Anesthetic-dependent excitability changes in the hypothalamic ventromedial nucleus of the rat

EXPERIMENTAL

67, 524-538 (1980)

NEUROLOGY

Anesthetic-Dependent Excitability Changes in the Hypothalamic Ventromedial Nucleus of the Rat HUGO F. CARRERAND HORACIO FERREYRA' Institute

de InvestiguciBn Cusilla de Correo

Received

March

MGdica. Mercedes 389, 5000 C6rdoba.

19, 1979; revision

received

y Martin Ferreyra, Argentina October

IS, 1979

In acute experiments in rats anesthetized with urethane, the field potentials, population spike, and unit activity evoked in the hypothalamic ventromedial nucleus (HVM) by amygdaloid stimulation are significantly increased with respect to control when preceded by a conditioning volley at 20- to lOO-ms intervals. Under pentobarbital anesthesia, in contrast, the evoked responses were inhibited by the conditioning stimulus for similar interstimulus intervals. In unanesthetized animals chronically implanted with stimulating and recording electrodes, a facilitation of responses by a conditioning stimulus was observed when they were awake or anesthetized with urethane. When the same animals were anesthetized with pentobarbital the HVM evoked response was inhibited by a conditioning pulse. Frequency facilitation and post-tetanic potentiation of HVM responses were markedly enhanced under urethane, whereas in pentobarbital-anesthetized animals inhibition predominated. Picrotoxin reversed the inhibition under pentobarbital to facilitation. These results suggest that the HVM neuron population is under both excitatory and inhibitory influences from the amygdala, the former being predominant in awake and urethane-anesthetized animals and the latter being expressed under pentobarbital anesthesia and is probably mediated by y-aminobutyric acid.

INTRODUCTION The ventromedial nucleus of the hypothalamus (HVM) has been shown to mediate a variety of functions pertaining to reproduction (16), caloric Abbreviations: HVM-ventromedial nucleus of the hypothalamus, PSTH-poststimulus time histogram, EEG-electroencephalogram, GABA--y-aminobutyric acid. ’ This work was supported by the Consejo National de lnvestigaciones Cientificas y Tecnicas of Argentina through an institutional grant and through a personal grant, Argentine-U.S.A. Cooperative Research Project 7363175, to H.F. and by a personal grant from Programa Latinoamericano de Investigaciones en Reproduction Humana, PLAMIRH 81.156.1.77 under the auspices of Corporation Centro Regional de Poblacion (Bogota, Colombia) to H.F.C. 524 0014.4886/80/030524-15$02.00/O Copyright All

rights

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Academic tn any

Press. form

Inc. reserved

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homeostasis (24), somatic growth (2). and emotional behavior (5). Lesions or stimulation of the HVM affect behavioral (3), endocrine (I, 14, 19), or autonomic aspects of those functions, suggesting a complex integrative role for these cells. Although significant advances have been made concerning the cytology, (23, 37), anatomy (6, 8, 22, 33). and synaptic organization of the HVM and its connections (1 I, 29-3 1, 36). much is still to be learned if the function of this brain region can be described in terms of its input, output, and intrinsic synaptic activity. Quantitatively, amygdaloid afferents to the HVM are the most important of its afferent fibers (7,22): moreover, participation of the amygdala in most aspects of the HVM functions ennumerated above has been suggested. Indeed, medial or lateral amygdaloid influences exerting opposite effects on luteinizing hormone (I, 4), adrenocorticotrophic hormone (21). and somatostatin and growth hormone (20) release as well as sexual, aggressive, and appetitive behavior were described (15). For these reasons the amygdala input was repeatedly chosen to study HVM function with neurophysiological techniques. Unfortunately, variable and often contradictory evidence was obtained, making it difficult to achieve a unifying concept on this matter. The results presented in this report indicate that at least part of these divergent results may be explained by the differential effects of the two most frequently used anesthetic agents (pentobarbital and urethane) on the response of HVM neurons to amygdaloid stimulation. MATERIALS

