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LIBRARY
Michigan Stan
University .

This is to certify that the
thesis entitled

UNIT ACTIVITY IN I‘HE SEPI‘AL NUCLEI
DURING WATER DEPRIVAI‘ION. DRINKING.
AND REHYDRATION

presented by

John G. Bridge

has been accepted towards fulfillment
of the requirements for

Ph.D. degnPhl Psychologv

W

Major professor
Date w

0-7639

 

 

ABSTRACT
UNIT ACTIVITY IN THE SEPTAL NUCLEI

DURING WATER DEPRIVATION. DRINKING.
AND REHYDRATION

By
John G. Bridge

To investigate changes in the firing rates of septal
cells during and after drinking, single and multiple unit
activity in the septal nuclei of unanesthetized and unre-
strained rats was monitored with multiple electrodes. In
rats adapted to a 23.5 hour water deprivation schedule,
cells discharged nearly twice as fast before the 0.5 hour
drink period as they did afterward. Electrical activity
recorded for one hour in a control group of rats on ad
libitum food and water did not change significantly. Sim-
ilarly, in a group adapted to a 23 hour food deprivation
schedule, electrical activity recorded during 23 hour
deprivation, one hour of eating. and 15 minutes of food
satiation showed no significant changes. Septal unit
activity changed markedly during drinking if the animal had
undergone 23.5 hours of water deprivation, but not if it
had drunk water on an éQ.llD schedule: unit activity in
rats deprived of food did not change significantly during
eating.

Sensory stimuli were relatively ineffective in alter-

ing firing rates of septal neurons. except in the gustatory

fflfiég John G. Bridge
(9‘53
mode with hypertonic saline as the stimulus. Units also
appeared responsive to proprioceptive feedback from
swallowing.

The finding that septal cells are much more active
during dehydration than rehydration supports the hypothesis
generated by studies using anesthetized preparations: that
one septal role in water regulation is to stimulate during
dehydration supraoptic cells which then release more anti-
diuretic hormone, causing the animal to conserve water.
Changes in septal activity during drinking suggest that
septal neurons influence lateral hypothalamic units not
only as a consequence of the hydration conditions, but also
during the drinking behavior per se. The nature of this
influence is yet unknown. since septal units may either

increase or decrease discharge rates during drinking.

UNIT ACTIVITY IN THE SEPTAL NUCLEI
DURING WATER DEPRIVATION. DRINKING,
AND REHYDRATION

By
- .{N - ‘- I:
John G .° Bridge

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of
DOCTOR OF PHILOSOPHY

Department of Psychology
1974

ACKN O WLEDGMEN TS

I wish to thank my committee. Dr. Glenn I. Hatton
(chairman), Dr. John I. Johnson, Dr. Lawrence I. O'Kelly,
and Dr. Rudy A. Bernard. And special thanks to Keven Bridge

for help in statistics. editing, and typing.

ii

TABLE OF CONTENTS

Page
LIST OF TABLES. . . . . . . . . . . . . . . . . . . .iv
LIST OF FIGURES. . . . . . . . . . . . . . . . . . . .v
INTRODUCTION. . . . . . . . . . . . . . . . . . . . . 1
METHOD. . . . . . . . . . . . . . . . . . . . . . . . 6
RESULTS. . . . . . . . . . . . . . . . . . . . . . . 10
DISCUSSION. . . . . . . . . . . . . . . . . . . . . .30
APPENDICES

APPENDIX A
HiStOlogy. O O O O O O O O C O O O O O O O OLJ'O

APPENDIX B
Equipment and Suppliers. . . . . . . . . . .47

APPENDIX C
Construction of a Multiple Electrode for
Unit Recording from Unanesthetized and
Unrestrained Animals. . . . . . . . . . . . 48

APPENDIX D
Raw Data 0 o o o e o o o o o o o o o o o o .65

LIST OF REFERENCES . . . . . . . . . . . . . . . . . 71

iii

Table
1.

LIST OF TABLES

Page

Effects of Sensory Stimulation on Firing
Rates of Septal Cells . . . . . . . . . . . . . 24

Confirmed Recording Sites 1 - 22 . . . . . . . 40

Raw Data 0 O O O O O O O O O 0 O O O O 0

iv

Figure

LIST OF FIGURES

Page

Indicated are mean discharge rates per second
expressed as a percentage of the mean discharge
rate for all data points (within animals). On
the abscissa is the recording time in minutes.
Panel one (H20 DEP) shows 15 minutes recorded
when rats were water-deprived for 23.5 hours.
Panel two (DRINK) shows 30 minutes recorded when
rats were allowed free access to water. Panel
three (REH) shows a 15 minute rehydrated period
recorded after the 0.5 hour drink period. Below
the abscissa are the sample sizes of each group
N 0 O O O O O O I O O O O O O O O 0 O O O O O O 11

Panel A indicates the distribution of changes at
the onset of drinking in firing rates of cells in
rats deprived of water for 23.5 hours. Panel B
shows the distribution of changes in firing

rates at the onset of drinking in rats with food
and water available ad lib. . . . . . . . . . . .13

Indicated are mean discharge rates of the chew—
ing and swallowing components of eating by one
rat. One swallow lasts approximately 700 ms. . .16

Indicated are mean discharge rates per second
expressed as a percentage of the mean discharge
rate for all data points (within animals). On

the abscissa is the recording time in minutes.
Panel one (FOOD DEP) shows a 15 minute period
recorded when animals had been deprived of food
for 23.0 hours. Panel two (EAT) shows a one

hour period recorded when food was available

ad lib. Panel three (SAT) shows a 15 minute post-
eat period. N = 11 during non-consummatory be—
havior: during eating. N = 6. . . . . . . . . . .18

Indicated are firing rates of one septal unit
during different components of grooming.. . . . .21

Indicated are responses of septal units to a
gustatory stimulus of hypertonic saline. admin-
istered by substituting the saline bottle for

V

LIST OF FIGURES (Cont.)

Figure

12.
13

Page

the regular water bottle. Each data point

shows the mean of 10 seconds. Open circles
represent unstimulated non-consummatory be-

havior. Closed circles show the rat's drinking

of 1.2% saline solution. (Rat 20. tape 45). . . .26

Indicated are confirmed recording sites 1 - 6. . 41
Indicated are confirmed recording sites 7 - 18. .43
Indicated are confirmed recording sites 19 - 22. 45
Basic female Amphenol socket . . . . . . . . . . 51
A: Recording electrodes and ground electrode

have been soldered to the beveled pins of the
female socket. Bx Wires have been drawn to-

gether through a PE-5O tube. straightened. and
clipped to an appropriate length for recording. .53
Leads in recording position. . . . . . . . . . . 57
Top trace: unit activity recorded with 62.5me
electrode. Bottom trace: the same activity as

the t0p trace filtered through a 22 picofarad
capacitor. Both sweep durations are 10 seconds. 63

vi

INTRODUCTION

Extensive evidence implicates the septal nuclei in
consummatory behavior. Lesions of the septal area cause
hyperdipsia in rats (Harvey and Hunt. 1965: Lubar. Schaefer.
and Wells, 1969; Blass and Hanson. 1970). Electrical
stimulation of the septal area reduces the water intake
both of rats with water available ad libitum and of those on
23 hour water deprivation schedules (Wishart and Mogenson,
1970). Carbachol stimulation of the septal area increases
drinking (Fisher and Coury. 1962). and atropine blockage of
the medial septal nucleus reduces drinking (Grossman, 1964).
According to Bridge and Hatton (1973). septal unit activity
in rats anesthetized with urethane is usually faster during
and after stimuli which are associated with or which induce
dehydration (water deprivation: subcutaneous and carotid
injections of hypertonic saline) than during and after
stimuli which are associated with or which induce hydration
(the 0.5 hour drink period of a 23.5 hour water deprivation
schedule: carotid injections of hypotonic saline: stomach
loads of tap water).

The septal area also appears to be involved in eating
and/or in controlling food intake. though not to the extent
that it is involved in drinking. When the lateral septal
area is stimulated with carbachol. rats eat more than before

1

stimulation. Food deprivation (or water deprivation) in-
creases septal self-stimulation rates in cats and rats
(Brady. Boren. Conrad, and Sidman, 1957). According to
Johnson and Thatcher (1972), as food deprivation time in-
creases. septal lesioned rats increase lever-pressing rates
for food reward significantly faster than do unlesioned
control rats. Although hyperdipsia has been reported as a
consequence of septal lesions more often than has hyper—
phagia. one study (Stoller, 1971) found that septal lesioned
Holtzman rats eat more but do not drink more than sham
lesioned or unlesioned controls.

The septal nuclei also appear to influence various
components of sexual behavior. MacLean and Ploog (1962)
found that septal stimulation elicits penile erection;
electrical self-stimulation of the human septal region
produces a sensation of sexual gratification (Heath. 1963).

Though researchers have studied several ways in which
septal activity is related to consummatory behaviors, how-
ever. the mass of septal stimulation or septal lesion data
still does not allow us to describe the unit activity during
these behaviors and. therefore. to construct and support
logical hypotheses about the septal role in them. Soulairac,
Tangapregassom, and Tangapregassom (1972) recorded with gross
electrodes slow wave potentials in the septal area during
dehydration. drinking, and rehydration, but their effort
did not suggest how the septal nuclei function. Bridge and

Hatton (1973) measured septal unit activity during different

hydration states in rats anesthetized with urethane. But
although their technique measured the vegetative aspects of
water regulation, it could not evaluate septal activity
during the behavioral act of drinking. Furthermore.
Calaresu and Mogenson (1972) have found that the effects of
septal stimulation on cardiovascular response can depend in
part on the anesthetic used (chloralose versus urethane).
thus casting doubt on the extent to which a urethane prep-
aration reflects events in a normal, awake animal. And
while Ranck (1973) has reported an extensive examination of
unit activity in the hippocampus and the septal region. this
experiment focuses on the hippocampus and on non—consummatory
behaviors. When Yamaoka and Hagino (1974) measured the
diurnal rhythms of septal unit activity in awake and un-
restrained animals, they found that spontaneous activity
decreases shortly after lights go off and increases shortly
after lights are turned on: they did not report septal
activity during eating or drinking. however. None of these
experiments, then, adequately describes septal unit activity
before. during, and after consummatory behaviors.

