STUDY OF UNITARY ACTIVITY IN THE SUPRAOPTIC OF THE *

Isamu SUDA,Kiyomi KOIZUMI** AND Chandler McC.BROOKS

Departments of Physiology,Kobe Medical College,Kobe,Japan, and State University of New York,Downstate Medical Center, Brooklyn,New York

It has been well established that the supraoptic nucleus of the hypothalamus is concerned with the production and output of antidiuretic (ADH). The hypothalamic-hypophyseal system maintains an ADH discharge appropriate to the needs of the body for water conservation and it has been concluded that both neural and humoral agencies can modify ADH release. VERNEY1),HARRIS2), and others have studied the effects of various circumstances and stimuli on antidiuretic hormone output,urine and plasma concentrations3,4).The ability of atrial receptors to affect ADH release has recently been investigated5)and a number of physiologists have attempted to record activity within the supra- optic nuclei.Von EULER6)recorded slow potentials from the supraoptic,CROSS and GREEN7)and BROOKS,USHIYAMA and LANGE8)recorded activities of within and near to the supraoptic nuclei by means of microelectrodes.In their very extensive work,CROSS and GREEN observed that cells within the paraven- tricular and supraoptic nuclei and some neighboring cells were induced to fire more frequently by intracarotid injection of hypertonic solutions.Tactile and light stimuli,on the other hand,affected only a few nuclear cells while aug- menting activity in neighboring neurons more profoundly.BROOKS,et al.8) stimulated specific nerves and the reticular formation as well as administering injections of hypotonic and hypertonic solutions.Definite inhibition and ex- citation of units within the nuclei was observed.In the present study the neural and humoral control of the supraoptic-hypophyseal system was further investigated and the effects of isolating the region of the hypothalamus con- taining these nuclei from normal neural connections were determined.A further elaboration of the significance of this project will be given in the discussion.

Received for publication April 12, 1963. *Presented in part at the XXII International Congress of Physiology , Leiden, September, 1962. **須 田 勇 , 小 泉 喜 代 美

374 ACTIVITY IN SUPRAOPTIC NUCLEUS 375

METHODS

Cats anesthetized with a-chloralose(35mgm/kg)were used except in a few ex- periments in which decerebrate preparations or Nembutal-anesthetized animals were employed.Decerebration by mid-collicular transection was performed under ether or a short-acting barbiturate.

Instead of using a stereotaxic instrument in a search for units of the supraoptic nuclei,as was done in previous work8),a hemispherectomy was performed and the hypothalamus of the opposite side was brought into clear view.Anterior,middle and posterior cerebral arteries were tied off on the hemispherectomized side,carotid arteries were occluded for a few minutes during operation but only in those cases in which it seemed necessary.Following this operation the cats generally remained in good con- dition for more than 30 hours.They were kept warm by an infra-red lamp and a heating pad.To prevent dehydration intraperitoneal injections of Ringer's solution or intravenous injections of Dextran were administered when the experiments were of long duration.A constant infusion of Dextran solution was maintained in those ex- periments in which urine output was measured. Thus the basal urine production was kept relatively constant for long periods of time.Fluid administration was halted and no injections given during intervals of potential recording in order to avoid drastic change of blood volume or of the constituents of circulating blood since such changes are known to affect output of antidiuretic hormone5). Peripheral nerves of the contralateral f are and hind legs were prepared for stimu-

lation and a concentric electrode was so placed as to permit localized excitation of the midbrain reticular formation.These structures were stimulated by single or

multiple rectangular pulses(0.1 to 0.5 msec in duration)of a strength only slightly-

above threshold for initiating or affecting reflex action.The effects of chemical stimu-

lation of the cerebellum were determined by injecting minute amounts of (0.01ml of two molar sodium glutamate)into the paramedian lobe.

Recording from units(individual cells or a few synchronously firing cells)within the supraoptic nuclear region was by means of small steel electrodes.These electrodes

were made from dental broaches,the tips of which were reduced to a diameter 1 to 2ƒÊ by grinding and electrolysis9).The insulated tips,approximately 7mm long,were

soldered to fine insulated wire.An indifferent electrode was placed on a saline soaked

pledget in the cranial cavity.Since the optic tract and the hypothalamus were seen clearly,it was not difficult to place the recording electrodes in the supraoptic nucleus

and their weight was such that they remained in the same position for many hours as a floating electrode.Movement of tissue in this area was also very small and

interfered little with recording.After each experiment current was passed through

the recording electrode to mark position of its tip.The locus of the marking lesion and the completeness of all surgical isolations were determined histologically after in

situ fixation of tissues.Section of the frozen or celloidin embedded material were

studied.Another positional check used was to advance the microelectrode until it

penetrated the optic tract;this was easily detected by observing response to light. From the movement required one could estimate how far distant the electrode tip had been and thus the possibility of its locus in the supraoptic nucleus.

