Microinjection of GABA antagonists into the posterior hypothalamus elicits locomotor activity and a cardiorespiratory activation

It is well known that electrical stimulation of an area (subthalamic locomotor region, STLR) of the posterior hypothalamus evokes locomotion as well as increases in cardiorespiratory activity. Uncertainty exists over whether these responses are due to stimulation of STLR neurons or to activation of fibers of passage originating outside this area. The purpose of the present study was to determine if stimulation of STLR neurons alone would elicit the cardiorespiratory and locomotor responses. Neurons were stimulated by microinjections of gamma-aminobutyric acid (GABA) antagonists (picrotoxin and bicuculline) into the posterior hypothalamus of anesthetized cats. Both picrotoxin and bicuculline produced increases in arterial pressure, heart rate and minute ventilation which were accompanied by locomotor movements of the limbs. Increases in arterial pressure, heart rate and phrenic nerve activity were also caused by picrotoxin microinjections in paralyzed, ventilated cats. Microinjections of GABA or a GABA agonist (muscimol) reversed all of these responses. In contrast, microinjection of GABA or muscimol into the STLR without a prior antagonist injection had only small, transient effects upon cardiorespiratory activity. However, microinjection of muscimol prevented the responses to a subsequent injection of bicuculline. These results indicate that: (1) stimulation of cell bodies alone in the subthalamic locomotor region of the hypothalamus produces all the cardiorespiratory and locomotor responses evoked by electrical stimulation; (2) the responses evoked by picrotoxin and bicuculline are due to an interaction with GABA receptors and (3) a GABAergic mechanism exerts a tonic depressive influence over the cardiorespiratory and locomotor systems by an action in the posterior hypothalamus.     

Cardiovascular responses to blockade of GABA synthesis in the hypothalamus of the spontaneously hypertensive rat

Previous studies have suggested that a decreased inhibitory input onto neurons within the posterior hypothalamus (PH), a known pressor area, may contribute to hypertension in the spontaneously hypertensive rat (SHR). Recent experiments from this laboratory have shown that neurons in the PH of the SHR have an altered and elevated discharge frequency compared to those in the normotensive rat. In addition, biochemical studies have reported that there is a decreased concentration of the inhibitory neurotransmitter, GABA, in the hypothalamus of the SHR. The objective of the present study was to assess any variations in GABAergic modulation of cardiovascular activity in SHRs compared to normotensive Wistar-Kyoto (WKY) rats and Sprague-Dawley (SD) rats. Arterial pressure and heart rate responses to microinjections of the GABA synthesis inhibitor 3-mercaptopropionic acid (3-MP) into the posterior hypothalamic area of anesthetized young (6–8 weeks) and mature (11–16 weeks) hypertensive and normotensive rats were recorded. Microinjection of 3-MP elicited increases in arterial pressure of 17.4 ± 3.9 mmHg, 18.1 ± 7.8 mmHg, 16.9 ± 6.4 mmHg, and 10.4 ± 3.5 mmHg in the mature WKY, mature SD, young WKY, and young SHR, respectively. In addition, heart rate was elevated by 33.2 ± 21.9 beats/min, 70.0 ± 25.3 beats/min, 56.3 ± 15.0 beats/min and, 45.9 ± 10 beats/min in the mature WKY, adult SD, young WKY, and young SHR groups, respectively. In contrast, microinjection of 3-MP into the posterior hypothalamus of adult SHRs produced no significant change in arterial pressure (−5.0 ± 1.8 mmHg) or heart rate (+5.3 ± 6.1 beats/min). In three of the adult SHRs, cardiovascular responses to electrical stimulation in the PH were compared to responses elicited by microinjection of 3-MP into the same PH site. Electrical stimulation produced large increases in both arterial pressure and heart rate; however, microinjection of 3-MP produced no significant changes in cardiovascular activity. These results indicate that spontaneously hypertensive rats have a deficiency in the tonic GABAergic input onto posterior hypothalamic neurons. This alteration may contribute to the maintenance of the elevated blood pressure in spontaneously hypertensive rats.     