AND METHODS

Adult female albino rats were used. HVM responses to amygdaloid stimulation were recorded in acute and chronic preparations. In acute experiments, either long-term ovariectomized animals injected with 100 pug estradiol benzoate/kg body weight 48 h before or intact estrous female rats were used. They were anesthetized with urethane (1 g/kg) or sodium pentobarbital (40 mg/kg). A large craniectomy was made to allow the stereotaxic introduction of two stimulating electrodes directed to different nuclei of the amygdaloid complex (stereotaxic coordinates: A 5.0 to A 6.2, L 3.0 to 5.0, V - 3.0). Care was taken to avoid damage to the stria terminalis, using an oblique angle for the penetration of medially directed electrodes. Stimulating electrodes were made of a pair of twisted stainless-steel wires (150 pm in diameter) insulated except for the tips. Recording electrodes directed to the HVM were of two kinds: electrolytically etched tungsten wires insulated with glass as described by Levick (17) or glass micropipets filled with 3 M NaCl (5 to 20 mR at 1000 Hz). Usually one or exceptionally two penetrations were made in the same animal (stereotaxic coordinates: A 5.0 to 6.2, L 0.6, V -3.0). The recording microelectrode

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was lowered through the HVM by means of a hydraulic microdrive while monitoring evoked potentials and unitary responses to amygdaloid stimulation. In five animals anesthetized with pentobarbital, recordings were made before and at various times after picrotoxin (1 mg/kg; i.p.). Recorded activity was amplified and displayed on an oscilloscope for monitoring and photography. When studying evoked potentials the amplified signal was fed to a signal averager module which accumulated 16 or 32 sweeps; the averaged evoked potentials thus obtained were fed to a strip chart recorder. Unitary spikes were also studied. At sites where evoked neuron firings were observed, poststimulus time histograms (PSTHs) were constructed using a time histogram module. These PSTHs were either transferred to the strip chart recorder or photographed from the oscilloscope screen. In chronic experiments, recordings of HVM potentials were made in three different conditions. The first recording was made under pentobarbital anesthesia on the same day that the electrodes were implanted. Stimulating and recording electrodes were identical to the stimulating electrodes used in acute recordings. When the evoked potential was studied, the electrodes were secured to the skull with dental cement. Stainlesssteel screws with soldered leads were fixed through holes in the parietal and occipital bones for electroencephalograms (EEGs). The animal was grounded through a screw fixed to the nasal bone. A plastic socket (Amphenol Tiny-tim) housing the contacts for all electrodes was fixed to the animal’s head with dental cement. Forty-eight hours after the implant had been made, recordings were repeated while the animal was awake using identical stimulus parameters. The recording and stimulating electrodes were lead through coaxial, low-noise, flexible wires. After recordings in the unanesthetized condition were completed, the animals received 1 g urethane/kg body weight and recordings were repeated. In both acute and chronic experiments, series of 16 conditioning and test responses were averaged and recordings repeated with different conditioning-test intervals. Stimuli were square wave pulses of 0.5 ms duration administered to the electrodes placed in the amygdala, at 0.4 to 0.8 Hz. Intensity was about 1.5 times threshold; the threshold was determined by progressively increasing stimulus intensity until a barely detectable response was obtained. The effect of varying intensity and frequency of stimulus administration on the conditioning-test paradigm was also studied. The EEG was continuously monitored throughout the procedure. After completing the acute or chronic experiments, stimulation and recording sites were marked by passing 100 WA of cathodal DC for 10 s. When micropipets were used the animal was fixed by cardiac perfusion with 10% Formalin and the micropipet left in situ 12 to 14 h. so that

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recording sites could be readily identified in frontal serial sections of the brain. In all other cases the animals were killed with ether, the brain was fixed in Formalin and subsequently cut in a freezing microtome for exact placement of electrode tracts and coagulation lesions in unstained, serially cut sections. RESULTS Field Potentials

