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Spontaneous and osmotically-stimulated activity in slices of rat hypothalamus.

Single unit activity was recorded from 400-500 mu m thick slices of rat hypothalamus, using either NaCl- or horseradish peroxidase-filled glass micropipettes. Spontaneous activity was present in the following hypothalamic loci: anterior hypothalamic-preoptic area, nucleus circularis, nucleus of the diagonal band of Broca, paraventricular accessory nucleus, paraventricular nucleus (all portions), periventricular regions of the anterior hypothalamus, and the suprachiasmatic nucleus. The supraoptic nucleus was the only major cell group studied to exhibit no spontaneous activity. Cells of the paraventricular and circularis nuclei were spontaneously active, displayed firing rates and patterns of activity similar to those recorded in vivo for magnocellular elements of the hypothalamus, and in some cases responded to increases in the osmolality of the bathing medium with altered firing rates and/or patterns of activity. Many cells in these preparations were characterized by phasic, bursting patterns of activity. Slow, irregular and regular, continuous activity was also frequently observed, as is typical in vivo. Median firing rates were in the range of 4-6 spikes/sec, somewhat faster than the rates usually reported for anesthetized in vivo preparations. These rates are more similar to those observed in unanesthetized monkeys or rats with diencephalic islands. Extracellular HRP marking provided a high degree of localization for many of the recorded cells. These results indicate that the hypothalamic slice preparation is useful for studies in which it is desirable to eliminate extrahypothalamic connections and in which it is necessary to exercise a fine degree of control over the extracellular environment of the cells.

Animals↗

Mutual connections of the raphe system and hypothalamus in relation to fever.

Previous studies in our laboratory have shown that lesions in certain brain stem regions prevent elevations of body temperature after administration of bacterial pyrogen. In the present experiments wer examined the morphology of the connections of these "brain stem thermoregulatory centers" which are represented in all raphe and paraphe nuclei of the brain stem reticular formation. The methods of anterograde degeneration and tracing of retrograde transport of horseradish peroxidase were used to identify afferent and efferent connections within this "thermoregulatory field." Abundant mutual connections between the raphe nuclei and hypothalamus were found. All nuclei of the raphe system receive afferents from the medial and lateral hypothalamus. All raphe nuclei have efferent projections to the medial reticular formation, and the raphe nuclei of the pons and mesencephalon provide ascending fibers to the hypothalamus. A lesion of any part (origin, course, termination field) of this mutual raphe-hypothalamic pathway system will prevent development of fever in response to bacterial pyrogen.

Animals↗

Effects of neuropeptide Y on norepinephrine and serotonin metabolism in rat hypothalamus in vivo.

A double-barrelled cannula and microdialysis system were used to inject 100 pmoles of neuropeptide Y (NPY) and to collect from the local microenvironment samples of norepinephrine, 3-methoxy-4-hydroxyphenylglycol (MHPG), serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA) and dihydroxyphenylacetic acid (DOPAC) a metabolite of dopamine. Following the administration of 100 pmoles of NPY into the ventromedial hypothalamus of conscious rats, there was a significant reduction in the local concentration of norepinephrine and an even greater reduction in the concentration of its metabolite, MHPG, as compared to saline-injected controls. The concentration of serotonin and its metabolite 5-HIAA as well as DOPAC were also reduced in the ventromedial hypothalamus following local injection of NPY. When NPY was injected into the lateral hypothalamus and monoamines measured from areas adjacent to the injection of NPY there was a significant increase in norepinephrine release and a significant increase in the concentration of DOPAC. Serotonin was significantly decreased and there was an increase in the ratio of 5-HIAA to serotonin. NPY is known to stimulate food intake when injected into either area. The present data are consistent with the concept that a reduction in concentration of serotonin is involved in the regulation of food intake following injection of NPY.

3,4-Dihydroxyphenylacetic Acid↗

Profile of NE, DA and 5-HT activity shifts in medial hypothalamus perfused by 2-DG and insulin in the sated or fasted rat.