Autonomic impairment in tetanus: delayed baroreflex involvement

Autonomic nervous system impairment plays an important role in the clinical course of tetanus and is thought to be responsible for life-threatening complications. It is believed to be associated with predominance of sympathetic activity. Direct baroreflex involvement has not yet been reported. We hypothesized that impaired baroreflex may contribute to the autonomic cardiovascular dysregulation in tetanus. In a patient with tetanus baroreflex sensitivity was measured on the first 5 consecutive days non-invasively using a Finometer device. Baroreflex gain was calculated as sequential cross-correlation between heart rate and blood pressure. Short-time pulse interval standard deviations (SDNN) were derived. Additionally, heart rate and arterial blood pressure were monitored and recorded continuously. Baroreflex gain values and SDNN were compared to a sex- and age-matched control subject. Compared to the control subject the patient with tetanus initially did not show a significant difference in baroreflex gain values (mean 3.68 vs 3.15, p=0.1). However, in the course of the disease an almost complete baroreflex failure occurred (mean 1.0 vs 3.15 and 0.97 vs 3.15, both p<0.0001). No correlation was found between the dynamics of baroreflex gain values and blood pressure or heart rate variability expressed by standard deviation and variance. All 5 measurements in the tetanus patient showed decreased short-time SDNN when compared to the control subject and healthy standards. In our patient we found baroreflex impairment as a part of complex autonomic dysfunction in tetanus. Furthermore, baroreflex impairment occurred only delayed. Blood pressure instability could not be explained by baroreflex dynamics. We suggest that a shift towards sympathetic activity possibly overruled the effects of decreased baroreflex sensitivity on blood pressure regulation.     

Cardiovascular regulation through hypothalamic GABA(A) receptors in a genetic absence epilepsy model in rat

PURPOSE: Gamma-aminobutyric acid (GABA) plays a vital role in both central cardiovascular homeostasis and pathogenesis of epilepsy. Epilepsy affects autonomic nervous system functions. In this study, we aimed to clarify the role of GABAA receptors in hypothalamic cardiovascular regulation in a genetically determined animal model of absence epilepsy. METHODS: Nonepileptic Wistar rats and genetic absence epilepsy rats from Strasbourg (GAERS) were instrumented with a guide cannula for drug injection and extradural electrodes for EEG recording. After a recovery period, iliac arterial catheters were inserted for direct measurement of mean arterial pressure and heart rate. Bicuculline, a GABA(A)-receptor antagonist, was injected into the dorsomedial (DMH) or posterior (PH) hypothalamic nuclei of nonepileptic control rats or GAERS. Blood pressure, heart rate, and EEG recordings were performed in conscious unrestrained animals. RESULTS: Bicuculline injections into the hypothalamus produced increases in blood pressure and heart rate of both control rats and GAERS. The DMH group of GAERS showed a twofold increase in the blood pressure and the heart rate compared with those of control rats. Pressor responses to bicuculline, when microinjected into the PH, were similar in the nonepileptic animals and GAERS. Conversely, the amplitude of tachycardic responses to the administration of bicuculline into the PH was significantly higher in GAERS compared with those of control rats. CONCLUSIONS: The bicuculline-induced increases in blood pressure and heart rate were more prominent when given in the DMH of GAERS. These results indicate an increased GABA(A) receptor-mediated cardiovascular response through the DMH in conscious rats with absence epilepsy.     

