Interaction of neural systems which control body water

Btam Research, 49 (1973) 323-347

323

~('~ Elsevier Scientific Pubhshmg Company, Amsterdam - Printed m The Netherlands

I N T E R A C T I O N OF N E U R A L SYSTEMS W H I C H C O N T R O L BODY WATER*

RAIMOND EMMERS D,,partment oJ Phystologv, College of Physzctans attd Surgeon~, Cohonbta UmverstO'. New York, N Y 10032 ( U S A )

(Accepted August 1st, 1972)

SUMMARY The method of electrophyslologlcal unit recording was used to study the Interaction of neurons among the following nuclear masses: (1) the nucleus semilunaris accessorlus thalami (nSA), which relays gustatory and splanchnlc afferents in the thalamus, (2) the nucleus supraopticus hypothalaml (nSoH) and the nucleus paraventricularis hypothalami (nPv) which are known to control water retention, and (3) the area lateralis hypothalami (ALH) and the nucleus entopedunculans (nEp) which are implicated in the control of water intake. Neuronal activity of these nuclei was altered by intracarotld infusions of a 3 %oNaC1 solution or distilled water, or by electrical pulses applied via a concentric stimulating electrode to the nSA or to neurons of the A L H - E p or the nSoH. It was found (Fig. 9) that neurons of the nSoH and nPv function as osmoreceptors (Fig. 9.1, 9.2) whereas those of the ALH-Ep do not. The activity of the latter depends on the interaction of tonic excitatory and inhibitory influences (Fig. 9.3, 9.4) which are relayed to the A L H - E p neurons via the nSA Therefore, oropharyngeal stimulation during water Intake will gradually alter the activity of the A L H - E p neurons. This function can serve as a metering device of water consumption. A reverberatory negative feedback circuit (Fig. 9.5, 9.6) provides for interaction between the two hypothalamJc control systems suggesting the explanation for many interesting phenomena observed with the regulation of water balance.

* Portions of thts study were reported at the meetings of the Federatton of American Socieues for Expemmental Biology in 1969 and the Amemcan Physlologlcal Society m 1971.

324 1N ' F R O I ) U C

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It ~s well k n o w n that two systems exist which provide a neural ~,ontrol I~)~ ~i~c regulation o f body' water -Fhe p a r a v c n m c u l a r s u p r a o p t i c o - h y p o p h y s l a l s~ten~ E' influences p r l m a r d y the r e a b s o r p u o n o f water m the collecting ducts o f the kldncs ~, whereas a n o t h e r g r o u p o f neurons scattered t h r o u g h o u t the lateral hypothalamu~ ''~ controls water c o n s u m p t i o n . As the acUv~ty o f both systems correlates web ,s~th relatively slight changes m the c o n c e n t r a t i o n o f osmotically active substances w.~thm the extracellular fluid, questions have been raised as to whether this correlation might be the expression ol a causal r e l a u o n s h l p 3". If neurons o f these c o n t r o l systems f u n c t m n as o s m o r e c e p t o r s , then their osmoreceptlvlty should remain unchanged m l s o l a t m n . [ h i s criterion, a l t h o u g h technically l b r m l d a b l e , has been fulfilled ~ n h ,he s u p r a o p t m o - h y p o p h y s m l system 20 3a. However, no similar a t t e m p t s have 13cen r e p o r t e d with the system which controls water intake. Direct apphcat~on o f o s m o t l ~ M ly active substances to n e u r o n s of the latter system m the intact a m m a l has not clarified the issue. A l t h o u g h d r i n k i n g has been induced by this method m some e~,pcr> ments ~8.31, no effect was observed w~th others ~4,22 Apparently,, experiments o( this type are complex, revolving the actlwty o f m a n y neural s~stem3, including taste. To what extent taste participates m the control o f water c o n s u m p t i o n has ne~cr been clearly, evaluated, because a total destruction o f the sense o f taste has not been techmcally feasible. Denervation o f the tongue, lesions destroying the sohtary nu,.lel. o r a b l a t i o n o f the cortical recelwng areas for taste will raise the threshold for gustatory d i s c r i m i n a t i o n o f various substances ~'7 but these p r o c e d u r e s will not abolish taste completely.. A p p a r e n t l y , taste receptor~ are located m other buccal structures besides the tongue, a n d taste p a t h w a y s m the brain stem were not fully k n o w n k in the thal,tmus, however, n e u r o n s o f the gustatory' system are s~tuated within a relativel 3 .,mall nucleus 1,{a it receives input from buccal taste receptors and possibly also from some o t h e r p e r i p h e r a l receptors -,,~ revolved m the m o m t o r m g o f the c o m p o s m o n of body fluids. Therefore, destruction o f this nucleus should disrupt the central relay t,l the majority ol chemosensltlve aflerents and thus assist m the evaluation o f their role m the control o f water mtakc. W i t h o u t brain surgery tt la possible to arrange a situation m which a rat can obtain water by squirting it directly into the s t o m a c h a'l Suctl an a r r a n g e m e n t , however, can only delay, not exclude the p a r t m l p a t m n of taste m the regulation o f water balance, because taste as a system is stimulated not only by, solutions a p p l i e d to the tongue, but also by substances a b s o r b e d or rejected Into thc c~rculatory system ~s The present series o f experiments d e v e l o p e d from a relatively simple ~tud) 'l° designed to answer the question as to whether e l e c m c a l stimulat,on o f the thalamlc taste nucleus wall Influence the activity o f those h y p o t h a l a m l c neurons which control the r e g u l a t m n o f body water. When such an influence was discovered, a d d l u o n a l e x p e r i m e n t s were p e r f o r m e d to ascertain whether the actlwty o f these neurons ~ ould be altered a l t e r the d e s t r u c t i o n o f the t h a l a m i c taste nucleus. D a t a o b t a i n e d from these experiments m d m a t e d that neurons o f the hypothalam~c system which control water retention l u n c t m n as o s m o r e c e p t o r s , whereas neurons which c o n t r o l water intake do

NEURAL CONTROL OF BODY WATER

325

not; their activity is dependent on the excitatory and inhibitory drive of the thalamlc taste neurons. Furthermore, a definite interaction was demonstrated to exist between the two neural control systems. METHODS

