Diencephalic and hippocampal mechanisms of motor activity in the rat: Effects of posterior hypothalamic stimulation on behavior and hippocampal slow wave activity

BRAIN RESEARCH

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D I E N C E P H A L I C A N D H I P P O C A M P A L M E C H A N I S M S OF M O T O R ACTIVITY I N T H E RAT: EFFECTS OF P O S T E R I O R H Y P O T H A L A M I C STIMULAT I O N ON B E H A V I O R A N D H I P P O C A M P A L SLOW WAVE ACTIVITY

BRIAN H. BLAND* AND C. H. VANDERWOLF

Departments of Psychology and Physiology~ University of Western Ontario, London, Ont. (Canada) (Accepted February llth, 1972)

INTRODUCTION

A number of studies have demonstrated that locomotor activity can be initiated in animals by electrical stimulation of areas in or near the posterior hypothalamus4, 5, 10,11,1s,3z. The behavior produced is not stereotyped or 'forced' and can vary in accordance with the environmental situation. Studies using acute preparations have shown that posterior hypothalamic stimulation has a facilitatory effect on cortically induced movement and spinal reflexes23,25, 37, an effect which may be due to the increase in gamma efferent activity demonstrated by Granit and Kaada 6 during such stimulation. An important role of the basal diencephalon in the control of movement is also demonstrated by the profound depression of spontaneous movement (akinesia) which results from lesions in this area12,1v, 2°. Other investigations have shown that the posterior hypothalamus exerts a powerful activating influence on the hippocampus, with ascending impulses acting via a relay in the septal nuclei. Stimulation of the posterior hypothalamus produces rhythmical slow activity (RSA or 'theta rhythm') in the hippocampus; large lesions of the hypothalamus prevent the appearance of such activity. Other subcortical structures (midbrain reticular formation, medial thalamus) apparently also exert a control over hippocampal electrical activity, but are less essential than the posterior hypothalamus8,13,14,30. Hypothalamic~control of movement and of hippocampal electrical activity may be closely related. According to VanderwolfZl, 32 spontaneous hippocampal activity is related to the details of the movements occurring during a behavioral sequence, rather than arousal, learning, attention, motivation, etc. as had previously been thought. This report describes relations between hippocampal mass activity and behavior during stimulation of the posterior hypothalamus in conscious rats. * Present address: Institute of Neurophysiology, University of Oslo, Karl Johans Gt. 47, Oslo 1, Norway. Requests for reprints should be sent to C. H. Vanderwolf, University of Western Ontario, London, Ontario, Canada. Brahl Research, 43 (1972) 67-88

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B. H, BLAND AND C. H. VANDERWOLF

METHODS

The subjects of the experiments were 51 male hooded rats, weighing 250-350 g, supplied by the Quebec Breeding Farms. Under sodium pentobarbital (50 mg/kg) anesthesia, bipolar electrodes were imp/anted in various brain areas and fixed rigidly to the skull with steel screws and dental cement. Standard stereotaxic procedures were used to locate the position of each electrodO 5. All electrodes were made from two nichrome wires, each 0.250 mm in diameter and twisted together (Plastic Products Company, Roanoke, Va. MS-303-0.010). Stimulating electrodes were cut so the tips were the same length, the only uninsulated surface being the cross-sectional area of the tips. Recording electrodes were cut so that one tip was 0.5 mm shorter than the other. In addition, the tips were separated laterally 0.5-1.0 ram, and each tip was bared of 0.5 mm of insulation. Placement of such an electrode with one tip above the pyramidal cell layer and one below it permitted the recording of clear large amplitude activity with very little artifact. The animal was grounded via a male Winchester subminiature component soldered to a jeweller's screw fixed in the skull. Electrical activity was recorded with an ink writing polygraph or a dual beam oscilloscope and Polaroid camera. Usually both high and low frequency activity was attenuated; half amplitude points were usually set at 1 and 35 c/sec during behavioral testing. In some experiments, heart rate recordings were taken with platinum needle electrodes inserted subdermally on either side of the thorax. Electrical stimulation was carried out by means of a Grass $4 square wave stimulator and a Grass SIU5 isolation unit. Stimulus parameters, checked with an oscilloscope, were the following: voltage, 0-15 V; pulse duration, 0.1 msec; frequency, 1-100 pulses/sec; pulse train duration, 1-60 sec. Behavior was recorded concurrently with electrical activity by means of manually operated signal markers attached to the polygraph, or by a movement sensing device of the type described by Griffiths et al. 9. Animals from the chronic experiments were sacrificed and their brains perfused with normal saline and 1 0 ~ formalin. The brains were then embedded in celloidin, sectioned at 20 # m (saving a minimum of every fifth section) and stained with thionin. Prior to the main experiments, a series of acute experiments (10 rats) was conducted to localize the hypothalamic area where stimulation produces a maximal effect on hippocampal activity. Rats were anesthetized with chloral hydrate (300 rag/ kg) and placed in a stereotaxic instrument. One recording electrode was placed in the frontal neocortex and two recording electrodes were lowered into the dorsal hippocampus until the RSA produced by pinching the skin of the hind foot could be recorded. A stimulating electrode was lowered 7.0 mm from the surface of the skull into the medial hypothalamus 0.5 mm lateral to the midline and 2-3.5 mm posterior to bregma. Stimulation began at this level and was repeated in 0.5 mm steps until a depth of 9 mm had been reached. At the termination of the experiment a low intensity DC current was passed through the stimulating electrode, in some rats at points which produced hippocampal RSA and in other rats at points which failed

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to yield RSA. The brains were fixed in formalin, frozen, sectioned at 40 # m intervals, and stained with thionin.