Medial prefrontal cortical lesions modulate baroreflex sensitivity in the rat

Previous neuroanatomical studies in rats have demonstrated that the medial prefrontal cortex sends projections to the nucleus of the solitary tract which also receives the bulk of baroreceptor information from primary afferents within the IXth and Xth cranial nerves. The present study examines the influence of the prefrontal cortex on baroreceptor heart rate reflex in conscious rats. Baroreceptor reflex activity was examined in rats with bilateral excitotoxin (N-methyl-d-aspartate)-induced lesions of the medial prefrontal cortex and in control rats (artificial cerebrospinal fluid). Seventeen to eighteen days after lesioning, reflex heart rate responses were recorded following intravenous bolus doses of the pressor agent phenylephrine and the depressor agent sodium nitroprusside. Baroreceptor reflex parameters i.e., maximum and average baroreceptor reflex gain (or sensitivity): minimum and maximum heart rate plateaus; heart rate range; upper and lower reflex thresholds were determined by sigmoidal computerized curve-fitting. Lesioning the medial prefrontal cortex did not affect resting mean arterial pressure and heart rate. However, the lesion reduced maximum and average baroreceptor reflex gain and produced a small reduction in lower reflex threshold. The other parameters were unaffected by the lesion. These observations suggest that although the medial prefrontal cortex does not exert a tonic influence or brainstem vasomotor neurons, there may be a descending excitatory projection from this brain region to medullary neurones involved in the baroreceptor reflex arc.     

The role of amygdala and hypothalamus in GABAA antagonist bicuculline-induced cardiovascular responses in conscious rats

γ-Aminobutyric acid (GABA) is known to play an important role in the central control of cardiovascular functions. GABAergic agonists and antagonists elicit blood pressure and heart rate changes when injected into the brain. It was demonstrated here that bicuculline methiodide (BMI), a GABA_A antagonist, caused dose-dependent increases in both blood pressure and heart rate in conscious rats when injected intracerebroventricularly. The roles of the central nucleus of the amygdala (CeA), the paraventricular nucleus (PVN) and the dorsomedial nucleus (DMH) of the hypothalamus in BMI-induced blood pressure and heart rate changes were investigated in this study. The pressor effect of BMI was significantly attenuated by the electrolytic ablation of DMH and PVN, whereas it was only slightly, but insignificantly reduced by CeA lesions. The microinjection of BMI into the DMH and the PVN elicited significant pressor and tachycardic responses whereas only a slight increase was observed in rats injected BMI into the CeA. The BMI-induced increases in both blood pressure and heart rate were more prominent when given into the DMH. These results indicate that the DMH plays an important role in GABAergic control of cardiovascular functions. The PVN and CeA seem to have a minor part in this respect.     

Starvation reduces norepinephrine activities in both hypothalamus and heart in rats

Norepinephrine (NE) activities in both hypothalamus and heart were simultaneously assessed in rats after food-deprivation for 2 days. The technique of gas chromatography-mass spectrometry was employed for the analysis of NE and its primary neuronal metabolite, 3,4-dehydroxyphenylethylene glycol (DHPG), and the ratio of DHPG to NE was used as an index of NE activity. Hypothalamic DHPG/NE ratio was significantly decreased by fasting and was completely reversed by a single day of refeeding. These changes in hypothalamic DHPG/NE ratio were parallel to those in cardiac DHPG/NE ratio. Supporting the concept in which hypothalamic NE neurons play an important role in modulating the sympathetic outflow, it is suggested the decrease in hypothalamic NE activity contributes to the reduction in cardiac NE activity during fasting.     

Further evidence that extracellular serotonin in the rostral ventrolateral medulla modulates 5-HT(1A) receptor-mediated attenuation of exercise pressor reflex