There are two inconsistencies in the literature about
the septal area and consummatory behavior. First, while
studies showing that lesions often produce hyperdipsia
(Harvey and Hunt. 1965), stimulation can reduce water or
food intake (Wishart and Mogenson, 1970), and unit activity
is greater during dehydration than during hydration (Bridge

and Hatton. 1973) imply that the septum is hyperactive

 

during deprivation and relatively inactive during satiation.
other studies indicate the septal area is a potent site of
self-stimulation (Stein and Ray, 1959). If electrical stim-
ulation activates septal cells and if active septal cells
are associated with deprivation, why then would animals
eagerly self-stimulate? Another discrepancy is that while
septal lesions produce hyperdipsia and electrical septal
stimulation reduces water intake, carbachol stimulation of
the septal area also results in hyperdipsia (Fisher and
Coury, 1962). At present it is impossible to infer how the
septal nuclei function during water regulation and other
consummatory behaviors. That is. are septal cells relative-
ly active during hydration. as the self-stimulation and
carbachol stimulation studies suggest: or are the septal
units relatively active during dehydration. as the electrical
lesion and stimulation experiments indicate? A description
of septal unit activity during naturally occurring consum-
matory behavior may help us to understand these apparent
discrepancies and related phenomena. such as the effects of
stimuli and anesthetics on septal activity.

To observe septal unit activity during consummatory
behaviors requires a recording system capable of sensing
cellular discharges in an awake and freely moving animal.
Septal cells can fire at extremely slow rates, and to avoid
a sampling error of missing slow cells, a multiple electrode
assembly would be superior to a single electrode. In the

present experiment, such an apparatus was developed and used

 

to record septal unit activity before. during. and after
consummatory behaviors (with particular attention to

drinking).

METHOD

Thirty-two adult male Holtzman rats were each implant-
ed with an assembly of six recording electrodes of either
62.5}lm diameter nickel-chromium conductor insulated with
enamel except at the tip or 25,1m diameter platinum-iridium
wire insulated. except at the tip. with teflon. One 125)um
diameter nickel-chromium ground electrode insulated with
enamel except for approximately 0.5 mm at the tip was low-
ered to a position just above the septal area. See Appen-
dix C for a detailed description of microelectrodes and
their construction. Rats were housed singly in cylindrical
plexiglas cages of approximately 35 cm diameter. The light-
dark cycle in the colony room was 14 hours light: 10 hours
dark, and to ensure that changes in electrical activity
during a recording session could not be attributed to the
rat's prior contact with a female rat. no females were
housed in this colony room.

Rats scheduled to be tested in water-deprived or food-
deprived conditions were adapted to a 23.5 hour water
deprivation schedule or a 23.0 hour food deprivation
schedule for 10 days before the implantation surgery.
Maintained on water or food deprivation. rats were allowed
a minimum of 10 days of post-operative recovery before the
first recording day. Rats scheduled for ad lib recording

6

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had food available ad lib for at least 10 days before surgery
and were allowed 10 days of post-operative recovery with
food and water available continuously.
A recording session began when the rats were two hours
into the light portion of their light-dark cycle. Home
cages served as recording chambers. and the procedure in—
volved carrying the rat in his home cage to the recording
room. removing the rat from his cage. plugging the leads
from the recording apparatus into the implanted socket, and
returning the rat to the cage. Electrical activity at each
electrode tip was observed on a dual-beam oscilloscope. and
the activity of electrodes with good signal-to-noise ratios
were recorded on channel one and/or channel two of a stereo
tape recorder. Most units were recorded bipolarly, using
one of the remaining five electrodes as a reference electrode.
Impulses from the brain that were sensed by the record-
ing electrode went to an off-lesion-record junction box via
three-strand phono wire. In the lesion position. the
junction box could supply current to the electrode tip for
a marking lesion: in the recording position, impulses from
the electrode went to channel one and channel two. resPec-
tively, of a stereo tape recorder via a low level pre-
amplifier set to amplify times 1000. Impulses were monitor-
ed with a loudspeaker. and an on-line counter was available
to record the rates of impulses whose amplitude exceeded
the threshold of the oscilloscope trigger.

Recording sessions which focused on drinking and on

the consequences of water intake lasted approximately one
hour: 15 minutes of 23.5 hour water-deprived base rate
activity were recorded. then the 30 minute drink period.
and. finally. a 15 minute rehydration period. During the
half hour drink period. approximately half the rats were
allowed to eat: the food was removed from the cages of the
remaining rats during this period. Recording sessions under
ad 11p conditions lasted one hour. To observe the effects
of food deprivation and eating on septal unit activity
required 90 - 120 minutes of recording: 15 minutes of food-
deprived base rate activity. a one hour eat period. and a
15 minute satiated period. Recording sessions began at
9:00 A.M. (two hours into the light period of the light-dark
cycle).

On some days additional activity was recorded: these
data included up to 0.5 hours of exposure to a female rat,
up to 0.5 hours of sensory stimulation, and (on the day the.
animal was to be sacrificed) up to 0.5 hours of ether or
urethane anesthetic. Sensory stimuli included tactile
(touch. pinch. poke. or stroke). visual (house lights on or
off). auditory (claps, shouts). gustatory (saline. sucrose.
or quinine solutions: cold tap water). and olfactory (alcohol,
ether. or litter from the tray of a female rat's cage) stimuli.

Recording sites were marked with direct current lesions
ranging from 8)¢A for 6 seconds to 30)JA for 15 seconds.
Animals were perfused transcardially with 0.9% saline

solution followed by a mixture of one part 37% formaldehyde

and nine parts saline. To confirm the location of the
marking lesion, brains were frozen and sectioned at 50lxm
intervals and the cells stained with cresyl violet.

The data from each recording session were considered
those of a unique cell or a unique group of cells, even when
an electrode in one particular rat was recorded from on two
successive days and appeared to be monitoring the same cell
on each day. That is. units recorded on different days
from the same rat and the same electrode are considered
related events. but not the same unit. Winer (1962) has
suggested expressing repeated measures as a percentage of
the total behavior or score. This method has an advantage
that eXpressing activity as a percentage of a base rate does
not: by assigning the value of 100% to base rates of each
subject. the latter method loses the variability among them:
the method Winer recommends preserves the variability among
base rates. The data are described with means and standard
errors and analyzed with repeated measure (treatment by
subjects) design because this statistic is robust against
deviations from the normal distribution by the population

sampled (Welkowitz, Ewen. and Cohen, 1971, p. 141).

RESULTS

When unit activity was measured during dehydration,
drinking. and rehydration. the cells characteristically
fired at a faster than average rate during the dehydrated
period (Figure 1). When the animals drank, the mean firing
rates did not change significantly, but the variance of this
mean increased markedly. That is, some cells fired much
more rapidly and others slowed considerably, while the mean
did not change significantly; approximately half the cells
increased and half decreased firing rates during the initial
drink period. The mean absolute change of discharge rates
(without regard to direction) from the water-deprived base
to the first prolonged drinking bout of the 0.5 hour drink
period showed the largest change (45%) measured in any
sequential comparison between non-consummatory and consum-
matory behavior (see Figure 2). When the rat terminated
its initial drinking bout. discharge rates returned during
the non-consummatory period that followed to near deprived
levels. Discharge rates during non-consummatory behavior
then decreased until they reached approximately 57% of the
water-deprived base rate. When the rats were allowed to
eat during the drink period. the non-consummatory discharge

rates only decreased to 67% of the water-deprived base rate.

10

11

Figure 1.--Indicated are mean discharge rates per second
expressed as a percentage of the mean discharge
rate for all data points (within animals). 0n
the abscissa is the recording time in minutes.
Panel one (H20 DEP) shows 15 minutes recorded
when rats were water-deprived for 23.5 hours.
Panel two (DRINK) shows 30 minutes recorded when
rats were allowed free access to water. Panel
three (REH) shows a 15 minute rehydrated period
recorded after the 0.5 hour drink period. Below
the abscissa are the sample sizes of each group

N .

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Figure 2.--Panel A indicates the distribution of changes at
the onset of drinking in firing rates of cells in
rats deprived of water for 23.5 hours. Panel B
shows the distribution of changes in firing
rates at the onset of drinking in rats with food
and water available ad_lib.

 

 

 

 

 

 

 

 

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Under all conditions. the mean changes in firing rates
while rats were eating were much smaller than those during
drinking. even when animals had been deprived of food. Dur-
ing the eating period. it was possible to measure the dis-
charge rates of components of the eating behavior: rooting
about in the food pile. picking up and carrying food.
chewing. and swallowing. Cells often fired faster during a
specific component of this behavioral sequence, usually
swallowing (see Figure 3). Cells also fired faster during
swallowing in the ad 119 periods and after 23.0 hours of
food deprivation. The lick-swallow sequence of drinking
was too rapid to measure each component separately.