Open field and wheel running The open field apparatus was a wooden box measuring 122 cm × 58 cm × 31 cm in height, and filled with sawdust to a depth of about 5 cm. Food pellets were scattered randomly about, along with several pieces of wood, and water bottles were mounted along the sides of the box. The stimulating and recording leads were suspended from an overhead support located in the middle of the box, allowing the rat to move to any area of the open field. Two weeks after surgical preparation, rats were placed in this apparatus and the effect of hypothalamic stimulation was observed at least 25 times under various conditions. Different voltage levels were presented randomly and the most effective polarity, in terms of eliciting m o t o r behavior, was determined for all rats for use in subsequent experiments (i.e., it was often found that more m o t o r activity could be elicited when the current flowed in one direction than when it flowed in the opposite direction). L o c o m o t o r behavior was measured in an activity wheel, 61 cm in diameter and 8 cm in width, constructed of wood and plexiglass. The wheel turned on an axle and ball bearings (fixed to one wall so that the axle did not protrude into the interior) and would rotate when a weight of about 50 g was placed at the circumference. Running was measured by radially protruding struts (located at 20 cm intervals around the circumference) which activated the movement sensing unit as they passed by it, producing spikes in one channel of the polygraph. Recording and stimulating leads were suspended from a stationary arm located in the middle of a 10 cm diameter opening in the center of one wall of the wheel. When the open field testing was completed, all rats were placed in the running wheel for 15 min on two consecutive days to habituate spontaneous locomotion. Following this procedure, the rats were given one stimulation trial each day for 15 days. A stimulation trial lasted 1 min, during which time one voltage level was used. The rats received a range of 0-15 V, presented in 1 V steps in an ascending order on successive daily trials. The dorsal hippocampal electrical activity was recorded on all trials. Trials were discontinued if abnormal E E G activity appeared in the hippocampus or if abnormal behavior occurred, such as jumping or twisting of the trunk. At the termination of testing the rats were assigned two voltage levels chosen randomly, ranging from 1 to 15 V, and retested.

Jump avoidance and passive avoidance The j u m p avoidance apparatus consisted of a 30.5 cm × 30.5 cm × 30.5 cm plywood box mounted on foam rubber blocks. A grid floor of light steel bars, placed 28 cm below the top of the box, could be electrified by means of a Harvard inductorium driven by a 1.5 V dry cell. A plywood shelf, 6 cm wide, was located around the outside of the box 1.3 cm below the upper edge. The rats could j u m p out of the box,

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B. H . BLAND AND C. H . VANDERWOLF

catch the raised edge with their forepaws, and pull themselves up onto the outside shelf. A 5 cm × 11 cm metal plate mounted on the outside of the box 15 cm above the grids activated the movement sensing unit described earlier, making it possible to record the jumping movements of the rat. Training was started by allowing a rat 10 rain to explore the box. On the succeeding training trials the rat was placed on the grid and given shock (to a m a x i m u m of 4 sec) after 10 sec had elapsed. Training was continued until a criterion of 10 successive avoidance responses (jumps prior to delivery of shock) was reached on each of 2 successive days. The intertrial interval was about 30 sec. On the test days, recording and stimulating leads were attached and the rats received an alternating sequence of 5 non-stimulation trials and 5 stimulation trials. On the first trial, the rats were placed in the box and the latency for avoidance recorded. Following the 30 sec intertrial interval the rats were again placed in the b o t t o m of the box and given stimulation of the hypothalamus. Stimulation was terminated as soon as the rat reached the ledge. Stimulation was 5 V on one day and the next day rats received the highest voltage which they had received in the running wheel experiment (7-15 V). The passive avoidance apparatus consisted of a gray plywood box, 39 cm × 39 cm with walls 30 cm high. The floor of the box was a grid of 3.3 m m diameter stainless steel rods set about 1.3 cm apart. The grid could be connected to a 'matched impedance' power source 2 which delivered a current of about 1 mA. An insulated gray wooden platform, 13 cm square and 5.4 cm high, was located in the center of the grid floor. A clear plexiglass sheet with a 2.5 cm diameter hole in the center (to admit wire leads) was used for a lid. The rats were first allowed 10 min adaptation in the apparatus, with recording and stimulating leads attached. Immediately following the adaptation period, the rat was placed on the platform and the latency for stepping off recorded. U p o n stepping off the platform, the rat received footshock for 4 sec and was then placed back on the platform. This procedure was repeated until the rat stayed on the platform for 1 rain. Further such training was given the next day. On day 3 the rats received no further grid shock, but instead were run on an alternating sequence of 3 trials without stimulation and 3 trials with hypothalamic stimulation. Trials consisted of 60 sec periods separated by a 30 sec intertrial interval. On one test day stimulation was 5 V; on a second test day it was the highest voltage used in the preceding experiments (7-15 V). Latencies for moving offthe platform were recorded with a stopwatch.

