of the ori—
ginal syringes with a syringe containing the third solu-
tion. When this occurred, it was necessary to flush not
only the chamber, but also the plastic tube between the
syringe and the chamger; this was accomplished by four
successive flush injections of 0.05 cm3.

Once a septal unit was isolated, a two minute base
rate was recorded. Injections lasted for 10 seconds; a
0.2 cm3 injection, for example, was injected at 0.02 cm3/
second. By using the auditory one of the click of the
on—line printer at the beginning of a 10 second time bin
and a watch with a second hand, an injection could be con-
tained in a single 10 second bin. Because no best concen-
tration, volume, or sequence of injections had been deter-
mined, a variety of combinations was used, and each of the
five recording sessions was a unique procedure. Only in
sessions three and four—-sessions in which two cells were

isolated simultaneously——were two units subjected to iden-

tical manipulations. Because of the procedural differences

42

among sessions no data grouping was done, and effects of
the injections on the firing rate of each cell was examined

individually.

Results

The volume, tonicity, and sequence of each injec-
tion, and the effects of the injection on the discharge
rate of each cell are shown in Figures 10 through 16.

Since effects of an injection were not confined to the

10 second bin in which it was administered, the next two

10 second bins are included in the means representing an
injection; each non—injection mean represents six 10 second
bins.

Hypertonic injections increased firing rates more
consistently and to a greater extent than did isotonic
or hypotonic injections. There appeared to be a strong
tendency of the septal cells to habituate to these injec-
tion stimuli: the first injection usually had the greatest
effect regardless of the tonicity, and if a second large
reaction occurred it usually came later in the recording
session rather than shortly after the first injection.

Flush injections had little or no effect on discharge rates.

ADDENDUM

In figures 10-16 (Experiment III), F = flush
injection, A = hypertonic injection, B == isotonic
injection, and C =. hypotonic injection. For concentration

and size of these injections, see Appendix B.

43

Figure 10.--Indicated are mean (i S.E.) discharge rates
during base rate periods, hypotonic, and
hypertonic NaCl injections into the left
carotid artery. Base means are six 10
second time bins; injection means are
three 10 second time bins.

44

IMINUTES

 

 

e 1 0
—— -—LL
—0— -co-<t
— . (D
+_ c2.)
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o: — “""L‘a’
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0. «2 N
'- -O O

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45

Figure 11.--Indicated are mean (i S.E.) discharge rates
during base rate periods, hypertonic, and
hypotonic NaCl injections into the left
carotid artery. Base means are six 10
second time bins; injection means are
three 10 second time bins.

46

mzo_._.om...z_

0&4“...

 

d - d

. . h

< n. .
$52.5. m. w w o. _.

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47

Figure 12.--Indicated are mean (i S.E.) discharge rates
during base rate periods, hypertonic, and
hypotonic NaCl injections into the left
carotid artery. Base means are six 10
second time bins, injection means are
three 10 second time bins.

CAR-3 (LG)

48

 

 

 

 

 

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49

Figure l3.--Indicated are mean (i S.E.) discharge rates
during base rate periods, isotonic, hyper-
tonic, and hypotonic NaCl injections into
the left carotid artery. Base means are
six 10 second time bins; injection means
are three 10 second time bins.

50

«20:09:2—

<<mouumu 0.... < (no

.. .
mm...3z_2_ _ _ _ _NN_ _ n. _ _o_ _
‘dd

 

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51

Figure 14.--Indicated are mean (i S.E.) discharge rates
during base rate periods, isotonic, hyper-
tonic, and hypotonic NaCl injections into
the left carotid artery. Base means are
six 10 second time bins; injection means are
three 10 second time bins.

