Characterization of beta-endorphin-related peptides in the caudal medulla oblongata and hypothalamus of the prenatal, postnatal and adult rat.

A comparison was made of beta-endorphin (B-END) concentrations versus post-translation products during the perinatal period in the hypothalamus and the caudal medulla oblongata. The concentration of B-END-like immunoreactivity did not differ statistically between embryonic day 21 (E21) and postnatal day 1 (P1) in either area. There were significant differences in forms, with a shift from larger precursors at E21 to smaller peptides at P1, with the predominant form of B-END being the 31 residue form at E21 in both regions. B-END varied between the two regions at P1, the 27-26 residue predominant in the hypothalamus, and the 31 residue in the caudal medulla.     

Coexistence of substance P- and enkephalin-like peptides in single neurons of the rat hypothalamus

The distribution of substance P- and enkephalin-like immunoreactivity in single cells were examined by the double immunofluorescence method. Substance P- and leucine-enkephalin-like compounds coexisted within individual neurons of some hypothalamic areas such as the medial preoptic area, anterior hypothalamic area, perifornical area, lateral hypothalamic area, premammillary nuclei and posterior hypothalamic nucleus, although they did not coexist in the majority of immunoreactive cells.     

Characterization of alpha-MSH-related peptides released from rat hypothalamic neurons in vitro

Reverse-phase high-performance liquid chromatography analysis, coupled with a sensitive radioimmunoassay for alpha-melanocyte-stimulating hormone (alpha-MSH), was used to characterize the alpha-MSH-related peptides stored in the rat hypothalamus or released from perifused hypothalamic slices. Four peaks of alpha-MSH-like immunoreactivity (alpha-MSH-LI) co-eluting with synthetic des-N alpha-acetyl alpha-MSH, alpha-MSH and their respective sulfoxide derivatives were resolved and quantified. In hypothalamic extract, deacetyl alpha-MSH which was the predominant peptide represented 94.4% of total alpha-MSH-LI content, while the relative amount of alpha-MSH was only 5.6%. Analysis of alpha-MSH-related peptides contained in effluent perifusates showed that deacetyl alpha-MSH and its oxidized form were the major peptides released from neurons in basal conditions or under KCl-induced depolarization (50 mM KCl for 75 min). However, the proportion of acetylated peptide was 3-4 times higher in the perifusion medium than in hypothalamic extracts. Our data indicate that acetylation of des-N alpha-acetyl alpha-MSH may occur during the process of exocytosis. Since acetylation of alpha-MSH markedly increases the behavioural potency of the peptide, these results suggest that regulation of the acetyltransferase activity could be a key mechanism to modulate the bioactivity of alpha-MSH-related peptides in the brain.     

Slice cultures of rat hypothalamus examined by immunohistochemical staining for neurohypophyseal peptides and GFAP

Organotypic cultures were prepared from slices of neonatal rat hypothalami and were immunohistochemically stained for the neurohypophyseal peptides vasopressin and oxytocin, their associated neurophysins, and for glial fibrillary acidic protein (GFAP). Both glial and neural elements survived and matured within the cultures, expressing cellular morphologies and retaining a topographic organization similar to that found in vivo. Neurones producing peptides were readily identified and such peptidergic neurones elaborated processes with an appearance characteristic of beaded axons. These presumptive axons grew in a selective and specific manner over certain regions in the slice cultures while avoiding other regions in a manner similar to that found in vivo. In cocultures of hypothalamus and neurointermediate lobe tissue, peptidergic axons found and grew over the neurointermediate lobe tissue and elaborated extensive terminal arborizations. Thus, it appears that at least some of the cues used for appropriate axonal guidance are maintained in these cultures. Organotypic cultures retain many in vivo characteristics as regards cellular morphology and cellular interactions, yet provide an in vitro environment useful for the study of morphology, physiology, cell biology and neurone-target interaction of hypothalamic neurones.     

