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Hypothalamic unit activity. I. Visceral and somatic influences
ELECTROENCEPHALOGRAPHY AND CLINICAL NEUROPHYSIOLOGY
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HYPOTHALAMIC UNIT ACTIVITY. I. V I S C E R A L A N D S O M A T I C I N F L U E N C E S I D. G. STUART,PH.D ~, R. W. PORTER,M.D., PH.D., W. R. ADEY, M.D. AND Y. KAMIKAWA, M.D. a Departments of Anatomy and Physiology, University of California, Los Angeles, Calif. and Veterans Administration Hospital, Long Beach, Calif. (U.S.A.) (Received for publication: December 18, 1962) (Resubmitted: March 11, 1963)
INTRODUCTION
Clinical and experimental findings have illustrated that a major role of the hypothalamus involves integration of somatic and visceral mechanisms in a variety of patterns (Hess 1957; lngram 1960; Ranson and Magoun 1939). Experimental work in this part of the brain has mainly been concerned with the effects of lesions and electrical stimulation on its effector activity. Less is known of the effects of ascending influences on such activity. Recent attempts to delineate the afferent input to the hypothalamus have demonstrated long latency potentials in response to sciatic nerve stimulation in the posterior region similar to those in the midbrain reticular formation,and short latency potentials indicative of input from leminiscal collaterals (Feldman et al. 1959). Additionally, pronounced changes in the synchrony of posterior hypothalamic, ventral thalamic and rostral midbrain electrical waves were shown to accompany alterations in urinary bladder pressure (Porter and Bors 1962). Such results illustrate that interactions of somatic and visceral modalities on hypothalamic structures are quite feasible, utilizing both fast and slow afferent pathways. Similarities between posterior hypothalamic and rostral midbrain responses have a sound anatomical basis (Nauta 1 Aided in part by grants B-1883 and A F 61-81 from the United States Public Health Service and the U.S. Air Force Office of Scientific Research, respectively. 2 Medical Research Fellow, Bank of America-Giannini Foundation. a Appointment supported by the International Cooperative Administration under the Visiting Research Scientist Program administered by the National Academy of Sciences of the U.S.A.
and Kuypers 1958) and are becoming increasingly evident in the physiological literature (Granit and Kaada 1952; Sayers 1957; Sawyer 1958). Less is known of the anatomical and physiological relationships of ventral thalamic and dorsal hypothalamic structures although both lesion and stimulation studies have shown the difficulty of separating these structures on functional grounds (Hess 1957; Starzl et al. 1951a and b; Stuart et al. 1961). The relationships between somatic and visceral inputs on the same hypothalamic structures are obscure. Additionally, there is a paucity of information concerning the firing pattern of single hypothalamic neurons during any but osmotic (Cross and Green 1959), thermal (Birzis and Hemingway 1957; Nakayama and Eisenman 1961) and combined amygdaloid-sciatic (Wendt 1961) stimuli. For these reasons, this study has concerned itself with analysis of the firing patterns of single units within hypothalamic and ventral thalamic regions during increases in urinary bladder pressure both independent of, and combined with, sciatic nerve stimulation. MATERIALS AND METHODS
Experimental results are based on sixteen acutely prepared cats. Surgical procedures were completed before discontinuing ether anesthesia, which was then replaced by immobilization with gallamine triethiodide (Fiaxedil). All wound margins and pressure points were infiltrated with 1 per cent procaine hydrochloride, and regional blocks were performed on the supraorbital and maxillary divisions of the trigeminal nerve at their points of emergence from the skull. Additional infiltration was carried out in the anterior and posterior wails of the external auditory meaElectroenceph. clin. Neurophysiol., 1964, 16:237-247
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tus. This regional anesthesia was repeated every 2 h. Artificial respiration was controlled by a Harvard p u m p at a rate of 20 to 25 breaths per min, and the stroke volume was adjusted to permit a 4 per cent COz level in each expired breath. This parameter was monitored by passing a small sample of the expired air through an infrared CO2 analyzer, Rectal temperature was maintained near 38°C with an electronic temperature controller. The surgical procedures included tracheal intubation, median vein and urinary bladder cannulation (described previously, Porter and Bors 1962), exposure of the left sciatic nerve in the popliteal fossa and craniotomy. A bipolar Sherrington electrode was applied to the exposed sciatic nerve, which was then covered with mineral oil. Craniotomy included sufficient bone removal to expose the dorsal surface of the brain on both sides between Horsley-Clarke coordinates Fr 6 to 14 and L 0 to 5. The dura was removed from this area and the surface of the brain covered with a 5 to 10 per cent solution of agar in 0.9 per cent saline solution. This minimized brain pulsation while permitting micro-electrode insertion. Three hours elapsed after discontinuing ether anesthesia before commencement of recording. Preliminary experiments convinced us that use of anesthetic agents such as pentobarbital sodium or alpha chloralose, at dose levels only a fraction of that necessary to induce anesthesia (e.g. 5 mg/ kg Nembutal i.v.) drastically reduced the number and firing rate of spontaneously active hypothalamic units. Moreover, such units were then unresponsive to the stimulation procedures. Visceral stimulation consisted of distention of the previously emptied urinary bladder with 25 to 35 ml of 0.9 per cent saline solution at 38°C. This volume was injected manually from a syringe such that filling, "full" and emptying phases each took 6 sec. Freely moving, unanesthetized cats exhibited no evidence of pain or discomfort when identical distention of the bladder was induced at the same rate via an indwelling Foley catheter. The parameters of sciatic nerve stimulation were adjusted to minimize A-delta and C fiber activation, i.e., 10/sec square wave pulses, 5 V intensity, 0.5 msec duration delivered from a Grass stimulator (S4-A) and isolation transform-
er. Previous studies in our laboratory have indicated that such a stimulus applied through chronically implanted electrodes to the sciatic nerve of freely moving cats is not associated with evidence of a painful experience (Lindsley and Adey 1961). Stainless steel (Adey and Dunlop 1960) or carbon steel micro-electrodes with tips less than 1 # were used. The latter were prepared by electropolishing dental broaches (S.S. White, Smooth Broach No. 42, fine) in an acid bath with 60 cycle A.C. and an E.M.F. of 1-3 V. The acid bath consisted of 34 ml of sulphuric acid (95 to 98 per cent concentrated), 42 ml of orthophosphoric acid (85 per cent concentrated) and 48 ml of distilled water. The electrodes, insulated with two to four coats of an epoxy-resin (EpiREZ 285-19, Jones-Dabney Co., Los Angeles) 1 were positioned stereotactically in various hypothalamic regions. Recording of unit activity usually began in ventral thalamic structures, 10 to I1 m m above the interaural line. Unipolar extracellular recording was referred to an indifferent electrode in the neck muscles. Potentials were led into the cathode-follower probe of a Grass model P5 R.C. pre-amplifier, then displayed on a Tektronix model 502 oscilloscope and recorded with a Grass Kymograph camera. Our observations on the positive-negative spike configuration, 1.0 to 2.0 msec duration and 0.5 to 10 mV amplitude of hypothalamic units were similar to previous reports (Cross and Green 1959; Wendt 1961). Usually the responses of single neurons to the stimulation procedures were observed prior to photography. Thus, positive results were based on at least two trials. With dubious responses, the same stimulus was often applied several times. In this way, chance correlations that could have arisen with single trials were minimized. During paired stimulation the sciatic stimulus started 6 to 12 sec before bladder distention and continued 3 to 12 sec after the bladder was emptied. Artifact reduction was successfully achieved by use of a Wagner ground system. Variations in stimulus artifact level, in the text figures, were due to manipulation of this ground system. At the conclusion of each experiment the animal was sacrificed and the brain perfused in situ through a cannulated carotid artery with a 5 per cent solu1 We are indebted to Dr. H. Batsel for this technique of micro-electrode construction.
Electroenceph. clin. Neurophysiol., 1964, 16:237-247
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tion of potassium ferrocyanide in 10 per cent formalin. The brain was then removed, fixed in formalin and later sectioned every 80/z. The sections were stained with neutral red for identification of Prussian blue spots that had been electrolyticaUy deposited at the tip site when each electrode had been driven to its deepest position. The loci of recorded units along the same track were calculated by reference to these spots. In the figure legends of Fig. 2 to 5, the numbers in brackets give each unit's stereotactic coordinates according to the atlas of Jasper and Ajmone Marsan. RESULTS
I. Location o f units responsive to bladder distention and sciatic stimulation The firing rates of 291 ventral diencephalic units were analyzed during bladder distention. The response of 214 of these neurons to sciatic TOTAL B
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Fig. 1 Schemata of number and location of single neurons whose firing rates were analyzed during bladder distention (B) and sciatic stimulation (S). Unshaded sector: no. of unresponsive units; stippled sector: units with augmented firing rates during stimulus; hatched sector: units with reduced firing rates during stimulus. Horizontal and vertical co-ordinates on the midsagittal schema indicate mm anterior and dorsal to the interaural line. All units within 3.5 mm of the midline. Abbreviations: AC: anterior commissure; CC: corpus callosum; OC: optic chiasm; M: mamillary body; MI: massa intermedia; PC: posterior commissure; Spt: septum.