For food-deprived rats. the apparent trend of septal
unit activity during eating and during the post-eat period
was not statistically significant (Figure 4). On some
occasions rats were still eating at the end of the one hour
eat period and appeared to be not yet satiated. Discharge
rates of cells in these rats did not differ from the rates
of those that had finished eating. And discharge rates
during eating did not consistently differ from those during
non-consummatory behavior. Food-deprived rats drank in
short bouts which occurred at such different times through-
out the recording session that representing them on single
data points was impossible. Generally. the firing rates of
these food-deprived animals changed slightly to moderately:
the direction of the change was consistent within but not

among animals.

16

Figure 3.--Indicated are mean discharge rates of the chew-
ing and swallowing components of eating by one
rat. One swallow lasts approximately 700 ms.

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Figure 4.--Indicated are mean discharge rates per second
expressed as a percentage of the mean discharge
rate for all data points (within animals). 0n
the abscissa is the recording time in minutes.
Panel one (FOOD DEP) shows a 15 minute period
recorded when animals had been deprived of food
for 23.0 hours. Panel two (EAT) shows a one
hour period recorded when food was available
ad 1gp. Panel three (SAT) shows a 15 minute post-
eat period. N = 11 during non-consummatory be-
havior: during eating. N = 6.

19

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During grooming. the discharge rates of cells usually
increased or did not change: rarely did they decrease.
Firing rates changed when the rat was licking its pelt or
rubbing its head with its forepaws (see Figure 5). In a
sample of the first two grooming behaviors in 10 recording
sessions, 20% of the cells discharged faster than during
non-consummatory behavior. 5% of the cells slowed. and 75%
did not change.

Firing rates of septal cells in rats with food and
water available ad 115 did not change significantly over
the course of the one hour recording session. Nor was
there a trend within recording sessions: no mean discharge
rate of the final 15 minutes had changed more than 12% from
its firing rate during the first 15 minutes. With the onset
of drinking. the firing rates of water-deprived animals
changed more than those of water-replete animals. The mean
absolute change (without regard for direction) was less
under ad Ida conditions than when rats were adapted to a
23.5 hour water deprivation schedule: mean absolute change
for water-replete animals = 17%; mean absolute change for
water-deprived animals = 45%, p4<.001.

To avoid overlooking cells which were not discharging
during the condition at the start of the recording (such as
water deprivation) but which might fire when the condition
changed (to drinking. for example). background activity
(multiple units with small signal-to-noise ratios) was

recorded for one hour sessions in one of two ways: 1) when

21

Figure 5.--Indicated are firing rates of one septal unit
during different components of grooming.

22

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23

only one electrode was yielding good signal-to-noise ratios.
two electrodes that received good background noise but no
large units were recorded on the second channel and. 2) when
no electrodes yielded good signal-to-noise ratios. data from
two electrodes were recorded on both channels. Twenty-six
hours of this sort of activity was recorded. One recording
showed two cells that never discharged during consummatory
behavior and rarely discharged during non-consummatory
behavior. When they did discharge, it was usually during
rapid neck movements. No activity was observed during the
other 25 one-hour recordings.

After six recording sessions (of three rats in two
sessions each). a female Holtzman rat was placed in the
cage of the male. and additional units were recorded from
the male's septum. None of these encounters resulted in
successful copulation or even in mounting or lordosis, nor
did the animals mate on numerous other occasions. even
without the recording leads attached. Because Holtzman
rats are notoriously hyposexual, this line of investigation
was discontinued after recording sessions in which four of
the units increased in firing rates. one did not change.
and one decreased while the female was in the cage. During
the session in which the discharge rates decreased. the
male rat sat immobile. without attempting to mount the
female. In the other five sessions. the male repeatedly
attempted to mount the female.

After many of the drinking. eating, or ad lib recording

24

sessions, additional recordings were made during non-consum-
matory behavior to observe septal unit activity in response
to sensory stimuli. Because previous sensory stimulation

of rats anesthetized with urethane (Bridge and Hatton. 1973)
indicated that septal cells habituate rapidly. the modes,-
intensities. and types of the stimuli in the present
experiment were varied rather than repeated. Yet. in
general. septal units did not respond to sensory stimuli
(Table 1).

Table 1.--Effects of Sensory Stimulation on Firing Rates of
Septal Cells

St ulus

 

Responses to sensory stimuli were measured for
10 seconds and compared to activity during the one minute
unstimulated period of non—consummatory behavior preceding
the stimulus. A change was defined as an increase or

decrease of 50% or more. To establish a control, 10 second

25

unstimulated periods were compared to the one minute of
unstimulated unit activity that preceded them. Control

data came from five measures of eight rats: stimuli data
came from a varying number of measures of 14 rats. (Figure 6
shows the response of an individual septal unit to sensory
stimulation.)

Panel A of Table 1 lists the effects of all the
sensory modes. Auditory stimuli consisted of claps or
shouts: olfactory stimuli included 95% ethanol, ether. and
litter from the cage of a female rat: house lights. on or
off. were the visual stimuli; a tactile stimulus was a
stroke or a poke with a pencil or finger. Panel B lists the
gustatory stimuli in detail. Because the rats would lick
aversive fluids for no more than a second voluntarily. their
tongues were bathed with quinine and 5.0% NaCl while the
animals were anesthetized with urethane (immediately before
sacrificing). As the table indicates. the gustatory mode
was the most effective stimulus in producing change: within
the gustatory mode. sodium chloride appeared most effective.

When rats were anesthetized just before they were to
be sacrificed. the effects of ether or intraperitoneal
urethane on septal unit discharge rates were observed. Unit
activity was measured when the animal appeared anesthetized
to a level appropriate for surgery (e.g., the animal did not
respond to pinches or pokes). During ether anesthesia unit
activity decreased to 58% of the non-consummatory activity

rate prior to the anesthetic (p<:.02, N = 6, two-tailed

26

Figure 6.--Indicated are responses of septal units to a
gustatory stimulus of hypertonic saline. admin-
istered by substituting the saline bottle for
the regular water bottle. Each data point
shows the mean of 10 seconds. Open circles
represent unstimulated non-consummatory be-
havior. Closed circles show the rat's drinking
of 1.2% saline solution. (Rat 20, tape 45)

27

m mmaol

ON.

mozoowm

0m

O¢ o

 

 

- d d

mo_><Imm
.EEDwZOonzoz n O ..

602 .\.-m._ oz_xz_mo u o

o
S
d
0. mm
2..
S
.0
ONE
no
8
OMB
O
O
N
O¢G

 

 

28

t-test). During urethane anesthesia discharge rates dropped
to 61% of pre-anesthetic levels (p<:.02. N = 6, two-tailed
t-test). The effects of both drugs appeared related to

the dosage.

On three occasions unit activity was recorded for
90 minutes after the rat had been deprived of both food and
water. In general. the effects of total deprivation were
intermediate to those observed in rats adapted either to
water or to food deprivation. For example. if cells at the
electrode tip had increased firing rates during eating (after
food deprivation) and decreased firing rates during drinking
(after water deprivation). even after a totally deprived
animal had eaten and drunk. the cells continued to fire at
approximately the same rate as when the rat was totally
deprived. In totally deprived animals. discharge rates
during drinking did not change as greatly (from non-consum-
matory discharge rates that preceded the drinking) as when
the animals were deprived only of water.

Electrode size (62.5;4m or 25)Am) did not produce
observable differences. When more than one unit was record-
ed on a particular electrode. two characteristics were
observed: 1) Drinking occasionally affected the units
differently (one might increase and the other decrease its
discharge rate). but rehydration usually affected cells at
a given electrode site in the same manner. 2) Units with
larger action potentials discharge more slowly than those

with smaller action potentials. This was true in over 90%

29

of the multiple unit recordings. and in each exception the
electrical activity was judged to be recorded from fibers
rather than cell bodies. Rise time and spike duration were
used as criteria to determine whether an electrode was re-
cording from an axon or from a cell body. (When recording
from a cell body. the rise time is slower and the Spike
duration longer than when recording from an axon: Chow and
Lindsley. 1964). Only one type of response depended on
locus within the septal area: some units that decreased
discharge rates during rehydration would occasionally fire
rapidly in "rushes" of 1 - 5 seconds at rates not reached
even during dehydration. This type of response was

observed only in the medial septal area.

DISCUSSION

The two strongest findings in this eXperiment were the
changes in septal unit activity in water regulation and in
drinking behavior. As water-deprived rats became rehydrated
during the 0.5 hour drink period. septal unit activity
decreased significantly. And during the initial drinking
bout by water-deprived rats, individual septal units changed
markedly: many increased and many decreased firing rates,
although the mean remained relatively stable.

The 23.5 hour water deprivation schedule was chosen
for this experiment because it is a natural sequence of
dehydration and rehydration, because many other researchers
use this schedule. and because other studies have reported
the details of its osmotic and volemic correlates. Hatton
and Bennett (1970) have shown that as rats adapt to a 23.5
hour water deprivation schedule over a 10 day period: 1) the
animals drink their fill progressively earlier in the half
hour session until, by the 10th day. they drink in the first
15 minutes nearly all the water they will drink, and 2) the
difference between pre-drink and post-drink osmotic pressure
increases as a function of days of deprivation. The differ-
ence between pre-drink and post-drink plasma protein did
not change over days of adaptation. Hatton and Bennett also
reported that by the time rats stopped drinking. their

30

31

osmotic pressure had drOpped to ad lip levels or below.
After 6 minutes of access to water. rats are still hyper-
osmotic: from 8 - 10 minutes, osmotic pressure is near ad
lip levels. and by 13 minutes the osmotic pressure has
drOpped below ad Ida levels.