We determined changes in extracellular levels of glutamate, serotonin (5-HT), norepinephrine (NE), and dopamine (DA) within rostral ventrolateral medulla (RVLM) during 5-HT_1A-receptor stimulation-mediated inhibition of cardiovascular responses to static muscle contraction using anesthetized rats. In ten rats, muscle contraction significantly increased (P<0.01) mean arterial pressure (MAP) by 29±4 mmHg, heart rate (HR) by 25±3 bpm, and glutamate levels by 4.5±0.8 ng/5 μl. Microdialysis of a 5-HT_1A receptor agonist, 8-OH-DPAT (10 mM), into the RVLM for 30 min attenuated cardiovascular responses to muscle contraction and had no effect on glutamate levels. A subsequent administration of 10 mM WAY100635, a 5-HT_1A antagonist, into the RVLM antagonized the attenuating effects of 8-OH-DPAT. In another ten rats, muscle contraction significantly increased (P<0.01) MAP and HR by 20±2 mmHg and 25±8 bpm, respectively. In addition, levels of 5-HT, NE, and DA in the RVLM significantly increased (P<0.01) by 3.6±0.3, 3.2±0.3, and 3.3±0.4 pg/10 μl, respectively. Administration of 8-OH-DPAT (10 mM) into the RVLM for 30 min attenuated cardiovascular responses to muscle contraction and had no effects on NE and DA levels. However, the drug significantly attenuated 5-HT levels following a muscle contraction. Microdialysis of 10 mM WAY100635 into the RVLM reversed both cardiovascular and 5-HT changes. These results suggest that stimulation of 5-HT_1A-receptors within the RVLM attenuates cardiovascular responses to static exercise via a reduction of extracellular 5-HT concentration and most likely not through changes in glutamate, NE or DA levels.     

Cardiovascular effects and changes in midbrain periaqueductal gray neuronal activity induced by electrical stimulation of the hypothalamus in the rat

The effects of low-intensity electrical stimulation of sites in the hypodalamus and zona incerta (ZI) on mean blood pressure (MBP), heart rate (HR), and neuronal activity in the midbrain periaqueductal gray (PAC) were investigated in rats. Long-lasting depressor responses were elicited from 67 sites in the hypothalamus and ZI. Effects on HR were variable, except for the ZI where bradycardic responses were evoked. The amplitude of the depressor responses was significantly correlated with baseline MBP on stimulation of the dorsomedial hypothalamic nucleus (DMH) or the dorsal hypothalamic area. Extracellular single-unit recordings were made from 94 PAG neurons. Most units were boated in the ventral half of the PAG (62/94), where spontaneous firing rates were significantly higher than in the dorsal half: 12.5 ± 1.4 spikes/s as compared to 6.0 ± 0.9 spikes/s. Changes in PAG neuronal activity to both ipsi- and contralaterel hypothalamic stimulation were observed. Most neurons were inhibited or unresponsive. There was no site specificity: responsive and unresponsive neurons were scattered throughout the PAG. Inhibition was most effectively evoked by stimulation of the DMH (25 out of 39 neurons) and the Zl (9 out of 15 neurons). In most neurons, the inhibition strictly followed the time course of hypothalamic stimulation. The results suggest that PAG as well as nonPAG pathways participate in the hypothalamic control of cardiovascular function.     

Facilitation of baroreflex-induced bradycardia by stimulation of specific hypothalamic sites in the rat

Hypothalamic stimulation generally inhibits baroreflex-induced bradycardia. However, we have noted discrete areas of the rat hypothalamus which facilitate reflex bradycardia. The effects of hypothalamic stimulation on baroreflex-induced changes in heart rate were investigated in urethane-anesthetized rats (1.2 g/kg, i.p.; n = 6) instrumented with femoral arterial and venous catheters. Bipolar electrodes (250 micron diameter) were implanted stereotaxically in the hypothalamus. Baroreflex-induced bradycardia was elicited by phenylephrine (PE) injection (8-20 micrograms/kg). Responses to stimulation (STIM) (50-150 microA, 80 Hz, 0.5 ms), PE, and Stim + PE were studied for 1 min. In the ventral medial and anterior hypothalamus, STIM caused transient increases in blood pressure and no changes in heart rate. Peak blood pressure was lower during STIM + PE than during PE (144 +/- 5 vs 164 +/- 3 mm Hg; P less than 0.05). However, STIM + PE resulted in a lower heart rate compared to PE (194 +/- 22 22 vs 270 +/- 17 bpm; P less than 0.05). At 1 min, the heart rate in STIM + PE rats remained lower than in PE rats (205 +/- 37 vs 319 +/- 16 bpm; P less than 0.05). Atropine administration indicated that the facilitation was primarily parasympathetic in nature. These results identify specific hypothalamic regions which facilitate baroreflex-induced bradycardia by parasympathetic mechanisms.     