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stimulation was also observed. Fig. 1 indicates that the visceral stimulus exerted as powerful an effect on these units as the somatic stimulus. Thirty per cent of all units analyzed were responsive to bladder distention, the firing rate of 19 per cent being augmented and 11 per cent reduced, abbreviated hereafter to A and R respectively. Thirty-five per cent of the units were responsive to sciatic stimulation (27 per cent A, 8 per cent R). Despite the small sample, the results indicated that both stimuli more commonly influenced units within ventral thalamic and dorsal posterior hypothalamic regions than units within the anterior or ventral posterior hypothalamus. Of the 77 dorsal posterior hypothalamic units observed during bladder distention, 37 were within 1.5 mm of the midline, the remaining 40 being 1.6 to 3.5 mm from the midline. Of the former, fourteen responded to bladder distention (10A, 2R) while in the latter group twelve responded (8A, 4R) to the stimulus. Thus, no distinction could be made between medial and lateral neurons for this stimulus nor, after the same analysis, for the somatic stimulus. Similarly, no distinction could be drawn between the behavior of the more rostral neurons (Fr 11 to 10) and the more caudal ones (Fr 10 to 8). One hundred and sixteen ventral thalamic units were analyzed, 25 from the posteromedial aspect of the ventrolateral nucleus (VL) and 38 from the ventromedial nucleus (VM). Six VL units responded to bladder distention (1A, 5R) while 18 VM units responded (15A, 3R) to the same stimulus. Influenced by sciatic stimulation were fourteen VL units (9A, 5R) and eighteen VM units (15A, 3R). The remaining 53 responsive thalamic units were distributed in a variety of medial thalamic structures. I[. Nature of visceral stimulation Sensory volleys during bladder distention could arise not only from proprioceptive afferents in ves[cal musculature but also from other visceral receptors due to the increase in intraabdominal pressure associated with bladder distention. Additionally, somatic activity could not be excluded since bladder distention mildly stretches the anterior abdominal wall. Our results suggested that whereas all three were possible, the stimulus was predominantly visElectroenceph. clin. Neurophysiol., 1964, 16:237-247
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ceral and furthermore that vesical proprioceptive afferents alone were capable of modulating the firing patterns of these units. In only two instances did units responsive to bladder stretch also respond to gentle stroking of the anterior abdominal wall. However, this stimulus was not used throughout all experiments. In three experiments a rubber balloon of approximate bladder size was inserted intra-abdominally in juxtaposition to the bladder. The firing rates of 52 units ,
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Fig. 2 Three types of augmented firing rate during bladder distention. Unit I (Fr 9.5, L 0. 5, H minus 0.6) was evoked into activity from a silent background during the initial phase of bladder filling. Unit 1I (Fr 9.0, L 1.0, H minus 3.6) was tonically active and also responded to the early phase of filling whereas Unit III (Fr 9.5, L 2.0, H minus 2.9) typified those neurons whose firing rates "peaked" in the late "'full" phase. Abbreviations for this and subsequent figures: Fi: begin filling bladder; Fu: "full" bladder; W: commence withdrawal of fluid from bladder; E: empty bladder. Photographs of unit discharges in this and subsequent figures show negative d~flection downward. The letters ,4, B, C and D relate these photographs to specific epochs shown in the graph. For this and subsequent graphs the ordinate is firing rate of units in spikes/2 sec.The abscissa is time in seconds.Vertical calibrations: 1 inV.
Electroenceph. clin. Neurophysiol., 1964, 16:237-247
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ing to the control level in the "full" phase. Some such units were evoked into activity f r o m a silent background (Unit I, Fig. 2), but more often spontaneous activity was present (Unit II, Fig. 2). In the second major group (22 units), the increase in firing rate did not reach a maximum until the end of the "full" phase. Rarely was there a steady rise in firing rate but rather, during the filling and early"full" phases, one to three bursts of increased firing rate occurred that nearly reached the m a x i m u m rate attained (Unit III, Fig. 2). The
in a 38°C solution of 0.9 per cent saline so that its distention could not alter intra-abdominal pressure or the tension on the anterior abdominal wall. Fourteen units were analyzed, six of which responded to bladder distention (2A, 4R). II1. Patterns o f response to bladder distention The firing rates of 55 neurons were augmented by bladder distention. Two predominant patterns emerged. In the first (23 units) the firing rate rose briefly and transiently in the filling phase, return-
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firing rate of each unit in this group returned to the control level within 20 sec of the bladder being emptied. In the case of four neurons, the peak or near peak increase in frequency was maintained for as long as 2 min after bladder distention. There were two distinct peaks in the firing rate of six other units, one during initial filling and the other during initial emptying. Despite this second peak the firing rate quickly regained the control level after the bladder was emptied. As with augmented responses, patterns of reduced responses, demonstrated by 33 neurons during bladder distention, fell into two major categories. Most typical (19 units) were gradually reduced firing rates reaching a peak suppression in the "full" phase (Unit I, Fig. 3). Less typical (10 units) were brief reductions in firing rates during the filling phase, with control rates returning in the "full" phase. For both groups, a suppressed firing rate was often followed by a 1 to 5 sec burst at a higher frequency than the control rate. Of particular interest was the behavior of the remaining five neurons wherein a reduction in firing rate occurred in the early emptying as well as the early filling phase (Unit II, Fig. 3). In none on these units did the reduction in firing rate during bladder distention persist more than 20 sec after the bladder was emptied. IV. Bilateral effects o f sciatic stimulation Of the neurons analyzed during sciatic stimulation, 112 were ipsilateral and 102 were contralateral to the stimulated nerve. In the former group 37 units were influenced by stimulation (30A, 7R) and in the latter group 37 units also responded (28A, 9R). In one experiment eleven units were studied during left and right side sciatic stimulation. Seven units were responsive; four being equally affected by both stimuli (2A, 2R). In another case the response to contralateral stimulation was greater while in the remaining two cases the ipsilateral stimulus exerted the stronger effect. Ventral thalamic neurons were similar to hypothalamic neurons in their response to ipsilateral sciatic stimulation. For example, of the VM neurons observed during sciatic stimulation, the stimulus was ipsilateral in 25 cases and contralateral in thirteen. Twelve of the former were influenced(10A, 2R) andsix of the latter (5A, 1R). For VL units, fourteen were observedduring
ipsilateral stimulation and eleven during contralateral stimulation. Eleven of the former responded (8A, 3R) and three of the latter (1A, 2R). V. Responses to combined visceral and somatic stimulation Of the neurons whose firing rates were analyzed during individual and combined bladder distention and sciatic stimulation, 109 were unresponsive to all three stimulation procedures. Twelve units were influenced by sciatic stimulation but not by the visceral or combined stimulus. Fourteen others responded unidirectionaUy to both individual stimuli (8A, 6R) but such augmentation or reduction in firing rate was not exaggerated by combined stimulation. The remaining 38 units displayed a variety of responses to combined stimuli that could be categorized as follows: (a) Thirteen units responded in opposite direction to individual stimuli. During paired stimulation either the rate remained at the control level or it followed the influence of one or the other stimulus in reduced amount. (b) Twelve other units that responded to bladder distention in a characteristic fashion illustrated no such response during concomitant sciatic stimulation even though firing rates were uninfluenced by individual sciatic stimulation (Fig. 4). (c) The response of the remaining thirteen units to bladder distention was augmented by simultaneous sciatic stimulation. Five of these responded in the early phase of bladder emptying during paired stimulation but not during individual visceral stimulation. The other eight units responded in the "filling" or "full" phases of the bladder distention stimulus. Three were mildly influenced by visceral but not somatic stimulation, then markedly accelerated by combined stimulation (Unit I, Fig. 5). With three others the individual stimuli had no effect on the firing rate which was then augmented by the paired stimuli (Unit 11, Fig. 5). In the remaining two cases, the response to paired stimuli was greater than the augmented responses to the individual stimuli.