This study was carried out in the unrestrained rat to determine the nature of the in vivo profile of monoamine neurotransmitters within the medial hypothalamus in response to the presence of a glucoprivic or metabolic challenge to neurons within this region. In these experiments, insulin or 2-deoxy-D-glucose (2-DG) was applied locally to the paraventricular nucleus (PVN), dorsomedial nucleus (DMN) and ventromedial hypothalamus (VMH). In each of 11 Sprague-Dawley rats, a guide cannula was implanted stereotaxically to rest just above these structures. Upon recovery, a concentric push-pull cannula system was used to perfuse an artificial CSF within a medial hypothalamic site. The CSF was perfused at a rate of 20 microliters/min with a 5.0 min interval intervening between the collection of each 100 microliters sample. After the rat was fasted for 20-22 hr, either 10 micrograms/microliters 2-DG or 4.0 mU/microliters of insulin was incorporated into the control CSF medium and perfused at the same locus. The aliquots of hypothalamic perfusate were assayed by high performance liquid chromatography with electrochemical detection (HPLC-EC) for the respective concentration in pg/microliter of norepinephrine (NE), dopamine (DA), serotonin (5-HT) and each of their major metabolic products. When the rat was sated, 2-DG enhanced significantly the mean efflux of NE from the medial hypothalamus in comparison to control CSF values. However, under the fasted condition, 2-DG augmented the turnover of both the catecholamine and 5-HT as reflected by elevated levels of MHPG and 5-HIAA, respectively. On the other hand, insulin perfused within the same medial hypothalamic sites evoked a significant increase in the synthesis and release of DA from the sated rat, but did not alter its turnover. Following the interval of fast, insulin produced no immediate alteration in transmitter activity; however, in the interval following insulin's perfusion, DA and 5-HT turnover were enhanced while the efflux of 5-HT was suppressed. An analysis of the proportional values of the levels of the amines to each other revealed marked shifts in the relationships between the catechol- and indoleamine transmitters following local perfusion with both 2-DG and insulin. Overall, NE synthesis and turnover exceeded that of 5-HT following 2-DG, whereas DA predominated over NE and 5-HT during insulin's perfusion.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

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)

3-Mercaptopropionic Acid↗

Decrease of norepinephrine and preservation of acetylcholine in the hypothalamus of VMH obese rats.

We investigated to find which types of neuronal disturbance in the hypothalamus are responsible for ventromedial hypothalamic nucleus (VMH) lesion-induced development of obesity. We found that in VMH-lesioned obese rats, the contents of norepinephrine (NE) and dopamine in the hypothalamus were selectively decreased, but that the serotonin and acetylcholine levels were unchanged from those in sham controls. Also, the content of NE in the lateral portion of the hypothalamus was decreased. Our results show that disturbance of the hypothalamic noradrenergic and dopaminergic neurons, but not of the serotonergic or cholinergic neurons, contributes to the development of VMH lesion-induced obesity.

Acetylcholine↗

Glucagon-like peptide-1(7-36)amide induces the release of aspartic acid and glutamine by the ventromedial hypothalamus of the conscious rat.

Glucagon-like peptide-1(7-36)amide (GLP-1(7-36)amide) and its own receptor have been found in the hypothalamus and brain stem of the rat. In an attempt to gain further insight into the role of this peptide in brain functioning we investigated the effects of GLP-1 (7-36)amide on the release of excitatory amino acid neurotransmitters by the ventromedial hypothalamus using an experimental microdialysis approach. GLP-1(7-36)amide produced an immediate increase in the extracellular concentrations of aspartic acid and glutamine, p < 0.01 and p < 0.05, respectively. By contrast, extracellular concentrations of glutamic acid, alanine, threonine, and tyrosine were unaffected. The results of this study show a stimulatory effect of GLP-1(7-36)amide on the release of aspartic acid and glutamine by the ventromedial hypothalamus of the rat.