Shuttlebox self-stimulation experiment Self-stimulation in the dorsomedial-posterior hypothalamus was tested in 6 rats using a shuttlebox (Harvard Instrument Company) measuring 20 cm × 23 cm x 21 cm. The floor was pivoted at the center so that the rat's weight on one side operated relay equipment which (1) started the stimulator, (2) operated counters, (3) started a cumulative clock which gave the total brain stimulation time, and (4) gave a positive report on an event recorder of the rat's location in the shuttlebox

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plus the duration of each individual shuttle. The end of the shuttlebox which would initiate these events could be alternated by means of switches. The stimulation lead came from a mercury c o m m u t a t o r located above the middle of the shuttlebox. The apparatus was housed in a sound-proofed chamber (Industrial Acoustics) measuring 147 cm × 147 cm × 203 cm and containing an observation window. Test sessions were always 15 min/day. Results of a pilot study indicated the importance of having the animals well trained in the shuttlebox before the actual experimental treatments began. Therefore, the rats were given one session per day in the shuttlebox with a moderate stimulation level (6 V) for a period of 2 weeks. During this time the end of the shuttlebox controlling the stimulation was switched in a random fashion. Next, all rats were given 2 sessions without stimulation. The rats then received stimulation of 3, 4, 8 and 12 V, administered randomly to each rat over a 4 day period. In addition, the end of the shuttlebox controlling the stimulation was determined randomly with the restriction that at the end of the 4 days, stimulation was switched on twice on the right and twice on the left. Following this, each rat was run another 4 days, keeping the same order of voltage presentation and reversing the order of the end of the shuttlebox controlling the stimulation. RESULTS

The series of acute experiments (n = 10) showed that stimulation of the posterior hypothalamic nucleus or the dorsal part of the dorsomedial hypothalamic nucleus was particularly effective in producing RSA in the dorsal hippocampus. The RSA would persist beyond the period of stimulation at the higher stimulation intensities. Also, as stimulation intensity increased up to 15 V, the frequency of RSA at the onset of stimulation increased up to 10 c/sec, declining to a lower, more stable level of about 4 c/sec, even though stimulation continued. Stimulation of the pars ventralis portion of the dorsomedial nucleus would also produce RSA but the threshold for doing so was much higher than in the dorsomedialposterior areas. Stimulation of other medial hypothalamic areas such as nucleus premammillaris dorsalis, nucleus mammillaris medialis, and nucleus periventricularis did not produce RSA. Stimulation of the ventromedial nucleus was found to produce RSA a few seconds after stimulus offset, the RSA persisting for periods up to several minutes. All hypothalamic areas gave rise to activation of the cortical E E G regardless of whether RSA was produced or not. H e a r t rate and the rate and depth of respiration were also found to increase with stimulation of the dorsomedial-posterior hypothalamus.

Open fieM Observation of the chronically prepared rats in the open field revealed consistent correlations between the electrical activity of the dorsal hippocampus and spon-

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B. It. BLANDAND C. H. VANDERWOLF

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Fig. 1. Hypothalamic stimulation placements affectingbehavior and the hippocampal EEG. V, dorsomedial-posterior hypothalamic points producing hippocampal RSA and voluntary movement upon stimulation (diagrammatic serial sections, left to right); Q, points having less effect on hippocampal RSA and voluntary movement (see text). Drawings and abbreviations from K6nig and Klippe115. Some relevant abbreviations are: hpv, nucleus periventricularis (hypothalami); hvmc, nucleus ventromedialis (hypothalami) pars centralis; FMT, fasciculus mammillothalamicus; hdv, nucleus dorsomedialis (hypothalami) pars ventralis; hdd, nucleus dorsomedialis (hypothalami) pars dorsalis; re, nucleus reuniens; pv, nucleus premammillaris ventralis; hp, nucleus posterior (hypothalami); FMP, fasciculus medialis prosencephali; pd, nucleus premammillaris dorsalis; mml, nucleus mammillaris medialis pars lateralis; mmm, nucleus mammillaris medialis pars medialis. taneous behavior. Rhythmical slow activity (RSA) accompanied walking, running, rearing, head movements, postural shifts, and manipulatory movements of the paws. Behaviors such as face washing, chewing, lapping at a water spout, and alert immobility were accompanied by large amplitude irregular activity (LIA). It was found that electrodes in the dorsomedial posterior hypothalamus (n = 12), the region producing good RSA when stimulated in acute preparations, produced a behavioral syndrome of head movements, rearing, walking and running. The other sites produced different behaviors (see Figs. 1 and 2). Therefore, the effects of stimulation of these areas will be described separately. Dorsomedial-posterior placements. The behaviors induced by stimulation of the dorsomedial-posterior hypothalamus were correlated with hippocampal electrical activity in the same way as spontaneously occurring behavior. Stimulation at supra-

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EFFECTS OF HYPOTHALAMIC STIMULATION ON BEHAVIOR

5

Fig. 2. Photomicrograph showing a stimulating placement in the medial posterior hypothalamus. Rat 34. Arrow indicates the tip of the electrode tract, x 17. threshold voltages produced head movements, rearing, walking, and running, and was always accompanied by RSA in the dorsal hippocampus. Furthermore, as the voltage was raised the onset frequency of the RSA increased, and the rats also ran faster. During stimulation at intensities of 4 V and above, the rats were never observed to eat, drink, groom, or sit still. When the animal was already engaged in one of these behaviors, stimulation would result in an immediate transition to walking and rearing. The threshold was the same regardless of whether the preceding behavior was immobility, face washing, eating, etc. The rats would also not attack or bite while being stimulated, unless they were forcibly restrained. Shivering was never observed. I f the rats were lifted up, with the feet hanging free, stimulation produced vigorous struggling. In one test, rats were placed in a corner of the box and obstacles constructed around them using wooden boards placed in the sawdust. Stimulation in this situation demonstrated that the rats were not forced to run in any particular direction, and could make turns to the left or right, back up, etc. That is, the pattern of locomotion was controlled by environmental stimuli, rather than being rigidly controlled by the central stimulation. In a supplementary experiment, several rats were placed in a water tank and the effect of posterior hypothalamic stimulation on swimming was observed. Normally when a rat swims, it will tuck its forepaws up under the chin, propelling itself mainly with the hind legs. During hypothalamic stimulation all 4 limbs were used in swimming, giving the impression of 'running' in the water. Brain Research, 43 (1972) 67-88