VI. Visual and auditory responses At the conclusion of visceral and somatic stimulation procedures, the responses of many units to brief and prolonged flashes of light, Electroenceph. clin. Neurophysiol., 1964, 16:237-247
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Fig. 4 Combined somato-visceral stimulation producing greater changes in unit firing than either stimulus alone. Bladder distention induced a mild increase in the firing of Unit I (Fr 9.0, L 1.0, H minus 4.3) as indicated by continuous line. Paired stimuli, indicated by a broken line, greatly augmented this effect. Sciatic stimulation began 6 sec before bladder filling and ended 6 sec after emptying. It was without effect except during bladder distention. Unit II (Fr 8.0, L 3.5, H minus 4.5) was uninfluenced by either stimulus alone, but the firing rate increased during paired stimuli. Here, sciatic stimulation extended before and after the epoch of bladder distention for 10 sec. Paired unit records (marked A, B and C) relate these unit discharges to specific epochs shown in the graphs before, during and after bladder distention. The upper trace of each pair shows the effects of bladder distention alone. The lower trace (marked S) shows the effects of bladder distention during concomitant sciatic stimulation. Ordinates: spikes/2 sec; Abscissae: time in seconds. Vertical calibrations: 1 mV. clicks a n d extremely loud noises were noted. Only nine of 178 n e u r o n s were influenced by auditory s t i m u l a t i o n (7A, 2R) a n d three of 171 by visual s t i m u l a t i o n (1A, 2R). As in other studies (Cross a n d G r e e n 1959; W e n d t 1961), responsive units were widely dispersed t h r o u g h o u t the hyp o t h a l a m u s . We f o u n d n o n e in ventral thalamic
nuclei. Five of the responsive units were also affected by visceral stimuli b u t no attempts were made to pair visceral with visual or auditory stimuli. DISCUSSION There were essential similarities in the patterns Electroenceph. clin, Neurophysiol., 1964, 16:237-247
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of responsiveness of neurons in the posterior hypothalamus and ventral thalamus to the three stimulation procedures used here. This is in good general agreement with previous studies showing similar alterations in the synchrony of electrical waves recorded from both regions during increases in urinary bladder pressure (Porter and Bors 1962). Similarities in the form and latency of potentials evoked in both regions by single shock stimuli to the contralateral sciatic nerve have also been observed (Starzl et al. 1951b; Feldman et al. 1959; Feldman 1962). In the present study VL, VM and hypothalamic units responded to both ipsilateral and contralateral sciatic stimulation as has recently been observed for VL units using other somatic stimuli (Kruger and Albe-Fessard 1960). The large number of responsive units at the j unction of the ventral thalamus and dorsal posterior hypothalamus is of interest for three reasons. First, the concepts of Hess (1957) are exempli-
fled, in that neurons in this "tween-brain" region exhibited inseparable relationships to somatomotor and visceral functions and that the responsive zone lacked clearly defined boundaries related to classical anatomical schemes. Second, the region (Fr 10 to 8, L 0 to 3, H 0 to minus 3, according to the atlas of Jasper and Ajmone Marsan) exerts many central and peripheral effects. It has been termed the ergotropic zone of the diencephaLon in that its stimulation evokes increased respiratory activity, pupillary dilation, increased motor excitability, and sometimes flight (Ranson and Magoun 1939; Hess 1957). It has been implicated in temperature regulation (Stuart et al. 1961), activation of gamma motoneurons (Granit and Kaada 1952; Stuart et al. 1962), cerebral cortex (Starzl et al. 1951a and b)andthe midbrainreticular formation possibly determining the latter's responsiveness to peripheral stimuli (Adey and Lindsley 1959; Lindsley and. Adey 1961). Since bladder distention exerts such a powerful effect Electroeneeph. clin. Neurophysiol., 1964, 16:237-247
HYPOTHALAMIC UNITS. I.
on units within this region, it might be expected that all the above mentioned parameters, concerned with gross alterations in body homeostasis, are subject to modulation by this and other forms of visceral activity. Third, the region contains two-way connections between the midbrain and the basal ganglia and rhinencephalon (Adey 1959; Bodian 1946; Nauta 1958). Numerous references indicate that rhinencephalic influences on somato-viscerat mechanisms are secondary to a primary hypothalamic control (cf. Gloor 1956). However, information concerning afferent visceral inputs to the rhinencephalon is sparse (Dell and Olson 1951; Dunlop 1958; Feldman 1962; Machne and Segundo 1956). To insure stable recording in the present study, units were recorded wherein the initially negative wave showed a positive-negative configuration. Closer approach of the micro-electrode to the cell membrane, with positive dominance in spike configuration, often resulted in a ruptured cell membrane. A limited number of extremely brief and purely negative spikes were recorded. In some instances they responded to the stimulation procedures. Their recorded characteristics did not preclude axonal structures. In view of the potential origin of such spikes in cells lying outside the hypothalamus, including the rhinencephalon, data drawn from these units were not systematically collected nor have they formed the basis of conclusions drawn from this study. It is difficult to evaluate the latency between bladder distention and acceleration of responsive hypothalamic units. Intraluminal bladder pressure was not recorded. Even this parameter does not indicate localized changes in vesical motility that influence the firing rate of the "in series" stretch receptors (Iggo 1955). Additionally, so little is known of the pathway to the hypothalamus (Amassian 1952; Ruch 1960) that any discussion of latency is purely speculative. Most frequently, the accelerated responses began 1-3 sec after beginning bladder distention although shorter latencies were observed in a few cases. Since spike amplitudes and configurations were rarely altered during distention, such accelerated responses could hardly be due to blood pressure changes mechanically displacing the neuron (Adey and Dunlop 1960). Many units that reached a peak firing rate late in the "full" phase of the
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visceral stimulus accelerated one to three times prior to the peak rate. This suggested that the stimulus exerted more than one central effect. Reduction of firing rate in hypothalamic unit discharge was observed almost as frequently as augmentation. Moreover, the paradigms of stimuli patterns associated with increased firing rates were essentially mirrored by an equivalent incidence of reduced neuronal firing, both in degree and duration of change. These induced patterns of changed firing, considered in conjunction with the preponderance of seemingly uninfluenced and spontaneously active units in the same nuclear domains, are in good general agreement with similar studies of a variety of cortical and subcortical neuronal populations (Evarts 1960; Huttenlocher 1961; Jasper et al. 1958; Jung 1958; Adey and Walter 1963). A major finding in this study was that the majority of units that responded to bladder distention also responded to sciatic stimulation, or the firing rate during bladder distention was modified by concomitant sciatic stimulation. Although there was no way of telling if such unitary responses were related to descending or ascending pathways, it was evident that responses in the hypothalamus to visceral stimulation could be accentuated or attenuated by somatic influences. The significance of such interactions with respect to psychosomatic mechanisms is selfevident. Our failure to observe many unitary responses to visual and auditory stimuli is in contrast to the results of others and illustrates a major limitation of the micro-electrode technique as applied to the hypothalamus. Evoked responses to click and sciatic stimuli have been recorded from the same posterior hypothalamic region (Starzl et al. 1951b) within which we found many units responsive to visceral and somatic, but not auditory stimuli. Recently, evoked potentials to single light flashes and "photic driving" up to 20 flashes/ sec were recorded in midline hypothalamic structures (Massopust and Daigle 1961) in which we recorded many units unresponsive to visual stimuli. However, our experience and that of others (Cross and Green 1959) is that hypothalamic units can fire as slowly as 1/10 sec or not be tonically active. The former are overlooked if they happen to be silent as the micro-electrode passes by. The Electroenceph. clin. Neurophysiol., 1964, 16:237 247
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latter are often provoked into aberrant activity when grazed b~ the micro-electrode. A unit analysis of a.liypothalamic region is then limited to those units firing tonically at a rate of about 1/sec or g~eater. Hence the failure to observe a unitary response to a given Stimulus does not imply that such units and. such responses are non-existent. SUMMARY
1. In sixteen acute experiments the firing rates o f single units within hypothalamic and ventral thalamic structures were analyzed during individual and paired visceral and somatic stimulation. Visceral stimulationinvotveddistention oftheurinary bladder. Somatic stimulation consisted of electrical sti.mulation of the sciatic nerve, adjusted to minimize A-delta and C fiber volleys. Responses to auditory and visual stimuli were also noted. 2. Thirty per cent of the units responded to bladder distention and 35 per cent to sciatic stimulation. During these stimuli, a reduction in firing rate was encountered nearly as frequently as an augmentation in rate. lpsilateral sciatic stimulation exerted as strong an effect as contralateral stimulation on units within both ventral thalamic nuclei and the hypothalamus. 3. Anterior and ventral posterior hypothalamic units were not as responsive to visceral and somatic stimuli as were dorsal posterior hypothalamic and ventral thalamic units. The pattern o f response o f the majority o f ventral thalamic units was similar to that of the hypothalamic units. Few units, dispersed widely through the hypothatamus, were responsive to visual and auditory stimuli. 4. Over 80 per cent of the units that responded to bladder distention also responded to sciatic stimulation or their firing rate during bladder distention was modified by simultaneous sciatic stimulation. The :authors wish to express appreciation to Mr. J. Cummings and Mrs. C. Penrod for their technical assistance, to Mrs. E. Baum and Mr. T. Dodge for preparation of the figures, and to Mrs. I. Austin for preparation of the manuscript. REFERENCES ADEY, VV. R. ',,, cent stt.dic ~)f the rhinencephalon in rela'don to t~:mr~ora! lobe epilepsy and behavioural dis:rders. 1~: ~'v. Neurobiol., 1959, 1: 1-46.