Animals↗

Methanol-induced monoamine changes in hypothalamus and striatum of albino rats.

This paper suggests that like ethanol, methanol also produces certain changes in the steady state level of monoamines in hypothalamus and striatum of albino rats. Though, the toxic manifestations of methanol are attributed to the metabolic end product of methanol viz. formic acid by several workers, we report here that the methanol-induced brain monoamine changes, at least, could be attributed to the direct action of methanol rather than to its metabolic end products like formaldehyde or formate. Studies in the steady state level of rat brain monoamines have shown that after methanol administration (3 g/kg), there is severe depletion of dopamine level in striatum but a significant increase in the level of dopamine, serotonin and 5-hydroxy indole acetic acid in hypothalamus. At the same time, norepinephrine and epinephrine levels are reduced in hypothalamus as well as in striatum. These effects do not seem to be induced by metabolic acidosis. The changes in monoamine levels are very well correlated with the blood and brain level of methanol as evidenced by maintaining a higher methanol level either by simultaneous administration of ethanol or by blocking methanol metabolism by pretreatment with 4-methyl pyrazole and 3-amino-1,2,4-triazole. It is thus postulated that monoamine changes induced by methanol appear to be the direct effect of methanol per se on the monoaminergic neuronal membranes.

Amitrole↗

Species differences in epinephrine concentration and norepinephrine N-methyltransferase activity in hypothalamus and brain stem.

1. The concentration of epinephrine, norepinephrine and dopamine, and the activity of norepinephrine N-methyltransferase, the epinephrine-forming enzyme, were determined in hypothalamus and brain stem in several species. 2. Epinephrine concentration in hypothalamus, a nerve terminal region, varied in the order frog greater than turtle greater than chicken greater than cat greater than dog greater than pigeon greater than rat greater than ferret greater than hamster greater than mouse, with concentrations being undetectable in rabbits, horses and guinea pigs. 3. Epinephrine concentration was lower than norepinephrine concentration in all species except the frog. 4. NMT activity was detected in all species except guinea pigs. 5. Epinephrine concentration was lower in brain stem, a cell body region, than in hypothalamus in all species. Only in the frog brain stem was there more epinephrine than norepinephrine. 6. No epinephrine or NMT activity was detected in either brain region in guinea-pigs.

Animals↗

Occurrence of secretogranin II in the prolactin-immunoreactive neurons of the rat lateral hypothalamus: an in situ hybridization and immunocytochemical study.

The occurrence of secretogranin II in a neuron population of the rat lateral hypothalamus specifically detected by an anti-serum to ovine prolactin was examined. As this population was previously reported to synthesize dynorphin, the distribution of neurons recognized by ovine prolactin-, dynorphin B- and secretogranin II anti-sera was investigated on adjacent sections of hypothalami. The prolactin immunoreactive neurons were the only cells in the lateral hypothalamus to be stained by secretogranin II anti-serum. Moreover, coupling immunocytochemical detection and in situ hybridization with an oligonucleotide probe complementary to secretogranin II mRNA showed that these neurons expressed the secretogranin II gene. These new findings should help to study the physiological role of the prolactin immunoreactive neurons of the lateral hypothalamus.

Animals↗

Interaction of chlorphenvinphos with cholinergic receptors in the rabbit hypothalamus.