Cardiovascular instability and baroreflex activity in a patient with tetanus

In a patient with tetanus we tested the hypothesis that the hyperadrenergic cardiovascular instability might be due to impairment of the baroreceptor reflex by the tetanus toxin. Baroreflex sensitivity assessed with the phenylephrine method was found to be normal. Changes in arterial pressure correlated inversely with relative changes in plasma volume but not with plasma catecholamine levels. There were both extreme hypo- and hyperadrenergic episodes. We conclude that sympathetic overactivity in tetauns temporarily overrules a functionally intact baroreflex leading to severe blood pressure instability with episodes of hypertension.     

GABA modulates baroreflex in the ventral tegmental area in rat

There are some reports demonstrating the cardiovascular functions of the ventral tegmental area (VTA). About 20–30% of the VTA neurons are GABAergic, which might play a role in baroreflex modulation. This study was performed to find the effects of GABA_A, GABA_B receptors and reversible synaptic blockade of the VTA on baroreflex. Drugs were microinjected into the VTA of urethane anesthetized rats, and the maximum change of blood pressure and the gain of the reflex bradycardia in response to intravenous phenylephrine (Phe) injection were compared with the preinjection and the control values. Microinjection of bicuculline methiodide (BMI, 100 pmol/100 nl), a GABA_A antagonist, into the VTA strongly decreased the Phe‐induced hypertension, indicating that GABA itself attenuated the baroreflex. Muscimol, a GABA_A agonist (30 mM, 100 nl), produced no significant changes. Baclofen, a GABA_B receptor agonist (1000 pmole/100 nl), moderately attenuated the baroreflex, however phaclofen, a GABA_B receptor antagonist (1000 pmole/100 nl), had no significant effect. In conclusion, for the first time, we demonstrated that GABA_A receptors of the VTA strongly attenuate and GABA_B receptors of the VTA moderately attenuate baroreflex in rat. Synapse 69:592–599, 2015 . © 2015 Wiley Periodicals, Inc.     

Sex differences in GABA turnover and glutamic acid decarboxylase (GAD65 and GAD67) mRNA in the rat hypothalamus

GABAergic neurons are estimated to make up more than half of the neuronal population of the hypothalamus and they likely account for some of the structural and functional sexual dimorphisms observed in the mammalian brain. We previously reported sex differences in the rate of GABA turnover in discrete hypothalamic structures of adult rats. In the present study, we extended our search for sex differences in GABA turnover to additional structures, and further determined whether these differences were associated with differences in GAD₆₅ and or GAD₆₇ mRNA levels. Utilizing the GABA transaminase inhibition method, we determined GABA turnover in 14 microdissected brain regions. The rate of GABA turnover was about 2-fold greater in male than in diestrous day one (D₁) female rats in the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis [DBB(ovlt)], anteroventral periventricular nucleus (AVPv), median eminence (ME), and dorsomedial portion of the ventromedial nucleus (VMNdm). A sex difference also was noted in the DBB(ovlt) for GAD₆₅ mRNA determined by microlysate RNase protection assay. Here, GAD₆₅ levels were almost 2-fold greater in male rats, which suggests that differences in the activity of this GAD enzyme isoform contributes to the difference in turnover in this area. Additionally, in the dorsomedial nucleus (DMN), the GAD₆₅ mRNA level was significantly higher in female rats, and in the medial amygdaloid nucleus (Am), GAD₆₇ mRNA was higher in male rats. These data reveal striking sexual dimorphisms in the rate of GABA turnover and in GAD mRNA levels in specific populations of hypothalamic GABAergic neurons. The functional relationships between these GABAergic neurons and sexually dimorphic phenotypes associated with these structures, such as gonadotropin secretion, reproductive behaviors, seizure threshold and others, warrant further investigation.     