52

 

 

 

 

 

 

4 4 m m mumWZOWOWazf 4 h. o ...
35.........._._.”.“_....._......._.__
___ ...... ____ __ _ U...
_ _ .
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ONOOBS 83d SEMIdS

53

Figure 15.—-Indicated are mean (i S.E.) discharge rates
during base rate periods, hypertonic, and
hypotonic NaCl injections into the left
carotid artery. Base means are six 10

second time bins; injection means are three
10 second time bins.

mm...3z_2 m.

w20_.—.om_..2_
o u <

 

 

 

m-m<o

 

 

 

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ONOOBS 83d SBXIdS

00.
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55

Figure l6.--Indicated are mean (t S.E.) discharge rates
during base rate periods, isotonic, hypo-
tonic, and hypertonic NaCl injections into
the left carotid artery. Base means are
six 10 second time bins; injection means
are three 10 second time bins.

56

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352.: _ mm
...

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57

After two of the sessions (an unreported pilot
session and session five) blood was extracted from the
carotid artery into the chamber and syringe barrel. Small
increases in discharge rates were observed following both
these extractions.

All cells but 1 in experiment 3 were in the la—
teral septal nucleus. For individual recording sites, see

Appendix B.

DISCUSSION

The results obtained in experiments 1, 2, and 3
did not diSplay any differential effects along the
anterior—posterior, lateral—medial, or dorsal-ventral
dimensions. This, however, does not contradict the report
of Lubar, gt_al. (1969) that lesions in the anterior por-
tion of the posterior third of the septum cause the
greatest increment in water intake, while lesions in the
anterior septum cause the least. In the present study
such a great prOportion of the recording sites were in the
middle third of the septum that no sound inference can be
made as to differential effects along the anterior-posterior
dimension. In fact, Lubar, §£_a1. reported moderate incre—
ments in water intake following middle septal lesions; the
moderate effects found in the present eXperiments may have
been greater had the recording sites been in a more pos-
terior location. These effects were considered "moderate"
in a number of respects. For example, the 2—3.5 cm3 group
in eXperiment 1 (Figure 4) did not differ from base rates

58

59

or discharge rates of control cells. In the 5 cm3 extract
group the 5 minute means of the extract and post-extract
periods differed significantly when measured by percent

of base rate (Figure 3), but not when measured as an abso-
lute firing rate (Figure 5). Also, the septal cells of
23.5 hour water deprived rats did not discharge signifi-
cantly faster than cells in water satiated rats (eXperi-
ment 2). Bennett (1971) found that anterior hypothalamic
(AH) cells in 23.5 hour water deprived rats fire approxi—
mately 50%.faster than AH cells in ad lib_rats, and supra-
Optic (SON) cells in 23.5 hour water deprived rats dis-
charge 400% faster than cells in §g_;;p rats. Although
Bennett compared discharge rates of cells in 23.5 hour
water deprived rats to those in ad lib rats while the
present study compared cells in deprived and satiated rats,
AH and SON cells apparently are more responsive to extended
(23.5 hour) periods of deprivation than are septal cells.
Bennett also applied the deprived base--stomach load——
hypertonic saline injection series of manipulations (iden—
tical to the DEP group of experiment 2) to rats while re-
cording from SON and AH cells. Interestingly, under these
conditions of rapid alterations of hydration states sept

and AH cells appeared less reSponsive to the stimuli than

60

did the septal cells of the present experiment. (See page
for discussion of response of septal cells to novel
stimuli.)

Comparisons among the present eXperiments must be
made with caution, since distribution of recording sites
was not the same for each eXperiment. For example, 13 of
18 cells in the first experiment were in the medial septum,
14 of 16 cells in the second eXperiment were in the lateral
septum, and 6 of 7 cells in the third eXperiment were
lateral septal cell. In addition to being more in the
lateral septum, cells in the second and third experiments
were slightly more dorsal than were those in the first ex-
periment. When the control cells for all groups in eXper—
iment 1 (during the first five minutes of base rates) were
divided into lateral or medial cells, the ad lib firing
rates per second were:

6

lateral septum: 2.63 =
1.35, N = 11

medial septum: 5.82

H H
o
(D
N
2'.