The purpose of this study was to find out whether chlorphenvinphos (CVP), an organophosphorous pesticide, interacts with the muscarinic cholinergic receptors in CNS. To attain this goal, the effects of intrahypothalamic injections of oxotremorine (Ox), a muscarinic agonist, and physostigmine (Phys), a carbamate anticholinesterase, were compared with those produced by intrahypothalamic injections of CVP in the rabbit. It was found that the infusion of Ox (20 micrograms) as well as Phys (200 micrograms) into the anterior hypothalamus leads to an increase in the 4-7 Hz theta rhythm in the hippocampus and to the appearance of behavioral symptoms suggestive of a threat response. In the case of Ox, the effects could be prevented by injections of 20 micrograms scopolamine, a muscarinic antagonist. Pretreatment of the hypothalamus with 100 micrograms hemicholinium (HC-3) did not prevent the effects of Phys injected 2 h later. (HC-3 prevents the resynthesis of acetylcholine by blocking choline reuptake. This leads to a gradual depletion of ACh stores and to an inhibition of the cholinergic transmission). It suggests that Phys activates directly postsynaptic muscarinic receptors. Intrahypothalamic injections of CVP in doses of up to 1360 micrograms produced no overt changes in behavior nor in the hippocampal EEG of the rabbit and did not prevent the effect of subsequent injections of Ox. This suggests that CVP is neither an agonist nor antagonist of the muscarinic receptors in the rabbit hypothalamus.

Acetylcholinesterase↗

Epilepsy and hormonal regulation: the patterns of GnRH and galanin immunoreactivity in the hypothalamus of epileptic female rats.

Endocrine and reproductive alterations are frequently reported to occur in women with temporal lobe epilepsy as well as in female rats in different experimental models of limbic seizures. We have recently observed that rats with structural damage of limbic structures induced by sustained convulsions triggered by systemic administration of pilocarpine develop spontaneous seizures after a mean latency of 15 days. In order to investigate the possible substrate of endocrine alterations in epilepsy, changes of the gonadotropin-releasing hormone (GnRH) and the neuropeptide galanin (GAL) were studied in the hypothalamus of epileptic female rats after pilocarpine treatment. Female rats injected with pilocarpine (320-350 mg/kg, i.p.) and control cases injected with saline were killed 10-20 h, 10-15 or 60-90 days following treatment. In some of these animals colchicine was injected in the lateral cerebral ventricle 24 h before death. GnRH immunopositivity was observed in the hypothalamus in neuronal cell bodies, fibers and punctate elements of both epileptic and control cases. A striking reduction of the density of GnRH-immunoreactive fibers and puncta was observed in the hypothalamus of the epileptic female rats killed 10-15 or 60-90 days following pilocarpine administration. No significant differences were observed in the number and size of GAL-immunoreactive perikarya of epileptic and control cases. The present findings suggest that a substantial rearrangement of GnRH-containing efferents, and in particular a loss of their terminal branches, occurs in the epileptic rat brain. Comparable regressive changes could account for alterations in endocrine and reproductive functions observed in temporal lobe epilepsy.

Animals↗

Testosterone metabolism in the avian hypothalamus.

Many central actions of testosterone (T) require the transformation of T into several metabolites including 5 alpha-dihydrotestosterone (5 alpha-DHT) and estradiol (E2). In birds as in mammals, 5 alpha-DHT and E2, alone or in combination, mimic most behavioral effects of T. The avian brain is, in addition, able to transform T into 5 beta-DHT, a metabolite which seems to be devoid of any behavioral or physiological effects, at least in the context of reproduction. By in vitro product-formation assays, we have analyzed the distribution, sex differences and regulation by steroids of the 3 main T metabolizing enzymes (aromatase, 5 alpha- and 5 beta-reductases) in the brain of the Japanese quail (Coturnix c. japonica) and the zebra finch (Taeniopygia guttata castanotis). In the hypothalamus of quail and finches, aromatase activity is higher in males than in females. It is also decreased by castration and increased by T. The activity of the 5 alpha-reductase is not sexually differentiated nor controlled by T. The 5 beta-reductase activity is often higher in females than in males but this difference disappears in gonadectomized birds and no clear effect of T can be observed at this level. The zebra finch brain also contains a number of steroid-sensitive telencephalic nuclei [e.g. hyperstriatum ventrale, pars caudale (HVc) and robustus archistriatalis (RA)] which play a key role in the control of vocalizations. These nuclei also contain T-metabolizing enzymes but the regulation of their activity is substantially different from what has been observed in the hypothalamus. Aromatase activity is for example higher in females than in males in HVc and RA and the enzyme in these nuclei is not affected by castration nor T treatment. In these nuclei, the 5 alpha-reductase activity is higher in males than in females and the reverse is true for the 5 beta-reductase. These sex differences in activity are not sensitive to gonadectomy and T treatment and might therefore be organized by neonatal steroids. We have been recently able to localize aromatase-immunoreactive (AR-ir) neurons by ICC in the brain of the quail and zebra finch. Positive cells are found in the preoptic area, ventromedial and tuberal hypothalamus. AR-ir material is found in the perikarya of cells and fills the entire cellular processes including axons. At the electron microscope level, immunoreactive material can clearly be observed in the synaptic boutons. This observation raises questions concerning the mode of action of estrogens produced by central aromatization of T.

