Light and electron microscopic immunocytochemistry of GRF-like immunoreactive neurons and terminals in the rat hypothalamic arcuate nuclues and median eminence

Growth hormone-releasing factor (GRF) synthesizing neuronal perikarya and terminals were investigated by light and electron microscopic immunocytochemistry using rat hypothalamus. Immunoreactive neuronal perikarya were located mainly in the ventrolateral part of the arcuate nucleus. They contained well developed cell organella such as mitochondria and rough surfaced endoplasmic reticulum with some expansion. They also contained immunoreactive dense granules (80-120 nm in diameter). On the surface of the immunoreactive neuronal perikarya were frequently found non-immunoreactive axo-somatic synapses. Therefore, the GRF-like immunoreactive neurons were assumed to receive neuronal inputs from other neurons on their neuronal soma. In the external layer of the median eminence large numbers of immunoreactive terminals were distributed particularly around the capillaries of the portal vessel. Electron microscopic immunocytochemistry revealed large numbers of immunoreactive terminals containing immunoreactive dense granules, synaptic vesicles and mitochondria in the vicinity of the basement membrane of the pericapillary space of the portal vessel. Therefore, we concluded that GRF-like immunoreactive substances are released into the portal capillaries from the nerve terminals, which originate from the neuronal perikarya in the ventrolateral part of the arcuate nucleus, and act on growth hormone release in the anterior pituitary. We also suggest that GRF-like immunoreactive neurons have abundant terminal arborization in the external layer of the median eminence.     

Some cellular characteristics of somatostatin neurons and terminals in the periventricular nucleus of the rat hypothalamus and median eminence. Electron microscopic immunohistochemistry

Somatostatin neuronal perikarya and their processes, presumably dendrites, in the periventricular nucleus of the rat hypothalamus and terminals in the median eminence were observed by electron microscopic immunohistochemistry. Neuronal perikarya and processes contained immunoreactive dense granules (100-120 nm in diameter) and other cellular components such as polysomes, rER membranes occasionally showed high electron density. Few axo-somatic terminals were found on the somatostatin neurons, but we could detect a number of preterminal axons on immunoreactive processes, presumably dendrites. Therefore, we considered that somatostatin neurons receive mainly neuronal input through axo-dendritic synapses rather than through axo-somatic ones. In the somatostatin terminals in the external layer of the median eminence immunoreactivity was completely restricted on the granules.     

Light and electron microscopic immunocytochemistry of neurotensin-like immunoreactive neurons in the rat hypothalamus

Neurotensin-like immunoreactive neuronal perikarya, fibers and terminals in the rat hypothalamus, particularly in the arcuate nucleus, the paraventricular nucleus and the median eminence, were investigated by light and electron microscopic immunocytochemistry. The main distributional areas of immunoreactive neuronal perikarya were found to be the arcuate nucleus, the periventricular nucleus and the paraventricular nucleus by light microscopic immunocytochemistry. Immunoreactive neuronal perikarya showed a characteristic distributional pattern in the arcuate nucleus. In the paraventricular nucleus they were distributed in both the magnocellular and parvocellular portions. A large number of immunoreactive terminals were observed throughout the external layer of the median eminence, particularly its lateral portion. A moderate number of immunoreactive terminals were also observed in the internal layer of the median eminence. By electron microscopic immunocytochemistry immunoreactive neuronal perikarya both in the arcuate and paraventricular nuclei showed generally well-developed cell organelles such as mitochondria, r-ER, and Golgi complex. In addition, immunoreactive dense granules were dispersed throughout the perikarya. A large number of immunoreactive terminals containing immunoreactive dense granules, clear vesicles and mitochondria were observed in the vicinity of pericapillary spaces of the external layer of the median eminence. This observation strongly suggests that neurotensin-like immunoreactive substance is released into the portal capillaries.     

Distribution of thyrotropin-releasing hormone (TRH)-containing cells and fibers in the human hypothalamus

In the present study, we describe for the first time the distribution of thyrotropin-releasing hormone (TRH)-containing cells and fibers in the human hypothalamus using brain material obtained with a short postmortem delay. Following fixation in paraformaldehyde, glutaraldehyde and picric acid, excellent staining was obtained with two different TRH antisera. Many TRH-containing neurons were present in the paraventricular nucleus (PVN), especially in the dorsocaudal part of this nucleus. They were mostly parvicellular, but a few magnocellular TRH-positive neurons were observed as well. The PVN also contained a dense network of TRH fibers. The supraoptic nucleus (SON) did not show any TRH immunoreactivity, excluding the possibility of cross-reactivity of the antiserum with neurohypophysial hormones or their precursors. In addition, TRH cells were found in the suprachiasmatic nucleus (SCN), which is the circadian clock of the brain, in the sexually dimorphic nucleus (SDN) and dorsomedially of the SON. We observed small numbers of TRH cells throughout the hypothalamic gray in all subjects studied. A high density of TRH-containing fibers was seen not only in the median eminence but also in other hypothalamic areas, e.g., in the ventromedial nucleus (VM) and in the perifornical area. The results generally agree with earlier data in the rat, with the exception of the absence of TRH cells in the SON. The large number of sites of TRH-containing fiber terminations on neurons suggests important physiological functions of this neuropeptide as a neurotransmitter or neuromodulator in the human brain in addition to its role as a neurohormone in pituitary secretion of thyroid-stimulating hormone (TSH).     