Adult female cats were used for the experiments. An intraperitoneal injection of 35 mg/kg of Nembutal induced a sufficiently deep surgical anesthesia to prepare the animal for electrophysiological recording of the activity of hypothalamic neurons. An additional dose (10 mg/kg) of the same drug was administered approximately half an hour before the recording began. Usually this maintained an adequate level of anesthesia until the termination of the experiment. The left common carotid artery was cannulated with a polyethylene tubing for the infusion of substances and m 6 preparations the femoral vein was cannulated for the withdrawal of blood samples. The head of the animal was then fixed rigidly in the Stoelting quadruple stereotaxic instrument, the skull was opened between stereotaxic values of A 13.0-A 2.0 and L 1 0-L 8.0. The dura was slit and retracted but no part of the brain was removed. A tungsten microelectrode with a tip diameter of 1-3 # m was inserted through the cortex at A 8.0, L 4.0, H 0.5 to localize thalamlc neurons which could be acUvated by mechanical stimulaUon of the tongue. If such neurons were not encountered on the first penetration, additional penetrations were made at 0.25 mm distances m the anterlor-posterlor (AP) and left-right (LR) directions until they were localized; their position was used as a reference point for the localizaUon of the nucleus semilunaris accessorius (nSA) which serves as the thalamic relay of taste 9 and localization of neurons in the hypothalamus involved in the regulation of body water. After this point of reference was located, the microelectrode was withdrawn and a stimulating electrode was positioned 1.2 m m medial and 0.5 mm deeper from the reference point to reach the nSA. The stimulating electrode was a double barrel concentric steel electrode with a total diameter of approximately 1 m m and the core sharpened to a tip of approximately 0.1 mm. Such a tip was chosen to provide as localized stimulation as technically feasible. The mlcroelectrode was used to explore the activity of neurons in the nucleus supraopticus hypothalaml (nSoH), the area laterahs hypothalami (ALH), the nucleus entopeduncularis (nEp), and the nucleus paraventriculans hypothalaml (nPv). The procedure was the following. After a neuron of a p a m c u l a r hypothalamic target region was electrophysiologically isolated, its activity was observed on the oscilloscope for approximately 15 min. If its spontaneous firing rate remained fairly stable during this period of time, a control count of this firing was accumulated on a computer (CAT model 400 C of Technical Instruments). It was programmed for digital accumulation of spike potentials in 400 separate addresses. These were scanned in 1 sec. Consequently, 2.5 msec were alloted for counting spikes which fell in a particular address. Samples of the firing, each 1 sec in duration, were superimposed in the computer 1000 times and spikes added in any of the 400 addresses during the accumulation were typed out by a model 535 teletype-punch-read umt of Technical Instruments. In addition, they were plotted as dot-like events on an

326

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a b s o l u t e c o u n t - t n n e scate On those occasions when a stmlulu~ ~a~ applaud' neurons o f a particular nucleus, the c o m p u t e r was triggered by a Grass_ $8 stmmJ,tl~ .. a n d a pulse o f 0.3 msec d u r a t i o n wa<~ repeated at I,sec with the inomcnt ot ~tJn~ **., tlon s u p e r n n p o s e d on the c o m p u t e r time base I000 times. A c c u m u l a t i o n ~,c ,:,c s p o n t a n e o u s liring countb lrom a particular neuron was repeated after each c\pc~ Jmental m a n i p u l a t i o n , such as l n t r a c a r o t i d (ICy infusions o f a 3 ",, N a ( l solution 121 ,,I at l ml/min), distilled water (2 ml), or a 0.9" 0 sahne solution (2 ml). destructio~ ,~t the nSA, etc. The effect ot a particular experimental m a n i p u l a t i o n was never evaluated with a c o m p a r i s o n o f spike counts obtained front <~eparate neurons I1", after ,,,me e x p e r i m e n t a l m a m p u l a t i o n , technical difficulties prevented the c o n t i n u a t i o n ol reco~ cling spike activity o f the n e u r o n from which a control couint had been obtained, recording from a n o t h e r neuron was begun again with a control count, not w~th a contmt,,,, N a C l , (4) 1 mln after all i ( ' infusion ol 2 ml o f distilled water. (5) I nlin after a repeat o f the NaCI infusion. (6) 1 mm alter electrolytic destruction o f the nSA, (7) ~vdh a repeat o f the electrical stimulation ol l l~e nSA, (8) I m m after a repeat o f the I(" infusion o f distilled water, and t9) 1 mm altci a r e p e a t o f the IC infusion o f the N a ( I solution A l t e r the selection o f the initial infusion, the o r d e r o f the IC infusions of hypertonic saline or distilled water was varied, a n d in some cases two or m o r e infusions o f the NaCI solution were administered m series to test the response o f the neuron to such c u m u l a t i v e infusions If a particular h y p o t h a l a m i c n e u r o n did not r e s p o n d to an IC lntusion with a definite change m its firing rate, two a d d i t i o n a l infusions o f the same type were given before the neuloii was declared unresponsive. When a change in the firing rate was p r o d u c e d , the change was usually more than 20",, o f the a p p r o p r i a t e control count. In fact, this change c o u l d be readily noticed b} watching the spike activity displayed c o n t i n u o u s l y on i l~e oscilloscope screen W i t h no effect, the c o u n t deviated from the control only' by a icv~ counts. S e l d o m the changes in firing rates were more subtle than this ~,s a rule, the5' occurred with a partial destruction o f the n S A and with an incomplete elimination ,>1" afferent input into the nSA. A particular test m a n i p u l a t i o n p r o d u c e d a change ot the firing rate always in one and the same d i r e c t i o n , only in two individual cases c \ c u p tions to this were noted Moreover, any experimental effects could be readily a p p r a i s c d by utilizing sham infusions (0 9°<, saline), sham destruction o f brant tissue m the vicinity o f the effective sites, or b~ v a r l a t m n in the poSltlOn o f the stimulating electrode Because o f these experimental controls, statistical evaluation o f the d a t a a d d e d little to I '