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B. H. BLAND AND C. H. VANDERWOLF

As stimulation frequency increased from 10 to 100 pulses/sec the voltage necessary to elicit motor activity decreased. At the most effective frequency (100 pulses/ sec) the threshold for movement was about 4 V. Higher frequencies were not tested. Other medial hypothalamic placements. Stimulation at 100 pulses/sec and 4 V resulted in a behavioral pattern of head turning, sniffing, and some shifting of posture, followed by grooming or quiescence, in 18 rats. During high intensity stimulation (up to 15 V), 12 of the rats would show some sporadic locomotor activity and sniffing, and then begin to groom themselves or sit still. The remaining 6 rats showed only head movements, sniffing and postural changes, at all voltage levels. In general, the type of hippocampal electrical activity recorded from the animals with stimulating electrodes outside the dorsomedial-posterior hypothalamus depended upon what the rat was doing during the stimulation, just as it did in recording from rats during spontaneous behavior. If the rat was motionless or engaged in grooming, hippocampal activity was irregular. During head movements, postural shifts and walking, RSA could be recorded from the hippocampus. For the rats in which high intensity stimulation elicited locomotor activity, there were no consistent relations between stimulus onset and the appearance and frequency of RSA, but there was a good relation between RSA and movement. Locomotion was sporadic, during which time RSA was present at a frequency of about 9 c/sec, interspersed

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Fig. 3. Hippocampal EEG during wheel running behavior induced by dorsomedial-posterior hypothalamic stimulation. Rat 40. Varying height of the spikes indicating motion of the wheel is due to the variation in the distance of the struts from the movement sensor. Stimulus parameters: 7 V, 0.1 msec pulse duration, 100 pulses/sec.

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EFFECTS OF HYPOTHALAMICSTIMULATIONON BEHAVIOR

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with periods of alert immobility, during which time the hippocampal electrical activity was irregular. Two rats showed more consistent behavior patterns during the low intensity (4 V) stimulation periods, one rat (with an electrode in the ventromedial nucleus) showing piloerection and chattering of the teeth, while the other rat (with an electrode in the lateral hypothalamus, dorsomedial to fornix) engaged in eating. Stimulationinduced piloerection, teeth chattering, and chewing food were not associated with RSA but handling food, moving the head, etc. were associated with RSA, The rat with the lateral hypothalamic placement would eat during high intensity stimulation (12 V) but made an abnormally large number of manipulative movements of the paws and general 'fidgeting' (shifts of posture, etc.), never crouching motionless while chewing as is normally the case. These 'fidgety' movements were accompanied by almost continuous RSA.

Wheel running Dorsomedial-posterior placements. At stimulation intensities of 4 V and above the rats started to run immediately at stimulus onset and the hippocampal electrical activity changed from irregular activity (LIA) to RSA (Fig. 3). Running was not continuous at 4 V, however; the rats sometimes reversed directions or showed a mixed pattern of rearing and running. At 5 V or more, the rats ran throughout the entire stimulation period, although some rats reversed direction occasionally. RSA persisted in the dorsal hippocampus throughout the stimulation period. The RSA frequency was initially high and then gradually declined to a stable level of about 8 c/sec as stimulation continued (Fig. 4). The decline in frequency took longer the higher the voltage used. Brain Research, 43 (1972) 67-88

B.H. BLAND AND C. H. VANDERWOLF

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RSAONSETFREQUENCY(Hz) Fig. 5. Relations among intensity of dorsomedial-posterior hypothalamic stimulation, RSA onset frequency, and running speed. There were relations between stimulus voltage, running speed and RSA onset frequency (defined as the mean frequency of RSA during the first second of stimulation). As stimulus voltage increased, the running speed of the rat increased and the frequency of RSA at stimulation onset increased (see Figs. 4 and 5). Frequencies of RSA above 12.5 c/sec were correlated with aberrant behavior such as leaping and twisting of the trunk. I f the rat was already running steadily in response to hypothalamic stimulation and the voltage was suddenly increased, running speed increased abruptly. This Brain Research, 43 (1972) 67-88

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EFFECTS OF HYPOTHALAMIC STIMULATION ON BEHAVIOR

acceleration was associated with a rise in RSA frequency followed by a decline back to the original stable level of about 8 c/sec. After the main experiments were completed, the rats were stimulated at a voltage picked at random from 0 to 15 V. The resulting running speed and RSA frequency did not differ from that observed during the same intensity stimulation administered earlier. It would appear therefore, that the behavioral and hippocampal responses to a given stimulus voltage were not altered by experience with the hypothalamic stimulation and the wheel running. Relations between stimulation parameters, RSA and behavior were extremely reliable and in individual rats were virtually identical on all trials. An interesting effect was observed in one rat that had a poor hippocampal recording placement; that is, little RSA could be recorded when the animal walked about spontaneously. High intensity stimulation of the dorsomedial-posterior hypothalamus resulted in very fast running, during which time very clear, large amplitude RSA appeared at the recording electrode. RAT 28