These values were considerably greater than firing

rates during water deprived (0.68 t 0.14 spikes per second,

N 8) and water satiated (0.51 I 0.23 spikes per second,

N 8) conditions found in eXperiment 2. It is difficult

to believe that cells in deprived and satiated states can

61

discharge at similar rates while ad lib cells fire signif—
icantly faster. While the differential sampling of the

ad lib group (mostly medial) and the satiated group (mostly
lateral) can account for most of the greater firing rate
of the ad lib cells, three other sources of this discrep—
ancy are possible. First, different electrodes were used
for the hypovolemia group (from which the ad lib samples
were drawn) than were used for the SAT and DEP groups.

How different electrodes can effect firing rates or bias
sampling is not known. Apparently, when an electrode is
introduced to the immediate vicinity of a cell (or actually
touching the cell) the cell can be stimulated, causing it
to discharge faster. Another possible alteration of dis-
charge rates by the introduction of the electrode could

be a result of separating a cell from synapses on that
cell. Because a different population of electrodes was
used for each eXperiment, inconsistencies in physical
properties (size, shape, conductivity, etc.) of electrodes
among groups was realized. It is possible, then, that a
differential effect of the introduction of the electrode
has occurred across groups (although neither the direction
of this effect nor the underlying principle is understood).

The second possible source of this discrepancy is that the

62

CIBA—supplied urethane used in the hypovolemia experiment
was twice the concentration of the urethane mixed in our
laboratory and used in the SAT and DEP eXperiments. How-
ever, instead of requiring twice as great a dosage, these
latter rats required only about 150%.as much as the HV
rats, and therefore had only approximately 75%.as much
urethane anesthetic. Third, the SAT and DEP rats had
stomach load tubes inserted prior to recording.

Another major inconsistency cannot be resolved
anatomically. Hypovolemia, a characteristic of the water
deprived state, caused cells to decrease in firing rates
in experiment 1. But experiments 2 and 3 indicated that
septal cells fire faster during the hypertonic state: the
post-injection period is characteristically more active
than the SL and the P—SL periods, and hypertonic carotid
injections increase firing rates considerably more than
carotid injections of hypotonic saline. Why would one
component of deprivation (hypovolemia) decrease firing
rates, while another (hypertonicity) increase the dis-
charge rate?

All of the 5 cm3 extract cells were located in the
medial septum: if the consistent decrease in discharge

rate characteristic of this group were due to the

63

anatomical location rather than the size of the extraction,
the HV group with the intermediate size extracts (2.0 and
3.5 cm3) should show reSponses indicating that the medial
septal cells responded as the 5.0 cm3 group did, and the
lateral cells as the control group did. The data did not
support this. Two of the four lateral septal cells and

one of the two medial septal cells increased, while half
of both lateral and medial septal cells decreased in firing
rate, indicating that the decrease was not related to the
anatomical location of the cell.

A 5.0 cm3 loss of blood——especially following loss
of approximately 0.5-1.0 cm3 in femoral vein surgery and
1.0—2.0 cm3 in the skull surgery--is considerable. Ganong
(1967) described hypovolemia of 5—15 ml/kg (about 2-6 cm3
for a 400 g rat) as a "moderate" stress. One of the hy-
potheses entertained to explain why the septal cells re—
Spond differently to hypovolemia and hypertonicity was that
the decrease in discharge rate during hypovolemia was due
to hemorrhagic shock. The heart rates did not support
this hypothesis. No subject showed the rapid pulse that

Ganong (1967) and Wright (1965) regard as symptomatic of

hemorrhagic shock.

64

Cells in the carotid injection eXperiment whose
firing rates did change almost always responded to the
injection by increasing discharge rates. Although greater
and more frequent changes were elicited by hypertonic in—
jections, even hypotonic injections often were accompanied
by increased firing rates. If this is interpreted as a
response to the volume component of the injection stimulus,
then the carotid injection eXperiment corroborates the
hypovolemia study—~an injection slightly distends the ar—
tery, momentarily stimulating hypervolemia, and increases
the discharge rate, the expected reaction if hypovolemia
decreases the discharge rate.