NEURAL CONTROL OF BODY WATER

327

their interpretation but confirmed those conclusions which could be drawn immediately after the termination of each experiment. This report is based on the results of 78 experimental preparations. With 35 of them work was carried out on the effects of the IC infusions and nSA stimulation on the activity of neurons in the ALH, nEp, nSoH, and nPv. Another 15 dealt with these effects observed before and after the destruction of the nSA. In 13 preparations the interactions between the A L H - E p and the nSoH neurons were studied by a stimulatingrecording arrangement. In 5 of these experiments a stimulating electrode was posmoned in the A L H or the nEp to evaluate the influence these nuclei can exert on the nSoH. In 3 others, this stimulating-recording arrangement was reversed. Additional preparations were used to disrupt this interaction by a lesion in the nSoH. In 3 preparations results were obtained from unilaterally decerebrate animals. Decerebrat~on was effected by negative pressure aspiration of brain tissue approximately 2 mm in w~dth via a transfer semi-micropipette at A 2.0. In 4 experiments the responses of hypothalamlc neurons to the IC infusions were recorded before and after hgation of the lingual-chorda and the IXth nerves. Additional experiments dealt with the influence of the splanchmc nerve activity on such responses of the A L H - E p neurons. Afferent excitations conveyed via the splanchnic nerves centrally were eliminated by a spinal transection at Th 5. Laminectomy of the thoracic region at Th 4, 5, and 6 was performed prior to recording. Spinal transection was done while monitoring o f a hypothalamic neuron activity was in progress and control data on that neuron had been obtained. Finally, a study was made of the effects of rinsing the tongue with hypertonic NaCl or distilled water on the firing of the A L H - E p and the nSoH neurons. The concentration of Na + in blood was determined wah an Advanced Flame Photometer. The exact position of the tips of all electrodes as well as lesions were ascertained postexperimentally from histological sections of the brains.

RESULTS

A total of 89 hypothalamic neurons were studied: 64 in the ALH-Ep, 16 m the nSoH, and 9 in the nPv.

Rate of spontaneous firing Spontaneous activity of a hypothalamic neuron found in an area imphcated in the control of water intake is shown in Fig. IA, A1. It was localized in the A L H approximately 1.1 mm above the optic tract. Its stereotaxic AP level was 1.0 mm posterior to that shown in Fig. 5 on which the recording sites of two other similar neurons are indicated. The A L H merges with the nucleus Ep at the AP level of Fig. 6. The extent of the area in which such neurons were localized can be seen best in Fig. 8 (ALH, Ep) with its schematic replica in Fig. 9; From the 64 neurons studied in this region, 34 of them did not respond to any of the experimental manipulations. Eighteen of the 34 were found at the borders of the A L H - E p and were instrumental in producing the outline of this region in Fig. 9; the others were located among the 30 which did

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Fig I A, A1, Samples ofextracellularly recorded spontaneous activity of a neuron m the nucleu~ l:p over a 2 sec period. B, B1, Samples of the actJ,~ty of the same neuron of A, A 1, except that a stunulus of 0 3 msec duraUon was apphed to neurons of the nucleus SA at I/see as indicated by dots over the shmulus artifacts Abbrevtatlon~ oJ dlu~trat~om. ADH, antldmretlc hormone, ALH, area laterah~ hypothalam~, Amg, amygdala, Cd, nucleus caudatus, ChO, ch~asma nervorum opt~corum, f I, capsula mterna; CM. centrum medmnum thalami, Fx, formx, GL, corpus gemculatum medmle: GP, globus palhdus, H, nucleu~ habenulans eplthalaml, M, nucleus corpons mamd[aHs, MD. nucleus medmhs dorsahs thalami; (n)Ep, (nucleus) entopedunculans; (n)Pv, (nucleus) paraventnculans hypothalami, (n)SA, (nucleus) semdunans accessorms thalamh (n)SoH, (nucleus) supraoptlcus hypothalaml, PC, pedunculus cerebn, PIT, pedunculus lnfertor thalami, Put, putamen, SM, stria medullans thalamh S[, nucleus subthalam~cus, TO, tractus optlcus, TR, lractus retroflexus (Meynert). VA, nucleus ~entrahs anterior thalami, VPL, nucleus ventrahs posterior lale~ah~ thalami, VPM. nucleus ventrahs posterior med~ahs thalami, I11. ventnculus tertms

respond to particular experimental m a m p u l a t i o n s . With I1 o f t h e m IC mfusions were given before and alter a parttal or a total destruction o f the n S A ; w~th others~ the 1C infusion tests were repeated after h g a ti o n o f the t o n g u e afferents or stimulation o f the n S o H . A c o m p u t e r r e a d - o u t o f the s p o n t a n e o u s activity dlustrated in Fig. 1A, A I ~s shown in Fig. 2A It revealed that the firing rate o f this n e u r o n was 5,460/1000 sec before any experimental tests were begun N e u r o n s which belonged to the c o n t r o l system for water retention were f o u n d to possess an inherently slower rate o f firing than the A L H - E p n eu r o n s (on the average

NEURAL CONTROL OF BODY WATER

329

3,967/1000 sec for the nSoH, 3,659/1000 sec for the nPv neurons). Moreover, their firing was rather irregular. Often several seconds of total silence interrupted their activity but because of the sampling technique used to accumulate control counts, the computer read-outs of their firing gave dot distributions which were similar to those of the A L H and nEp. Electrophyslologlcal isolation of neurons in the nSoH and nPv was extremely difficult which was most likely related to the fact that neurons of these nuclei are packed more closely together than those in the ALH and nEp All 16 nSoH neurons responded to the IC infusions and 7 of the 9 nPv neurons were responsive. With 4 neurons IC infusion tests were performed before and after the destruction of the nSA and with 2 nSoH neurons a study was made of their responsiveness to stimulation of the tongue with a hypertonic NaC1 or distilled water rinse.

The e/feet of 1C infusions No more than 5 IC infusions were made per animal, and an interval of 25 min or more elapsed between two successive infusions. If the initial firing frequency of a neuron was slow, 2 ml of the 3 ~o NaC1 solution was infused in the carotid artery at a rate of I ml/mln; if It was fast, distilled water was used. This procedure for the initial IC infusion was adopted because on several occasions an IC infusion of distilled water decreased the rate of a slowly firing neuron to zero and thus not only truncated the experimental value but also terminated further experimentation with this neuron. As soon as an experimental test series was begun, however, the order of IC Infusions of NaCI or distilled water was varied irrespective of the firing rate of a neuron. Physiological saline solution was Infused in some preparations to provide for an additional control but since its effect on the firing frequency was practically nil, ItS consistent use was discontinued to shorten the experimental procedure. The computer accumulation of spike firings was commenced l rain after the conclusion of any infusions and collectlng of the data for a particular infusion terminated with a teletype print-out of the computer's count. This required 6 rain in addition to the time required for accumulation of the spike count If a neuron responded to the IC infusions at all, infusion of the NaCI solution Invariably raised its firing frequency. The average increase was higher for neurons of the A L H - E p system (Table I) than for neurons of the nSoH (an increase by 2,308 counts/1000 sec; P < 0.01 of the t-test) or the nPv (an increase by 1,670 counts/1000 sec; P ~ 0.01 of the t-test). Repeated infusions of the NaC1 solution gave progressively diminishing returns, although the second infusion sometimes was as effective as the first (Table 1). Infusion of distilled water decreased the firing rate more effectively than the NaCI solution raised it. This paralleled the change in NaC1 concentration in blood samples. They were taken during the teletype print-outs for a particular infusion and indicated that infusion of the NaC1 solution did not raise the NaCI concentration as much as distilled water lowered it (Table I). I f a neuron responded to these IC infusions, it was also possible to mftuence its firing by stimulation of the nSA, and conversely, if its firing was modified by nSA stimulation, it responded also to the IC infusions. Stimulation of the nSA and IC infusions were either both effective or both ineffective in modifying the firing of a particular A L H - E p or nSoH neuron. Therefore, it became possible to predict the behavior of a neuron to the IC