Field potentials were evoked in the HVM by single-shock stimulation of corticomedial, basolateral, or lateral amygdaloid nuclei. The evoked responses to medial amygdaloid nucleus stimulation were similar in latency and shape to the triphasic responses previously reported by Renaud (29) in the rat: onset latency at 10 to 12 ms for the negative wave, peak 15 ms: positive wave peak 18 to 2 1 ms, late negative wave 50 to 60 ms. In 35% of the animals in which a reliable evoked response was recorded, the initial positive wave was split into two secondary positive deflections by a sharp, negative going wave with the general characteristics of a population spike as shown in Figs. IA and B. We suggest that the latter represents the quasi synchronous discharge of HVM neurons on the following basis: (i) The population spike was found only within the boundaries of the HVM cell population. (ii) Differentiated records showed that multiunit or individual spike discharges were temporally associated with it (Figs. IC, D) and (iii) the mean onset latency of transynaptically driven cells (by corticomedial amygdaloid stimulation) coincided with the mean peak of the population spike (respectively 18.1 ? 3.1 and 19.7 ? 1.2 ms mean latency 2 SE). Frequency

rrnd Post-Tetanic

Potentiation

Stimulation delivered to the amygdaloid nuclei at different frequencies produced amplitude changes in the HVM that differed according to the anesthetic used. In 12 rats anesthetized with urethane the HVM response to repetitive medial amygdaloid nucleus stimulation at different frequencies was studied. In 10 cases an enhancement in the amplitude of the response was observed during delivery of pulses as the frequencies were varied from 0.4 to 28 Hz in steps of 4 Hz, the increase ranging from 60 to 400% (frequency potentiation, Figs. 2A. B). Frequency potentiation was found in one rat in the pentobarbital group (N = 5) but only to 12 Hz; higher frequencies led to inhibition of responses. Post-tetanic potentiation was also observed in urethane-anesthetized rats. A train of 10 to 20 Hz and 5 s duration was administered through the

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128 SWEEPS

F

n FIG. 1. Representative gross evoked responses in the ventromedial nucleus of the hypothalamus (HVM) to amygdala stimulation in four animals in which population spikes were present. A and B-HVM-evoked response to medial amygdala stimulation. The sharp negative peak splitting the gross response into two secondary waves was present in A (upper third of VMH) and in B. 700 pm ventral to A. Peak latency was 21 ms in A and B. C-poststimulus time histogram of VMH units recorded at B; maximum probability of discharge coincides in time with the peak latency of the population spike shown in A and B. D-upper record. undifferentiated HVM gross response to medial amygdala stimulation: lower. differentiated recording showing multiunit firing synchronous with population spike. E-upper record. HVM evoked response from medial amygdala stimulation at subthreshold intensity for population spike generation; lower, a conditioning pulse facilitated the production of a population spike in the test response at a conditioning-test interval of 100 ms; facilitation was obtained between 40 and 500 ms of conditioning-test interval. A through E are records obtained from animals anesthetized with urethane. F-pentobarbital anesthesia. Conditioning and test stimuli at suprathreshold intensities for population spike activity. Inhibition of the test gross evoked response and of the population spike at 75 ms conditioning-test interval. Calibration marks: A and B. horizontal 50 ms, vertical 125 I.LV; D, 30 ms, 600 FV: E, 50 ms. 500 PV: F. 35 ms, 500 *V.

electrode in the amygdala and potentials evoked by single-shock evident increase was observed in frequency inhibition were found in with urethane, whereas post-tetanic

immediately afterward recording of stimulation at 1 Hz was resumed. An 9 of 10 rats (Fig. 2C). Post-tetanic and only 2 of 12 animals (16%) anesthetized and frequency inhibition were found in

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FIG. 2. Effect of varying the stimulation frequency on the amplitude of an evoked gross response in a ventromedial nucleus of the hypothalamus (HVM) of a urethane-anesthetized animal. A-control evoked response in HVM to lateral amygdala stimulation. B-frequency potentiation at 8 Hz stimulation, I .2 times threshold voltage. Camera shutter was maintained open during the first 4 s of stimulation onset. C-response obtained after tetanic stimulation (20 Hz; 5-s duration ; 8 V. 0.3-ms square pulses) delivered to a bipolar stimulating electrode in the lateral amygdala. Seven superimposed oscilloscope sweeps. Photograph was obtained 1s after end of tetanus. A marked enhancement of the early positive wave and of the small, late positive wave seen in A (post-tetanic potentiation) occurred after high-frequency stimulation of the lateral amygdala.