Animals↗

EST-based identification of genes expressed in the hypothalamus of adult tilapia, Oreochromis mossambicus.

The hypothalamus is involved in many physiological functions in teleosts. To accelerate the molecular analysis of hypothalamic functions, a list of transcripts expressed in the hypothalamus of adult tilapia, Oreochromis mossambicus, was compiled using the expressed sequence tag (EST) strategy. Of 161 clones, 130 clones were unique genes and 31 clones were found to be redundant. Of 130 unique genes, 32.3% (42/130 ESTs) were identified as known genes and 67.7% (88/130 ESTs) as unknown genes. The functional categorization of the known genes was analyzed. Bioinformatic analysis revealed that 62 of 88 unknown genes (62/130 ESTs, 47.7%) showed a significant homology to neither nucleotide nor translated peptide sequences in the public database. These genes might be particularly expressed in the tilapia hypothalamus.

Amino Acid Sequence↗

Involvement of nuclei in the hypothalamus in cardiac sympathoexcitatory reflexes in cats.

The hypothalamus is considered to be an important area in the central regulation of cardiovascular function. However, its role in processing excitatory cardiovascular reflexes induced by stimulation of cardiac afferents has not been established. In the present study, using c-Fos immunoreactivity, we located neurons in the hypothalamus activated by inputs from cardiac sympathetic afferents. Following bilateral barodenervation and cervical vagotomy in anesthetized cats, bradykinin (BK, 1-10 microg, in 0.1 ml; n=7) was applied repetitively (6x, every 20 min) to the anterior epicardial surface of the left ventricle. This chemical stimulation caused consistent excitatory cardiovascular reflexes characterized by increases in blood pressure (BP) and heart rate (HR), while the vehicle for BK (0.9% saline, n=6) produced no such responses. Compared to control cats, c-Fos immunoreactive cells were significantly increased (P<0.05) in the arcuate nucleus (ARC), dorsal hypothalamic area (HDA), dorsomedial nucleus, paraventricular hypothalamic nucleus (PVN) and periventricular nucleus in the BK-treated animals. More neurons double-labeled with c-Fos and nitric oxide synthase (NOS) were observed in the PVN following epicardial application of BK (P<0.05). There was no significant increase in co-localization of these two labelings in the other nuclei. These results suggest that several nuclei in the hypothalamus respond to activation of cardiac sympathetic afferents, leading to sympathoexcitatory reflexes. Nitric oxide (NO) may function as a neurotransmitter or as a neuromodulator in the PVN during these cardiac-cardiovascular responses.

Afferent Pathways↗

Leptin inhibits norepinephrine efflux from the hypothalamus in vitro: role of gamma aminobutyric acid.