Rats in the present eXperiment that were adapted to
23.5 hour water deprivation and received no food during the
drink period were under conditions similar to those analyzed
by Hatton and Bennett. By the end of the drink period.
discharge rates in this group had decreased significantly.
though they drOpped more slowly than osmotic pressure
decreased or volume increased in the Hatton and Bennett
study. Unit activity in the group for which food was
available during the drink period decreased less markedly
(Figure 1), and firing rates of cells in the ad lip group
did not change. These results indicate that, in general,
septal cells are hyperactive during hyperosmotic (dehydrated)
conditions and hypoactive during hypo-osmotic (rehydrated)
stimuli: they corroborate an earlier report (Bridge and
Hatton. 1973) of this effect in urethane-anesthetized rats.

Firing rates changed considerably during the first
drinking bout by rats adapted to 23.5 hours of water dep-
rivation. These changes were smaller later on in the 0.5
hour drink period and when rats were on an ad lip. rather
than a water-deprivation schedule. Furthermore, when rats
are deprived of water for only 2 - 3 hours. and not adapted

to this schedule, the changes observed during drinking are

32

also small (Ranck. J. B.. personal communication). Therefore,
these results strongly suggest an interaction between drive
state (water deprivation) and the effect drinking has on
septal unit activity.

That the septal units were relatively unresponsive
to sensory stimuli except when the mode was gustatory and
the particular stimulus hypertonic saline suggests that
when their discharge rates change during drinking. units
are responding to proprioceptive feedback of the behavior
per se. It was not possible to detect whether firing in—
creased during licking or swallowing. since both occurred
very rapidly. During eating. septal cells responded most
frequently during the swallowing component. If it is true
that swallowing water also alters septal activity. then the
greater frequency of swallowing during drinking than during
eating may account for the greater changes in firing rates
during drinking. The greater frequency of swallowing
during drinking may also contribute to the consensus among
researchers that the septal area is more involved in water
regulation than in food ingestion.

But while other sensory modes were relatively ineffect-
ive in the present study. other literature shows that
sensory stimulation can change septal activity. although it
also suggests that septal cells habituate frequently to this
sort of stimulation. Cross and Green (1959) have reported
that many septal units respond for 2 - 5 seconds to tactile.

visual. and auditory stimuli and frequently habituate to

33

them. Bridge and Hatton (1973) found that 10 second carotid
infusions of hypertonic saline often activated septal units:
these units frequently habituated after only one or two
infusions. Brown and Remley (1971) found septal lesioned
rats hyperreactive to thermal. sound. shock stimuli. but
not to taste and light. According to Hayat and Feldman
(1974). photic. contralateral sciatic. and acoustic stimuli
alter discharge rates of most medial septal and diagonal
band of Broca cells. Although their results report only
responses up to 256 ms. they reported studying responses up
to 5 seconds.

In the present study all stimuli except auditory were
presented for the duration of the 10 second response measure-
ment. The changes in firing rates after saline stimulation
of the tongue generally lasted longer than those Bridge and
Hatton reported (1973). perhaps because rats in this study.
which presented most sensory stimuli after rehydration. were
hypo-osmotic when stimulated with saline. Since a rat's
daily colony room life presents many visual, tactile. and
auditory stimuli similar to those presented in this study.
septal units may have habituated to these stimuli before the
experiment. Often firing rates would increase initially
after sensory stimulation. drOp below base rate for 2 - 3
seconds. and then return to pre-stimulus rates. as Figure 6
shows. This evidence. in effect. corroborates both the re-
port of Hayat and Feldman (1974) that septal cells respond

for very short durations to sensory stimulation and the

34

strong habituation effect first reported by Cross and
Green (1959).

Why do septal cells respond to hypertonic solutions
in contact with the tongue. and how does this mechanism
serve the rat in normal daily existence? As a recipient of
information from exteroceptors. the septal area apparently
inhibits responses to environmental stimuli. as numerous
reports of the septal startle response or hyperreactivity
phenomenon suggest (see Lubar and Numan, 1973). Septal
lesioned rats drink more of palatable solutions than do
control rats (isotonic saline: Donovick, Burright. and
Lustbader. 1969: saccharine: Beatty and Schwartzbaum.
1967). They also drink less of aversive solutions (hyper-
tonic saline solutions: Donovick, Burright. and Lustbader.
1969; quinine: Beatty and Schwartzbaum. 1967). Thus one
septal function may be to moderate or inhibit responses to
aversive or rewarding gustatory stimuli.

0n the other hand. taste buds also respond to chemicals
in the blood (Bradley, 1973). By recording from the chorda
tympani nerve, Bradley showed that when solutions were
perfused into the tongue. units responded to sodium. but
not to other solutions. such as sucrose and glucose.
Furthermore. cells habituate to sodium stimulation when the
solution is perfused into the tongue, but not when it is
delivered to the surface of the tongue. Responses of septal
cells often habituated to carotid infusions of hypertonic

saline (Bridge and Hatton. 1973). yet in the present study.

35

cells habituated less frequently to hypertonic saline
applied to the exterior of the tongue.

When considered together. the eXperiments on the
responses of septal cells to gustatory stimulation suggest
that information about the salinity of the external and
internal environments may reach the septal area via the
chorda tympani nerve. Stimulation of the chorda tympani
could activate the septal area. which would modify or in-
hibit behavior by decreasing or increasing the intake of
substances containing sodium. The response is mediated via
the lateral hypothalamus (Miller and Mogenson. 1971; see
below).

Changes in the animal's behavior or reSponses could
also elicit the charateristic response of unit activity to
sensory stimuli: to increase immediately after stimulation
and then decrease for 2 - 3 seconds before returning to
base rate. Often when a rat changed behaviors. by beginning
to groom. for instance. cells fired faster immediately after
the change. decreased in rate below the previous mean. then
returned to levels near the mean. Thus septal cells appear
to fire in reSponse to a novel situation. whether it is a
change in stimuli or a change in behavior. Septal lesioned
rats are severely impaired in their ability to habituate to
novel stimuli (Feighley and Hamilton. 1971). One role of
the septum may be to transmit to the hippocampus information
that a stimulus or a response has just been initiated.

Hippocampal cells are able to respond to Specific novel

36

stimuli. for instance to novel water-related stimuli, but
not to novel photic stimuli (Ranck. 1973).

Ranck (1973) has also estimated that 75% of the lateral
septal units are ”neck movement cells" which fire when the
rat changes the position of his head. These cells did not
fire when Ranck moved the head and neck of the rat. and the
neurons did not react to specific objects as did hippo-
campal cells. In the present experiment. responses similar
to those of the neck movement cells were observed in units
which were primarily in the lateral septum.

Hayward and Smith (1963) found that septal stimulation
decreases urine flow by. they suggested. activating supra-
optic neurons and increasing the release of antidiuretic
hormone. The rapid discharge rates of septal cells during
dehydration and their slower discharge rates during re-
hydration found in the present study strongly support this
hypothesis. The septal area may also influence water
regulation by two other routes. Septal stimulation can
decrease blood pressure (Covian and Timo-Iaria. 1966;
Calaresu and Mogenson. 1972). If the activity of the septal
area generated by water deprivation causes hypotension of
the renal artery. both sodium and water will be retained.
Septal stimulation reduces corticosteroid levels in adrenal
venous blood (Endroczi and Lissak, 1963), and septal lesions
increase ACTH secretion (Bohus. 1961). In this system
septal stimulation by dehydration would reduce ACTH, which

would in turn lower aldosterone. thereby reducing the

37

retention of sodium and water.

But the septal area is not involved only in water-
saving: it is also involved in the behavior of drinking.
Wishart and Mogenson (1970) found that septal stimulation
can inhibit ongoing drinking; Miller and Mogenson (1971)
reported that septal stimulation can alter discharge rates
of lateral hypothalamic neurons. but the effect depends
on the rate of the lateral hypothalamic unit. If the unit
is in a fast phase. septal stimulation usually decreases its
discharge rate and septal stimulation activates the firing
of a lateral hypothalamic cell in a slow phase. That is.
septal stimulation moderates the discharge rates of lateral
hypothalamic neurons. This dual effect makes the septal
function in drinking behavior difficult to understand. It
is interesting that the septal area also moderates the
intake of aversive solutions (see above. p. 34; Donovick,
Burright. and Lustbader. 1969 : Beatty and Schwartzbaum. 1967) .
Unfortunately. the bi-directional change of septal discharge
rates during drinking complicates matters more. and no
simple explanation is available.

The present eXperiment indicates there are two distinct
responses of septal units in water regulation. First.
septal units fire significantly faster during water
deprivation than during rehydration. Second. at the onset
of drinking the absolute change in septal firing rates is
greater when rats are water-deprived than when rats are on

ad lib food and water or are food-deprived. Because the

38

septum has two distinct roles in water regulation. the
apparently contradictory results of electrical stimulation
and lesion experiments versus chemical stimulation and
blockage studies are not necessarily incompatible. The
septum's excitation of the supraoptic nucleus most
parsimoniously explains why electrical septal stimulation
reduces water intake while electrical septal lesions increase
drinking. When activated. the supraoptic nucleus releases
antidiuretic hormone. which causes the kidney to reabsorb
water. In one role. then. the septum governs water
retention. But changes of septal firing rates during
drinking may also influence lateral hypothalamic cells

and may be mediated by cholinergic synapses. In this role.
the septum influences water ingestion.

While this experiment has shown that urethane and
ether reduce firing rates of septal cells. there is evidence
that unit activity recorded in unanesthetized and unre-
strained animals may be slower than in animals with no
electrodes implanted. This evidence comes from the curious
phenomenon that when two cells are recorded on the same
electrode. the larger one (the unit with the largest
voltage) almost always discharges more slowly than the
smaller one. This phenomenon has also been observed in
septal (Bridge and Hatton. 1973) or hypothalamic (Bennett.
0. T., personal communication: Walters. J., personal com-

munication) units under urethane anesthetic. Apparently,

the electrode is a heat sink, and cells in its vicinity

39

are subjected to mild hypothermal anesthesia. The cell
with the larger action potential is usually closer to the
electrode and will be cooled more. causing it to discharge

more slowly.

