Decrease in glutamic acid decarboxylase level in the hypothalamus of spontaneously hypertensive rats.

BACKGROUND: A reduction in gamma-aminobutyric (GABA)-mediated inhibition of pressor sites in the caudal hypothalamus of spontaneously hypertensive rats compared with that of normotensive Wistar-Kyoto rats has recently been demonstrated. OBJECTIVE: To determine whether the reduction in GABA-mediated inhibition of the caudal hypothalamus of the spontaneously hypertensive rats results from reductions both in the number of GABA-synthesizing neurons and in the amount of the GABA-synthesizing enzyme, glutamic acid decarboxylase messenger RNA (mRNA). DESIGN AND METHODS: A polyclonal antibody (Chemicon) for the 67 kDa isoform of glutamic acid decarboxylase (GAD67) was used to immunocytochemically label GABAergic neurons in the caudal hypothalamus of spontaneously hypertensive and Wistar-Kyoto rats that had been treated beforehand with colchicine. The labeled cells were counted for both strains by a blinded analysis and compared. Caudal hypothalamic tissues from spontaneously hypertensive and Wistar-Kyoto rats were analysed for GAD67 mRNA by Northern blotting. The signal intensities of the radioactive probe specific for GAD67 for the two strains were analyzed by using a phosphorimager and compared. Control areas for the immunocytochemical (zona incerta) and Northern blotting (cortex, midbrain, cerebellum, and brain stem) experiments were used to determine regional differences in expression of GAD67. RESULTS: Both the hypothalamus and cerebellum of spontaneously hypertensive and Wistar-Kyoto rats contained GAD67-immunoreactive neurons; however, there were 42% fewer GAD67 neurons in the caudal hypothalamus of spontaneously hypertensive rats than there were in that of Wistar-Kyoto rats. Furthermore, a 33% reduction in the amount of GAD67 messenger RNA in the caudal hypothalamus of spontaneously hypertensive rats compared with that for Wistar-Kyoto rats was demonstrated. Analysis of the expression of GAD67 in the cortex, midbrain, cerebellum, brain stem, and total brain revealed no difference between spontaneously hypertensive and Wistar-Kyoto rats. CONCLUSIONS: Our findings demonstrate that the spontaneously hypertensive rat has fewer neurons synthesizing GABA and less GAD67 mRNA in the caudal hypothalamus than do Wistar-Kyoto rats. This deficit in the GABAergic system in the caudal hypothalamus, a well-known cardiovascular regulatory site, could contribute to the essential hypertension in this animal model.

In vitro effects of GABA and hypoxia on posterior hypothalamic neurons from spontaneously hypertensive and Wistar-Kyoto rats.

Recent studies from this laboratory have shown that neurons in this hypothalamic region are stimulated by hypoxia in vivo and in vitro. In addition, GABAergic activity is depressed in the posterior hypothalamus of the spontaneously hypertensive rat compared to the normotensive rat. The major purposes of the present study were to: a) evaluate if posterior hypothalamic neurons respond differently to GABA in the hypertensive rat compared to the normotensive rat; and b) examine the possibility that hypothalamic neurons from spontaneously hypertensive rats respond differently to hypoxia than those from normotensive rats. In addition, the effects of GABA on hypoxia-sensitive neurons was recorded. Extracellular single unit recordings of hypothalamic neurons were performed in a rat brain slice preparation. Neuronal responses to hypoxia (10% O2/5% CO2/85% N2) and to GABA were recorded from slices taken from both Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Administration of three different concentrations of GABA evoked a dose-related decrease in discharge rate in similar percentages of neurons from both SHR and WKY rats. The magnitude of the depression elicited by GABA did not differ significantly between the neurons from SHR and WKY rats. Hypoxia increased the firing rate of 75% and 69% of the SHR and WKY neurons, respectively; no differences (p > 0.05) were noted in the magnitude of the response or in the percentage of neurons responding to hypoxia between the two strains of rats. The discharge rate of most of these neurons fell to below control level following removal of the hypoxic stimulus. A significant percentage of SHR (75%) and WKY (75%) neurons that were stimulated by hypoxia were inhibited by exogenously applied GABA. These results indicate that a) an altered sensitivity of hypothalamic neurons to GABA does not contribute to hypertension in the SHR and b) the depressed respiratory response to hypoxia in the SHR is not due to a decreased responsiveness of hypothalamic neurons to hypoxia.