Leptin, a hormone secreted by adipocytes, produces a number of central and neuroendocrine effects, the mechanisms behind which are not completely understood. Hypothalamic norepinephrine (NE) is involved in many of the neuroendocrine effects that are associated with leptin. Therefore, we hypothesized that leptin could affect hypothalamic NE activity to bring about its central and neuroendocrine effects. Because gamma aminobutyric acid (GABA) is known to affect the release of NE, we also tested the possibility that leptin-induced changes in NE could be mediated through GABA. The mediobasal hypothalami from adult male rats were incubated in an in vitro incubation system for four consecutive incubation periods of 60 min each at 37 degrees C in Krebs Ringers Henseleit (KRH) solution in an atmosphere of 95% O(2) and 5% CO(2.) After determining the basal release, the hypothalami were challenged with 0, 0.1, 1 or 10 nm of leptin, bicuculline (a GABA-A receptor antagonist; 10 microM) and bicuculline (10 microM) +10 nM of leptin during the second incubation period. Residual effects of leptin were measured in the third incubation where tissues were incubated with KRH alone, and the viability of tissues was determined in the fourth incubation when tissues were exposed to high K(+) KRH. NE levels in the incubation medium were measured using high-performance liquid chromatography with electrochemical detection (HPLC-EC). Leptin inhibited NE efflux from the hypothalamus in a dose-dependent manner. Moreover, incubation of hypothalami with 10 nM of leptin and bicuculline, a completely blocked the leptin-induced decrease in NE efflux. These results demonstrate for the first time that leptin could act directly on the hypothalamus to inhibit NE efflux through GABA. It was concluded that leptin could probably produce its central and neuroendocrine effects by modulating NE and GABA levels in the hypothalamus.

Animals↗

Effect of subdiaphragmatic vagotomy on bacterial DNA-induced IL-1beta expression in the mouse hypothalamus.

We investigated whether bacterial DNA (CpG-DNA)-induced IL-1beta expression in the mouse hypothalamus is mediated via afferent vagus nerve. Subdiaphragmatic vagotomy did not modify the CpG-DNA (i.p.)-induced IL-1beta expression in the hypothalamus, indicating that CpG-DNA-induced IL-1beta expression is independent of the afferent vagus nerve originating from the subdiaphragmatic organs. On the other hand, we observed the Toll-like receptor 9 mRNA expression in the hypothalamus, suggesting that circulating CpG-DNA acts directly in the brain.

Animals↗

Mode of leptin action in chicken hypothalamus.

While there have been many studies in various species examining the mode of central leptin action on food intake, there is however a paucity of data in birds. We have, therefore, addressed this issue in broiler chickens because this strain was selected for high growth rate, hence high food intake. Continuous infusion of recombinant chicken leptin (8 microg/kg/h) during 6 h at a constant rate of 3 ml/h resulted in a significant reduction (49-57%) of food intake in 3-week-old broiler chickens (P < 0.05). The effect of leptin within the central nervous system (CNS) was mediated via selective hypothalamic neuropeptides. Leptin significantly decreased the expression of its receptor (Ob-R), neuropeptide Y (NPY), orexin (ORX), and orexin receptor (ORXR) (P < 0.05), but not that of agouti-related protein (AgRP) (anabolic/orexigenic effectors) in chicken hypothalamus. However, the catabolic/anorexigenic neuropeptides namely proopiomelanocortin (POMC) and corticotropin-releasing hormone (CRH) mRNA levels remained unchanged after leptin treatment. Despite the absence of leptin effect on AgRP (the antagonist of melanocortin receptor MCR) and POMC (the precursor of alpha-melanocyte stimulating hormone which is a potent agonist for MCR), leptin significantly decreased the expression of MCR-4/5 gene in chicken hypothalamus (P < 0.05) suggesting that leptin acts directly (as ligand) or indirectly (via other ligands) on MCRs to regulate food intake in birds. Additionally, leptin down-regulated the expression of fatty acid synthase (FAS) gene in chicken hypothalamus, indicating an additional pathway of leptin action on food intake such as described for FAS inhibitors. These findings provide new insight into the mechanism of leptin control of food intake in chickens.

Agouti-Related Protein↗