APPENDICES

APPENDIX A

Histology

Table 2 lists confirmed recording sites (1 - 22).
Columns two and three identify the 28 electrodes listed at
these 22 sites by rat number and electrode number. The
location of each site is shown on one of the six atlas
sections on the three following pages (Figures 7. 8, and 9).
The sections are redrawn from Konig and Klippel (1970). and
the numbers next to each refer to the distance in microns
anterior to ear bar zero these sections appear in the Konig

and Klippel atlas.

Table 2.--Confirmed Recording Sites 1 - 22.

 

Electrode Site Rat Electrode
1 25 b
2 5 b.d.f
3 30 e
4 32 a
5 6 a
6 28 a
7 22 a.d
8 22 e
9 21 c

10 32 d
11 6 f
12 17 c
13 21 a.f
14 3 f
15 3 e
16 2 a
17 23 e
18 28 e.f
19 24 e
20 20 a.b
21 18 f
22 26 b

 

4O

41

 

Figure 7.--Indicated are confirmed recording sites 1 - 6.

 

43

7
Sl

d g

r

r

d

nd

.

4L:

 

 

 

 

 

45

 

Figure 9.--Indicated are confirmed recording sites 19 - 22.

 

 

   

APPENDIX B

Equipment and Suppliers

Eguipmant and suppliers for microelectpode construction:

1.

5.

 

Female Amphenol hexagonal 7-pin socket. $.86. and
male Amphenol hexagonal 7-pin plug. $.97. Amphenol
Corp.. Endicott. N.Y.

Nickel-chromium enamel-insulated wire. 62.5;1m and
125;4m dia.. ”Nichrome" brand. Driver-Harris Corp..
Harrison. N.J. .

Platinum-iridium teflon-insulated wire. 25)Jm dia..
10 ft. for $41.00. Medwire Corp.. Mt. Vernon. N.Y.

Three-strand phono wire. 30% shielding. 10 ft. for
$1.69. Belding Corp.. Chicago. Ill.

Liquid Tape. $1.09. CG Electronics. Rockford. Ill.

Equipment and supplieps f0; bioelectpic data pecozding and

analysis:

1.

2.

Stereotaxic instrument. $750. Stoelting Co.. 424 N.
Homan Ave.. Chicago. Ill.

Preamplifier (122). $165: power supply (125). $335:
dual beam oscillos00pe (502A). $13958 Tektronix.
Inc.. P.O. Box 500. Beavertown. Oregon.

Tape recorder (1028. $1196. Magnacord. Main Elec-
tronics. 5558 S. Pennsylvania Ave.. Lansing. Mich.

Electronic counter (5512A). $1050. Hewlett Packard.
E. Hartford. Conn.

Audiomonitor with speaker (AM8). $157. Grass Elec-
tronics. Quincey. Mass.

47

APPENDIX C

Construction of a Multiple Electrode for Unit Recording from

Unanesthetized and Unrestrained Animals

Abstzac;

This appendix describes in detail the construction of
a single and multiple unit recording system using either
62.5,am or 25,1m diameter wires as recording electrodes and
a 125}4m diameter wire as an in-brain ground electrode.
The wires are soldered to the pins of an Amphenol 7-pin or
9-pin socket. then insulated with Epoxylite or Insul-X. and
the assembly affixed to the skull with dental acrylic and
anchor screws: a 7 or 9-pin plug is inserted into the
socket during recording sessions. and impulses reach the
recording equipment via 3-cable phono wire. Comparison of
recordings from the septal area using the two electrode
sizes showed that electrodes of 62.5)Am diameter yielded
a higher proportion of records with acceptable signal-to-
noise ratios than did the 2511m diameter electrodes. while
the 25;«m electrodes generally maintained acceptable

records for a longer time.

48

 

49

Introductipn

This electrode assembly has evolved from a number of
previously described chronic unit recording systems. par-
ticularly that of Johnson. Clemens. Terkel. Whitmoyer. and
Sawyer (1972). who developed an in-brain ground wire prep-
aration and have compared the performances of floating and
rigid electrodes. and that of Chorover and Deluca (1972).
who developed a technique for implanting wire so flexible
that it essentially floats with the brain. Constructing
the present multiple electrode is so simple that. after
practice. fabricating a 7-electrode system requires only an
average of 46 minutes: there is virtually no waste: and a
novice can manufacture perfect electrodes from the start.
The accompanying equipment (leads. etc.) is also inexpen-
sive. reliable. and simple both to fabricate and to use.
The animal. merely plugged into the recording apparatus and
unplugged at the end of the recording session. experiences
very little stress. Because the system uses either the
62.5 )um diameter nickel-chromium or the 25 )um diameter
platinum-iridium wire with equal facility. their recording
properties can be compared. Although either the 7 or 9-pin
Amphenol sockets may be used. the 7-pin apparatus. along

with the 62.51pm wire. is described here as an example.

 

50

Constzuction

A female Amphenol socket (Figure 10) is chosen into
which the male Amphenol plug (Amphenol Corp.. Endicott.
N.Y.) to be used on the lead will easily fit. Usually the
individual sockets are out of round when purchased. so that
the fit is too tight for recording purposes. In this case.
a metal probe is inserted into the individual sockets. and.
by moving the probe back and forth. the sockets are rounded
out until the plug fits easily into the socket. Then six

2 - 4 cm lengths of 62.51pm diameter. enamel-coated. nickel-

 

chromium wire and one length of 125)1m diameter wire
(Driver-Harris Corp.. Harrison. N.J.) are cut. Using a
scalpel. 1 - 2 mm of the enamel insulation are scraped off
one end of each wire. and the scraped ends of these wires
are soldered to the beveled ends of the pins on the Amphenol
socket (Figure 11. A): the 1251Pm ground is soldered to the
center pin.

The integrity of the assembly depends in part on three
alterations of the Amphenol socket: (1) to form a single.
rugged unit. the beveled pins. which are loosely held by the
hexagonal plastic case. must be made rigid: (2) the pins and
solder must be electrically insulated: and. (3) so that
subdermal fluids will not flow into the plug-socket contact
area and short the circuit. the junction of the individual
sockets with the plastic which houses them must be made

watertight. These transformations are all accomplished by

51

 

Figure 10.--Basic female Amphenol socket.

52

 

do._.

>

00 ON

 

00 O on

 

mo 04

 

2 3%.;

 

 

 

 

me

%

 

 

 

 

 

 

 

 

 

53

Figure 11.--A: Recording electrodes and ground electrode
have been soldered to the beveled pins of the
female socket. B: Wires have been drawn to-
gether through a PE-50 tube. straightened. and
clipped to an appropriate length for recording.

55

the following steps: a small portion of modeling clay is
spread thinly about the base of each pin. and both the end
surface of the plastic casing and the pins are covered with
Epoxylite or Insul-X. Insul-X usually performs satisfac-
torily. requires only a few layers. and needs no baking.
but it sometimes bubbles and does not form as hard an
insulating cover: although Epoxylite requires numerous
layers (often 10 - 15) which must be baked individually. it
forms a superior surface.

To draw them together (Figure 11. B). the wires are
inserted into a 1 mm length of PE-50 plastic tubing. which
is drawn down to the base of the 125)Am wire at the center
pin. At this point the ground wire is cut to a length
which will allow the tip. when implanted. to reach a point
just above the target nucleus. (The end of the PE-5O tube
that is distal to the pin is flush with the interior sur-
face of the skull after implantation.) Approximately 1 mm
of enamel is scraped from the distal end of the 125 pm
ground electrode. By holding the PE-50 tube near the base
of the ground wire. it is possible to straighten the record-
ing wires to a nearly parallel position. A small drop of
Elmer's Glue-All is placed at the edge of the PE-50 tube
distal to the pins (Figure 11. B: point c): the dr0p
will be drawn into the tube and. after the glue is hard.
further straightening of the wires is possible. With a

pair of very sharp scissors. the recording wires are cut to

 

56

an appropriate length. depending on the depth of the target
area. If 25/Mm wires (Medwire Corp.. Mt. Vernon. N.Y.) are
used. they must be fused to the beveled pins with electro-
conductive cement. Using a ring lamp with a 2X magnifying
lens. it is a simple matter after 10 minutes practice to
scrape with a scalpel approximately 1 mm of the teflon in-
sulation from the end of each 25)am wire to increase the
conductive area which will contact the electroconductive
cement. Before implantation. the electrodes are collectively

coated with melted dextrose: the dextrose hardens when

 

cooled and allows the electrodes to be implanted as a rigid
unit. yet after implantation the dextrose dissolves and
disperses. See Chorover and Deluca (1972) for a complete
description.

The leads are made from four 30 - 35 cm lengths of
3-strand phono wire with 30% shielding (Beldon Corp..
Chicago. 111.). two Amphenol plugs. and one Amphenol socket
combined as Figure 12 shows. The six conductive strands
found in the pair of phono wires are soldered to the beveled
prongs of plug pins A - F. the shields are soldered to the
center prong. and the prong and solder are covered with
Liquid Tape (CG Electronics. Rockford. Ill.). The shield
wire goes to ground. and the six phono wires go to the
recording equipment. To reduce noise that the two strands
would create if they rubbed together. they are held apart
with lengths of masking tape spaced 3 - 5 cm apart. If the

57

 

Figure 12.-— Leads in recording position.