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Fig. 2. A, A computer read-out of an accumulation of 1000 samples each of I sec duration as shown in Fig 1A. B, A computer read-out of an accumulation of 1000 samples each of I sec duration as shown in Fig 1B. The stimulus was delayed 100 msec and the moment at which 2t was apphed to the nSA was superimposed on the computer 1000 t~mes as indicated by an arro~ below the time base Stimulation of the nSA fired the neuron of the nEp 975 Umes out of 1000 with a latency of 2 -5 mse~ Following this, the actwity of the neuron was characterJzed by alternate inhibitory excitatory oscillations.

infusions by observing the effects which the nSA exerted on its activity. Behavior of the nPv n e u r o n s did not follow this rule; their activity was uninfluenced bv the nSA

Effects of nSA stimulation A single square wave pulse (3.0 V, 0,3 msec) a p p h e d to neurons o f the nSA (Fig. 7) at l/sec p r o d u c e d definite effects on the actwity o f the A L H - E p and the n S o H neurons. These effects were ~psdateral only. They are illustrated m Figs 2B and 3B, C. T h e influence o f the nSA on the A L H - E p neurons was m o r e c o m p l e x than its influence on the n S o H neurons. The c o m p l e x i t y seemed to arise from a c o m p e t m o n between an excitatory and inhibJtory drive with unequal timing circuits. The effects d l u s t r a t e d m Figs. 2B a n d 3B, C were o b t a i n e d w~th a m a x i m a l l n t e n s a y o f stimulation; a further increase in the stimulus intensity did n o t alter the response characteristics. There was a short-latency firing o f the A L H - E p n e u r o n which was followed by an interval (100-150 msec) d u r i n g which s p o n t a n e o u s activity was inhibited Then there emerged a relatively high surge o f firing which was i n t e r r u p t e d by an inhibitory pemod o f lesser d u r a t i o n a n d severity than the first This in turn was followed by a n o t h e r p e a k o f increased activity. The oscillation between the i n h i b i t o r y - e x c i t a t o r y ( l - E ) phases lasted for a p p r o x i m a t e l y l sec after the a p p l i c a t i o n o f the stimulus to the nSA. These effects d e p e n d e d to a great extent on the stimulus intensity and the location o f the s t i m u l a t i n g electrode. In some o f the earlier experiments the stimulat-

2 ml H 2 0 , IC Partial n S A - x

Total n S A - x 2 ml, 3 o/0 NaC1, IC after total n S A - x

2 ml HzO, IC after total nSA-x

2 ml, 3°o N a C I , IC after t o n g u e nerve h g a t i o n 2 ml, 3 °.o~NaC1, IC after Th5-x

8,686 9,641

7,553 5,996

6,846

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Number of neurons f r o m 30

NEURONS

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row.

T h 5 - x ' spinal t r a n s e c t i o n at Th5. T h e average o f this row was c o m p a r e d to 2,210 o f the 2nd

Since t h e s e n e u r o n s were tested w~th 2 ml, 3 °o NaCI, IC before nSA-x, th~s average was c o m p a r e d to 2,210 o f the 2nd row Since this was the 1st H 2 0 i n f u s i o n , t h e average o f 1,542 was c o m p a r e d to 3,947 o f the 3rd row Before t o n g u e nerve hgatton ~ 2,602

n S A - x designates n S A - e c t o m y ; the effects o f IC infusions r e m a i n e d unaltered.

This was t h e 2nd successive IC m f u s l o n o f NaCI

Remark~

*** On the average, an IC infusion o f the NaCI solution raised the NaC1 c o n c e n t r a t i o n in a peripheral v e n o u s sample f r o m 144 8 m E q u l v / 1 by 4.6 m E q u i v / 1 a n d an infusion o f dlstdled ~ a t e r lowered it by 7 5 m E q m v / 1 .

** Except as indicated, all P values refer to the p r o b a b i h t y o f observing the c h a n g e m firing rates u n d e r the null-hypothesis (one-sided t-test).

* T h e control values a p p e a r e d to be related to t h e m o m e n t a r y p l a s m a c o n c e n t r a t i o n o f N a C I which was n o t u n d e r direct e x p e r i m e n t a l control.

6,504

4,266

R i n s i n g t h e t o n g u e w~th 3 % NaCI R i n s i n g t h e t o n g u e with H 2 0

2 ml, 3 o o NaCI, I C * * * a d d m o n a l 2 ml, 3°0 NaCI, IC

5,399 7,260

4,899

Experimental mampulatton

30 A L H - E p

Control*, average colmts/lO00 sec

AN ANALYSIS OF THE FIRING RATES OF

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Fig. 3. A, A computer read-out of the activity of a neuron m the nucleus S o i l , and (B, C) similar readouts of the activity of another neuron in the same nucleus shorting the influence on the probabihty of spontaneous firings imme&ately after stimulation of the nEp m A and st~mulauon of the nSA at 200 m~ec intervals (m B and C) Records of A and C were accumulated after an mtracarotld infusion of a 3 of NaC1 solution (2 ml).

ing electrode was positioned, for the purpose of experimental control, in the nucleus ventralis posterolateralis of the thalamus. N o effect was observed on the firing of ALH-Ep neurons when the stimulus was applied to this thalamic site. In later experiments it was learned that a change in the position of the stimulating electrode from the nSA by as little as 0.8 mm made it impossible to fire the ALH-Ep neuron with a shortlatency spike and the I-E oscillations were less pronounced. The short-latency firing o f the neuron was apparently dependent on a near perfect match between the parttcular stimulation and recording sites; although the I-E phasing was obtained with all o f the ALH-Ep neurons which were studied, only 13 o f them responded with the shortlatency spikes. With some of these neurons the stimulus intensity was progressively decreased. On those occasions it was observed that the computer count of the shorllatency spikes diminished to the point that no short-latency firing was recorded and the I-E phasing became less distinct. Finally, a period of reduced probability of firing was the only stimulus related effect that remained, until this too was erased by bull further decreasing the intensity o f stimulation. The effect exerted by the nSA on the nSoH was relatively uncomplicated. It consisted of a simple inhibitory period Its

333

NEURAL CONTROL OF BODY WATER

duration depended on the stimulus intensity and the location of the stimulating electrode, and it could be shortened by IC infusion of the NaC1 solution (Fig. 3C). On two occasions a slight I-E oscillation was observed with maximal intensity of nSA stimulation; short-latency firing was never obtained.