ADEY, W. R. and DUNLOP, C. W. Amygdaloid and peripheral influences on caudate and pallidal units in the cat with investigation of the effects of chlorpromazine. Exp. Neurol., 1960, 2: 348-363. ADEY, W. R. and LINDSLEY~D. F. On the role of sub-thalamic areas in the maintenance of brainstem reticular excitability Exp. Neurol., 1959, 1: 407-426. "AoEY, W. R. and WALTER, D. O. Application of phase detection and averaging technics in computer analysis of EEG records in the cat. Exp. Neurol., 1963, 7: 186-209. AMASSIAN, V. E. Fiber groups and spinal pathways of cortically represented visceral afferents. J. Neurophysiol., 1951, 14: 445-460. BIRZlS, L. and HEMINGWAV, A. Efferent brain discharge during shivering. J. Neurophysiol., 1957, 20: 91-99. BOOIAN, D. Studies on the diencephalon of Virginia opossum. J. comp. Neurol., 1946, 72:207 298. CROSS, B. A. and GREEN, J. D. Activity of single neurones in the hypothalamus: effects of osmotic and other stimuli. J. Physiol. (LoAd.), 1959, 148: 554-569. DELL, P, et OLSON, R. Projections thalamiques, corticales et c6r6belleuses des aff6rences visc6rales vagales. C. R. Soc. Biol. (Paris), 1951, 145: 1084-1088. DUNLOP, C. W. Viscero-sensory and somato-sensory representation in the rhinencephalon. Electroenceph. clin. Neurophysiol., 1958, 10: 297-304. EVARTS, E. V. Effects of sleep and waking on spontaneous and evoked discharge of single units in visual cortex. Fed. Proc., 1960, 19: 828-837. FELDMAN, S. Neurophysiological mechanisms modifying afferent hypothalamo-hippocampal conduction. Exp. Neurol., 1962, 5 : 269-291. FELDMAN, S., VAN DER HEIDE, C. S. and PORTER, R. W. Evoked potentials in the hypothalamus. Amer. J. Physiol., 1959, 196:1163-1167. GLOOR, P. Telencephalic influences on the hypothalamus. In W. S. FIEtDS (Editor), Hypothalamic-hypophyseal interrelationships. C. C. Thomas, Springfield, lll., 1956:74-113. GRANIT, R. and KAADA, B. R. Influence of stimulation of central nervous structures on muscle spindles in the cat. Acta physiol, scand., 1952, 27: 130-160. HEss, W. R. The functional organization of the diencephaIon. Grune and Stratton, New York, 1957, 180 p. HUTTENLOCHER, P. R. Evoked and spontaneous activity in single units of medial brainstem during natural sleep and waking. J. Neurophysicl., 1961, 24: 451-468. IGGO, A. Tension receptors in the stomach and the urinary bladder. J. Physiol. (Lomt.), 1955, 128: 593-607. INGRAM, W. R. Central autonomic mechanisms. In J. FIELD, H. W. MAGOUN and V. E. HALL (Editors), Handbook of physiology, Sect. 1, Vol. II. Amer. Physiol. Soc., Washington, 1960: 951-978. JASPER, H. H. and AJMONE MARSAN. C. ,4 stereotaxic atlas of the diencephalon of the cat. National Research Cou ncil of Canada, Ottawa, 1954. JASPER, H., RICCl, G. F. and DOANE, B. Patterns of cortical neuronal discharge duringconditioned responses. In G. E. W. WOLS'rENHOLME and C. M. O'CONNOR (Editors), Neurological basis ofbehaviour. Little, Brown and Co., Boston, 1958: 277-290.
Electroenceph. olin. Neurophysiol., 1964, 16:237-247
HYPOTHALAMIC UNITS. I. JUNG,R. Excitation, inhibition and coordination of cortical neurons. Exp. Celt 2es., 1958, 5: 262-271. KRUGER, L. and ALBE-FESSARD, D. Distribution of responses to somatic afferent stimuli in the diencephalon of the cat under chloralose anesthesia. Exp. Neurol., 1960, 2: 442-467. LINDSLEV, D F. and ADEV, W. R. Availability of peripheral input to the midbrain reticular formation. Exp. Neurol., 1961, 4: 358-376. MACHNE, X. and SEGUrqDO,J. P. Unitary responses to afferent volleys in amygdaloid complex. J. Neurophysiol., 1956, 19: 232-240. MASSOPUST, L. C. and DAIGLE, H. J. Hypothalamic and anteroventral mesencephalic photic responses in the cat. Exp. NeuroL, 1961, 3: 476-486. NAKAVAMA, T. and EISENMAN, J. S. Recording activity in the anterior hypothalamus during periods of local heating. Fed. Proc., 1961, 20: 334. NAUTA, W. J. H. Hippocampal projections and related neural pathways to the midbrain in the cat. Brain, 1958, 81: 319-340. NAUTA, W. J. H. and KUYPERS, G. J. M. Some ascending pathways in the brainstem reticular formation. In H. H. JASPER,L. D. PROCTOR,R. S. KNIGHTON,W. C. NOSHAY and R. T. COSTELLO (Editors), Reticular formation of the brain. Little, Brown and Co., Boston, 1958: 3-3(/. PORTER, R. W. and BORS, E. Modulation of brainstem electrical activity by visceral (urinary bladder) distent i o n Electroenceph. clin. Neurophysiol., 1962, 14: 527534. RANSON, S. W. and MAGOUN, H. W. The hypothalamus. Ergebn. Physiol., 1939, 41: 56-163. RUCH T. C. Central control of the bladder, in: J. FIELD,
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H.W. MAGOUN and V. E. HALL (Editors), Handbook of physiology, Sect. 1, Iiol. H. Amer. Physiol. Soc., Washington, 1960:1207-1223. SAWYER, C. H. Activation and blockade of the release of pituitary gonadotropin as influenced by the reticular formation. In H. H. JASPER, L. D. PROCTOR, R. S. KNIGHTON, W. C. NOSHAY and R. T. COSTELLO (Editors), Reticular formation of the brain Little, Brown and Co., Boston, 1958: 223-230. SAVERS, G. Factors inhibiting the level of ACTH in the blood. In G. E. W. WOLSTENnOLMEand C. M. O'CoNNOR(Editors), Ciba Foundation Colloquia on Endocrinology, Little, Brown and Co.,Boston, 1957, 9: 138-146. STARZL, T. E., TAYLOR, C. W. and MAGOUN, H. W. Ascending conduction in reticular activating system, with special reference to the diencephalon. J. NeurophysioL, 1951a, 14: 461-477. STARZL, T. E., TAYLOR, C. W. ~ MAGOUN, H. W. Collateral afferent excitation of reticular formation of brainstem. J. Neurophysiol., 1951b,.14: 479-496. STUART, D. G., EEDRED, E., HEMINGW~Y,.A. and KgWAmura, Y. The neural regulation of the rhythm of shivering. In C. M. HERZFELD(Editor), Temperature-its measurement and control in science and industry. Vol. 111, Part. 3. Reinhold, Washington, 1963: 545557. STUART, D. G., KAWAMURA, Y., HI~MINGWAY, A. and PRICE, W. M. Activation and suppression of shivering during septal and hypothalamic stimulation. Exp. Neurol., 1961, 4: 485-506. WENDT,R. Amygdaloid and peripheral influences upon the activity of hypotha[amic neurones in the cat. Unpublished Ph.D. dissertation, U.C.L.A., Los Angeles, 1960.
Reference: STUART,D. G., PORTER,R W., ADEY,W. R. and KAMIKAWA,Y. Hypothalamic unit activity. I. Visceral and somatic influences. Electroenceph. clin. Neurophysiol., 1964, 16: 237-247.