Almli (1970) noted that a 2.2% increase in osmo-
lality follows hemorrhagic hypovolemia (5.0 cm3 from a
tail cut) when measured at initiation of drinking (approx—
imately 53 minutes after the hemorrhage). In the present
eXperiment, post-recording blood samples did not differ
significantly in plasma osmolality from blood extracted
during the recording session. However, two minor hemor-
rhages preceded recording (femoral vein surgery approxi-
mately 30 minutes prior to recording and skull surgery

about 15 minutes before recording), and the recording

65

extract and the post recording sample were separated by
only 50-52 minutes.

The only significant difference in osmotic pressure
found was between samples taken after the DEP (306.2 f 1.7
mOsm) and SAT (297.3 f 3.8 mOsm) recordings, p < .01.
Osmotic shifts concomitant with the manipulations of the
SAT and DEP groups are of interest. In a free intake
period of 30 minutes, Hatton and Thornton (1968) found that
rats drink 9-10 ml in response to a 1.0 cm3 subcutaneous
injection of 16%.NaCl. In the present experiment, the in-
jection and the stomach load, then, approximately offset
each other. The total exogenous solution administered was
approximately 3%.body weight of 1.5%.NaCl_(l.0 cm3 16%
NaCl injection plus 10 cm3 water). This was not a severe
challenge: rats are able to regulate internally (i.e.,
without drinking) up to 2.0% NaCl solutions (2.0%.body
weight, stomach loaded: Hatton and O'Kelly, 1966).

A 1.0 cm3 injection of 16%.NaCl is sufficient to
initiate drinking at approximately six minutes post-
injection (Hatton and Thornton, 1968). Following a 23.5
hour water deprivation period rats stOp drinking after
9-10 minutes, with osmotic decreases and volume increases

occurring at approximately 8 and 10 minutes, respectively

66

(Hatton and Bennett, 1970). Examination of individual
firing rates (see Appendix B) indicates that only a few
cells reSponded in temporal patterns closely coincidental
to these behavioral and physiological indices.

Cells in the DEP group tended to increase firing
rates as a function of time, although from Figure 2 it is
apparent that even control cells tend to increase discharge
rates throughout the recording session. Throughout the
deprived base rate, stomach load, and post-stomach load
periods, this pattern closely resembles the pattern of the
control cells (see Figure 2); not until the post-injection
period did the DEP group cells begin to fire faster than
the control cells.

The paradoxical result of the hypovolemic manipu-
lation decreasing discharge rates (eXperiment 1) while
hypertonic manipulation increased discharge rates (exper—
iments 2 and 3) has not been resolved by the present ex—
periment. An examination of individual records in eXperi-
ment one shows that of the three cells that increased dur—
ing the post extract period (two 2.0 cc and one 3.5 cc
extracts), two were quite similar to the control group
while the other kept increasing far above the mean control

rate. These data are consistent with the hypothesis that

67

there is a threshold of hypovolemia (unattained in the
recording sessions of the two intermediate extracts that

fired in control—like patterns) necessary to increase

 

septal single unit firing rates (this threshold was at-
tained by the one cell that fired considerably faster than
control cells); and a second threshold-—a stress component
not registered by the heart rate monitor—-which decreases
septal unit activity (this threshold was attained by all
the 5.0 cc extract animals and those in the intermediate
group that decreased in discharge rate). This interpre—
tation would render all three eXperiments consistent and
corroborate the theory offered in the introduction: that
septal cells are more active during deprivation, and stim-
ulate the supraOptic-hypophysial system to increase ADH
release. However, with only one cell responding at an
increased rate, this interpretation is highly Speculative.
But this possibility strongly suggests new research: a
replication of the present HV eXperiment with small (per—
haps 1.0-1.5 cm3) blood extracts. A chronic preparation
would eliminate effects of the femoral vein surgery. The
large extracts (up to 5.0 cm3) of the present eXperiment
were used to facilitate a comparison with the results of

the Almli study (1970) in which he used 5.0 cm3 extract,

68

and to avoid masking such changes by the hypovolemia (1.0—
2.0 cm3) of the surgery.