Changes m spontaneous actii'ity and effects oJ IC infusions following destruction of the nSA After a neuron of a particular hypothalamic nuclear region was tested with IC infusions and stimulation of the nSA, a 1.2 mA direct current was passed through the stimulating electrode for approximately 30 sec to destroy the nSA. Since the nSA of the cat is relatively small (approximately 2 mm in AP, 1.5 mm in LR, and 1 0 mm m the H dimension) this procedure destroyed the entire nucleus in l0 preparations (6 when recording from ALH-Ep, 2 with nSoH, and 2 with nPv) and partially damaged it in 5 others. Approximately 1 rain after the destruction of the nSA the computer accumulation of spontaneous firing from the same neuron was resumed. With a pama[ destruction of the nSA the average decrease of the spike count of the ALH-Ep neurons was small (Table I) and not all of the cases exhibited a decrease in count. With a total destruction of the nSA the decrease was relatively large In fact, the spontaneous activity of most ALH-Ep neurons decreased below their rate of firing recorded at the outset of the experiment. In comparison to this, changes In spontaneous actwlty of the nSoH and the nPv neurons after a total destruction of the nSA was shght and variable, mimicking the effect of a partial destruction of the N S A on the ALH-Ep neurons 100

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334

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When IC lnluslorls, ~ t l a which a given neuron was tested prlo~ to the nS,~, ,~c structlon, ~ e r e repeated alter the deslrLlUilon, a most unexpected result was obta~rw~!. An IC infusion o f the N a ( ' l solutmn instead o f ral~mg the firing rate ol the a~kll-~ ~ neurons, no~ loitered it (-I able 1) (_ tmversely, an 1( infusion o f distilled water f,,~cd their firing frequency, not~ ~thstandmg the lact that the NaC1 c o n c e n l l a n o n ol b h , , < was found to have increased with the NaCt infusion and decreased with the mfusum ,,1 d~stllled \~ater With the nSoH and nP~ neurons, however, the 1( ~nluslon t,t Na( 1 after nSA destruction still raised their lanng rate and the infusion o~ distilled ~a~c~ lowered it m a c o m p a r a b l e range ol ~alues observed before the destruction ol the n!~ ~, The extstt'n~ e o[ a nek~att t e/eedhacA ctrcutt between the 4 L H - E p and tire nSo H neur~tt

The results cited a b o v e suggested that 3 factors acted in c o n j u n c t m n with each other, and that their mterrelationsh~ps determined the o u t c o m e observed alte~ ,~ p a m c u l a r experimental m a m p u l a t i o n . These factors were" ( l ) the great d e p e n d e n c e o f the A L H - E p neurons on the activity o f the nSA, (2) the relative independence ol ~lle nSoH neurons from the nSA, and (3) a reciprocal r e l a u o n s h l p between the A L H - L p and the nSoH neurons If the nSoH neurons could exert an inhibitor3 influence on the A L H - E p neurons, then an IC infusion o f the NaCI solutmn would not only raise Ihe firing rate o f the nSoH neurons but would also, by way o f the inhibitory c o n n e c u o n (Fig. 9.6), decrease the A L H - E p neuron actlwty A l t e r a total destruction of the n b A , the effects o f this inhibitory ~,onnecuon would be u n o p p o s e d by the excitatory d i n e o f the nSA on the A L H - E p neurons (Fig. 9 3) and, therefore, a NaCI infusion after nS \ destruction would result m the obser~,ed decrease o f the A L H - E p neuron acm~l~ W i t h the systems intact, a sudden increase m the nSA firing (due to electrical stnnulatlon) could discharge some A L H - E p neurons and initiate a series ol I E oscfllanon~ ~la the n S o H , p a m c u l a r l v iI" the nSoH m h l b l t m n o f the A L H - E p neurons would bc reciprocated b3 e \ c l t a t m n (Ftg 0.5) The existence o f on'trots labeled as 5 a n d 6 m F~g. 9 were tested in several wass, and the results o f these tests are Illustrated in Figs 3A and 4A, B (I) Stlmulat,on ol the A L H - E p neurons at the A P level o f Fig. 6 readily discharged neurons o f the n S o H . Each stimulus (0.8 V, 0.3 reset, I/sec) ehcfled one to several spikes o f a single n S o H neuron with a latency o f 5-8 reset. These n S o H neurons res p o n d e d also to the IC m f u s m n o f h y p e r t o m c NaCI and &stilled water as described p r e w o u s l y The b u r s t - h k e response to the electrical stxmulus was particularly prominent alter the lC infusion o f the NaC1 s o l u n o n (Fig. 3A) The effecuve sites o f stimulation extended as far as 7 mm laterally from the midhne at the A P level o f Fig. 6 and were situated between 0 5 and 2 0 mm abo~e the optic tract W i t h d r a w i n g o f the stimulating electrode lk~r a greater distance a b o v e the optic tract, while recording from a n S o H neuron was m progress, rendered the s n m u l u s incapable o f d r l w n g the n S o H neuron Therefore, the effective area o f s n m u l a n o n c o r r e s p o n d e d well to that A L H - E p nuclear area where neurons were f o u n d which r e s p o n d e d to IC Infusions, o f the NaCI solutmn. The r o s t r o m e d m l b o u n d a r y o f the effective stimulation sites could not be d e t e r m i n e d because o f the physical limitations encountered when placing the stimulating and recording electrodes at progressively closer loci.