Enzyme immunoassays for thyroliberin (TRH) and TRH-elongated peptides in mouse and rat hypothalamus

Enzyme immunoassays (EIAs) for Thyroliberin (TRH) and TRH-elongated peptides were developed. Three haptens less than E-H-P-NH2 (TRH). Less than E-H-P-OH (TRH-OH), and S-K-R-Q-H-P-G-K-R-F (P10) were conjugated by the use of different heterobifunctional cross-linking agents either to sun-flower globulin as carrier or to acetylcholinesterase as tracer. For a same hapten, the same chemical group in the peptide was used to prepare the immunogen and the enzyme conjugate. These EIAs were performed with a second antibody solid phase technique using acetylcholinesterase as label. They permitted the measurement of TRH and TRH-elongated peptides with a sensitivity threshold of 10 fmol/well for TRH and 2 fmol/well for P10. TRH EIA only detected authentic TRH whereas TRH-OH EIA detected TRH and TRH peptides elongated on C terminal part. Anti-P10 serum was specific of TRH peptides elongated both on C and N terminal parts and no cross reactivity was observed with TRH. Using these assays, TRH and TRH-elongated peptides were determined in crude or chromatographed mouse and rat hypothalamus tissular extracts. Several TRH extended forms were identified by P10 EIA, whereas TRH-OH EIA permitted detection of both TRH and TRH-elongated peptides in chromatographed extracts. Authentic TRH was measured by TRH EIA both in crude and chromatographed hypothalamic extracts. These assays can permit the study of the processing and maturation of TRH.     

Regulation of proopiomelanocortin messenger RNA concentrations by opioid peptides in primary cell cultures of rat hypothalamus.

The effects of opioid peptides on a 1.1-kb long proopiomelanocortin messenger RNA (POMC mRNA) have been investigated in rat hypothalamic cells maintained in culture. Most opioid peptides exerted an inhibitory control on POMC mRNA steady-state concentrations. beta-Endorphin caused a 65% maximal inhibitory effect (IC50 = 6.1 x 10(-9) M) while slightly less inhibition was caused by Met- and Leu-enkephalin, dynorphin A and DADLE ([D-Ala2,D-Leu5] enkephalin). The effects of beta-endorphin and of Met-enkephalin were completely reversed by the delta opioid antagonist ICI 174,864 while the kappa-receptor specific antagonist binaltorphimine or the sigma-receptor specific antagonist DTG (1,3-di(2-tolyl) guanidine) respectively blocked the inhibitory actions of dynorphin A and of DADLE. The mu-receptor specific agonist DAGO ([D-Ala2,N-Me-Phe4,Gly5-OL]enkephalin) did not affect POMC mRNA levels. The failure of the dopaminergic D2 antagonist haloperidol to modify the inhibitory effects of opioid peptides argues for a direct inhibitory opioid peptide modulation of hypothalamic POMC mRNA levels mediated by the delta-, kappa- and sigma- (but not mu-) receptors in vivo.     

Effect of opioid peptides onl-noradrenaline-stimulated cyclic AMP formation in homogenates of rat cerebral cortex and hypothalamus

Morphine and the opioid peptides leucine-enkephalin (leu-enk), methionine-enkephalin (met-enk) and beta-endorphin had no effect on basal cyclic AMP levels in rat cerebral cortex and hypothalamus, but each inhibited noradrenaline (NA)-stimulated cyclic AMP formation in both brain regions. This inhibition was reversed by naloxone. Naloxone did not reverse phentolamine- or propranolol-induced inhibition of NA-stimulated cyclic AMP formation. The increase in cyclic AMP formation induced by NaF or MnCl2 was unaffected by met-enk or morphine. These data suggest that in rat cerebral cortex and hypothalamus opiates bind to opiate receptors and that the opiate-receptor complex interferes with noradrenergic receptor activity.     

Characterization of alpha-melanocyte-stimulating hormone (alpha-MSH)-like peptides in discrete regions of the rat brain. In vitro release of alpha-MSH from perifused hypothalamus and amygdala

The neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) is synthesized by discrete populations of hypothalamic neurons which project in different brain regions including the cerebral cortex, hippocampus and amygdala nuclei. The purpose of the present study was to identify the alpha-MSH-immunoreactive species contained in these different structures and to compare the ionic mechanisms underlaying alpha-MSH release at the proximal and distal levels, i.e. within the hypothalamus and amygdala nuclei, respectively. The molecular forms of alpha-MSH-related peptides stored in discrete areas of the brain were characterized by combining high-performance liquid chromatography (HPLC) separation and radioimmunoassay detection. In mediobasal and dorsolateral hypothalamic extracts, HPLC analysis confirmed the existence of a major immunoreactive peak which co-eluted with the synthetic des-N alpha-acetyl alpha-MSH standard. In contrast, 3 distinct forms of immunoreactive alpha-MSH, which exhibited the same retention times as synthetic des-, mono- and di-acetyl alpha-MSH, were resolved in amygdala nuclei, hippocampus, cortex and medulla oblongata extracts. The proportions of acetylated alpha-MSH (authentic alpha-MSH plus diacetyl alpha-MSH) contained in these extrahypothalamic structures were, respectively, 78, 80, 60 and 92% of the total alpha-MSH immunoreactivity. In order to compare the ionic mechanisms underlaying alpha-MSH release from hypothalamic and extrahypothalamic tissues, we have investigated in vitro the secretion of alpha-MSH by perifused slices of hypothalamus and amygdala nuclei. High potassium concentrations induced a marked increase of alpha-MSH release from both tissue preparations. However, a higher concentration of KCl was required to obtain maximal stimulation of amygdala nuclei (90 mM) than hypothalamic tissue (50 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Lateralization of LH-RH in rat hypothalamus

An asymmetrical LH-RH distribution in rat hypothalamus has been found. In Wistar rats LH-RH content in the right hypothalamus exceeds that in the left one; in albino rats a contrary distribution is observed. LH-RH lateralization changes during a 24-h period. Unilateral castration or cold stress lead to a shift in LH-RH distribution in the hypothalamus.     

Comparative distribution of bombesin/GRP- and substance-P-like immunoreactivities in rat hypothalamus

Immunohistochemical localization of bombesin/gastrin-releasing peptide ( GRP )-like immunoreactivity (BN/ GRP -LI) and substance P-like immunoreactivity (SP-LI) in consecutive sections of rat hypothalamus was studied. Bombesin/ GRP -like immunoreactivity in the hypothalamus was partially characterized by gel filtration chromatography followed by radioimmunoassay. In the hypothalamus, SP-LI was more widely distributed than BN/ GRP -LI. Only the anterior and medial parvocellular parts of the nucleus paraventricularis and the nucleus suprachiasmaticus contained numerous cell bodies which exhibited BN/ GRP -LI. Neurons in these areas did not exhibit SP-LI. In contrast, cell bodies exhibiting SP-LI were numerous in the nucleus preopticus medialis and lateralis, nucleus anterior, nucleus ventromedialis and dorsomedialis, nucleus lateralis, nucleus arcuatus, and nucleus premamillaris ventralis and dorsalis. Only occasional cell bodies in these areas exhibited BN/ GRP -LI. It is concluded that the neuronal systems in the hypothalamus containing BN/ GRP -LI and SP-LI are separate, though the terminal fields in many areas overlap. Two peaks of BN/ GRP -LI were detected after gel filtration chromatography from extracts of the rat nucleus paraventricularis. The high molecular weight form coeluted with synthetic GRP (1-27), and the small molecular weight form eluted after synthetic bombesin. Thus, the endogenous BN/ GRP -LI is probably not authentic bombesin.     

Beacon immunoreactivity in the rat hypothalamus

Beacon (BC) is a peptide of 73 amino acids, whose gene expression was first reported in the hypothalamus of Psammomys obesus (or Israeli sand rat). To appreciate better the functional role of BC in normal rats and sand rats, the distribution of BC immunoreactivity (irBC) and its subcellular localization were studied in the brain of Sprague-Dawley rats. In the hypothalamus, intense staining was present in neurons of the supraoptic (SO), paraventricular (PVH), and accessory neurosecretory nuclei and in cell processes of median eminence. Double labeling of the hypothalamic sections with mouse monoclonal oxytocin (OT) antibody and rabbit polyclonal BC antiserum revealed that nearly all OT-immunoreactive cells from SO, PVH, and accessory neurosecretory nuclei were irBC. Double labeling of the sections with guinea pig vasopressin (VP) antiserum and BC antiserum showed that a population of VP-immunoreactive neurons was irBC. By immunoelectron microscopy, immunoreactive product was associated with mitochondrial membranes or appeared as electron-dense bodies in many PVH and SO neurons. Most of the neurosecretory granules were unstained for BC. Taken together, our results indicate the presence of beacon in the OT-containing neurons and a population of VP-containing neurons, mostly associated with mitochondrial membrane. Insofar as the amino acids sequence of beacon is identical to that of ubiquitin-like 5, it is possible that the distribution of BC immunoreactivity noted in our study is that of ubiquitin-like 5 peptide in the rat hypothalamus.     