Bradycardic and hypotensive responses to microinjection of -glutamate into the lateral aspect of the commissural NTS are blocked by an NMDA receptor antagonist

Baroreflex activation by phenylephrine infusion produces a bradycardic response while microinjection of -glutamate into the most lateral aspect of the commissural nucleus tractus solitarius (NTS, 0.8 mm lateral to the midline) produces bradycardic and hypotensive responses. In the present study we investigated the role of NMDA receptors in the lateral aspect of the commissural NTS (0.8 mm lateral to the midline) in the bradycardic and hypotensive responses to microinjection of -glutamate as well as in the processing of the bradycardic response to the baroreflex activation. The hypotensive and bradycardic responses to -glutamate microinjection into the NTS were blocked by methyl-atropine (intravenous, i.v.), indicating that the hypotensive response was secondary to the bradycardia. Microinjection of -glutamate (1 nmol/50 nl) into the NTS was performed before and after microinjection of increasing doses of phosphonovaleric acid (AP-5, a selective NMDA antagonist) at the same site. The microinjection of AP-5 [0.5 (n=9), 2.0 (n=8) and 10.0 nmol/50 nl (n=7)] into the NTS (0.8 mm lateral to the midline) produced a dose-dependent blockade of the bradycardic and hypotensive responses to -glutamate. In a specific group of rats the microinjection of 10 nmol/50 nl of AP-5 produced a significant reduction in baroreflex sensitivity 2 min after microinjection into the lateral NTS [gain=−1.48±0.12 vs. −0.5±0.2 beats/mmHg, (n=5)], which was reversible. The data show that the bradycardic responses produced by microinjection of -glutamate into the most lateral aspect of the commissural NTS or by activation of the baroreflex were blocked by microinjection of AP-5, indicating that the neurotransmission of the parasympathetic component of the baroreflex in the neurons of the lateral aspect of the commissural NTS involves NMDA receptors.     

Further evidence that extracellular serotonin in the rostral ventrolateral medulla modulates 5-HT1A receptor-mediated attenuation of exercise pressor reflex

We determined changes in extracellular levels of glutamate, serotonin (5-HT), norepinephrine (NE), and dopamine (DA) within rostral ventrolateral medulla (RVLM) during 5-HT_1A-receptor stimulation-mediated inhibition of cardiovascular responses to static muscle contraction using anesthetized rats. In ten rats, muscle contraction significantly increased (P<0.01) mean arterial pressure (MAP) by 29±4 mmHg, heart rate (HR) by 25±3 bpm, and glutamate levels by 4.5±0.8 ng/5 μl. Microdialysis of a 5-HT_1A receptor agonist, 8-OH-DPAT (10 mM), into the RVLM for 30 min attenuated cardiovascular responses to muscle contraction and had no effect on glutamate levels. A subsequent administration of 10 mM WAY100635, a 5-HT_1A antagonist, into the RVLM antagonized the attenuating effects of 8-OH-DPAT. In another ten rats, muscle contraction significantly increased (P<0.01) MAP and HR by 20±2 mmHg and 25±8 bpm, respectively. In addition, levels of 5-HT, NE, and DA in the RVLM significantly increased (P<0.01) by 3.6±0.3, 3.2±0.3, and 3.3±0.4 pg/10 μl, respectively. Administration of 8-OH-DPAT (10 mM) into the RVLM for 30 min attenuated cardiovascular responses to muscle contraction and had no effects on NE and DA levels. However, the drug significantly attenuated 5-HT levels following a muscle contraction. Microdialysis of 10 mM WAY100635 into the RVLM reversed both cardiovascular and 5-HT changes. These results suggest that stimulation of 5-HT_1A-receptors within the RVLM attenuates cardiovascular responses to static exercise via a reduction of extracellular 5-HT concentration and most likely not through changes in glutamate, NE or DA levels.     