NEURAL CONTROL OF BODY WATER

335

Fig. 5 A photomicrograph showing a portion of a coronal section of a cat brain cut approximately at A 12.3 The location of two recording sites is indicated by arrows in the ALH Direct current (0 2 mA, 5 sec) was passed through the recording mlcroelectrode to mark the site located dorsally to the first iecordlng s~te Calibration line: I mm

(2) W h e n the s t i m u l a t m g - r e c o r d m g a r r a n g e m e n t was reversed, the firing of an A L H - E p n e u r o n could be inhibited for approximately 100 msec following the apphcatlon of a stimulus (0.8 V, 0.3 msec, l/sec) to the n S o H n e u r o n s (Fig. 4A) The locus of the effective s t i m u l a t i o n sites was very restricted. It mctuded the n S o H and it extended for approximately 0 5 m m in the dorsomedlal s u r r o u n d of this nucleus. The posterolateral b o u n d a r y of this locus could not be determined for the physical h m l t a t i o n of stimulating-recording electrode proximity. The A L H - E p n e u r o n s which were inhibited by n S o H stimulation responded also to the IC infusions of the NaCI solution as described previously.

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NEURAL CONTROL OF BODY WATER

337

the n S A a n d to some extent also on the activity o f the n S o H , the question was raised as to how the n S o H a n d the n S A are activated Previous studies e°,:~a as well as the p r e s e n t e x p e r i m e n t s indicated t h a t the n S o H n e u r o n s can r e s p o n d d~rectly to changes o f the N a C I c o n c e n t r a t i o n in the extracellular fired. A r e neurons o f the n S A also responsive to such changes o f the N a C I c o n c e n t r a t i o n , or is there some o t h e r mechanism whereby an IC infusion o f a N a C I solution can excite the n S A n e u r o n s ?

Fig 7 A p h o t o m i c r o g r a p h showing a portion of a coronal section of a cat brain at the level of the nucleus SA The m a r k made by the tip of the stimulating electrode in thJs nucleus is indicated by an arrow CahbratJon hne 1 m m

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NEURAL CONTROL OF BODY WATER

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The criterion for the evaluation of osmosensltivlty of the nSA neurons should be the same as that applied to neurons of the nSoH and the ALH-Ep. Namely, If they function as osmoreceptors, their osmoreceptivity should remain unchanged after deafferentiatlon. Such a deafferentiatlon of the nSA can be done most effectively by decerebration. As the gustatory system 2.9 as well as the system of Fig. 9 are uncrossed, bilateral decerebration was not necessary, particularly when general anesthesia was maintained throughout the experiment. Moreover, the method of decerebration with a negative pressure aspiration via a transfer seml-microplpette produced a very mimmal damage to the vascular system; all the blood vessels at the base of the brain remained intact. Nevertheless, with a damage of brain tissue at a 4 mm distance from the nSA, some test was required to ascertain whether or not neurons of the nSA had remained functional, so that their unresponsiveness to an IC infusion of hypertomc NaCI could be interpreted as due to lack of osmoreceptlvity rather than due to some injury inflicted on them during decerebratlon The I-E oscdlatlons ol the ALHEp neurons induced by electrical stimulation of the nSA after decerebratlon provided for such a test. In 3 of the 4 preparations which were attempted, the nSA neurons were capable of modulating the A L H - E p neuron activity in the same manner as illustrated in Fig. 2B, and consequently, in these preparations the nSA was adjudged viable. It was expected that in case the nSA neurons function as osmoreceptors, an 1C infusion of hypertonlc NaC1 would now result m an increased driving of the AEH-Ep neurons by the neurons of the nSA. If, however, the nSA would be osmotically unresponsive under the present conditions, the A L H - E p firing frequency should decrease because of the osmotic response of the nSoH neurons and their inhibitory effect on the A L H - E p neurons via circuit 6 of Fig. 9. The latter was, m fact, observed. In all 3 preparations the results mimicked those of Table I hsted with total nSA-x, except for the fact that m the decerebrate preparations the nSA was intact and viable as demonstrated by the electrical stimulation test In order for the nSA neurons to remain viable for many hours after decerebration, they must have received adequate blood supply. However, whether or not the effectiveness of the NaCI solution was mlmmlTed because of some ~mpairment of the cerebral circulatKon cannot be answered with absolute certamt). Therefore, additional work was undertaken to evaluate the importance of peripheral input involved in the activation of the A L H - E p neurons via the nSA. Most of the gustatory input into the nSA can be ehminated by sectioning or by hgation of the llngual-chorda tympani bundle and the IXth nerves, whereas input into the ventral thalamus from the splanchnic area ~'9can be eliminated by a spinal transection at Th 5. q-o provide for adequate experimental controls, data on each A L H - E p neuron were obtained before and after hgatlon of these nerves or secUomng of the spinal cord. It was found that ligation of the tongue nerves ipsdaterally to the recording sites appreciably reduced the effect of IC infusions on the firing of the AEH-Ep neurons. In fact, the change of firing due to the infusions (NaC1 hsted in Table I) after hgation of the nerves was not significantly different from the corresponding control counts. Transection of the spinal cord at Th 5 had a more pronounced consequence than ligation of the tongue nerves; it resulted in a reversal of the NaCI (Table I) and distdled water infusion effects, approximating those observed with decerebrate

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p r e p a r a t i o n s . A similar cord transecUon at L1 prior to the transection at Th 5 was inconsequential to the IC infusion effects. As the IC Infusions did not change the A L H - E p n e u r o n firing m the same direction as m the intact a n i m a l s when most o f the p e r i p h e r a l i n p u t into the n S A was ehmmated, it became clear that neurons o f the nSA similar to those o f the A L H - E p did n o t funcUon as o s m o r e c e p t o r s and that the act~vaUon o f the n S A - A L H o r the n S A - E p systems was b r o u g h t a b o u t by peripheral m e c h a m s m s . It is p r o b a b l e that these mecha m s m s are b o t h o s m o u c 2~ and g u s t a t o r y I7

NEURAL CONTROL OF BODY WATER

341

In order to evaluate further the gustatory influence on the firing of the ALH-Ep neurons, accumulation of spike counts was obtained from an ALH-Ep neuron while the cat's tongue was steadily rinsed with the NaC1 solution. Under these circumstances, the spike count increased significantly over the control count (Table I). Moreover, rinsing of the tongue with distilled water during the accumulation of the spike count significantly decreased the count (Table I). The nSoH neurons could be also influenced by the rinsing of the tongue with hypertonic NaCI or water. The NaCI solution raised their firing rate; water reduced it by only slightly smaller values than the corresponding IC infusions. Conchlsions