four of five animals (80%) anesthetized with pentobarbital. Frequencies that produced frequency inhibition of HVM responses in the latter group (i.e., 12, 16, 20, and 24 Hz) when tested in the same animal in the awake condition or under urethane anesthesiaresulted in potentiation of the HVM gross evoked potentials. Recovery

Cycle of Amygdala

-HVM

System

The results described above suggested that excitability of the amygdala-HVM system could be increased or decreased depending on the anesthetic used. To test this possibility and to explore the functional characteristics of the synapses involved in the different responses obtained, the recovery cycle of the evoked response was investigated by means of paired shocks delivered at various time intervals. Under urethane anesthesia a conditioning stimulus consistently (five of five rats) facilitated the response to a second (test) stimulus at intervals from 20 to 3000 ms. The results are summarized in Fig. 3 showing that maximal facilitation occurred at 60 ms of conditioning-test intervals. The same experimental method applied to another group (N = 7) of animals under pentobarbital anesthesia disclosed a clear inhibitory effect after the response evoked by

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L-l FIG. 3. Excitability cycle of evoked potentials in the ventromedial nucleus of the hypothalamus measured by the amplitude of the test response relative to amplitude of the conditioning response (paired-shock analysis). Data points are mean ? SE. Upper half shows facilitation observed under urethane anesthesia (N = 5). Insert shows photograph of five superimposed oscilloscope tracings with conditioning-test interval of 40 ms. Lower half shows inhibition observed under pentobarbital anesthesia (N = 7). Inserts show photographs of five superimposed oscilloscope tracings with conditioning-test intervals of 37 ms (left) and 275 ms (right). Arrows point to stimulus artifact. Calibration marks: upper, vertical 250 pV, horizontal 25 ms: lower vertical 500 pV, horizontal 25 ms left and 150 ms. right. Test response versus conditioning response, P < 0.05 for intervals 20 to 100 ms in both urethaneor pentobarbital-anesthetized animals: randomization test for matched pairs.

lateral amygdaloid stimulation (Fig. 3). Maximal inhibition (49% of control) was observed at 40 ms of conditioning-test interval, although significant differences were still present at 400- to 500-ms intervals. Paired-Shock

Analysis

in Animals

with Chronically

Implanted

Electrodes

The results described above indicated the need to compare the HVM response to amygdaloid stimulation in a condition that would eliminate variations due to size or placement of electrodes as well as individual

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variations in response characteristics so as to detect differences in excitability due exclusively to anesthetic effects. For these reasons electrodes were chronically implanted and HVM responses evoked by amygdaloid stimulation were studied in three different conditions in the same animal (see Materials and Methods). As Fig. 4 shows, under pentobarbital, a conditioning stimulus consistently produced a depression of the test response with conditioning-test intervals to 1000 ms, independent of minor variations in electrode position (Fig. 5). On the other hand, when the same animals were awake or anesthetized with urethane, the test response was facilitated to a similar conditioning-test interval.

l -*Urethane

50 -

oFIG. 4. Excitability cycle of evoked potentials in the ventromedial nucleus of the hypothalamus measured by the amplitude of the test response relative to amplitude of conditioning response (paired-shock analysis). The same animals (N = 6) were used to study the response in three different conditions (see Materials and Methods). Data points are mean f SE. P < 0.05 for intervals 30 to 100 ms in awake or urethane-anesthetized animals and for intervals 30 to 70 ms in pentobarbital-anesthetized animals; randomization test for matched pairs.

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FIG. 5. A through F-schematic representation of frontal cuts of the brain showing placement of the recording electrode in the ventromedial nucleus (v, upper drawings) and stimulating electrode in the amygdala (0, lower drawings) in animals with chronically nucleus. ACEimplanted electrodes. ABL-basolateral nucleus, ABM-basomedial central nucleus, ACD-cortical nucleus, AL-lateral nucleus. DMH-dorsomedial nucleus, FX-fornix. VMH-ventromedial nucleus.