 

58

TO
RECORDING
APPARATUS

9

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 12

TO
IMPLANTED
SOCKET

 

21.33::—

v

 

59

counterweight is set slightly heavier than the combined
weights of the plug and the wires. the animal draws the
wires out of the way when he stands on his hind feet. and
the only weight the rat supports is the 5.5 g implanted

portion.

Discussion

The complete sysem is designed for simplicity and
minimal eXpense. For example. this lead apparatus replaces
the more expensive and cumbersome mercury commutator swivel.
If a continuous record is required. then the leads used here
should go to a mercury commutator. for if the rat turns in
one direction five or six times more than in the opposite
direction. the leads will have to be unwound by separating
them at the plug-socket junction near the counterweight
string and untwisting the wires. The wires usually tangle
about twice an hour and require approximately 10 seconds to
unravel. If a completely continuous record is not necessary.
the present lead system is quite sufficient.

Prefabricating the 62.5 pm electrodes requires less
time and cost than constructing the 25,um.electrodes. But
since the latter is a floating system. it retains good
single and/or multiple unit activity for a more extended
period of time than does the more rigid 62.51pm wire. Thus
the 62.5}1m wires are advantageous for the sort of eXperi-
ment in which a large number of electrodes are implanted

for a relatively short period of time (such as that required

 

60

to map unit activity in an area of the brain during a
specific behavior): 25)um electrodes are superior if fewer
electrodes are to be implanted for a longer period of time
(such as that required to examine unit activity during a
complex learning sequence). To construct a 7-pin Amphenol
unit with six 62.5 pm recording electrodes costs approx-
imately $1.50. or $.25 per electrode: the 25pm system is
$2.50. or about $.42 per electrode.

As well as the purpose of recording. the cytoarch-
itecture and discharge rates of the cells in the target
nucleus may also influence the choice of an electrode.
Chorover and Deluca (1972) have reported that all 51 of
their 25)1m electrodes implanted in the olfactory bulbs
yielded acceptable unit data. and Norgren (1970) found
that 70% of the 62.5;um electrodes implanted in the hypo-
thalamus gave good data. In the present eXperiment.
electrodes were evaluated three to five times each. and
approximately one of four 25lum electrodes and one of three
62.5)Am electrodes yielded good signal-to-noise ratios.
Part of this discrepancy between these and other reported
results may be caused by the extremely slow spontaneous
base rates of septal cells: there are even "silent pockets"
in the septal area. where there is little or no activity at
all (DeFrance. J. F.. personal communication). In such
areas it seems reasonable that the larger electrode would
yield more data.

The use of this multiple electrode system can

 

61

circumvent a bias toward selecting relatively fast-firing
cells. a sampling error which can occur when an eXperimenter
lowers a single electrode and monitors feedback simultane-
ously to select a cell for recording. The electrode may
bypass a cell that discharges only once per minute. for
example. while the cell is inactive. This problem may be
avoided by recording on one channel of a two-channel tape
recorder from electrodes with good unit activity. while
recording on the other channel from electrodes with neural
background activity free from extraneous noise. but with no
units. Re-playing channel two of the whole eXperiment can
reveal extremely slow-firing cells or cells that fire under
one eXperimental condition but not another. Septal cells
firing as slowly as three times an hour (under urethane
anesthetic) and 15 times per hour (in an unanesthetized
rat) have been recorded in our laboratory.

Because Norgren (1970) reported that unit activity
during implantation did not correlate with that during
subsequent recording sessions. units in this investigation
were not monitored during implantation. However. a number
of implants showed activity which was acceptable during the
first recording session (on the 10th post-operative day).
but which diminished in quality until. by about the fifth
recording session. the data were unacceptable: this suggests
that monitoring electrical activity during implantation may
be worthwhile. at least if the target nucleus is the septal

area and the electrodes are 62.51pm. This problem of

 

62

diminishing quality of unit activity may be attenuated by
housing the rats in cylindrical Plexiglas cages. eliminating
dark corners into which rats can poke their heads (thus
bumping the electrode socket). Plexiglas can be shaped
easily by heating it to approximately 1170 C and cooling it
around a cylindrical object. Rounding out the individual
sockets with a metal probe before constructing th electrode
and implanting 25}1m rather than 62.51am.wires also prolonged
collection of acceptable data by reducing the loss of good

signal-to-noise ratios caused by repeated insertion and

 

removal of the plug. Since most artifact noise in this
system is considerably lower in frequency than is the
signal. it can be reduced significantly more than the
signal by filtering the current through a 22 picofarad high
pass capacitor (see Figure 13).

If the target area is relatively large. such as the
rat septum or hypothalamus. or if the target is small and
the eXperiment requires numerous control cells in the
vicinity of the target nucleus. then this multiple electrode
system is very satisfactory. But. especially when using the
flexible 25}Am wires. this system is difficult to implant
accurately. If a small area such as the supraOptic nucleus
is the target. then a movable microelectrode such as that
described by Teyler. Bland. and Schulte (1974) may be

superior.

63

Figure 13.--Top trace: unit activity recorded with 62.5/1m
electrode. Bottom trace: the same activity as
the top trace filtered through a 22 picofarad
capacitor. Both sweep durations are 10 seconds.

 

ma 3&2

 

APPENDIX D

Raw Data

Table 3 (below) lists raw data from this experiment
as entered on computer cards: the first card gives in-

structions for interpreting data.

Table 3.-4Raw Data

1 THFDE ADE F DATA FHTRIFQ PER CARP. EACH NTRY '" ' =
2 1-11. 14-25. 27-3q. Ao-sz. 53-65: 66-79. Ccm. 7&3833A83L¥A§F nos.
3 an 1 Is ACTIVITY 10. R = HARE NON— CnmsUM.~ n = navnx. F =‘EAT.
A . = GonnM. N = NOM- cor.uu. Col 2-s ADF TADE anTAGF. COL 6-9 AQF
5 “FAN qDIKEs pap sccown. COL 10 —1? ADF sTAMnARn Epnnes. COL l3 "
6 Is NO. OF 10 sec QINS WHICH couoosfi THE MEAN. DECIMALS HAVE ‘EFV
3 EZSS‘Q‘SHSB”:.LHSA‘JWFW5'9 “”"“”“‘”‘5“‘ ”Fm" ” ‘
- x : _. -A LACEs HAVE HE N DRODP u'
9 THE MEAN AND 3.5. DESPECTIVELY. E ED FRO
paw DATA

:3:§.:2...222.3:...229”2 3 3

. :3. PO} R0]7018173416ROI491790279690170720030 ago 9 a 1
n02202SA71416002501850?93600990106721lsno30s:n333alawnasoaoa7svismolegzéwgzggggg
NOSOSZZSOOOOZNOHRBZO332736N05682dl73166N090G2A00BPHEM067030Q04l§6N06°02513300305
N082531171856N0845226IPR46N3R7033003206N090033334366N09883l333786N105843R3483605
M116019502466 os
ESTIsAanngélng? 31;?91n52q “3LT! EL = F 2 2

-n . 1 09a 090810168030a10271036003471013OSth04 712A3 a
239::9233352232:gamma:'3:3avzzézmfixszaozaznw‘9°"~°°“6°°""’*"“73"'~233
- - c . _. r ,0 _ :0 a o

N114509810816N111006930676MIPAGOBH11903N175807838756N10 3 770676N111 0 qufiafig;
£3850?2721§?ZA°39(173° nrD vuLTI EL = F 2 2

u. no , 0091HWOIA1lthnQRhHOPOWIRl1098690?3816§01076R Q n a
382?;{gzggzgzgggag}Zéggggfggzgg}gaggzgsnnwzglsg7o77snn17818:20A26n833si212347283

.: hNOl ?A 43” 4° 2 '
N052024700001M05782330136300596221800020081618126302 n 5?0R5000?N"q0? 930000183
3.33%.:9...:2*33 3 2
. .. an 95046 OIPfifio1E907000096H0200066701SAROZZBI2000 76R0 6 7

PnZRQIIROO3A6003PRA[R007780u3R0A481052h0017:Aab7096hnn391APA30£AAnuz§A2017gl?2%g
0094427310476Mc4hqonou7003nnao:G SOHDOOAOOHOOEkhOUfiQHGHQ3l“7‘83099590'4r8?00000l10
N056009930216H655”33fi00)0300C7P7dnnnhnlmannl7HOURTHF084Oln1303??fl06§?nfi§0000?10
N06642402000?F0h750i))OIPHOOHQAA«banhfi7NO7lOPRh7OOO3Gn7PR§7aOI?79007R§4??ROQ§A10
Agééfifizmmfigv’miié‘fl232"3332‘;l§2‘:”‘:“'m”1“WW:wwvrveomcoow0......”
- ~ ' N ' . n s: unease? 46 .
N107020§00536N108511000275N112075330876N0 ‘ ‘1 ”10‘0'“l704’5N107526R30446}8

655

 

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1111111
169176
oqaooq
077007
01;:H00
0000031
0:. 00 (.41
415425
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2626:5616
10.03307
0000000
3070701.
33065603
0 7 0.569 0
07'6111
FHlXquqIO
7.470.963
.. Isa/1.701 ”7
00,000“
LRDEMCLNE
.fih 3.56.622
9500700
14?...)100
99.1.1000
F.1I709700

11421796

0959997
E3705737
90126801
A 000 001-1..
TRDPEDDD

III!