GABA receptor mediated suppression of defensive rage behavior elicited from the medial hypothalamus of the cat: role of the lateral hypothalamus

Recently, our laboratory has demonstrated that predatory attack behavior in the cat, elicited by electrical stimulation of the lateral hypothalamus, is suppressed following activation of the region of the medial hypothalamus from which defensive rage behavior is elicited [Han, Y., Shaikh, M.B., Siegel, A., Medial amygdaloid suppression of predatory attack behavior in the cat: II. Role of a GABAergic pathway from the medial to the lateral hypothalamus, Brain Res., 716 (1996) 72–83.]. The mechanism for this suppression is a direct GABAergic projection from the medial to lateral hypothalamus. The present study tested the hypothesis that the inhibitory relationship between these two regions of hypothalamus is reciprocal, namely, that a GABAergic neuron, which also projects from the lateral to medial hypothalamus, serves to suppress defensive rage elicited from the medial hypothalamus. Monopolar stimulating electrodes were implanted into lateral hypothalamic sites from which predatory attack behavior was elicited. In addition, cannula-electrodes were implanted into the medial hypothalamus for elicitation of defensive rage behavior and for microinjections of GABA compounds. Initially, in the absence of drug administration, the effects of dual stimulation of the lateral and medial hypothalamus upon response latencies were compared with those following single stimulation of the medial hypothalamus alone. Dual stimulation significantly (p<0.01) suppressed defensive rage behavior elicited from the medial hypothalamus. Then, administration of the GABA_A receptor antagonist, bicuculline (10–60 pmol), into medial hypothalamic sites from which defensive rage was elicited blocked the suppressive effects of lateral hypothalamic stimulation. The GABA_A receptor agonist, muscimol (0.3–30 pmol), microinjected into the medial hypothalamus, suppressed defensive rage elicited by single stimulation of the medial hypothalamus in a dose dependent manner. These suppressive effects of muscimol upon defensive rage were blocked following pretreatment with bicuculline (60 pmol). Administration of muscimol into adjoining regions of the lateral hypothalamus had no effect upon defensive rage, indicating its site specificity. Bicuculline (60 pmol) delivery into the medial hypothalamus had no effect upon defensive rage, suggesting the, presence of an episodic rather than tonic mechanism. A combination of immunocytochemical and retro grade tracing procedures were then employed to determine the origin of the putative GABAergic pathway projecting to the medial hypothalamus. In this experiment, the retrograde tracer, Fluoro-Gold (8%, 0.5 μl), was microinjected through a cannula-electrode in the medial hypothalamus from which defensive rage had been elicited. Following survival periods of 5–6 days, cats were perfused with 4% paraformaldehyde and brain tissue was processed for immunocytochemical staining of GABA neurons. Retrogradely labeled, immunopositively labeled, as well as Fluoro-Gold and GABA labeled cells, were identified in the lateral hypothalamus. Each type of neuron was distributed over wide regions of the lateral hypothalamus, extending from the area immediately caudal to the optic chiasm to the level of the posterior hypothalamus. Together, the behavioral pharmacological and anatomical data provide evidence of a direct inhibitory projection from the lateral to medial hypothalamus whose functions are mediated by GABA_A receptors. When coupled with our previous findings, these results reveal the presence of reciprocal GABAergic inhibitory pathways between the medial and lateral hypothalamus. The findings suggest that functions associated with either the lateral or medial hypothalamus, but not both, can be activated at a given time.     

GABAergic synaptic transmission modulates swimming in the ascidian larva

To examine the role of the amino acid GABA in the locomotion of basal chordates, we investigated the pharmacology of swimming and the morphology of GABA-immunopositive neurones in tadpole larvae of the ascidians Ciona intestinalis and Ciona savignyi. We verified that electrical recording from the tail reflects alternating muscle activity during swimming by correlating electrical signals with tail beats using high-speed video recording. GABA reversibly reduced swimming periods to single tail twitches, while picrotoxin increased the frequency and duration of electrical activity associated with spontaneous swimming periods. Immunocytochemistry for GABA revealed extensive labelling throughout the larval central nervous system. Two strongly labelled regions on either side of the sensory vesicle were connected by an arc of labelled fibres, from which fibre tracts extended caudally into the visceral ganglion. Fibre tracts extended ventrally from a third, more medial region in the posterior sensory vesicle. Two rows of immunoreactive cell bodies in the visceral ganglion extended neurites into the nerve cord, where varicosities were seen. Thus, presumed GABAergic neurones form a network that could release GABA during swimming that is involved in modulating the time course and frequency of periods of spontaneous swimming. GABAergic and motor neurones in the visceral ganglion could interact at the level of their cell bodies and/or through the presumed GABAergic fibres that enter the nerve cord. The larval swimming network appears to possess some of the properties of spinal networks in vertebrates, while at the same time possibly showing a type of peripheral innervation resembling that in some protostomes.     