237
HYPOTHALAMIC UNIT ACTIVITY. I. V I S C E R A L A N D S O M A T I C I N F L U E N C E S I D. G. STUART,PH.D ~, R. W. PORTER,M.D., PH.D., W. R. ADEY, M.D. AND Y. KAMIKAWA, M.D. a Departments of Anatomy and Physiology, University of California, Los Angeles, Calif. and Veterans Administration Hospital, Long Beach, Calif. (U.S.A.) (Received for publication: December 18, 1962) (Resubmitted: March 11, 1963)
INTRODUCTION
Clinical and experimental findings have illustrated that a major role of the hypothalamus involves integration of somatic and visceral mechanisms in a variety of patterns (Hess 1957; lngram 1960; Ranson and Magoun 1939). Experimental work in this part of the brain has mainly been concerned with the effects of lesions and electrical stimulation on its effector activity. Less is known of the effects of ascending influences on such activity. Recent attempts to delineate the afferent input to the hypothalamus have demonstrated long latency potentials in response to sciatic nerve stimulation in the posterior region similar to those in the midbrain reticular formation,and short latency potentials indicative of input from leminiscal collaterals (Feldman et al. 1959). Additionally, pronounced changes in the synchrony of posterior hypothalamic, ventral thalamic and rostral midbrain electrical waves were shown to accompany alterations in urinary bladder pressure (Porter and Bors 1962). Such results illustrate that interactions of somatic and visceral modalities on hypothalamic structures are quite feasible, utilizing both fast and slow afferent pathways. Similarities between posterior hypothalamic and rostral midbrain responses have a sound anatomical basis (Nauta 1 Aided in part by grants B-1883 and A F 61-81 from the United States Public Health Service and the U.S. Air Force Office of Scientific Research, respectively. 2 Medical Research Fellow, Bank of America-Giannini Foundation. a Appointment supported by the International Cooperative Administration under the Visiting Research Scientist Program administered by the National Academy of Sciences of the U.S.A.
and Kuypers 1958) and are becoming increasingly evident in the physiological literature (Granit and Kaada 1952; Sayers 1957; Sawyer 1958). Less is known of the anatomical and physiological relationships of ventral thalamic and dorsal hypothalamic structures although both lesion and stimulation studies have shown the difficulty of separating these structures on functional grounds (Hess 1957; Starzl et al. 1951a and b; Stuart et al. 1961). The relationships between somatic and visceral inputs on the same hypothalamic structures are obscure. Additionally, there is a paucity of information concerning the firing pattern of single hypothalamic neurons during any but osmotic (Cross and Green 1959), thermal (Birzis and Hemingway 1957; Nakayama and Eisenman 1961) and combined amygdaloid-sciatic (Wendt 1961) stimuli. For these reasons, this study has concerned itself with analysis of the firing patterns of single units within hypothalamic and ventral thalamic regions during increases in urinary bladder pressure both independent of, and combined with, sciatic nerve stimulation. MATERIALS AND METHODS
Experimental results are based on sixteen acutely prepared cats. Surgical procedures were completed before discontinuing ether anesthesia, which was then replaced by immobilization with gallamine triethiodide (Fiaxedil). All wound margins and pressure points were infiltrated with 1 per cent procaine hydrochloride, and regional blocks were performed on the supraorbital and maxillary divisions of the trigeminal nerve at their points of emergence from the skull. Additional infiltration was carried out in the anterior and posterior wails of the external auditory meaElectroenceph. clin. Neurophysiol., 1964, 16:237-247
238
D.G. STUARTet al.
tus. This regional anesthesia was repeated every 2 h. Artificial respiration was controlled by a Harvard p u m p at a rate of 20 to 25 breaths per min, and the stroke volume was adjusted to permit a 4 per cent COz level in each expired breath. This parameter was monitored by passing a small sample of the expired air through an infrared CO2 analyzer, Rectal temperature was maintained near 38°C with an electronic temperature controller. The surgical procedures included tracheal intubation, median vein and urinary bladder cannulation (described previously, Porter and Bors 1962), exposure of the left sciatic nerve in the popliteal fossa and craniotomy. A bipolar Sherrington electrode was applied to the exposed sciatic nerve, which was then covered with mineral oil. Craniotomy included sufficient bone removal to expose the dorsal surface of the brain on both sides between Horsley-Clarke coordinates Fr 6 to 14 and L 0 to 5. The dura was removed from this area and the surface of the brain covered with a 5 to 10 per cent solution of agar in 0.9 per cent saline solution. This minimized brain pulsation while permitting micro-electrode insertion. Three hours elapsed after discontinuing ether anesthesia before commencement of recording. Preliminary experiments convinced us that use of anesthetic agents such as pentobarbital sodium or alpha chloralose, at dose levels only a fraction of that necessary to induce anesthesia (e.g. 5 mg/ kg Nembutal i.v.) drastically reduced the number and firing rate of spontaneously active hypothalamic units. Moreover, such units were then unresponsive to the stimulation procedures. Visceral stimulation consisted of distention of the previously emptied urinary bladder with 25 to 35 ml of 0.9 per cent saline solution at 38°C. This volume was injected manually from a syringe such that filling, "full" and emptying phases each took 6 sec. Freely moving, unanesthetized cats exhibited no evidence of pain or discomfort when identical distention of the bladder was induced at the same rate via an indwelling Foley catheter. The parameters of sciatic nerve stimulation were adjusted to minimize A-delta and C fiber activation, i.e., 10/sec square wave pulses, 5 V intensity, 0.5 msec duration delivered from a Grass stimulator (S4-A) and isolation transform-
er. Previous studies in our laboratory have indicated that such a stimulus applied through chronically implanted electrodes to the sciatic nerve of freely moving cats is not associated with evidence of a painful experience (Lindsley and Adey 1961). Stainless steel (Adey and Dunlop 1960) or carbon steel micro-electrodes with tips less than 1 # were used. The latter were prepared by electropolishing dental broaches (S.S. White, Smooth Broach No. 42, fine) in an acid bath with 60 cycle A.C. and an E.M.F. of 1-3 V. The acid bath consisted of 34 ml of sulphuric acid (95 to 98 per cent concentrated), 42 ml of orthophosphoric acid (85 per cent concentrated) and 48 ml of distilled water. The electrodes, insulated with two to four coats of an epoxy-resin (EpiREZ 285-19, Jones-Dabney Co., Los Angeles) 1 were positioned stereotactically in various hypothalamic regions. Recording of unit activity usually began in ventral thalamic structures, 10 to I1 m m above the interaural line. Unipolar extracellular recording was referred to an indifferent electrode in the neck muscles. Potentials were led into the cathode-follower probe of a Grass model P5 R.C. pre-amplifier, then displayed on a Tektronix model 502 oscilloscope and recorded with a Grass Kymograph camera. Our observations on the positive-negative spike configuration, 1.0 to 2.0 msec duration and 0.5 to 10 mV amplitude of hypothalamic units were similar to previous reports (Cross and Green 1959; Wendt 1961). Usually the responses of single neurons to the stimulation procedures were observed prior to photography. Thus, positive results were based on at least two trials. With dubious responses, the same stimulus was often applied several times. In this way, chance correlations that could have arisen with single trials were minimized. During paired stimulation the sciatic stimulus started 6 to 12 sec before bladder distention and continued 3 to 12 sec after the bladder was emptied. Artifact reduction was successfully achieved by use of a Wagner ground system. Variations in stimulus artifact level, in the text figures, were due to manipulation of this ground system. At the conclusion of each experiment the animal was sacrificed and the brain perfused in situ through a cannulated carotid artery with a 5 per cent solu1 We are indebted to Dr. H. Batsel for this technique of micro-electrode construction.
Electroenceph. clin. Neurophysiol., 1964, 16:237-247
HYPOTHALAMIC UNITS. I.
tion of potassium ferrocyanide in 10 per cent formalin. The brain was then removed, fixed in formalin and later sectioned every 80/z. The sections were stained with neutral red for identification of Prussian blue spots that had been electrolyticaUy deposited at the tip site when each electrode had been driven to its deepest position. The loci of recorded units along the same track were calculated by reference to these spots. In the figure legends of Fig. 2 to 5, the numbers in brackets give each unit's stereotactic coordinates according to the atlas of Jasper and Ajmone Marsan. RESULTS
I. Location o f units responsive to bladder distention and sciatic stimulation The firing rates of 291 ventral diencephalic units were analyzed during bladder distention. The response of 214 of these neurons to sciatic TOTAL B
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Fig. 1 Schemata of number and location of single neurons whose firing rates were analyzed during bladder distention (B) and sciatic stimulation (S). Unshaded sector: no. of unresponsive units; stippled sector: units with augmented firing rates during stimulus; hatched sector: units with reduced firing rates during stimulus. Horizontal and vertical co-ordinates on the midsagittal schema indicate mm anterior and dorsal to the interaural line. All units within 3.5 mm of the midline. Abbreviations: AC: anterior commissure; CC: corpus callosum; OC: optic chiasm; M: mamillary body; MI: massa intermedia; PC: posterior commissure; Spt: septum.