3311311233
111111111]
6711—517
onnrfiqnn

00 00

En nrp

.140

000

0098013701860n
01690760000180
O3Huvfi3unofilHfl

03
006900A58u55460

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76216 C1
“69077 «.11. 0
97.00.66.034
00900090
7903.707 6.
60.971911:
317769401
100000000
4107QR§0
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pOIPEgL902
AOnXYUOO‘i;

TRRBDDDNN

6666666
1111111
666766
4R9037
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55.5.5.5.)
OR nqqo
04 .H 574

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an032769
3m046§66

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766qrfi44
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R77qu1la
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150.7..Hnfiw-I
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RDNJHVWE nu.
066,651.64
Ffidfirfi77 40
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7..5Q..H.))Hl.l.
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SOP3579.U
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9900“?”
7666.66.66.
R7480677
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97407?R
60097577
14665544
373?...900
67269066
p0234790
AonnOOOI
TRBDNNEN

77777777

11111111

Pogo—.5015
c.447qauls
=I..1C.6u.0.l
09000.11
LR”NH..F.NN
E6666366
3094367
0211565
4.15 4078
[equiv/493
7.17.2950?)
LII-10900
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13:6)nl
0000011..
thMEHN
96666666
F.‘n7 77(10
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A 4 .14 09 7 01).
903108815
44101771
741310917
r57 9 R q 366
7077.0. 0.. 444
1111100000
00557377
646773768
P02468901
AOOOOOO‘II
TRPDDGEEN

3888998888

RnnNGENNG
646666166
0079050rfi—b
793442044
320240000
738707028
468463976
qaq47?_000
959374729
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1241367891
000000011
RRDNMEHVM
26665666626
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6690698304
31051000100
32082299.:25

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P02345789OI
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90999999999
lllll‘llllll
6646966666
Q46R733009
1907670094
1011617....400
9730179877
06RC7G1§01
3328739944
4.35.5.347100
“21.000091.“
OlIOHRwIfi—H-I
7.145567890
0000000001

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6163666616
10640747QQ.
10471067018
20 0013311000
5070897nw7n57
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266133221666

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2971.036 4 3757

07.17.76.991: 46
646645771000

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=‘l7\1.urfi.h.7nfiq 07.

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080739., 0.5.16 372
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5.59 994488
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TRDFENNOENE

67

 

7722772 44 99999 99999 6666666666 272?8227 98988 9999 909 33133 4444 99999
7772772 77 ?2??7 ?72?7 7777277227 33333333 33333 3333 333 44444 4444 44444
666666 6 43366 63664 6411A66?9 68836666 6666 6726 66 6616 643 6?11
006796 3 80071 90930 760017708 136979?6 3747 6000 46 0308 346 3003
340986 2 40014 41087 230091401 97103939 3088 8008 09 4901 017 9007
1.1.7.000 1 90032 7.5109 000000000 00000000 1100 0000 71 1001 774 3031
877.789 7 00373 39049 7900119700 37303003 7177 0000 37 3700 797 1.000
498940 6 00119 9099? 810070919 3160R999 1161 0099 36 4109 676 3000
830060 0 91935 19073 339330311 78709161 4414 9193 64 4094 691 6116
333695 1 44111 72003 000000000 52317322 0000 0100 11 0110 111 ???2
949999 1 910?? 04798 ?QR946792 91R099n9 0900 9919 09 0009 040 9?59
439164 1 79335 44775 87QQI?Z66 74891441 0700 8980 07 4459 309 301
134680 3 74701 74801 134978401 13468902 3581 2468 38 2460 398 ?469
000001 0 00011 00011 000000011 00000011 0001 0001 00 0001 000 0000
FFFEFE D RNHFE RDMEF Rc.nDNFNGG RRHMENNN RDMN RDDN RN RDGN RDN RNNF
669664 6 64466 66666 67.761664? 66696636 6666 6666 64 6666 6164 6666
304141 0 09417 00901 009600490 07319702 4993 ?307 18 8811 ?016 A778
110676 3 77671 07843 301?0?490 441339?1 1?07 0?QR 00 1041 8000 9439
11?.001 5 43.971 77201.. 000000000 00000000 1111 1100 7.1 1011 107.1 7.7.51
370030 0 105503 108033 00n7n9939 07303073 3773 7707 230 0737 0000 3700
694360 0 0?7OR 90989 909011139 06393969 8113 6691 90 0131 9000 R199
R3708? 2 H7047? H787?Q 3?1191417 030C9811 3337 3077 H9? 7143 4870 09??
437.994 4 C47711 C7720]. 000000000 41.33.7337. 0000 00 00 C11 0100 1711 7379
730990 9 26806 70347 965979650 39005080 0000 0507 00 0595 5658 0309
10177d 4 43013 83013 367780959 41719310 9999 9903 97 0185 6607 3143
13499.0 1 ?46 01 14801 11.496.64.00 134670.07. 2.570 74.61 75 «(49.5 2.9.90 7.4.94.
000001 0 00011 00011 000000011 00001011 0001 0001 00 0000 0001 0000
FFUFFH G HUHFF QDHFF HHFMDCNNN QQUHFMGN RNMM RDMN 90 QUNN RGHH QUMF
16676636 36 394?66 366?66 7166646636 766646666 36066 36666 366 36666 36336 36846
7008096 6 70075 00076 073734151 84717825 1099 9005 28 2613 6763 2096
0430616 5 35043 00046 012911171 3414438? 344? 0394 40 1046 0674 0?19
171.1010 9 6.7011 1.70.1.5 000000000 00000000 1101 1101 71 1110 3011 1.717
780.5770 7 00037 600.43 077988.300 00703700 71.1.0 3370 1.1) 0000 71.37. 3707..
18569187 30 305581 319538 8227401076 359603100 36930 33be 338 35050 31336 33691
0947479 3 0910? 3496? 171411141 ARPIHGQI 94?4 3737 43 619? 3947 R?06
4334944 9 ?1?01 32311 000000000 44341??? 0000 0000 11 0100 1110 71?]
F6709999 AQ A04974 A10396 A3300910?0 CRRUU99QO FHUOO 90090 A09 ROORH A9099 RRUOH
9707906 1 7139?. 9.93.... 7. 156970744 009Q7104 0000 037?. 09 639G 0191. 6048
=024HIOI :1 :14h91 :11741 =134967+01 =194$7QOI :?R70 21440 :24 :1187 :DRhO :139!
0000011 O 00001 00001 OOOHDOOII 00000011 00H1 0001 00 0000 0001 000)
LFFDFENE L8 LHDUFE L8PDFF LDEFNFLGON LRQHNFHNN LHNNN LHDMN LPG LRDNN IRHFN LADMN
5.3663666 F.63 E66973 E66646 F.666266266 E66666666 E6666 .6666 E666. E66363 F6666 (.6673
9990177 60 19300 14100 714095096 3344??91 0347 3n76 497 HaAn7 9174 1101
8.97076...) 90 7.83.00 416.67 358011037. 4.3107773 12.49 096.3. 393 31876 48.11 410....
9013000 90 71400 D31?10 00000n000 00010040 1100 [11? 71? 11000 111? ??44
4H319n? 71 F03003 E709PO F?540073Sn 03751703 E3370 F0173 330 31703 1010 7700
1376M466 116 L08 4533 ”blidQS L994238135 15.861.56.08 1.83.50 L0318 188:. 138693. 1302.9 11690
77....)79750 .175 625011 73.849 077.83.102.31 184264802 03.354 041.34 1231 .14 903.3 17.2.89 T1610
L53??999 L9? M31317 097110 M000040000 L44343?33 M0000 H0100 L111 100109 11100 I731?
”9139099 H38 176702 248710 1099300094 001705900 10000 10909 0095 HOOE)9 ”0956 H9069
“94.40791. “.00 (6.64070 HOCOD7 C0??1679?7_ M967797Q4 9.59.99 940.50 MG10 M7A94J. M6747 .4461].
09197Q1 n9 11941 11700 134967401 fl?497401 1460 1&9fl 14? 11971 1460 1397
0000001 00 0.40"] 00)]... 00.7. d...........11 4.00....)091 On. On. H.001 4)l Air. .101 30...." 0900
DNcFHFFE RH QHHHF HWHFH DREFFFFLMN HHHHUFUN {HUN DHWM PM” HRGNH HDFH WTDN
F?n63fi66 966 9666H6 667?3 F167166366 966636666 66666 96676 0666 D6nfihh 96616 96666
09.00.0436 F14 F3911n: 9.00006 008n070169. F771Q4Q07 947709 F.09U6 F866 €99,974 F9609 F6398
314.4794 0.4? 07413.7 10.91..th 07).“...4 .71 h??411114 11661.4 1?.409 DAD? 0.09394 0.9304 0003.9
03113000 71 70R44 691006 0000000000 00030040 L100?0 1700 111 10101 170) 911?
05373050 000 063.000 N30007 0070055700 0032313030 33.003 0006.0 0777. 030330 00700 Orv/.13
09986597 ?00 719H90 180906 0078627050 ?033h1535 033900 20900 7156 719410 23139 70181
F967?7?7 H79 H1739U 999346 F134117352 H96?08?3? A3376? H4903 H?30 H74l34 H?010 HAOPQ
4373595 60 91416 3???? 973070000 44343223 UOUJ3 0010 111 00100 1331 83)]
9000999. 1...) 73973 8.9?71 .58 97.99909 40.740997 UUOLU 3040. 500 030 50 9.53? 4990
3189840 76 14149 63.3.9.9 740044500 4.5010971 00.030 0.81.... 980 97007 074.... 6100
9.08....4491 603 613.570 603660 6074967801 7 .17. 4.57891 814697. 0039/ 00.51 003-371 014 6... 001.47
00000001 000 000001 000001 0000000011 100000001 IUUOOI. 20000 7.001 700001 7.0000 .COUCU
QFFnFrE 80 9000” 908%” HHHHFnfiHH QRHDHFMU HHMHM QHDH RDH DRUM” 8360 .QnWV
T9669646 T66 T66266 T6£669 16621666763 T66AAF€66 TBA?66 T66?6 T566 TA4636 T6649 T41866
A79Q4QA6 A6? A92074 A03070 A91009070§7 AQ7n17343 A91076 A7309 A694 Ano?37 A3040 AR7K93
9??66362 946 991079 947955 97200111022 9154?5253 912085 99203 9048 943951 97800 996376
4110000 40 1303? 17110 0000000000 00000040 11000 0101 711 11111 ?1?0 31???
69709.50 77 1.0071. 73.639 9900700971. 03777000 00077 0303 337 31.30.... 3700 03037
3998??4 66 139963 <14380v 12335647477 93661090 90916 40399 9396 R1101 Rhnn 01941.
77094877 408 9?7Q?R 977979 61?2917171? 710104708 933743 0339? 0981 379199 43031 904743
23423995 790 757772 772100 20000000000 344333332 300000 30000 3101 400100 41112 437312
9909099 9? 0n890 06491 0904073737 60995900 00000 9990 009 000?9 09?8 80900
F1767908 E24 E47578 F31744 F9190734693 [10917366 E95955 F4390 E491 E48764 59719 E79667
90134690 903 903470 903580 9023567890? 902356890 903581 90347 9039 902461 90368 902469
40000001 ‘00 400001 400001 40000000011 400000001 400001 90000 4000 500001 40000 400000
TNFFFEEF 790 TRDDNN TRDNNE TRRDGFNFDND T8900NENN TRNDNN TRRNN TBRN TRRONN TRDND TRRDNN