Population Spike. Paired-shock analysis of HVM responses in which a population spike was present were consistent with the results obtained in the evoked-potential studies, that is, the population spike was facilitated in rats anesthetized with urethane (Fig. 1E) and inhibited in animals studied under pentobarbital (Fig. 1F).

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Effects ofPicrofoxin. Several reports (24,261 suggested that pentobarbital enhances inhibition mediated by y-aminobutyric acid (GABA): furthermore, this inhibitory neurotransmitter was found in some cells of the HVM of the rat ( 17). The possibility was thus considered that the inhibitory phase observed under pentobarbital may be mediated by GABA. To test this hypothesis paired-shock analysis was performed in five rats anesthetized with pentobarbital before and after administration of a noncompetitive GABA antagonist, picrotoxin (1 mg/kg body weight, i.p.). In three cases picrotoxin reversed the inhibition observed under pentobarbital. Figure 6 shows the results of one experiment. Thirty minutes after pentobarbital administration the usual inhibition of the test response was observed (Fig. 6A). Inhibition was partially suppressed 15 min after picrotoxin administration and reversed to facilitation at 30, 45. and 60 min (Fig. 6B). Facilitation was again reversed to inhibition after a second injection of pentobarbital. In one rat the inhibition observed after pentobarbital was decreased 30 and 60 min after picrotoxin and in one other experimental animal no change was observed after picrotoxin administration. Unit Rrcordings. Extracellular unit recordings were made on 49 neurons which by histological criteria were considered to be within the boundaries of the HVM. The majority of those neurons displayed little or no

t

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FIG. 6. Effects of picrotoxin on the amplitude of the test response evoked in the ventromedial nucleus by lateral amygdala stimulation in an animal under pentobarbital anesthesia. Open and hatched bars represent amplitudes of test responses relative to amplitudes of conditioning responses (paired-shock analysis). Insets: A-the control response recorded 30 min after induction of pentobarbital anesthesia (40 mgikg). B-45 min after an i.p. injection of picrotoxin (I mg/kg). The usual depression of the test responses observed in animals under pentobarbital was reversed to a conspicuous facilitation after picrotoxin. C-inhibition of the test response 30 min after an i.p. injection of pentobarbital(20 mgikg). Records shown in insets A-C are averaged evoked responses, I6 sweeps each. Stimulation in the lateral amygdala at 9 V. 0.5 ms. 0.5 Hz. 50 ms ofconditioning-test interval.

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spontaneous activity. In those which did show sufficient spontaneous discharges, a silent period of 50 to 150 ms followed single-shock stimulation of both the lateral and medial amygdaloid nuclei. The silent period was not necessarily preceded by enhanced unit activity, suggesting some form of onset-inhibition in HVM neurons. Forty-two neurons (85%) discharged a single orthodromic spike in response to stimulation of the amygdala and seven units (15%) were antidromically driven. Paired-shock analysis of 26 of the former units under urethane or pentobarbital disclosed response patterns similar to those in the evoked potential studies, i.e., units were most frequently facilitated by a conditioning shock under urethane (8 of 13; Figs. 7A and C, left) but were depressed or inhibited under pentobarbital (13 of 13; Figs. 7B and C. right). Conditioning-test intervals at which these

FIG. 7. Poststimulus time histograms (A and B) and sample records (C) of ventromedial nucleus (VMH) units in response to lateral amygdala stimulation under different anesthetic agents. A-rat under urethane anesthesia. Increased firing probability of VMH unit with respect to control. Conditioning and test stimuli (dots above vertical bars) were set at I .5 times threshold for spike discharge. B-animal anesthetized with pentobarbital. The poststimulus histogram shows the decreased firing probability of an VMH unit discharged by a test stimulus when preceded by a conditioning volley with a 60-ms interval. C-(left) shows facilitatory effects of a conditioning volley on a test response, in a urethane-anesthetized animal when both the conditioning and test shocks are delivered at subthreshold intensities for spike discharge: C-fright) shows suppression of spike discharges to the test stimulus in another animal under pentobarbital. Both shocks were delivered at 1.5 times threshold. Conditioning stimulus-test stimulus interval was 65 ms.