Stimulation of the was carried out by placing a small bipolar electrode made from paired dental broach tips on the stalk just above the neural lobe.

The placement of this electrode was done visually and its position was also checked

histologically after each experiment. When measurement of urine output was necessary,both ureters were cannulated 376 I.SUDA,K.KOIZUMI AND C.McC.BROOKS

with polyethylene tubes and the urine collected in small weighing bottles for 5 minute intervals and then weighed.Since urine output of the cat is quite small,we found it an easier and a more accurate procedure to estimate the urine output by weight rather than from the volume.

RESULTS "Spontaneous"discharges from units of the supraoptic nuclei of cats under chloralose anesthesia are shown in FIG.1.The frequencies of discharges seemed to be higher than those found in the previous work",possibly because the larger electrodes used recorded from units rather than from a single cell. Stimulation of a skin afferent(sural nerve)augmented the discharges recorded from the supraoptic nucleus in some animals(FIG.2),but in other instances activity was inhibited to some degree(FIG.3).These results were similar to those obtained from single cell recording".FIG.2 also shows a post- stimulation depression with recovery of the normal level of activity after a few seconds.Repetitive stimulation of the reticular formation also gave mixed effects.The functional significance of these changes produced in activity of supraoptic nucleus by peripheral or central stimulation is hard to explain; possibly any kind of afferent impulses strong enough to induce considerable activity within the central nervous system can alter the activities of cells in the supraoptic nucleus. In an attempt to obtain direct information concerning the nerve fiber con- nection between hypophysis and supraoptic nucleus,recordings were made from cells of the supraoptic nucleus during periodic stimulation of the pituitary stalk by bipolar electrodes.Though nothing resembling an antidromically-evoked potential was observed,an unexpected reaction did occur.Repetitive stimula- of the pituitary stalk at a rate of 1-2 shocks per sec.resulted in an augmented firing of neurons in the supraoptic nucleus.FIG.4shows that when the stalk was stimulated at a rate of 1 per sec.there was a deffiite increase in firing.In FIG.5 is shown the augrnentation of activity in the supraoptic nucleus as the frequency of pituitary stalk stimulation was increased from 1/sec.to 4/sec.It is difficult to explain this peculiar effect of antidromic stimulation.Spread of current either to adjacent tissues or to the nuclei them- selves might have occurred though relatively weak voltages were used.Histo- logically there seemed to be no structure other than the pituitary stalk which could have been stimulated in our preparations.The position of these stimu- lating electrodes on the stalk was confirmed in each instance by frozen section techniques.CAJAL did suggest an afferent connection from the pars neuralis; this would explain the result. In view of the findings that neurons in the supraoptic nucleus often respond to many influences such as afferent nerve excitation,reticular formation stimu- lation,injection of hyper- or hypotonic solution,injection of adrenalin,etc.8), ACTIVITY IN SUPRAOPTIC NUCLEUS 377

attempts were made to determine how reactions would change after exclusion of all possible influence from neural structures other than those of the hypo- .Decortication and elimination of the caudate nucleus of the same side did not modify cellular activities in the supraoptic nucleus to any detect- able degree,but a section separating the midbrain from the hypothalamus did cause increased activity in the supraoptic nuclei.Furthermore,when the small area surrounding an electrode,which was recording activities within the supra-

A B C D E

FIG.1.•@ Spontaneous discharge from units of the supraoptic nucleus.A and B,

discharges from the same unit at different sweep speeds.C,D and E show cellular discharges from different preparations.

A

B

C

D

FIG.2.•@ Augmentation in discharge of supraoptic nucleus cells during stimulation of sural nerve(B).Rate immediately after termination of nerve stimulation(C).

Level of activity before(A)and a few seconds after stimulation(D). 378 I.SUDA,K.KOIZUMI AND C.MCC.BROOKS

A B C D

FIG.3.•@ Inhibitory effects of sural nerve stimulation on unit activity.Rate of firing before(A),during(B),immediately after (C)stimulation and some minutes later(D).Read from bottom up.Vertical tracings are the continuous recordings.

A

B

C

FIG.4.•@ Effects of stimulating the pituitary stalk on activity of units in the

supraoptic nucleus.Activity before(A),during(B),and after a period of 1/sec. stimulation(C).Spikes retouched. ACTIVITY IN SUPRAOPTIC NUCLEUS 379

A B C D

FIG.5.•@ Activity in the supraoptic nucleus induced by various rates of stalk stimu- lation.(A)prestimulation state.Sections of records showing results of stalk stimu-

lation at rates of 1/sec.(B),2/sec.(C),and 4/sec.(D).Stimulation artefacts at left

of sweeps.