239
stimulation was also observed. Fig. 1 indicates that the visceral stimulus exerted as powerful an effect on these units as the somatic stimulus. Thirty per cent of all units analyzed were responsive to bladder distention, the firing rate of 19 per cent being augmented and 11 per cent reduced, abbreviated hereafter to A and R respectively. Thirty-five per cent of the units were responsive to sciatic stimulation (27 per cent A, 8 per cent R). Despite the small sample, the results indicated that both stimuli more commonly influenced units within ventral thalamic and dorsal posterior hypothalamic regions than units within the anterior or ventral posterior hypothalamus. Of the 77 dorsal posterior hypothalamic units observed during bladder distention, 37 were within 1.5 mm of the midline, the remaining 40 being 1.6 to 3.5 mm from the midline. Of the former, fourteen responded to bladder distention (10A, 2R) while in the latter group twelve responded (8A, 4R) to the stimulus. Thus, no distinction could be made between medial and lateral neurons for this stimulus nor, after the same analysis, for the somatic stimulus. Similarly, no distinction could be drawn between the behavior of the more rostral neurons (Fr 11 to 10) and the more caudal ones (Fr 10 to 8). One hundred and sixteen ventral thalamic units were analyzed, 25 from the posteromedial aspect of the ventrolateral nucleus (VL) and 38 from the ventromedial nucleus (VM). Six VL units responded to bladder distention (1A, 5R) while 18 VM units responded (15A, 3R) to the same stimulus. Influenced by sciatic stimulation were fourteen VL units (9A, 5R) and eighteen VM units (15A, 3R). The remaining 53 responsive thalamic units were distributed in a variety of medial thalamic structures. I[. Nature of visceral stimulation Sensory volleys during bladder distention could arise not only from proprioceptive afferents in ves[cal musculature but also from other visceral receptors due to the increase in intraabdominal pressure associated with bladder distention. Additionally, somatic activity could not be excluded since bladder distention mildly stretches the anterior abdominal wall. Our results suggested that whereas all three were possible, the stimulus was predominantly visElectroenceph. clin. Neurophysiol., 1964, 16:237-247
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D.G. STUARTet al.
ceral and furthermore that vesical proprioceptive afferents alone were capable of modulating the firing patterns of these units. In only two instances did units responsive to bladder stretch also respond to gentle stroking of the anterior abdominal wall. However, this stimulus was not used throughout all experiments. In three experiments a rubber balloon of approximate bladder size was inserted intra-abdominally in juxtaposition to the bladder. The firing rates of 52 units ,
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Electroenceph. clin. Neurophysiol., 1964, 16:237-247
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HYPOTHALAMIC UNITS. I.
ing to the control level in the "full" phase. Some such units were evoked into activity f r o m a silent background (Unit I, Fig. 2), but more often spontaneous activity was present (Unit II, Fig. 2). In the second major group (22 units), the increase in firing rate did not reach a maximum until the end of the "full" phase. Rarely was there a steady rise in firing rate but rather, during the filling and early"full" phases, one to three bursts of increased firing rate occurred that nearly reached the m a x i m u m rate attained (Unit III, Fig. 2). The
in a 38°C solution of 0.9 per cent saline so that its distention could not alter intra-abdominal pressure or the tension on the anterior abdominal wall. Fourteen units were analyzed, six of which responded to bladder distention (2A, 4R). II1. Patterns o f response to bladder distention The firing rates of 55 neurons were augmented by bladder distention. Two predominant patterns emerged. In the first (23 units) the firing rate rose briefly and transiently in the filling phase, return-
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firing rate of each unit in this group returned to the control level within 20 sec of the bladder being emptied. In the case of four neurons, the peak or near peak increase in frequency was maintained for as long as 2 min after bladder distention. There were two distinct peaks in the firing rate of six other units, one during initial filling and the other during initial emptying. Despite this second peak the firing rate quickly regained the control level after the bladder was emptied. As with augmented responses, patterns of reduced responses, demonstrated by 33 neurons during bladder distention, fell into two major categories. Most typical (19 units) were gradually reduced firing rates reaching a peak suppression in the "full" phase (Unit I, Fig. 3). Less typical (10 units) were brief reductions in firing rates during the filling phase, with control rates returning in the "full" phase. For both groups, a suppressed firing rate was often followed by a 1 to 5 sec burst at a higher frequency than the control rate. Of particular interest was the behavior of the remaining five neurons wherein a reduction in firing rate occurred in the early emptying as well as the early filling phase (Unit II, Fig. 3). In none on these units did the reduction in firing rate during bladder distention persist more than 20 sec after the bladder was emptied. IV. Bilateral effects o f sciatic stimulation Of the neurons analyzed during sciatic stimulation, 112 were ipsilateral and 102 were contralateral to the stimulated nerve. In the former group 37 units were influenced by stimulation (30A, 7R) and in the latter group 37 units also responded (28A, 9R). In one experiment eleven units were studied during left and right side sciatic stimulation. Seven units were responsive; four being equally affected by both stimuli (2A, 2R). In another case the response to contralateral stimulation was greater while in the remaining two cases the ipsilateral stimulus exerted the stronger effect. Ventral thalamic neurons were similar to hypothalamic neurons in their response to ipsilateral sciatic stimulation. For example, of the VM neurons observed during sciatic stimulation, the stimulus was ipsilateral in 25 cases and contralateral in thirteen. Twelve of the former were influenced(10A, 2R) andsix of the latter (5A, 1R). For VL units, fourteen were observedduring
ipsilateral stimulation and eleven during contralateral stimulation. Eleven of the former responded (8A, 3R) and three of the latter (1A, 2R). V. Responses to combined visceral and somatic stimulation Of the neurons whose firing rates were analyzed during individual and combined bladder distention and sciatic stimulation, 109 were unresponsive to all three stimulation procedures. Twelve units were influenced by sciatic stimulation but not by the visceral or combined stimulus. Fourteen others responded unidirectionaUy to both individual stimuli (8A, 6R) but such augmentation or reduction in firing rate was not exaggerated by combined stimulation. The remaining 38 units displayed a variety of responses to combined stimuli that could be categorized as follows: (a) Thirteen units responded in opposite direction to individual stimuli. During paired stimulation either the rate remained at the control level or it followed the influence of one or the other stimulus in reduced amount. (b) Twelve other units that responded to bladder distention in a characteristic fashion illustrated no such response during concomitant sciatic stimulation even though firing rates were uninfluenced by individual sciatic stimulation (Fig. 4). (c) The response of the remaining thirteen units to bladder distention was augmented by simultaneous sciatic stimulation. Five of these responded in the early phase of bladder emptying during paired stimulation but not during individual visceral stimulation. The other eight units responded in the "filling" or "full" phases of the bladder distention stimulus. Three were mildly influenced by visceral but not somatic stimulation, then markedly accelerated by combined stimulation (Unit I, Fig. 5). With three others the individual stimuli had no effect on the firing rate which was then augmented by the paired stimuli (Unit 11, Fig. 5). In the remaining two cases, the response to paired stimuli was greater than the augmented responses to the individual stimuli.