The rostral ventrolateral medulla mediates suppression of the circulatory system by the ventromedial nucleus of the hypothalamus

We recently reported that a train of episodic neural discharges within the ventromedial nucleus of the hypothalamus (VMH) associated with suppression of the circulatory system had been determined by monitoring multiple unit activity (MUA). Abrupt increases in neural activity (MUA volleys; 1 to 4 min in duration) accompanied transient decreases in heart rate (HR) and blood pressure (BP), and showed circadian rhythm, occurring every 15 to 30 min in the light phase but seldom in the dark phase. The present study was aimed to determine if neurons in the vasomotor area of the rostral ventrolateral medulla (RVL) are involved in this VMH-induced cardiovascular suppression. MUAs of the VMH and RVL were monitored simultaneously with HR and BP in urethane-anesthetized rats. In synchrony with each MUA volley in the VMH, spontaneous activity of RVL neurons significantly decreased, as well as HR and BP. These RVL neurons are most likely vasomotor neurons because MUA of the RVL was attenuated by baroreceptor reflex activation, and electrical stimulation of these cells through the MUA recording electrodes produced pressor responses. These data suggest that VMH neurons that show a train of episodic discharges suppress the circulatory system at least in part by inhibiting the excitability of vasomotor neurons in the RVL.     

Development of immunoreactive lhrh neurons in the fetal rat hypothalamus

The ontogenesis of immunoreactive (ir) LHRH neurons was investigated in rats applying the double-bridge PAP method of Vacca et al. (J. Histochem. Cytochem. 28, 297–307, 1980). Ir LHRH was first evident in the cell bodies confined in the ventromedial surface of the anterior part of the forebrain vesicles on day 16.5 of gestation. Only one or two cells, if any, were found in the brains examined; the cells are oval showing ir brown granules within the perikarya and also within the processes extended from both poles. On day 17.5 of gestation, a few ir cells appeared in the olfactory cortex, medial septum, medial preoptic area, diagonal band of Broca, and ventro-lateral surface of the anterior hypothalamus, and, with development, their numbers increased gradually. During the prenatal period, no ir cell exists in the arcuate nucleus or in the medial-basal hypothalamus. Beaded ir fibers appeared in the organum vasculosum of the lamina terminalis (OVLT) and in the external layer of the median eminence on 18.5 and 19.5 days of gestation, respectively. The present findings ascertain that the hypothalamic regulation of hypophysial gonadotrophic function exists during fetal period in rats.     

Injection of muscimol in the posterior hypothalamus reduces the pge,-hyperthermia in the rat

The firing rate of the nerves innervating interscapular brown adipose tissue PBAT), MAT and colonic temperatures (T_IBAT and T_c, heart rate, and oxygen (O₂) consumption were monitored in urethane-anesthetized male Sprague-Dawley rats. These variables were measured for 40 min before (baseline values) and 40 min after a 56 ng muscimol injection in the posterior hypothalamus and an intracerebroventricular administration of 500 ng prostaglandin E, (PGE₁). The same variables were monitored in other rats with muscimol injection or PEE₁ administration alone. No drug was Injected in control rats. The results show that muscirnol injection reduces the increases in firing rate, T_IBAT, T_c, heart rate, 02 consumption induced by PGE₁. These findings suggest that GABAergic tone in the posterior hypothalamus is important in the control of thermogenic changes induced by PGE₁.