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effects were obtained (40 to 100 ms) were similar to those at which maximal facilitation or inhibition of evoked field responses were found. Twenty-four transynaptically driven units were tested for differential responses to both lateral and medial amygdaloid nuclei stimulation. Ten neurons were discharged by medial but not by lateral amygdaloid nuclei stimulation; seven units were activated by lateral nucleus stimulation alone, and seven other neurons were orthodromically driven by both lateral and medial nuclei stimulation. Similarly, medial amygdaloid stimulation antidromically discharged five neurons whereas lateral amygdaloid stimulation antidromically activated one neuron. One other neuron was antidromically discharged from both amygdaloid nuclei. DISCUSSION Our findings demonstrate that the HVM gross evoked test response (in paired-amygdala shock analysis) was either facilitated or inhibited depending on whether the animals were anesthetized with urethane or pentobarbital, respectively. Frequency and post-tetanic potentiation results further support the suggestion that the overall excitability of the amygdala-HVM system is differentially modified by both anesthetic agents. Because the field potentials evoked within the HVM by amygdala stimulation in the cat (9, 11) and rat (29) are a reflection of the excitability patterns of single HVM neurons, the differential effect of both anesthetic agents can be extended to explain the general facilitatory effects observed on unit discharges and population spikes when recorded under urethane and the inhibition of neuron activity and of population spikes when recording in pentobarbital-anesthetized animals. Results from experiments using paired-amygdala shock analysis of the HVM field potentials do not support Renaud’s (29) claim that in the rat the test response is consistently reduced in amplitude. In fact, the opposite was true when using animals under urethane anesthesia, a finding which casts serious doubts on the validity of conclusions that have not taken into consideration the type of anesthesia used. Our results coincide with those of Stewart and Scott (35) who found that urethane enhanced and pentobarbital depressed the evoked test responses in the olfactory bulb. When using chronically implanted animals, the evoked field response was always facilitated in the awake animal or after anesthesia with urethane. This was taken as an indication that urethane did not interfere with the normal facilitatory action that amygdaloid stimulation exerts on HVM neurons, nor that it enhanced putative inhibitory mechanisms (11, 29), which, although probably present under this condition, were masked by the more powerful excitatory phenomenon. It would seem, therefore. that

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urethane should be considered the anesthetic agent of choice when carrying out neurophysiological studies on this particular system. On the other hand, the consistent inhibition of the test response seen under pentobarbital can be explained by the proposed dual effect of the anesthetic agent on synaptic transmission, that is a postsynaptic blockade of excitatory synapses-most probably mediated by decreasing the number of synaptic vesicles attached to the presynaptic membrane (14)-coupled with a postsynaptic enhancement of GABA-mediated synaptic inhibition. The latter assumption is substantiated by our results with picrotoxin and by further evidence from the literature: (i) Pentobarbital enhances the action of GABA on postsynaptic neurons, both in vitro (26, 27. 34) and in riro (13, 25), (ii) this inhibitory neurotransmitter is present in cells of the HVM nucleus of the rat (18); and (iii) microiontophoresis of GABA and glycine inhibits firing of HVM neurons in the rat ( 10, 28), whereas picrotoxin (intravenously or iontophoretically injected) antagonizes synaptic inhibition (28), and bicuculline, in addition, antagonizes the reduction in cell firing observed during the application of GABA (10). Furthermore, in situ application of picrotoxin enhances evoked sensory potentials in the caudate nucleus within a time period similar to that observed in our cases (32). An obvious similarity with our results can be drawn from these experiments, as nigral inhibition of striatum is apparently mediated by GABA-releasing interneurons ( 12). The fact that both medial and lateral amygdaloid nuclei stimulation transynaptically or antidromically discharged two different cell groups in the HVM which were only partially coextensive, suggests that the often quoted differential effects observed on hormone output (1, 4), behavioral performance (15), and sexual receptivity (Masco, D. H., an Carrer, H. F., in preparation) after stimulation or destruction of medial and lateral amygdaloid nuclei may be mediated by independent or discrete groups of functionally specified HVM neurons. This later possibility was in fact suggested on purely morphological basis (37). REFERENCES I.

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