(1) As the effect of extracellular NaCI concentration on the firing frequency of the ALH-Ep neurons was disrupted with locahzed destruction of the nSA, the ALHEp neurons were not functioning as osmoreceptors; apparently, their firing was determined by the interaction of excitatory (Fig. 9.3) and inhibitory (Fig. 9.4) influences relayed to them via the nSA. These influences were indicated by results of the stimulation experiments illustrated in Fig. 2B. (2) The excitatory influence (Fig. 9.3) appeared to be tonically active. It predominated over the inhibitory influences as evidenced by the fact that destruction of the nSA led to a severe reduction of the firing rate of the ALH-Ep neurons. The inhibitory influences (Fig. 9.4 and 9.6) exhibited a relative independence of each other as revealed by experiments illustrated in Fig. 4A, B. (3) A reverberatory negative feedback circuit was found to exist between the ALH-Ep and the nSoH neurons (Figs. 2B, 4B, 3A, and 4A). Although stimulation of the ALH-Ep or the nSoH neurons could activate some fibers in passage from an unknown origin, the experiment illustrated in Fig. 4B indicated that the circuit originating and returning to the nSA was completed via the nSoH. (4) The influence of the nSA on the firing of the nSoH neurons was characterized by a phasic type of inhibition. A single stimulus applied to the nSA inhibited the firing of the nSoH neurons for a certain period of time (Fig. 3B, C) but destruction of the nSA did not consistently increase the firing frequency of the nSoH neurons. Furthermore, the nSoH neurons responded to the IC infusions of hypertonic NaC1 by increasing their firing rate not only before but also after the destruction of the nSA, indicating their relative independence of the nSA and a possible osmoreceptivity of the nSoH neurons. Therefore, an increase in the actlvlty of the nSoH neurons should result m some reduction in the activity of the ALH-Ep neurons via the circuit of Fig. 9.6 independently of the action of the nSA. Under conditions in which the nSA was destroyed, such a reduction of the ALH-Ep neuron activity could lead to the 'reversal effect' observed with IC infusions. (5) There was no influence of the nSA on the firing of the nPv neurons. Their firing rate appeared to be a function of the extracellular NaC1 concentration. (6) As the effectiveness of the IC infusions was severely diminished or disrupted after ligation of nerves which innervate the tongue or after spinal transection at Th 5,

342

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the mechamsm for activation of the A L H - E p neurons must be of peripheral o n g m Apparently, the activation ol such a mechanism could be accomphshed by the NaC1 solution supplied to it via the vasculature or in part by topical apphcatlon ot hype)tonic NaC1 or distilled water to the tongue. In connectmn with the above conclusions it should be emphasized that ~hc illustration of F~g 9 does not represent a preconcewed theoretical model for which support was sought by gathering experimental data. It represents a functmnal relationship of factors deNved from expeNmental results. DISC USS1ON

Results of this study may help to clarify several issues fundamental to our understanding of the neural control of body water. (1) The anatomical location of neurons which belong to the control of water retention and water intake has been indicated by numerous stu&es :~'. The fact that neurons of the paraventricular and the supraoptico-hypophysial systems are arranged in distinct nuclear groups and fiber bundles~, 15 made it easy to describe their anatomical location m relatively precise terms. Neurons which control water retake, however. seemed to be scattered throughout the lateral portion of the hypothalamus, and, since the hypothalamic lesions of behavioral studies usually extend over several anatomically non-homogeneous regions, It has not been possible to assign to these neurons a specifIc nucleus as their singular locus. Moreover, localization of the hypothalamlc area revolved m the control of water retake by using electrical stimulation, lesions, and chemical stimulation must result in a detection of a spuriously large area, because this area will include not only the neurons which are directly involved in such a contl ol but also their neural connections, particularly those with the nSA. Lesions in the posterolateral portion of the hypothalamus which are known to produce permanent adipsia z4 are likely to destroy these connections. This is further indicated by a recent finding t2 that permanent hypodipsia can be produced also by a bilateral destruction of the nSA without injury to any portion of the hypothalamus. Neurons of the present series of experiments were localized individually by histological methods. Within the hypothalamic area implicated in the control o f water intake, they were arranged loosely m a fairly homogeneous nuclear region. Although this regmn was not wider than 1.5 mm, it extended for several mm along the dorsomedial border of the optic tract. A similar description of their anatomical location has been presented by other authors 16,°"1,~9. On a closer inspection, however, it became clear that the relatively large cells of this region (Figs. 6 and 8) belong to the nEp, and it is hkely that some cells of the nEp spill into the ALH. It must be emphasized, however, that many cells. particularly m the posterolateral region of the nEp, did not respond to the NaC1 Infusions. Apparently, they belonged to a different system, most likely, a system which controls food intake 11 (2) The supposition that neurons of the two control systems of body water are somehow excited by a &rect action of osmotically active substances at their osmoreceptive membranes has been expressed almost universally for two decades z~ 7he

NEURAL CONTROL OF BODY WATER

343

fact that under conditions of relatively complete isolation from other brain structures, the firing rate of the nSoH neurons correlates well with the extracellular concentration of osmotically active substances, seems to imply that these neurons might function as osmoreceptors2°, 33. With neurons which control water intake, however, a correlation between their firing frequency and osmotic pressure has been observed by using only intact preparations. Therefore, the idea that these neurons also function as osmoreceptors must be regarded as a mere conjecture. In view of the present series of experiments, ~t becomes clear that subcutaneous, lntraperitoneal, lntragastric, lntracarotad, intravenous, lntraventricular, or lntrahypothalamac injections of substances will eventually reach some peripheral sensory elements. And if a substance is excitatory to these sensors, they wall, in preparations with intact hypothalamic connections, change the level of firing of the neurons which control water intake The firing rate of the latter, therefore, is not dependent on the effective osmotic pressure of the extracellular fluid (although a posltwe correlation can be demonstrated) but on the activity of a peripheral mechamsm. More important, this correlation can be abolished by destruction of the nSA, by decerebration or by elimination of the peripheral input relayed vm the nSA to the A L H - E p neurons. H o w substances in the vasculature can excite a peripheral mechanism is a question for a further experimental inquiry. Recording action potentials lrom the chorda tympanl or the IXth nerve before and after 1C infusions of various solutions appears to provide the first step in such an inquiry. However, the finding that fluctuations m blood supply to the gustatory receptors profoundly influences their sensitwlty to topical stlmulatlon I7 would render interpretation of the results of such experiments very difficult, particularly when experimental surgery in the vicimty of the tongue impairs its vascular bed. Whatever the means whereby the peripheral mechanism as excited, osmotic or gustatory, it cannot alter the functioning of the systems as diagrammed In Fig. 9. It has been reported that the flowing of water over the tongue leads to an increase in the spontaneous activity of some chorda tympanl fibers in the cat 6. dog 23, rabbit 3v, calf 3, and monkey 38 but a decrease of such an actwity in man 7 and rat 37. Therefore, it would be of interest to ascertain whether the inhibitory connection between the nSA and the A L H - E p neurons (Fig 9.4) exists in man and rat or the reduction of the A L H - E p neuron activity is accomplished by a simple decrease in the firing of the nSA neurons. As a monitor of the extracellular fluid composition the gustatory system is very efficient. Recent findings lz indicate that if the ventral portion of the thalamic gustatory nucleus escapes experimental destruction on only one side of the brain, the animals will regulate their water intake adequately. With a total bilateral destruction of th~s nucleus, however, water consumption in response to dehydration or to subcutaneous injections of a hypertonic NaC1 solution no longer occurs. Since with intact animals, hypertonic saline imparted into the extracellular fired by whatever means will induce water consumption, it is puzzling that some authors 's,31 have found that a differentml effect on water consumption can be produced by a direct injection of hypertonic sahne solutions at preselected sites of the hypothalamus. If at a particular hypo-