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Fig. 6. Hippocampal EEG during spontaneous walking and during stimulation of a medial hypothalamic placement outside the dorsomedial-posterior area. Brain Research, 43 (1972) 67-88

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B. H. BLAND AND C. H. VANDERWOLF

Other medial hypothalamie placements. The majority of these rats did very little running compared to rats with electrodes in the dorsomedial-posterior hypothalamus. The running which did occur did not begin at stimulus onset, and was sporadic, accompanied by a mean RSA frequency of 9 c/sec at the beginning of the running, while the frequency of RSA at the onset of running for the dorsomedialposterior rats went as high as 12 c/sec. In this group of rats then, no consistent relationship was found between stimulation intensity and frequency of RSA. Hippocampal electrical activity changed depending upon what the animal was doing, regardless of stimulation intensity (e.g., see Fig. 6). RAT NO. 45

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Brain Research, 43 (1972) 67-88

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Jun~ avoidance After prolonged training, the act of jumping out of the jump apparatus became stereotyped; there was very little variation in the type of movement exhibited from one trial to another. During intertrial periods the rats would sit motionless on the side of the box, and this was accompanied by irregular activity (LIA) in the dorsal hippocampus. The jump response was preceded by a period of immobility (except for slight movements of the head and tensing of the body) while the rat crouched with forepaws off the floor. RSA was continuously present during this period, and also during the sudden thrust of the hind legs which propelled the rat to the edge of the box. The RSA had a frequency of 6-7 c/sec when it first appeared, but would increase regularly to a mean peak of 9-10 c/sec just before the jump. The RSA frequency declined rapidly as the rat assumed a motionless position on the ledge (Fig. 7), and LIA reappeared. Dorsomedial-posterior hypothalamic placements. When the hypothalamus was stimulated during avoidance performance, all rats in this group (n = 10) were able to jump and RSA accompanied the response, just as it does during a normal conditioned jump avoidance response. Stimulation at an intensity of 5 V did not result in jump response latencies different from those of control trials (mean latency and associated S.E. for control trials, 0.95 -4- 0.10 sec; for 5 V trials, 0.97 4- 0.09 sec) (data based on means of 5 trials for each of 10 rats). The mean RSA frequency for all rats during the 5 V jump response trials was 9-10 c/sec, just as it is during a normal (unstimulated) jump (see Fig. 7A and B). The mean latency on the high voltage (10-14 V) stimulation trials was less than on control trials (control trials, 0.87 4- 0.10 sec; high voltage trials, 0.63 4- 0.06 sec; t ~ 2.65, P < 0.05) (data based on means of 5 trials for each of 10 rats). In addition, these stimulation trials were accompanied by RSA frequencies 2-2.5 c/sec higher than those accompanying control trials, giving a mean frequency of 11.5-12 c/sec (see Fig. 7C). The most striking difference on the high voltage stimulation trials was one which was not necessarily reflected in the jump avoidance latency. That is, the rats were consistently observed to jump with more force than normal, regardless of their latency. Some of the rats actually overshot the edge of the box and landed on the table on initial trials. Short latency was not always correlated with this since some animals circled 45 ° as they prepared to jump. Other medial hypothalamic placements. Stimulation at these loci (n = 19) had no effect on jump latency in most cases, and prolonged the latency in other cases. The frequency of RSA in the majority of these rats was similar on all trials, regardless of whether latencies were unaffected or increased, or whether 5 V or high voltage stimulation was administered to the hypothalamus. The mean latency for control trials did not differ significantly from the mean latency for 5 V stimulation trials (control trials, 1.08 ± 0.06 sec; 5 V trials, 1.36 4- 0.33 sec) (data based on means of 5 trials for each of 19 rats). The mean latency for the higher voltage (8-15 V) trials was longer than the mean latency for control trials (control, 1.03 4- 0.07 sec; higher voltage trials, 7.78 4- 2.89 sec; t ---- 2.33, P < 0.05)

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B. H. BLAND AND C. H. VANDERWOLF

(data based on means of 5 trials for each of 19 rats). The longer latencies were due to the fact that some o f the rats moved around in the bottom of the box before jumping. The mean frequency of RSA for both the 5 V and higher voltage trials was usually 9-10 c/sec, but there were exceptions. Two control rats with electrodes in the pars ventralis portion of the dorsomedial nucleus had mean RSA frequencies of 11-12 c/sec on high voltage trials. This correlated with a reduced latency of jumping.