GRF Neurons in the rat hypothalamus

The growth hormone-releasing factor (GRF)-containing neuronal system was immunohistochemically studied in the rat hypothalamus. The immunolabeled cell bodies were determined by intraventricular administration of colchicine 24 h before killing. In intact animals, the neurons appeared in the ventral portion of the arcuate nucleus (group 1) and in the area surrounding the ventromedial nucleus (group 2). Most of the cell bodies also indicated immunoreactivity for tyrosine hydroxylase (TH). The immunoreactive fibers accumulated showing a palisade arrangement in the external layer of the median eminence. The rats treated neonatally with monosodium glutamate revealed group 2 neurons and a few immunoreactive fibers in the median eminence. Half-anterolateral deafferentation of the medial basal hypothalamus, which was performed to isolate group 1 neurons or both group 1 and 2 neurons from the other brain parts, did not remarkably affect the appearance of the fibers in the median eminence. However, the perikarya were hypertrophic and strongly immunolabeled for GRF and TH. It is concluded that the fibers containing GRF in the median eminence derive mostly from group 1 neurons, and that the neurons may be regulated by an inhibitory mechanism by other neurons on the outside of the deafferented hypothalamic islands. GRF synthesized in group 2 neurons may act on other neurons as a neurotransmitter-like substance.     

Electroconvulsive seizures modulate levels of thyrotropin releasing hormone and related peptides in rat hypothalamus, cingulate and lateral cerebellum

We have studied the neuroanatomic extent of electroconvulsive (ECS)-responsive prepro-TRH and TRH-related gene expression and its possible interaction with forced swimming. Young adult male Wistar rats were treated in a 2×2 Latin square protocol of swimming, no swimming, three daily ECS or sham ECS. Sixteen different brain regions were dissected and immunoreactivity measured for TRH (pGlu–His–Pro–NH₂); TRH–Gly, a TRH precursor; Ps4, a prepro-TRH-derived TRH-enhancing decapeptide, and EEP (pGlu–Glu–Pro–NH₂). ECS, in addition to elevating TRH–immunoreactivity (TRH–IR), TRH–Gly–IR, Ps4–IR and EEP–IR levels in the limbic regions, as we have previously reported, also significantly increased Ps4–IR levels in hypothalamus, posterior cingulate and lateral cerebellum, and increased TRH–Gly–IR levels in hypothalamus. Interestingly, the combination of ECS and swimming significantly reduced the levels of TRH–Gly–IR in the anterior cingulate compared to the sham ECS-no swim group. The combined use of high-pressure liquid chromatography and the EEP radioimmunoassay (RIA) revealed that pGlu–Tyr–Pro–NH₂ and/or pGlu–Phe–Pro–NH₂ occur in amygdala, anterior cingulate, frontal cortex, entorhinal cortex, lateral cerebellum and striatum and make a substantial contribution to the EEP–IR and TRH–IR. We conclude that ECS can alter the expression and secretion of TRH-related peptides in the hypothalamus, cingulate and lateral cerebellum. Such effects have not previously been reported in these limbic and extra-limbic regions which are increasingly implicated in the autonomic, behavioral and volitional changes which accompany severe depression and its treatment.     

Developmental expression of glucokinase in rat hypothalamus

Neurons in the hypothalamus sense changes in glucose concentration. Glucokinase (GK), a key enzyme for pancreatic (beta)-cell glucose sensing, was found in both the embryonic and adult hypothalamus. GK activity accounted for approximately 20% of total hexokinase (HK) activity in both embryonic and adult hypothalamus with no activity measured in cortical samples, indicating that glucose sensing in the hypothalamus initiates early in development and precedes the maturation of glucose signaling in liver.     