344

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thalamm site some bamers to the effecuve diffusmn of the NaCI solution x~ould .,~ encountered, one ~outd expect to obserxe a delay in the behavioral response A,. ,~ ~c,t for Iocahzatmn of hypothalam~c osmoreceptors th~s method ~s totally, lnadequ,tt< When, instead o f a sahne solutton, electrmal stimuli are used to activate neurons v~h ~ lead to water consumptmn, and at some s~te of bram st~mulatmn no behavioral response is elicited, no one would think that at that sae elecmcally mexc~table t~,~t~e has been encountered Snmlarly, with hypertomc sahne as the smnulus and ~ater consumption as the response, one cannot detect osmotically excitable and osmotically mexcitable neurons. This should be pamcularly obvious when the NaCI solution has a relatively high osmolallt) W~th lower NaCI concentrations the procedure becomes a test for the threshold sensmvzt) of neurons to osmotic st~mulanon 11 some peripheral receptors would have the lowest threshold, they, under natural circumstances, would always inmate the neural activity m a pamcular system Consequendy, u~e finding of certain tugh threshold differentially osmosensitive neurons In the hy'p~> thalamus by d~rect chemostlmulatlon would lose all physiologmal meaning. Results of the present serms of experiments indicate that the osmosensmv~t) of these h) pothalam~c neurons ~s inadequate to respond to relatively small changes m the e\t~acellular NaCI concentration when the nSA is deprived of its peripheral input (3) The interaction ol' the control systems for water retentmn and water retake with each other and with the gustatory system prowdes for relatively simple explanatmns of several interesting but puzzhng phenomena (a) It has been demonstrated "'5 that water flowing over the tongue of rata ~lll induce dmresls m these animals, whereas a similar procedure with h2ypertomc sahne i~ followed by antidmresls Fhese responses to oral stimulation do not need to be regarded as 'anticipatory reflexes', a term proposed by the author ~ , the explanation for these phenomena can be found m the circuitry of Fig. 9. Circmts 3 and 5 prowde the excitatory drive lor the nSoH. and consequently, for a release of the ant~dmrct~c hormone when the tongue Is washed with a hypertonic NaCI solution. Conversely the activatmn of circuit 4 will inhibit the nSoH when the tongue is rinsed with water, resulting m dluresls. Slmdarly, the 'taste units' recently found m the rat hypothalamus 26 appear by their locatmn to be neurons whmh control water retake; the) are activated via the gustatory system (b} The metering of water intake {ref. 36, pp. 149-162) can now be understood m the following sequence ol neuroph)siologmal events. Stimulatmn of the gustatory system during water consumptmn will reduce the activity of the ALH-Ep neurons which control water intake. At some level of actlwty of these control neurons the ammal will stop drinking It i,~ hkel) that this level of the A L H - E p neuron actiwt) matches the actlvtty level whmh Is maintained during adequate condmons of hydratmn. Since the momtormg of body water wa the thalamlc gustatory nucleus never stops, the activity of the ALH-Ep neurons remains low after the consumed water enters the circulatory system. When dehydratton sets In, receptors of the splanchmc area will excite the neurons for water retake and the gustatory system wdl monitor another round of water consumptmn it is also interesting to note that clrcmts 3 and 4 of lqg. 9 can account for the fact that a larger quantity of a given fluid is consumed when, for

NEURAL CONTROL OF BODY WATER

345

the purpose of rehydration, hypotonic sahne is drunk instead of water 19. The NaCI m water may not only suppress the activity of the chorda t y m p a m 'water fiber '6 but it may also keep the activity of the A L H - E p neurons at a higher level for a longer time than in case pure water is consumed, thus prolonging the consumatory response and increasing the volume of fluid intake. Moreover, it has been demonstrated s that hypotonic (0.85 %) NaC1 infused m the vasculature reduces the firing rate of the nSoH and nPv neurons below control firing levels. Therefore, the A L H - E p neurons, when uninhibited by orcuit 4 of Fig. 9, should increase their firing frequency due to a reduction of inhibition via circuit 6. In this connection it is ironical that another correlative type of function to dehydration and water intake, namely, salivation, has confounded investigators for a long time 36. Although the rate of sahvary flow is related to the state of hydration is, it ts more likely that the gustatory system Js stimulated by diffusion of the NaC1 from the intravascular to the interstitial fluid at the taste receptors, than by the constituents of the saliva. Similarly receptors in the portal vein-hepatic area can be activated by substances in the vasculature 29. (c) The present findings can also account for the postulate of an 'inhibitory drinking center' in the hypothalamus 30. This postulate was formulated in relation to the observation that after lesions disrupting the supraoptico-hypophysial system, water retake was greater than necessary to compensate for water loss. Therefore, the 'inhibitory drinking center' must have been destroyed by the lesions. Findings of the present study indicate that this center is the negative feedback loop. If, because of the destruction of the nSoH, the A L H - E p neurons would fire at some higher frequency than under control condition, the animal would consume more water than expected. The higher firing frequency would be due to the destruction of circuit 6 of Fig. 9. Thus, the present series of experiments provide data for the analysis of a control system which incorporates the oropharyngeal, splanchmc, and central factors of body water regulation. Most likely this system is slightly more complex than that shown in Fig 9 because structures other than hypothalamic and thalamic nuclei are known to influence body water balancea2, aS. The system, as outhned in Fig. 9, however, appears to be the one which is involved in water retention and intake most directly ACKNOWLEDGEMENTS This study was supported by Grant NS-03266 from the National Institute of Neurological Diseases and Stroke, National Institutes of Health, U.S.A. Dr. Valentln Corpus rendered excellent technical assistance during this study.

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