Passive avoidance Dorsomedial-posterior hypothalamic placements. Stimulation of the dorsomedial-posterior hypothalamus was not compatible with the restraint of motor activity required by the passive avoidance task. All rats in this group (n = 10) stepped down off the platform during the 5 V stimulation trials. The mean step down latency without stimulation was 60 J= 0 sec; with 5 V stimulation it was 3.27 ~ 0.94 sec (data based on means of 3 trials for each of 10 rats). On control trials, the rats remained on the platform and the hippocampal electrical activity was predominately irregular (LIA). RSA appeared when the rats made small head movements or postural adjustments. As in the previous experiments, RSA appeared in the dorsal hippocampus immediately at the onset of stimulation. Stimulation at an intensity of 5 V resulted in a mean RSA frequency of 9-10 c/sec. All rats again moved off the platform during the high voltage (7-15 V) trials, the latency for doing so being significantly less than the latency for the 5 V trials (at 5 V, 3.27 ~ 0.94 sec; high voltage trials, 0.96 ~ 0.10 sec; t = 2.53, P < 0.05) (data based on means of 3 trials for each of 10 rats). RSA appeared in the dorsal hippocampus at stimulus onset, the frequency, however, being higher (11-12 c/sec) than that accompanying the 5 V trials (9-10 c/sec). Other medial hypothalamie placements. The majority of these rats remained on the platform, even during high voltage trials. Fourteen of the 15 rats tested remained on the platform during the 5 V trials, the remaining rat having a mean latency of 7.8 sec for moving off. Correlating with the lack of movement, the hippocampal electrical activity remained mostly irregular, RSA appearing only when locomotion, head movements, or postural adjustments occurred. Nine o f the rats remained on the platform during the high voltage (7-15 V) trials. Even when the stimulation resulted in some initial motor activity such as circling and head movements, RSA frequencies accompanying this were only about 7 c/sec. Low frequency RSA was thus compatible with remaining on the platform while higher frequency RSA was always associated with running off. Six of the rats did move off the platform, their mean latency to do so being significantly longer than the latencies of the dorsomedial-posterior rats at equivalent voltages (t -- 3.56, P < 0.01). The frequency of RSA accompanying the act of moving off the platform was correspondingly lower in 5 o f the rats (mean frequency 9 c/sec). The sixth rat had a mean frequency of 12 c/sec, corresponding with a short latency of 1.5 sec for moving off the platform. Thus, in cases where stimulation led to RSA frequencies of 9 c/sec Brain Research, 43 (1972) 67-88

EFFECTS OF HYPOTHALAMICSTIMULATIONON BEHAVIOR

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Fig. 8. Relation of stimulus intensity (V) to various measures of self-stimulation behavior. Inset numbers in all graphs refer to individual rats.

or more, step down latency ranged from 1.5 to 9.3 sec, whereas if the RSA frequency reached only 7 c/sec, the rats remained on the platform.

Shuttlebox self-stimulation experiment All the dorsomedial-posterior hypothalamic rats engaged in self-stimulation. At 4 V the rats would run on to the 'hot' grid, turn around and run off (shuttle). At the lower voltages the rat groomed itself in the neutral end of the box between shuttles. This behavior ceased at the higher voltages, since the rats were shuttling constantly. That is, at the higher voltages the rats ran on to the 'hot' grid, then ran off very rapidly, only to run back on a m o m e n t later. This sequence was repeated throughout a 15 min test session. Some rats learned to sit at the center and 'see-saw' the grids. Ejaculations were often observed at the higher voltages. Fig. 8 shows that shuttle rate went up as voltage increased, the rate increasing sharply from 4 V upwards. The rapidity with which the animals shuttled was reflected in the amount of time spent on the brain stimulation side of the box. As voltage increased above 4 V, the time spent per shuttle in the brain stimulation end decreased, but the rate of shuttling increased to such an extent that total brain stimulation time increased, as shown in Fig. 8. DISCUSSION Electrodes yielding the clearest RSA had the tips placed one above and one below the hippocampal pyramidal cells, as might be expected from previous work s, 28,32,86. Recordings from electrodes with the tips displaced from this optimal orien-

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tation were found to contain either admixtures of slow and fast activity, or fast activity (15-50 c/sec), depending on their exact location. The fast activity recorded from the dentate area of the hippocampal formation was not found to vary in any observable fashion during any type of spontaneous motor behavior. However, RSA and LIA were found to be related to concurrent motor activity in a consistent way, confirming previous observations in rats 27,31,36, in the guinea pig 2s, and in Mongolian gerbils and cats 34. RSA and LIA could be recorded from the same electrode, the particular pattern varying with the ongoing locomotor behavior. Rhythmical slow activity (RSA) was found to accompany the voluntary motor patterns of walking, running, jumping, rearing, swimming, digging, postural shifts, head movements, and manipulatory movements of the paws. Similar relations between RSA and motor activity can be inferred from studies using the dog as a subject1,3,29, 38. Large amplitude irregular activity (LIA) was correlated with behavioral immobility and with the automatic motor patterns of face washing, chewing, lapping at a water spout, and scratching. Foot stomping behavior in the gerbil 16 and shivering in the rat a6 are other motor patterns correlated with LIA. The results of the acute experiments replicated earlier findings 7,37 that electrical stimulation of the medial hypothalamus produces synchronization (RSA) in the dorsal hippocampus. Increases in respiratory rate were also noted, in agreement with the findings summarized by Monnier 22. The most significant finding of the acute experiments was that not all areas of the medial hypothalamus produced RSA in the dorsal hippocampus with equal facility; in fact some medial hypothalamic sites would not produce any RSA at all upon stimulation. The latter areas would, nonetheless, produce neocortical desynchronization. The optimal areas for producing hippocampal RSA were found to be the posterior nucleus and the pars dorsalis portion of the dorsomedial nucleus, a finding which was confirmed in the chronic experiments. In addition, increases in the intensity of stimulation of these nuclei were also reflected in increases in the frequency of the hippocampal RSA at stimulus onset, the RSA declining to a more stable level with continued stimulation. In the chronic experiments, the behaviors induced by electrical stimulation of the dorsomedial-posterior hypothalamus were correlated with hippocampal electrical activity in the same way as spontaneously occurring behavior. Stimulation of these hypothalamic areas resulted in head movement, postural changes, rearing, walking, running, and jumping, and was always associated with RSA in the dorsal hippocampus. Behaviors which are not normally associated with RSA when they occur spontaneously were not observed to occur during stimulation at 4 V or more. The rats were never observed to eat, drink, groom, or sit still while the stimulation was being administered. The pattern of locomotion observed was subject to modification by environmental stimuli. That is, during stimulation a rat could turn to avoid obstacles, reverse direction, etc. Furthermore, as the stimulating voltage was raised, the speed of locomotion increased, along with an increase in the onset frequency of RSA. The latter relationships were investigated more formally in the wheel running. In this situation the rats started to run immediately at stimulus onset and the hippocampal electrical Brain Research, 43 (1972) 67-88