Ontogeny of procholecystokinin processing in rat hypothalamus

The concentration of procholecystokinin (pro-CCK) in the fetal hypothalamus was 126 +/- 41 pmol/g (mean +/- SEM; n = 20), 22 +/- 9 pmol/g at day 7 postpartum and 3 +/- 2 pmol/g in the adult. In contrast, the concentration of bioactive carboxyamidated CCK rose from 6 +/- 2 pmol/g in the fetal hypothalamus to 52 +/- 10 pmol/g in the adult. The concentration of glycine-extended processing intermediates first decreased from 21 +/- 5 pmol/g in the fetus to 5 +/- 1 pmol/g at day 21 postpartum. Subsequently, the concentration rose to 21 +/- 4 pmol/g in the adult. The results show that the CCK gene is well expressed in the fetal hypothalamus. However, only a small fraction of pro-CCK reaches maturation before weaning. We conclude that expression of the CCK gene in the hypothalamus as bioactive peptide to a large degree is regulated at the posttranslational level.     

Electroconvulsive seizures modulate levels of thyrotropin releasing hormone and related peptides in rat hypothalamus, cingulate and lateral cerebellum

We have studied the neuroanatomic extent of electroconvulsive (ECS)-responsive prepro-TRH and TRH-related gene expression and its possible interaction with forced swimming. Young adult male Wistar rats were treated in a 2x2 Latin square protocol of swimming, no swimming, three daily ECS or sham ECS. Sixteen different brain regions were dissected and immunoreactivity measured for TRH (pGlu-His-Pro-NH(2)); TRH-Gly, a TRH precursor; Ps4, a prepro-TRH-derived TRH-enhancing decapeptide, and EEP (pGlu-Glu-Pro-NH(2)). ECS, in addition to elevating TRH-immunoreactivity (TRH-IR), TRH-Gly-IR, Ps4-IR and EEP-IR levels in the limbic regions, as we have previously reported, also significantly increased Ps4-IR levels in hypothalamus, posterior cingulate and lateral cerebellum, and increased TRH-Gly-IR levels in hypothalamus. Interestingly, the combination of ECS and swimming significantly reduced the levels of TRH-Gly-IR in the anterior cingulate compared to the sham ECS-no swim group. The combined use of high-pressure liquid chromatography and the EEP radioimmunoassay (RIA) revealed that pGlu-Tyr-Pro-NH(2) and/or pGlu-Phe-Pro-NH(2) occur in amygdala, anterior cingulate, frontal cortex, entorhinal cortex, lateral cerebellum and striatum and make a substantial contribution to the EEP-IR and TRH-IR. We conclude that ECS can alter the expression and secretion of TRH-related peptides in the hypothalamus, cingulate and lateral cerebellum. Such effects have not previously been reported in these limbic and extra-limbic regions which are increasingly implicated in the autonomic, behavioral and volitional changes which accompany severe depression and its treatment.     

Immunocytochemical analysis of proenkephalin-derived peptides in the amphibian hypothalamus and optic tectum

[Met5]-Enkephalin-, [Met5]-enkephalin-Arg6-, [Met5]-enkephalin-Arg6-Phe7-, metorphamide- and BAM 22P-like peptides could be localized in the amphibian brain by immunocytochemistry. However, a [Met5]-enkephalin-Arg6-Gly7-Leu8-like peptide could not be detected in the brain of any anuran species with an antiserum that was capable of detecting this octapeptide in mammalian brain. A synenkephalin-like peptide also could not be detected in the anuran brain with an antiserum that was capable of detecting the antigen in bovine and porcine brain. Although the intensity of proenkephalin-like immunoreactivity depended on the antiserum used, its distribution appeared to be identical with all of the effective antisera. Antisera directed against somatostatin and corticotropin-releasing factor stained perikarya, nerve fibers and terminals in the anuran brain with a distribution that was different from antisera directed against proenkephalin-derived peptides. The distribution of proenkephalin-containing perikarya and nerve fibers in the regions of the anuran brain selected for study showed many similarities to the distribution of proenkephalin-containing perikarya and nerve fibers in the same regions of the amniote brain.