VI. Visual and auditory responses At the conclusion of visceral and somatic stimulation procedures, the responses of many units to brief and prolonged flashes of light, Electroenceph. clin. Neurophysiol., 1964, 16:237-247
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Fig. 4 Combined somato-visceral stimulation producing greater changes in unit firing than either stimulus alone. Bladder distention induced a mild increase in the firing of Unit I (Fr 9.0, L 1.0, H minus 4.3) as indicated by continuous line. Paired stimuli, indicated by a broken line, greatly augmented this effect. Sciatic stimulation began 6 sec before bladder filling and ended 6 sec after emptying. It was without effect except during bladder distention. Unit II (Fr 8.0, L 3.5, H minus 4.5) was uninfluenced by either stimulus alone, but the firing rate increased during paired stimuli. Here, sciatic stimulation extended before and after the epoch of bladder distention for 10 sec. Paired unit records (marked A, B and C) relate these unit discharges to specific epochs shown in the graphs before, during and after bladder distention. The upper trace of each pair shows the effects of bladder distention alone. The lower trace (marked S) shows the effects of bladder distention during concomitant sciatic stimulation. Ordinates: spikes/2 sec; Abscissae: time in seconds. Vertical calibrations: 1 mV. clicks a n d extremely loud noises were noted. Only nine of 178 n e u r o n s were influenced by auditory s t i m u l a t i o n (7A, 2R) a n d three of 171 by visual s t i m u l a t i o n (1A, 2R). As in other studies (Cross a n d G r e e n 1959; W e n d t 1961), responsive units were widely dispersed t h r o u g h o u t the hyp o t h a l a m u s . We f o u n d n o n e in ventral thalamic
nuclei. Five of the responsive units were also affected by visceral stimuli b u t no attempts were made to pair visceral with visual or auditory stimuli. DISCUSSION There were essential similarities in the patterns Electroenceph. clin, Neurophysiol., 1964, 16:237-247
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D . G . STUART et al.
Beginiilling
Bladder Empty
__1
A.
II Begin Emptying
B
,
"Full" Bladder
_.
_
il
.
.
.
BIGdder Empty
.
i
I
Begin Sciatic Stim.
Begin Filling D.
"Full" Bladder -
Bladder Empty
Begin E~nptying !
End Sciatic Stim.
E.
2 sec Fig. 5 An example of sciatic stimulation suppressing the response of a unit (Fr 8,0, L 0.5, H 0.5) to bladder distention. Traces A and B form a continuous record an show an augmentation of firing rate during bladder distention. The firing rate remained relatively constant during sciatic stimulation alone (C) and was not augmented during (D) or after (E) bladder distention. Note the appearance of a second smaller unit whose irregular discharge was not affected by the stimulation procedures. Vertical calibration: 1 mV.
of responsiveness of neurons in the posterior hypothalamus and ventral thalamus to the three stimulation procedures used here. This is in good general agreement with previous studies showing similar alterations in the synchrony of electrical waves recorded from both regions during increases in urinary bladder pressure (Porter and Bors 1962). Similarities in the form and latency of potentials evoked in both regions by single shock stimuli to the contralateral sciatic nerve have also been observed (Starzl et al. 1951b; Feldman et al. 1959; Feldman 1962). In the present study VL, VM and hypothalamic units responded to both ipsilateral and contralateral sciatic stimulation as has recently been observed for VL units using other somatic stimuli (Kruger and Albe-Fessard 1960). The large number of responsive units at the j unction of the ventral thalamus and dorsal posterior hypothalamus is of interest for three reasons. First, the concepts of Hess (1957) are exempli-
fled, in that neurons in this "tween-brain" region exhibited inseparable relationships to somatomotor and visceral functions and that the responsive zone lacked clearly defined boundaries related to classical anatomical schemes. Second, the region (Fr 10 to 8, L 0 to 3, H 0 to minus 3, according to the atlas of Jasper and Ajmone Marsan) exerts many central and peripheral effects. It has been termed the ergotropic zone of the diencephaLon in that its stimulation evokes increased respiratory activity, pupillary dilation, increased motor excitability, and sometimes flight (Ranson and Magoun 1939; Hess 1957). It has been implicated in temperature regulation (Stuart et al. 1961), activation of gamma motoneurons (Granit and Kaada 1952; Stuart et al. 1962), cerebral cortex (Starzl et al. 1951a and b)andthe midbrainreticular formation possibly determining the latter's responsiveness to peripheral stimuli (Adey and Lindsley 1959; Lindsley and. Adey 1961). Since bladder distention exerts such a powerful effect Electroeneeph. clin. Neurophysiol., 1964, 16:237-247
HYPOTHALAMIC UNITS. I.
on units within this region, it might be expected that all the above mentioned parameters, concerned with gross alterations in body homeostasis, are subject to modulation by this and other forms of visceral activity. Third, the region contains two-way connections between the midbrain and the basal ganglia and rhinencephalon (Adey 1959; Bodian 1946; Nauta 1958). Numerous references indicate that rhinencephalic influences on somato-viscerat mechanisms are secondary to a primary hypothalamic control (cf. Gloor 1956). However, information concerning afferent visceral inputs to the rhinencephalon is sparse (Dell and Olson 1951; Dunlop 1958; Feldman 1962; Machne and Segundo 1956). To insure stable recording in the present study, units were recorded wherein the initially negative wave showed a positive-negative configuration. Closer approach of the micro-electrode to the cell membrane, with positive dominance in spike configuration, often resulted in a ruptured cell membrane. A limited number of extremely brief and purely negative spikes were recorded. In some instances they responded to the stimulation procedures. Their recorded characteristics did not preclude axonal structures. In view of the potential origin of such spikes in cells lying outside the hypothalamus, including the rhinencephalon, data drawn from these units were not systematically collected nor have they formed the basis of conclusions drawn from this study. It is difficult to evaluate the latency between bladder distention and acceleration of responsive hypothalamic units. Intraluminal bladder pressure was not recorded. Even this parameter does not indicate localized changes in vesical motility that influence the firing rate of the "in series" stretch receptors (Iggo 1955). Additionally, so little is known of the pathway to the hypothalamus (Amassian 1952; Ruch 1960) that any discussion of latency is purely speculative. Most frequently, the accelerated responses began 1-3 sec after beginning bladder distention although shorter latencies were observed in a few cases. Since spike amplitudes and configurations were rarely altered during distention, such accelerated responses could hardly be due to blood pressure changes mechanically displacing the neuron (Adey and Dunlop 1960). Many units that reached a peak firing rate late in the "full" phase of the
245
visceral stimulus accelerated one to three times prior to the peak rate. This suggested that the stimulus exerted more than one central effect. Reduction of firing rate in hypothalamic unit discharge was observed almost as frequently as augmentation. Moreover, the paradigms of stimuli patterns associated with increased firing rates were essentially mirrored by an equivalent incidence of reduced neuronal firing, both in degree and duration of change. These induced patterns of changed firing, considered in conjunction with the preponderance of seemingly uninfluenced and spontaneously active units in the same nuclear domains, are in good general agreement with similar studies of a variety of cortical and subcortical neuronal populations (Evarts 1960; Huttenlocher 1961; Jasper et al. 1958; Jung 1958; Adey and Walter 1963). A major finding in this study was that the majority of units that responded to bladder distention also responded to sciatic stimulation, or the firing rate during bladder distention was modified by concomitant sciatic stimulation. Although there was no way of telling if such unitary responses were related to descending or ascending pathways, it was evident that responses in the hypothalamus to visceral stimulation could be accentuated or attenuated by somatic influences. The significance of such interactions with respect to psychosomatic mechanisms is selfevident. Our failure to observe many unitary responses to visual and auditory stimuli is in contrast to the results of others and illustrates a major limitation of the micro-electrode technique as applied to the hypothalamus. Evoked responses to click and sciatic stimuli have been recorded from the same posterior hypothalamic region (Starzl et al. 1951b) within which we found many units responsive to visceral and somatic, but not auditory stimuli. Recently, evoked potentials to single light flashes and "photic driving" up to 20 flashes/ sec were recorded in midline hypothalamic structures (Massopust and Daigle 1961) in which we recorded many units unresponsive to visual stimuli. However, our experience and that of others (Cross and Green 1959) is that hypothalamic units can fire as slowly as 1/10 sec or not be tonically active. The former are overlooked if they happen to be silent as the micro-electrode passes by. The Electroenceph. clin. Neurophysiol., 1964, 16:237 247
D . G . STUART et al.
246
latter are often provoked into aberrant activity when grazed b~ the micro-electrode. A unit analysis of a.liypothalamic region is then limited to those units firing tonically at a rate of about 1/sec or g~eater. Hence the failure to observe a unitary response to a given Stimulus does not imply that such units and. such responses are non-existent. SUMMARY
1. In sixteen acute experiments the firing rates o f single units within hypothalamic and ventral thalamic structures were analyzed during individual and paired visceral and somatic stimulation. Visceral stimulationinvotveddistention oftheurinary bladder. Somatic stimulation consisted of electrical sti.mulation of the sciatic nerve, adjusted to minimize A-delta and C fiber volleys. Responses to auditory and visual stimuli were also noted. 2. Thirty per cent of the units responded to bladder distention and 35 per cent to sciatic stimulation. During these stimuli, a reduction in firing rate was encountered nearly as frequently as an augmentation in rate. lpsilateral sciatic stimulation exerted as strong an effect as contralateral stimulation on units within both ventral thalamic nuclei and the hypothalamus. 3. Anterior and ventral posterior hypothalamic units were not as responsive to visceral and somatic stimuli as were dorsal posterior hypothalamic and ventral thalamic units. The pattern o f response o f the majority o f ventral thalamic units was similar to that of the hypothalamic units. Few units, dispersed widely through the hypothatamus, were responsive to visual and auditory stimuli. 4. Over 80 per cent of the units that responded to bladder distention also responded to sciatic stimulation or their firing rate during bladder distention was modified by simultaneous sciatic stimulation. The :authors wish to express appreciation to Mr. J. Cummings and Mrs. C. Penrod for their technical assistance, to Mrs. E. Baum and Mr. T. Dodge for preparation of the figures, and to Mrs. I. Austin for preparation of the manuscript. REFERENCES ADEY, VV. R. ',,, cent stt.dic ~)f the rhinencephalon in rela'don to t~:mr~ora! lobe epilepsy and behavioural dis:rders. 1~: ~'v. Neurobiol., 1959, 1: 1-46.