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activity changed from irregular to rhythmical slow activity (RSA). The rats ran throughout the entire stimulation period and, likewise, RSA persisted in the dorsal hippocampus throughout stimulation, although the frequency would decline from a high initial level (up to 12 c/sec) to a lower, more stable level (about 8 c/sec). As the voltage level was raised, the onset frequency of RSA increased and the speed of running also increased (the relationship between running speed and voltage of posterior hypothalamic stimulation was previously demonstrated by Gerben4). In all cases, RSA frequency declined to a lower level, despite the fact that the r,tt appeared to run at a relatively constant speed, related to the particular voltage level used. This conclusion is limited to some extent by the fact that the wheel used here is not an ideal measuring device since its inertia underestimates the 'true' speed a t the onset of stimulation, and may smooth out irregularities during continuous running. Further, the length of individual bounds during galloping is limited by the diameter of the wheel. However, these limitations are not serious and it would appear that steady movements, irrespective of speed, are associated with a constant RSA frequency, while increases in movement from a stable level (e.g., a change from sitting still to running, or a change from running at a constant speed to running at a higher speed) are accompanied by upward frequency shifts. This suggests that the dorsomedialposterior hypothalamus can 'trigger' a movement 'program', providing a specific level of facilitation for its initiation and continued maintenanceZL The decay of an RSA frequency increase occurs gradually, in the present study being complete, or nearly complete, at the end of 1 min. Such a decay would ensure that the hippocampus maintains a sensitivity to changes in diencephalic activity and motor behavior rather than a sensitivity to absolute levels. It is interesting that onset frequencies of RSA above 12.5 c/sec were correlated with aberrant behavior such as uncontrolled leaping, or twisting of the trunk. Yoshii et aL as also observed that frequencies above about 12 c/sec (which is approximately the normal upper limit in rats) were associated with postural disturbances. Possibly, such unphysiological effects indicate a failure of the normal control of brain stem motor mechanisms by the hippocampus. The jump avoidance experiment served to illustrate further that the type of motor response elicited by stimulation of the dorsomedial-posterior hypothalamus is determined by the situation. That is, the experiment showed that jumping as well as running is compatible with stimulation of this area, and that RSA accompanies this response, just as it does a normal conditioned jump avoidance response. Furthermore, increasing voltage levels again resulted in higher onset frequencies of RSA, in this case correlated with shorter jump latencies and increased force of the jump response. The passive avoidance experiment showed that training in a behavior incompatible with RSA would not modify the effects of stimulation of the dorsomedialposterior hypothalamus. The same relationships between the stimulation level, onset frequency of the RSA, and speed of locomotion that were demonstrated in the preceding experiments were also found in the passive avoidance experiment. The results of the passive avoidance experiment also confirm previous reports that a learned immobility response need not be accompanied by hippocampal Brain Research, 43 (1972) 67-88

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RSAa, z4. In general, the performance of learned behavior is accompanied by RSA only when certain movements are performed; there is no intrinsic association between RSA and learning, memory or recall. It might be thought that many of the results described here are due to aversive properties of the hypothalamic stimulation, i.e., the rats run or jump because the stimulation is painful or otherwise unpleasant. However, stimulation of the dorsomedial-posterior hypothalamus, which produces running behavior, was shown to be positively reinforcing, in agreement with a previous report by Gerben 5. In the test used, rats could select the duration of the trains of hypothalamic stimulation received. The preferred durations were usually between 1 and 2 sec, even at the highest voltages. Running or jumping was initiated by much shorter trains of stimulation (latency of jumping in the jump avoidance test averaged 0.63 sec during intense stimulation) indicating that the motor activity produced is not due to possible aversive properties of the stimulation. A further point is that a train of dorsomedial-posterior hypothalamic stimulation is not succeeded by freezing behavior such as occurs following peripheral electric shock. The fact that the above effects were specific to the dorsomedial-posterior hypothalamic area was revealed through control stimulation of other medial hypothalamic sites. Only two control animals showed consistent behavior patterns throughout the stimulus range. Stimulation elicited piloerection and chattering of the teeth in one of the rats and eating in the other. In both of these animals the hippocampal electrical activity (LIA) was the same as would be expected during the equivalent spontaneous behavior. The type of hippocampal electrical activity recorded from the remaining control animals depended upon what the rat was doing during the stimulation. In some rats, with electrodes close to the dorsomedial-posterior hypothalamic area, high intensity stimulation resulted in sporadic locomotion, particularly in two rats with electrodes in the pars ventralis area of the dorsomedial nucleus. The finding that stimulation of the mammil]ary bodies did not produce locomotion confirmed a study by Maire 19, in which he reported failure to produce locomotion by stimulating the mammillary bodies in cats. In the jump avoidance experiment the performance of the control rats, in contrast to the dorsomedial-posterior group, was either not facilitated or was actually disrupted during hypothalamic stimulation. The majority of the control rats were also able to perform passive avoidance behavior, even during high intensity stimulation. The above data suggest that the dorsomedial-posterior hypothalamic area and its connections to the hippocampus form a part of a system capable of initiating and maintaining performance of motor patterns which can be classified as higher order or voluntary in nature. More specifically, the movements which are elicited tend to be larger bodily movements such as those involved in walking, running, jumping, swimming, etc. Small manipulatory movements such as pressing a bar were not elicited even though such movements are normally accompanied by RSA. In fact, supplementary experiments (unpublished) showed that hungry animals pressing for food on an FR5 schedule (i.e., every fifth lever press was followed by a food pellet) would cease responding while receiving stimulation of the posterior hypothalamus