Evidence that septal cells increase firing rates in
response to blood hypertonicity comes from another, unquan—
tifiable source: during three recording sessions great
increases in background activity in reSponse to hypertonic
injections could be heard via the auditory monitor. This
occurred during the first injection for the CAR-5 cell and
the first injection of an unreported pilot cell, and during
the first and second hypertonic injections for CAR-3. This
suggests that in addition to the microelectrode approach,

a gross electrode study could be useful in detecting
changes in the activity of the septum.

Some interesting comparisons were found between the
Cross and Green (1959) study and the present one. Cross
and Green applied a number of stimuli (pinch, visual, audi-
tory) before they applied the osmotic stimulus. They re—
corded from the septum, but they declined to infuse the
hypertonic stimulus when a very slow cell was isolated,
because of the difficulty in statistical interpretation
and because many slow-firing neurones were passed by the
electrode tip while the cells were inactive. For these

reasons, Cross and Green did not apply the hypertonic

69

stimulus when a septal cell was isolated, but they did
apply the other stimuli frequently enough to observe that
the lateral septum was quite responsive to pinch stimuli
and the medial septum sensitive to light stimuli. They
also reported a strong tendency for septal cells to habi—
tuate to repeated stimuli. This corresponds to the inter-
pretations of the reactiOns of some of the cells in eXper—
iment 3. The septal response to novel stimuli and habit-
uation to repeated stimuli may be important septal char-
acteristics.

The effects of stomach loads of water and injec—
tions of 16%.NaCl on heart rates (Figures 8 and 9) are
interesting in comparison to two previous experiments.
Bennett (1971) found heart rate changes similar to those
for the DEP group. However, O'Kelly, Hatton, Tucker, and
Westall (1965) found that stomach loads of water increase
heart rates during and after the load, while stomach loads
of hypertonic saline increase heart rates during the load
but decrease heart rates after the load.

Indications by Blass and Hanson in 1970 that the
septal area of rats are receptive to volemic but not os—
motic stimuli was not supported by the present study.

Experiment 3 indicated there are septal cells that, in

7O

addition to responding to volemic stimuli, also react to

osmotic stimuli.

L IST OF REFERENCE S

LIST OF REFERENCES

Almli, C. R. Hyperosmolality accompanies hypovolemis: A
simple eXplanation of additivity of the stimuli for
drinking. Physiology and Behavior, 1970, 5, 1021—
1028.

Bennett, C. T. Electrical activity of supraOptic neurons
in water deprived rats during slow intragastric
infusions of water. Ph.D. Thesis, Michigan State
University, 1971.

Blass, E. M. & Hanson, D. G. Primary hyperdipsia in the
rat following septal lesions. Journal of Compara—
tive and Physiological Psychology, 1970, 70, 87-93.

Brady, J. V., Boren, J. J., Conrad, D., & Sidman, M. The
effect of food and water deprivation upon intra-
cranial self-stimulation. Journal of Comparative
and Physiological Psychology, 1957, 50, 134-137.

Cross, B. A. & Green, J. D. Activity of single unit neu—
rones in the hypothalamus: Effects of osmotic
and other stimuli. Journal of Physiology, 1959,
148, 554—569.

De Groot, J. The Rat Forebrain in Stereotoxic Coordinates.
Amsterdam: N. V. Noord-Hollandsche Uitgevers
Maatschappij, 1959.

Dyball, R. E. J. & Koizumi, K. Electrical activity in the
supraOptic and paraventricular nuclei associated
with neurohypOphysial hormone release. Journal of
Physiolggy, 1969, 201, 711-722.

 

Fisher, R. E. & Coury, J. N. Cholinergic tracing of a
central neural circuit underlying the thirst drive.
Science, 1962, 138, 691-693.

71

72

Ganong, William F. Review of Medical Physiology. 3rd Edn.
Los Altos: Lange Medical Publication, 1967, pp.
476-507.

Grossman, S. P. Effects of chemical stimulation of the
septal area on motivation. Journal of Comparative
and Physiological Psychology, 1964, 58, 194-200.