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).(ABSTRACT TRUNCATED AT 250 WORDS)

Augmented neuronal activity in the hypothalamus of spontaneously hypertensive rats.

Previous studies indicate a tonic GABAergic inhibitory mechanism in the posterior hypothalamus (PH) contributes to modulating cardiovascular activity. Blockade of GABA receptors on neurons in this area elicits an increase in sympathetic discharge, arterial pressure, and heart rate. It has been proposed that a deficit in this inhibitory system may be responsible for the elevated pressure in the spontaneously hypertensive rat (SHR). The purpose of this study was to determine if the spontaneous neuronal activity in the posterior hypothalamus of spontaneously hypertensive rats differs from that of age-matched normotensive Wistar-Kyoto rats (WKY). Single unit, extracellular recordings of posterior hypothalamic neurons were performed on both in vivo and in vitro preparations. The spontaneous firing rate of posterior hypothalamic neurons in the anesthetized adult SHR was significantly higher (3.66 +/- 0.55 Hz) compared to that of the anesthetized adult WKY rat (2.11 +/- 0.29 Hz). Moreover, more of the neurons in the anesthetized SHR (38%) had a bursting discharge pattern than in the WKY (16%). In order to exclude inputs from peripheral receptors or other brain areas, an in vitro preparation was used. Neurons from both young and adult SHRs also had an increased spontaneous discharge rate and higher percentage of burster-type cells in the posterior hypothalamus compared to neurons from age-matched WKYs in the brain slice preparation. Both the in vivo and in vitro findings support the possibility that an elevated neuronal activity in the posterior hypothalamus, a known pressor area, of the SHR contributes to the development and/or maintenance of hypertension in this animal model.

Hypothalamic GABAergic mechanism involved in respiratory response to hypercapnia.

Previous studies have demonstrated that suprapontine areas of the brain modulate the respiratory responses to hypoxia and hypercapnia. The purpose of the present study was to determine if neurons in the posterior hypothalamus are responsible for this modulation. The respiratory (monitored from diaphragmatic activity) and cardiovascular responses to hypoxia and to hypercapnia were examined in anesthetized rats before and after microinjection of a GABA synthesis inhibitor (3-mercaptopropionic acid, 3-MP) into the posterior hypothalamus. Unilateral micro-injection of 3-MP into the posterior hypothalamus elicited an increase in minute diaphragmatic activity (+54.9 +/- 15.8%), arterial pressure (10.5 +/- 3.2 mmHg) and heart rate (26.8 +/- 10.7 min-1) after a delay of 15-20 minutes. The respiratory responses to hypercapnia but not the cardiovascular responses were greatly accentuated after hypothalamic microinjections of 3-mercaptopropionic acid. In contrast, none of the responses (increases in diaphragmatic activity and heart rate; fall in arterial pressure) elicited by hypoxia were altered after microinjections of the GABA synthesis inhibitor into the posterior hypothalamus. These findings indicate that a GABAergic inhibition of posterior hypothalamic neurons modulates the respiratory response to hypercapnia.

Hypothalamic GABAergic modulation of respiratory responses to baroreceptor stimulation.

Little is known about possible hypothalamic modulation of the respiratory response to baroreceptor activation. The purpose of this study was to determine if the respiratory depression associated with the baroreceptor reflex is modulated by neurons in the posterior hypothalamus. Breathing frequency and tidal diaphragmatic activity were derived from diaphragmatic electromyographic recordings in anesthetized rats. Respiratory responses to baroreceptor activation were analyzed before and after unilateral microinjections of GABA antagonists (picrotoxin, bicuculline methiodide) or a GABA synthesis inhibitor (3-mercaptopropionic acid, 3-MP) into the posterior hypothalamus. Baroreceptor stimulation prior to microinjections elicited a decrease in both breathing frequency and tidal diaphragmatic activity. Microinjection of picrotoxin elicited an increase in respiratory activity. The decrease in tidal diaphragmatic activity evoked by baroreceptor stimulation was blocked after the microinjection. Furthermore, the baroreceptor-induced fall in breathing frequency was converted to an increase in breathing frequency. These effects of picrotoxin were reversed by microinjections of a GABA agonist (muscimol) into the same site. Microinjections of 3-MP also blocked the decrease in breathing frequency associated with baroreceptor stimulation. The GABA antagonist bicuculline methiodide elicited similar effects. These results indicate that a GABAergic mechanism in the posterior hypothalamus modulates the respiratory responses to baroreceptor stimulation.