A B C D

E F

FIG.6.•@ Continuous and periodic sweep recording of activity in supraoptic nucleus before(A)and after(B,C,D)production of a hypothalamic island containing the

nuclei.Records from another experiment showing activity before(F)and after iso-

lation from all neural connections(F). 380 I.SUDA,K.KOIZUMI AND C.McC.BROOKS optic nucleus,was isolated from the rest of the nervous tissue,the activities were enhanced(FIG.6 E and F particularly).Since hemispherectomized pre- parations were used throughout our experiments,this island of hypothalamus was connected to other regions of the brain only through blood vessels.When the island of isolated tissue was too small,less than 5mm.in diameter,the augmented activities did not last for more than an hour,but when the island was sufficiently large,i.e.,greater than 5mm.in diameter,the unitary activi- ties remained for many hours.In one instance spontaneous unit discharges were observed for some 40 hours after the isolation. These isolating procedures were performed without moving the recording electrode from its original position.The increased activity of supraoptic nucleus neurons was not due to a drop of blood pressure such as that which is known to cause an increased output of ADH from the pituitary gland10).The operation produced almost no bleeding and blood pressure did not fall but became somewhat elevated following these procedures.Furthermore,when 10cc hypertonic glucose solution(8%)was injected into a carotid artery the electrical activities of neurons in the isolated supraoptic nucleus region were increased(FIG.7).Thus this tissue retained its ability to respond to an osmotic or a local dehydrating effect of glucose.It seems unreasonable to assume that injury currents from damaged tissue could be responsible for the activity maintained in the island for many hours or that cells without an adequate blood supply could maintain their responsiveness to hypertonic injections. Enhanced activity in a hypothalamic island should promote the release of ADH from the .FIG.8 shows the changes in the urine output associated with the rate of discharge of neurons in the supraoptic nucleus. This increase in rate of discharge occurred when the region of the hypothala- mus surrounding the recording electrode was isolated from other tissues.The urine output which decreased to one quarter that of the control volume remained reduced for one hour and until the pituitary stalk was sectioned.The fact that section of the pituitary stalk was followed by an increase of the urine output to the control level provided additional evidence that the change of urine volume associated initially with the isolation of the supraoptic nucleus region might be due to increased release of ADH in association with the in- crease in neuronal discharges.Pituitary stalk section did not increase the urine output above the control level in this particular case shown in FIG.8.The absence of a might have been due to the dehydration of the preparation,since in this instance the animal had not received a constant infusion of fluid during the prolonged experiment(6 hours).The supraoptic nuclei might also have supplied sufficient ADH even in the absence of stalk and neural lobe. This augmentation of cellular activity and evidence of excess ADH output after isolation of the supraoptic region from neural connections suggests that ACTIVITY IN SUPRAOPTIC NUCLEUS 381

A B C D

FIG.7.•@ Unit activity,within an island of hypothalamic tissue containing the supraoptic nuclei,before(A),during intracarotid injection of 10cc.of a 10 per cent glucose solution(B),a few minutes(C)and 30 minutes(D)after completion of the injection.

FIG.8.•@ Activity within supraoptic nucleus(sweep records to left)and

urine production before and after isolation of these nuclei by making a

hypothalamic island.Increase in unit activity shown on sweeps at right . Section of the pituitary stalk abolished the apparent excessive antidiuretic

effect. 382 I.SUDA,K.KOIZUMI AND C.McC.BROOKS

FIG.9.•@ Increase in urine output due to stimulation of cerebellar cortical cells by application of Na-glutamate and reduction of effect by section of cerebellar peduncles.

This increase in urine output ascribed to an inhibition of ADH output. there are neural structures in the central nervous system which have an in- hibitory influence on the supraoptic nucleus.One such possible structure is the cerebellum since it was found that the excitation of cerebellar cells of the paramedian lobe by injection of minute amount of glutamic acid(0.01cc.of two molar sodium glutamate)"increased urine output and that following this increase,section of all cerebellar peduncles reduced the output(FIG.9).How- ever,we do feel that other structures of the midbrain,medulla and thalamus probably also have some control over the supraoptic nucleus and hypothalamus in general.The cerebral cortex was not shown to have any great influence, since decortication produced no obvious change in electrical activity within the supraoptic nucleus.Urine output was reduced by decortication but the volume output returned to a nomal level in 10 minutes.This temporary reduction could have been due to a transient disturbance of the neurons by the surgical procedures.The excitatory and inhibitory actions of higher centers on the supraoptic nuclei will be investigated further.