68

TAPF Ho QAT 21 H20 nrp quTI FL = c 3 2
800216100n696900496an307nhu037lH933nHRhH010H435007 7630132Hl3303°6R015833170H66H0
9019093930236q02l0ahshflh?6"n?WthfinnhHfiHOPfiHAAHU)916Q099?hfl§003?68031R36670336§0
0039H603305H600411376I on363uu35AHHTn32AnnAHn400034lHnnan7anH00236MUHlHaQ33l016§n
NOSPRH7R3n7Q600§n360fi00u06H0630439007R40061IHab7|173HOBQSH2670766Mo7[€42H00376qn
N07298433n996no7414H830476Mn7HAH4670736Nn7676433nH16H043u99000776NURH6§16302RRSO
N092052330736N097H anJOAhfiHl“306n33n426nl02Ra2670966Ml06092670396N10903900027650
N112266000606M113R6217O3H6HI13089170496 SO
TAPE 91 PAT 2? FOOD nFD CIHGIF EL = n 3 3
POO3H026708963007HOP330096u0lIHOPH00766HOIAHOPI709863010004001106G023004HOOQ°691
n026n031707nAq029903671.3w6uuu1206332193wca6004l70096M04000a000266N0H7H07nnl73391
0066606000003306933 330676Mn73008000001N076H0H0000010091005670673N0°2803H0088691
M09BHO3R30936H103566171306nlnFH0433102bnlIRHO7UOOOOI 51
TAPE 5? RAT 22 F000 nFP FIN NCLF EL = n 3 3
N0090040014160011006500002N01403 317114 6N030804001136M032§06001066 52
TAPE S3 PAT 22 ann DFP VHITI EL = A 3 3
80030423347460006062n0497690390H~33292690[Poa983l94690l5839335706R02096367343693
GOPlfiaonnnoanOPH)4300P1469620H1033263630320H3003Hg6flo37082173466F04HH37004076H3
F061053933346”0h996”lIPJP6uO7nOhfih7PAI3037PH47672755007606000000I€082§46933PR653
0098661502726M100961302944”!02799000001I31086assooooznl06546002003N12266767485353
TAPE $4 RAT 22 ann nFD uHITI EL 3 3
N010049173166N01IHAA333Q66MOPP3393319R6N0P6041401699M030042503lRAN033066672033SA
TAPE SH PAT 21 F000 nFo MHITI EL = A 3 3
8003024173636R007H2l6716QHH3!1H2380279H6H l6H2a0026363020018332236R026018672096HH
2028019172126803051967167600fia24167 171 600 QROAQZQIl140101043672636N10292800178455
TAPE S6 PAT 21 ann nEp MULTT FL = A 3 3
M016237006929n0297316326360n212PHHoooo 2w023n3]832H26nn37923333206004n02417166656
N047S300035250052926000'02NOH402733223 buoHSOPHl730760056220000001N058H2690305656
TAPE 6! PAT 21 F000 nFD MHITI EL = F 3 3
ROO3H2OHOI5763007QPHHUPQahHOI091990SaleO140230019163017519R318H6001802100000361
8021013672336 024H2PHOIAH6HOPAOP35021l6DOHAQPA33DQS3DOQQOP733118600010250012a661
N101729000001H1028PHuODnO[Ml046263321460102023831766nlo3429674103Nl150388000026]
TAPE 6? RAT 21 F000 OED MHLTI FL = F 3 3
N0010303332n3 OQan3lH3236600)6C3ISO3176M010029000002N012523203 89009283067121-62
005612QPHPSPOM057334‘30603 2H0‘3031332 '53(N0642P3001216N06832RA31 62
TAPE 66 RAT 23 HPO ”F0 MHITI FL = F 2 2
30022401839L69010742153F,OHHC]3Sao§§|6Q540[60439§?756Q0lR§34RV1936R020042G21§0664
30253343075780031H3H3HPHA6R.3119303u17hnnq3hanloH306nn37R39]00:46n0603379012366a
004733566053§nflfi373°633HH3flnuAQW7dnl351M”¢QHAISHPIOHHO5PO3Q3OIR76NOQQR3117176364
60557366000626056736300 08H‘975037000"WOHH2A3801953M060737022346M06PH3SaRIAQH6a
N064736131036H36753H7UPI76m06053u7721’6N07]330670736N073H3l271126DO7HH33H3IH0364
~076242100002007654IHnonnlM077A3PP6lH7HGn7Hn3Aa0[844N04003Pl22376N081033n3163664
N083929H01305Mo8H79163A nn6M.)°7H43P92Au6E09104613Pu96Fo°3042621616Eooa2a309105664
E0954300912F6~0968407HHQOAHI“003hannHAAGl0PP731SQuNl06234R8lnHHEIORHaono3nH664
6110040933026F11leIHfi3whhull30343013QHNIIAH3 7312686116932302887Nl1833589293664
N120036461416M12l034u22006 6a
TAPF 66 PAT 23 An L_IR MHLTI EL = E 2 2
30015172000768004219 21n76R007CPu07l23690102192312063013018780986901962122130666
R0182203H219630209216(2626R024619331236 90230)6h7l206qn32017P31Hl6n0337210H000266
N03S3IRPPO7AAH03~H17981nu6604201924|242M04a71R931336Mnu8317R£0APBNURIAIQA3IQ6666
N054816H611083056316333102MHH7OP£hHunthOFRPIQVHluS6F060017PSIPA6F0617IR7H000266
NO63H162206P6M066+I69UPAPSH1691197R]7Haw07t3llHfio1H66Mn73A16371006Nu75q15071a0565
N079018HQ.?006NOR?2[H320 360:,«4717230926u)37016231R3a603831IARRHIHNOQOPIRIHIPIH66
nn927237nnnan093hlu651a>6MO367lH60202660900l6490002ml004186§lthN103°lH6HlHP666
N10671970151361071237800n2ml)3717131606N1100 210500020111218300002Ml1231033OQH666
N1150162§21360117513'u000 UPN119019HrflnonlflllH 218000016119021200001N120H1763087666
N12301967l976 66
TAPE 67 QAT 2H H20 nFP HIHGLF Fl_ = P 3
80039617057qag0004.un 2A.36Hu|26.63397]6HOIHAAAH3HROAQQIRF 322349762021y3nnn300667
8023H3QH0616630263AO3%‘HHUPOHH3H3a7663031761672n35:101406617H2AHRU3HH601122H367
0037307672016PfluHJJH671+66dfi'2HnuunoHH60066Hol330Hohnna67nPHOOUVHnuaIHOPHnonnPh7
006820600000?IJHHHQ:uunn)lHanxv7H3finnPGOHPooRHnnnoP3oHHonH331363F06000133042667
F06SonPPQn7F7uU7AUQPqu,c’0071H0P7Hlllama/20n2000002N07HH0nA7’316M07Hnn317007667
~081001330406M032H0]l7)AG6HQHAH53nno63HDOH67o6onPn03H0H9)0A00P7IAM0800019H04RAB7
N0920007H0406F093H93 0))nnrounPUOHnn3a6Nl00HnPh71386mlOHHUPPHO63AN1IPH0333168667
N115303001246N118‘06671I63H1233nanPPH4MlPlIOPOOOOOPMIPBROPOOOHH6 6
TAPE 60 PAT 26 H20 HFD ”1““IF FL = u 3 3
R004067I7AHH60008036HO3176un|20H167AH2630|an36832696qn2ooaaa742hsnopanaal733966q
nn2953ann3396an33333nu17LHHU3HH3517>.1hnn38016001uannn39617672126n04272 00216660
00991263333R3HUA639 an fifloPUJHIIRUSOOHOPNOHn7PH l73u9600H2023nonnnPM0H6H2433PPh66°
90Hfl63333PMAHH06*:129H 2436 66anwo) )£736un727>;H9 gpARHM0323363330MAM\)3HF3333427660
096007H3I 36F0°9033751304C‘3131100 Pnawlt‘mHP l PQGHNIO PP‘OOOO?M\ 009 00267669
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LIST OF REFERENCES

LIST OF REFERENCES

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Wishart, T. B. & Mogenson, G. J. Reduction of water intake
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Yamaoka, S. & Hagino, N. Spontaneous septal neuron activity
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