 

Harvey, J. A. & Hunt, H. F. Effect of septal lesions on
thirst in the rat as indicated by water consumption
and Operant responding for water reward. Journal
of Comparative and Physiological Psychology, 1965,
59, 49-56.

Hatton, G. I. & Bennett, C. T. Satiation of thirst and
termination of drinking: roles of plasma osmo-
lality and absorption. Physiology and Behavior,
1970, 5, 479-487.

Hatton, G. I. & Thornton, L. W. Hypertonic injections,
blood changes, and initiation of drinking. Journal
of Comparative and Physiological Psychology, 1968,
66, 503-506.

Hatton, G. I. & O'Kelly, L. I. Water regulation in the
rat: consummatory and excretory responses to
short—term osmotic stress. Journal of Comparative
and Physiolggical Psychology, 1966, 61, 477-479.

 

Hayward, J. R. & Smith, W. K. Influence of limbic system
on neurohypOphysis. Archives of Neurology, 1963,
9, 171-177.

Kaada, B. R., Rasmusson, E. W., & Kvein, O. Impaired
acquisition of passive avoidance behavior by sub-
callosal, septal, hypothalamic, and insular lesions
in rats. Journal of Comparative and Physiological
Psychology, 1962, 55, 661-670.

Lubar, J. F., Schaefer, C. F., & Wells, D. G. The role of
the septal area in the regulation of water intake
and associated motivational behavior. Annals of
the New York Academy of Sciences, 1969, 157, 875-
893.

 

73

McCleary, R. A. ReSponse Specificity in the behavioral
effects of limbic lesions in the cat. Journal of
Comparative and Physiological Psychology, 1961,
54, 605-613.

 

O'Kelly, L. I., Hatton, G. 1., Tucker, L., & Westall, D.
Water regulation in the rat: heart rate as a
function of hydration, anesthesia, and association
with reinforcement. Journal of Comparative and
Physiological Psychology, 1965, 59, 159-165.

Suda, I., Koizumi, K., & Brooks, C. M. Study of unitary
activity in the supraOptic nucleus of the hypo-
thalamus.

Wishart, T. B., & Mogenson, G. T. Effects of food depri-
vation on water intake in rats with septal lesions.
Physiology and Behavior, 1970, 5, 1481-1486.

Wright, Samson. Samson Wright's Appiied Physiology. 11th
Edn. London: Oxford University Press, 1965,
p. 151.

APPENDIX A

Apparatus and Suppliers

75

Eggipment and sgppliers for microelectrode construction.

1.

20
3.
u.

S.

6.

Stainless steel tubing, 26 gauge, Superior Tube
CO., Norristown, Pennsylvania.

Tungsten wire, 0.005”, Sylvania, New York, N.Y.
Glass tubing, Corning Glass, Corning, New York.

Micrometer eyepiece, x8, Zeiss, Matheson Scientific,
1600 Howard St., Detroit, Michigan.

Stereozoom dissecting microscope, Nikon, E.H.
Sargent & CO., 8560 W. Chicago Ave., Detroit,
Michigan.

Microscope, Unitron BMLU NO. h202h, E. H.
Sargent & CO., 8560 W. Chicago Ave., Detroit,
Michigan.

E ui ment and suppliers for blood analysis, extractions,
and Efifusions.

1.
2.

3.

h.

5.

Centrifuge, International Equipment Company.

Refractometer, American Optical 00., Instrument
Division, Buffalo, N.Y.

Freezing point osmometer, Precision Systems,
Inc., at Rumford Ave., Waltham, Mass.

Plastic syringes, Plastipak, Apothecary ShOp,
Lansing, Mich.

Catheters, Dow Chemical, Midland, Mich.

Egpipment and suppliers for bioelectrical data recording

 

and analysis.

1.

2.

3.

Stereotaxic instrument, Stoelting CO., hZu
N. Homan Ave., Chicago, Ill.

Preamplifier, 122, Tektronix, Inc., P. 0. Box
500, Beaverton, Oregon.

Power supply, 125, Tektronix, Inc., P. 0. box
500, Beaverton, Oregon.