ADEY, W. R. and DUNLOP, C. W. Amygdaloid and peripheral influences on caudate and pallidal units in the cat with investigation of the effects of chlorpromazine. Exp. Neurol., 1960, 2: 348-363. ADEY, W. R. and LINDSLEY~D. F. On the role of sub-thalamic areas in the maintenance of brainstem reticular excitability Exp. Neurol., 1959, 1: 407-426. "AoEY, W. R. and WALTER, D. O. Application of phase detection and averaging technics in computer analysis of EEG records in the cat. Exp. Neurol., 1963, 7: 186-209. AMASSIAN, V. E. Fiber groups and spinal pathways of cortically represented visceral afferents. J. Neurophysiol., 1951, 14: 445-460. BIRZlS, L. and HEMINGWAV, A. Efferent brain discharge during shivering. J. Neurophysiol., 1957, 20: 91-99. BOOIAN, D. Studies on the diencephalon of Virginia opossum. J. comp. Neurol., 1946, 72:207 298. CROSS, B. A. and GREEN, J. D. Activity of single neurones in the hypothalamus: effects of osmotic and other stimuli. J. Physiol. (LoAd.), 1959, 148: 554-569. DELL, P, et OLSON, R. Projections thalamiques, corticales et c6r6belleuses des aff6rences visc6rales vagales. C. R. Soc. Biol. (Paris), 1951, 145: 1084-1088. DUNLOP, C. W. Viscero-sensory and somato-sensory representation in the rhinencephalon. Electroenceph. clin. Neurophysiol., 1958, 10: 297-304. EVARTS, E. V. Effects of sleep and waking on spontaneous and evoked discharge of single units in visual cortex. Fed. Proc., 1960, 19: 828-837. FELDMAN, S. Neurophysiological mechanisms modifying afferent hypothalamo-hippocampal conduction. Exp. Neurol., 1962, 5 : 269-291. FELDMAN, S., VAN DER HEIDE, C. S. and PORTER, R. W. Evoked potentials in the hypothalamus. Amer. J. Physiol., 1959, 196:1163-1167. GLOOR, P. Telencephalic influences on the hypothalamus. In W. S. FIEtDS (Editor), Hypothalamic-hypophyseal interrelationships. C. C. Thomas, Springfield, lll., 1956:74-113. GRANIT, R. and KAADA, B. R. Influence of stimulation of central nervous structures on muscle spindles in the cat. Acta physiol, scand., 1952, 27: 130-160. HEss, W. R. The functional organization of the diencephaIon. Grune and Stratton, New York, 1957, 180 p. HUTTENLOCHER, P. R. Evoked and spontaneous activity in single units of medial brainstem during natural sleep and waking. J. Neurophysicl., 1961, 24: 451-468. IGGO, A. Tension receptors in the stomach and the urinary bladder. J. Physiol. (Lomt.), 1955, 128: 593-607. INGRAM, W. R. Central autonomic mechanisms. In J. FIELD, H. W. MAGOUN and V. E. HALL (Editors), Handbook of physiology, Sect. 1, Vol. II. Amer. Physiol. Soc., Washington, 1960: 951-978. JASPER, H. H. and AJMONE MARSAN. C. ,4 stereotaxic atlas of the diencephalon of the cat. National Research Cou ncil of Canada, Ottawa, 1954. JASPER, H., RICCl, G. F. and DOANE, B. Patterns of cortical neuronal discharge duringconditioned responses. In G. E. W. WOLS'rENHOLME and C. M. O'CONNOR (Editors), Neurological basis ofbehaviour. Little, Brown and Co., Boston, 1958: 277-290.
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HYPOTHALAMIC UNITS. I. JUNG,R. Excitation, inhibition and coordination of cortical neurons. Exp. Celt 2es., 1958, 5: 262-271. KRUGER, L. and ALBE-FESSARD, D. Distribution of responses to somatic afferent stimuli in the diencephalon of the cat under chloralose anesthesia. Exp. Neurol., 1960, 2: 442-467. LINDSLEV, D F. and ADEV, W. R. Availability of peripheral input to the midbrain reticular formation. Exp. Neurol., 1961, 4: 358-376. MACHNE, X. and SEGUrqDO,J. P. Unitary responses to afferent volleys in amygdaloid complex. J. Neurophysiol., 1956, 19: 232-240. MASSOPUST, L. C. and DAIGLE, H. J. Hypothalamic and anteroventral mesencephalic photic responses in the cat. Exp. NeuroL, 1961, 3: 476-486. NAKAVAMA, T. and EISENMAN, J. S. Recording activity in the anterior hypothalamus during periods of local heating. Fed. Proc., 1961, 20: 334. NAUTA, W. J. H. Hippocampal projections and related neural pathways to the midbrain in the cat. Brain, 1958, 81: 319-340. NAUTA, W. J. H. and KUYPERS, G. J. M. Some ascending pathways in the brainstem reticular formation. In H. H. JASPER,L. D. PROCTOR,R. S. KNIGHTON,W. C. NOSHAY and R. T. COSTELLO (Editors), Reticular formation of the brain. Little, Brown and Co., Boston, 1958: 3-3(/. PORTER, R. W. and BORS, E. Modulation of brainstem electrical activity by visceral (urinary bladder) distent i o n Electroenceph. clin. Neurophysiol., 1962, 14: 527534. RANSON, S. W. and MAGOUN, H. W. The hypothalamus. Ergebn. Physiol., 1939, 41: 56-163. RUCH T. C. Central control of the bladder, in: J. FIELD,
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H.W. MAGOUN and V. E. HALL (Editors), Handbook of physiology, Sect. 1, Iiol. H. Amer. Physiol. Soc., Washington, 1960:1207-1223. SAWYER, C. H. Activation and blockade of the release of pituitary gonadotropin as influenced by the reticular formation. In H. H. JASPER, L. D. PROCTOR, R. S. KNIGHTON, W. C. NOSHAY and R. T. COSTELLO (Editors), Reticular formation of the brain Little, Brown and Co., Boston, 1958: 223-230. SAVERS, G. Factors inhibiting the level of ACTH in the blood. In G. E. W. WOLSTENnOLMEand C. M. O'CoNNOR(Editors), Ciba Foundation Colloquia on Endocrinology, Little, Brown and Co.,Boston, 1957, 9: 138-146. STARZL, T. E., TAYLOR, C. W. and MAGOUN, H. W. Ascending conduction in reticular activating system, with special reference to the diencephalon. J. NeurophysioL, 1951a, 14: 461-477. STARZL, T. E., TAYLOR, C. W. ~ MAGOUN, H. W. Collateral afferent excitation of reticular formation of brainstem. J. Neurophysiol., 1951b,.14: 479-496. STUART, D. G., EEDRED, E., HEMINGW~Y,.A. and KgWAmura, Y. The neural regulation of the rhythm of shivering. In C. M. HERZFELD(Editor), Temperature-its measurement and control in science and industry. Vol. 111, Part. 3. Reinhold, Washington, 1963: 545557. STUART, D. G., KAWAMURA, Y., HI~MINGWAY, A. and PRICE, W. M. Activation and suppression of shivering during septal and hypothalamic stimulation. Exp. Neurol., 1961, 4: 485-506. WENDT,R. Amygdaloid and peripheral influences upon the activity of hypotha[amic neurones in the cat. Unpublished Ph.D. dissertation, U.C.L.A., Los Angeles, 1960.
Reference: STUART,D. G., PORTER,R W., ADEY,W. R. and KAMIKAWA,Y. Hypothalamic unit activity. I. Visceral and somatic influences. Electroenceph. clin. Neurophysiol., 1964, 16: 237-247.