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at 2 V, which is 2 V below the threshold for consistently eliciting locomotion. These rats (n = 6) would begin to bar-press and eat as soon as the stimulation was terminated. Stimulation of other medial hypothalamic areas with up to 6 V did not cause a similar cessation of bar-pressing. Initially, we had expected that posterior hypothalamic stimulation at a low intensity might facilitate bar-pressing. This did not occur; such stimulation produced only head movements and sporadic locomotion. An interpretation of this is suggested by the observation that posterior hypothalamic stimulation tends to produce large amplitude RSA with a frequency of 8 c/sec or more, resembling the pattern seen during walking or running. It may be that the hypothalamic-hippocampal system is concerned mainly with the control of the neck and trunk musculature in locomotion, head movement, etc., while more discrete control of the distal musculature (required during bar-pressing) is effected via more recently evolved thalamic-neocortical mechanisms. Therefore, perhaps, bar-pressing is not facilitated by posterior hypothalamic stimulation. Whether this is true or not, it is worth noting that the RSA accompanying bar-pressing has a lower amplitude than RSA accompanying walking and has a frequency of only 6-7 c/sec. Kawamura and Domino 13 showed that RSA of this type can be elicited by medial thalamic stimulation while larger amplitude higher frequency patterns are produced by hypothalamic stimulation. Although the posterior hypothalamic area and its ascending connections to the hippocampus are not conventionally regarded as a part of the motor system, it is clear that this system must play an important role in the control of motor activity, particularly of the more voluntary types. On the basis of studies of the effect of electrical stimulation of the diencephalon, Hess10,11,22 proposed the existence of a dynamogenic zone which controlled somatomotor activity. This seems readily compatible with Penfield's26 concept of a centrencephalic system in which the final integrative processes that are prerequisite to planned voluntary action must take place. Penfield did not delineate the anatomical substrate of this system beyond saying that medial diencephalic structures must certainly be involved, a suggestion which is supported by the present investigation. It has been proposed21, 8~ that there are two distinct phases in higher level motor control. A programming mechanism (located in the cerebral hemispheres, hippocampus, etc.) must select from a large number of possible voluntary movements those which are appropriate in a given situation. The central representations of these movements must be maintained in a sub-threshold state of excitation for some time in order that they can be activated in a particular sequential order. A trigger mechanism will then fire off, at the appropriate time, whatever motor activities have been programmed. The data of the present study strongly suggest that the dorsomedial-posterior hypothalamic area and its connections with the hippocampus form a part of the anatomical substrate for such a mechanism. SUMMARY

Slow wave activity was recorded from the hippocampus in chronically prepared Brain Research, 43 (1972) 67-88

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rats. During spontaneous or learned behavior, rhythmical slow activity (RSA) accompanied walking, rearing, climbing, manipulation of objects with the forelimbs, isolated movements of the head or one limb and changes in posture (voluntary movement) but did not accompany alert immobility, licking, chewing, face-washing, or scratching (more automatic behavior). During stimulation of the hypothalamus similar b e h a v i o r - E E G relations were observed. Walking, running, jumping, and isolated head movements were always accompanied by RSA, but this waveform was absent during alert immobility, chewing, face washing, piloerection or chattering of the teeth. Stimulation of the dorsomedial posterior hypothalamus was especially effective in the elicitation of RSA in the hippocampus and also consistently produced head movement, running, or jumping when administered in conscious animals. Sites in the dorsomedial-posterior hypothalamus would also support self-stimulation behavior. Increases in the voltage of posterior hypothalamic stimulation produced increases in the frequency of the RSA during the first few seconds of stimulation, and a corresponding increase in running speed or in the force with which jumping was initiated. Relations between behavior and hippocampal activity were the same during tests of learning (active and passive avoidance) and during spontaneous behavior (e.g., exploration). It is suggested that an ascending hypothalamo-hippocampal mechanism plays a role in the control of voluntary movement. ACKNOWLEDGEMENTS

This research was supported by grants from the National Research Council (APB-118), the Medical Research Council (MA-4212), and a National Research Council Post-Graduate Scholarship. Portions of this research were presented in a paper given at the Eastern Psychological Association, 41st Annual Meeting, Atlantic City, New Jersey, April, 1970.

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