Oscilloscope, 502A, Tektronix, Inc., P. 0.
Box 500, Beaverton, Oregon.

76

5. Tape recorder, 1028, Magnecord, Main Electronics,
5558 S. Pennsylvania Ave., Lansing, Mich.

APPENDIX 8

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EXPERIMENT II

Plasma protein and osmolality.

 

 

Cell Protein Plasma
Ng. ggncentration, g/lOOml osmolality, mOsm/gg
DEPll 5.7 304
DEP2 5.4 313
DERh h.9 309
DEP6 5.3 306
DEP8 h.7 303
DEPlO 4.7 302
SATl 5.2 306
SATS 5.0 295
SATB )4. 9 300
SATIO 5 . 1 288
SAT2 5.2 288
SAT6 LI . 9 298

in
E3

Size

 

NaCl
conc. (fi)

 

85

EXPERIMENT II I

Inj
no.

 

and concentration of carotid infusions.

 

Size*

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* All injections took 10 seconds.

86

Indicated on the facing section are the following
confirmed recording sites:

Site Cell fig. Experiment fig.

 

HVh6
HVSB
HV37
HV60
HV21
HVSl

own¢nynoht
HHHHHH

8'7

-1 .--_-.__1 .

v.1 -

l'

"T'—

 

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--
-
l

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.
.

.
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_
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.
.
.

a

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I
l
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O
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I [III-IL I'lll‘ tlltll'L
II..- I

’é/
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W

*1
%

 

C----~‘~-

 

 

88

Indicated on the facing section are the following
confirmed recording sites:

 

 

 

Site Cell §_. Experiment 39.
1 HVZB 1
2 HVSO 1
3 HVSS 1
h HV29 1
S DEP 2
6 DEP 2

89

V
f!

 

 

___T___,T,____

T'

 

I.

 

0‘
--~-------

'. .I 7!.

   

..‘V..

”M”

////
W

I
4%
¢’
4

 

 

 

90

Indicated on the facing section are the following
confirmed recording sites:

Site Cell fig. Experiment fig.

 

DEP9
DEPlO
DEPll
DEP13
DEPlh
SATS

OARFflynch'
NNNNNN

92

Indicated on the facing section are the following
confirmed recording sites:

Site Cell §_. Experiment fig.

 

 

SAT
SAT
CAR-3
CAR-h
CAR-S

“4?me
wuwmm

93

 

 

 

 

  

 

 

 

J _ h . m
. P w n ' fl ( 1 w ‘o
_ u 4 a a 1 a d d
Lt.
TI
in
T
_ [-3
fl o
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In .4
_
- I
.3 1
...
Tu .
. I.
. I
/
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1,.
L.
r 1.
_
Lu
_
V 1.
TI J<
._

.l -ti' Lt

all '7‘ D

 

 

9'4

Indicated on the facing section are the following
confirmed recording sites:

U)
y.»
a
O
O
G
...:
H
'2
O
0

Experiment 33.

 

crvurcomna
:
...:
wNHHHH

95

 

«on

3:0

 

 

'I-‘ 'I’. II‘\||

 

96

Indicated on the facing section are the following
confirmed recording sites:

(D
p
d”
O
O
0
H
H
'2
0
0

Experiment 32.

 

HVh9
HV17

DEP6
SAT7
CAR-Y

O‘U'l-F'UNH
WNNHHH

97

 

 

    
       

  

   

 

.. ... \J t -
y/ L.
////x/// , ////%/¢, Ln
2% ,///// A‘ ////flx
//// 7%? ..

            

 

 

 

98

Indicated on the facing section is the following
confirmed recording site:

Site Cell 32. Experiment fig.

 

 

1 R-30 l

99

 

 

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,7//////////,/ I v -
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n. q...) Wvl/l/Ax/fl/ all .. «0: fix. . . ./ ... H .-. Wm. ./
.. ., ... K-,. x--. . ff \. \.\-- 2473/ . 1
a. x . II, 0 .\\\ .. / _.
~ I, “.u( . .ss 1“ N // x J.,,
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L.
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