DISCUSSION

The so-called "resting" or "spontaneous" discharges or neurons in the supraoptic nucleus was definitely present even when a small mass of hypo- thalamus containing the nucleus was isolated from the reset of the central nervous system.This suggests that neurons in the supraoptic nucleus are capable of maintaining their activity without impingement of impulses from ACTIVITY IN SUPRAOPTIC NUCLEUS 383

other areas in the central nervous system or from periphery through nerve pathways.HARE12)also reported the antidiuretic response to hypertonic saline injection still remained when all nervous paths to the from peri- pheral receptors were interrupted by section through the midbrain,transection of the cervical sympathetic trunks and the first 3 cranial nerves.Other areas of the central nervous system,however,can and do modify the activities of cells in the supraoptic region as has been shown. Since neurons in an isolated island were sensitive to osmotic change in circulating blood,the"resting"activity of the neurons might be maintained by a reaction to their humoral environment.It has been proposed that the osmolarity of the blood is a constant factor in regulating ADH secretion by the pituitary gland2).The results reported here tend to support this hypothesis since they supply direct evidence that the activity of supraoptic nucleus cells in isolation do react to osmotic changes.Some of the best evidence in support of the idea that the anterior hypothalamus though isolated from other than vascular connections can maintain its osmoregularity function comes from the work of MACHT and BARD13).They showed that an island of hypothalamic tissue maintained a normal water balance in chronic decerebrate cats. The relationship between the supraoptic nucleus and other areas in the central nervous system has not been well investigated.Our findings suggest that supraoptic nucleus activities are influenced by cerebellum,thalamus,mid- brain,and probably the medulla.Although direct fiber connections between celebellum and hypothalamus have not been found anatomically,much physio- logical evidences suggest a relationship between the structures14).MORUZZI15) found that stimulation of the cerebellum inhibited sham rage;this he attributed to a cerebellar influenced on the hypothalamus.BAN,et al.16)also found an inhibitory effect of the cerebellum on hypothalamic-induced autonomic responses. However,an inhibitory influence of the cerebellum on supraoptic nucleus cells has not been reported previously,to our knowledge. ANDERSON,et al.17)showed that following transection of midbrain or spinal cord in dogs,urine output was increased.They postulated the existence of a mesencephalic-hypothalamico-pituitary activating system.On the other hand, we observed an augmented activity of neurons in the supraoptic nucleus and a marked decrease in urine output when the midbrain was transected.Section of the pituitary stalk abolished this reduction,suggesting that the decrease in urine output was due to an increase in ADH discharge from the pituitary gland. The difference between our results and those of ANDERSON and his associates are thought merely to indicate the complexity of the control exerted over the supraoptic-hypophyseal system. Increased ADH output from the pituitary gland as a result of emotional stress or painful stimulus has been repeatedly observed1,2,18,19).It is generally accepted that this increase is a reflex response of the supraoptic nucleus to 384 I.SUBA,K.KOIZUMI AND C.McC.BROOKS afferent stimuli.In our anesthetized animal,stimulation of skin afferents caused changes in electrical activity of supraoptic neurons;in some there was augmentation of activity,in other there was a diminution.A reversal of effect after cessation of repetitive nerve stimulation was also seen in many cases. This instability of reversal may be due to activity in thalamic nuclei produced by the sensory impulses,since there are close connections between thalamus and supraoptic nucleus20) Another puzzling result obtained in these experiments was the antidromic activation of cells in the supraoptic nucleus.Stimulation failed to produce a typical antidromically-evoked response in the supraoptic neurons7,8),but"rest- ing"discharges were much augmented by the stimulation.The response had too short a latency to be the result of some systemic change such as a gene- ral or local dehydration2) or a vasopressor or depressor action.Furthermore, blood pressure remained at 100 to 130mmHg.Stimulation of the stalk did not produce augmented activity in these cells in every instance.There were many neurons in which no change was observed, but when there was a change,it was always an augmentation.The mechanism of interaction between supra- optic nucleus and pars neuralis of the hypophysis still requires study.

SUMMARY

Activity in hypothalamic supraoptic nuclear cells was recorded with micro- electrodes in hemispherectomized,chloralose-anesthetized cats.Repetitive stimulations of skin afferents augmented activity of some units but inhibited others.Activity of units was little affected by decortication and removal of the caudate nucleus but considerably increased by decerebellation,midbrain transection and separation from thalamic connections.Stimulation of the stalk augmented rate of firing of nuclear units.Complete isolation of the supraoptic nuclear region from all neural connections except with hypophysis resulted in an increase in cellular discharge rate which lasted for many hours.This aug- mented activity was associated with a decreased urine output until the pitui- tary stalk was sectioned.The supraoptic nuclear cells in the island retained their sensitivity to osmotic changes in the blood produced by intracarotid injec- tion of hypertonic glucose.Stimulation of the cerebellar cortex by application of glutamic acid increased urine output.These experiments provide evidence that the supraoptic nucleus cells in addition to being subject to excitation by both neural and humoral agents are under inhibitory control from structures within the cerebellum,midbrain and thalamus.

This work was supported by United States Public Health Grant B-847(C-6). ACTIVITY IN SUPRAOPTIC NUCLEUS 385

REFERENCES

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