Immunohistochemical mapping of neuropeptides in the premamillary region of the hypothalamus in rats

The topographical distribution of neuropeptide-containing cell bodies, fibers and terminals was studied in the premamillary region of the rat hypothalamus using light microscopic immunohistochemistry. Alternate coronal sections through the posterior third of the hypothalamus of normal and colchicine-treated male rats were immunostained for 19 different neuropeptides and their distributions were mapped throughout the following structures: the ventral and dorsal premamillary, the supramamillary, the tuberomamillary and the posterior hypothalamic nuclei, as well as the premamillary portion of the arcuate nucleus and the postinfundibular median eminence. Seventeen of the investigated neuropeptides were present in neuronal perikarya, nerve fibers and terminals while the gonadotropin associated peptide and vasopressin occurred only in fibers andn terminals. Growth hormone-releasing hormone-, somatostatin-, α-melanocyte stimulating hormone-, adrenocorticotropin, β-endorphin- and neuropeptide Y-immunoreactive neurons were seen exclusively in the premamillary portion of the arcuate nucleus. Thyrotropin-releasing hormone-, dynorphin A- and galanin-containing neurons were distributed mainly in the arcuate and the tuberomamillary nuclei. A high number of methionine- and leucine-enkephalin-immunoreactive cells were detected in the arcuate and dorsal premamillary nuclei, as well as in the area ventrolateral to the fornix. Substance P-immunoreactive perikarya were present in very high number within the entire region, in particular in the ventral and dorsal premamillary nuclei. Cell bodies labelled with cholecystokinin- and calcitonin gene-related peptide antisera were found predominantly in the supramamillary and the terete nuclei, respectively. Corticotropin-releasing hormone-, vasoactive intestinal polypeptide- and neurotensin-immunoreactive neurons were scattered randomly in low number, mostly in the arcuate and the ventral and dorsal premamillary nuclei. Peptidergic fibers were distributed unevenly throughout the whole region, with each peptide showing an individual distribution pattern. The highest density of immunoreactive fibers was presented in the ventral half of the region including the arcuate, the ventral premamillary and the tuberomamillary nuclei. The supramamillary nucleus showed moderately dense fiber networks, while the dorsal premamillary and the posterior hypothalamic nuclei were poor in peptidergic fibers.     

Centrally administered bombesin activates COX-containing spinally projecting neurons of the PVN in anesthetized rats

The paraventricular nucleus (PVN) of the hypothalamus has a heterogenous structure containing different types of output neurons that project to the median eminence, posterior pituitary, brain stem autonomic centers and sympathetic preganglionic neurons in the spinal cord. Presympathetic neurons in the PVN send mono- and poly-synaptic projections to the spinal cord. In the present study using urethane-anesthetized rats, we examined the effects of centrally administered bombesin (a homologue of the mammalian gastrin-releasing peptide) on the mono-synaptic spinally projecting PVN neurons pre-labeled with a retrograde tracer Fluoro-Gold (FG) injected into T8 level of the spinal cord, with regard to the immunoreactivity for cyclooxygenase (COX) isozymes (COX-1/COX-2) and Fos (a marker of neuronal activation). FG-labeled spinally projecting neurons were abundantly observed in the dorsal cap, ventral part and posterior part of the PVN. The immunoreactivity of each COX-1 and COX-2 was detected in FG-labeled spinally projecting PVN neurons in the vehicle (10 μl of saline/animal, i.c.v.)-treated group, while bombesin (1 nmol/animal, i.c.v.) had no effect on the number of these immunoreactive neurons for each COX isozyme with labeling of FG. On the other hand, the peptide significantly increased the number of double-immunoreactive neurons for Fos and COX-1/COX-2 with FG-labeling in the PVN (except triple-labeled neurons for FG, COX-2 and Fos in the dorsal cap of the PVN), as compared to those of vehicle-treated group. These results suggest that centrally administered bombesin activates spinally projecting PVN neurons containing COX-1 and COX-2 in rats.     

Hypothalamic projections to locus coeruleus neurons in rat brain

Locus coeruleus (LC) neurons respond to autonomic and visceral stimuli and discharge in parallel with peripheral sympathetic nerves. The present study characterized the synaptic organization of hypothalamic afferents with catecholaminergic neurons in the LC using electron microscopy. Peroxidase labeling of axon terminals that were anterogradely labeled from the paraventricular nucleus (PVN) was combined with gold-silver labeling of tyrosine hydroxylase in the LC. Approximately 19% of the anterogradely labeled axon terminals formed synaptic specializations with tyrosine hydroxylase-immunoreactive dendrites in the LC. Retrograde transport from the LC combined with immunocytochemical detection of enkephalin and corticotropin-releasing factor (CRF) suggested that most of the LC-projecting PVN neurons (30%) were CRF immunoreactive and few (2%) were enkephalin immunoreactive. Finally, dual retrograde tracing from the LC and median eminence revealed that PVN neurons that project to the LC are a population distinct from that projecting to the median eminence. The present data suggest that a population of hypothalamic neurons is poised to directly modulate the activity of LC neurons and may integrate autonomic responses in brain by influencing LC neurons. Moreover, PVN neurons that use CRF as a neurohormone are distinct from those that use CRF as a neuromodulator to impact on the LC.     

Specific expression of optically active reporter gene in arginine vasopressin-secreting neurosecretory cells in the hypothalamic-neurohypophyseal system

The anti-diuretic hormone arginine vasopressin (AVP) is synthesised in the magnocellular neurosecretory cells (MNCs) in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus. AVP-containing MNCs that project their axon terminals to the posterior pituitary can be identified using immunohistochemical techniques with specific antibodies recognising AVP and neurophysin II, and by virtue of their electrophysiological properties. Recently, we generated transgenic rats expressing an AVP-enhanced green fluorescent protein (eGFP) fusion gene in AVP-containing MNCs. In this transgenic rat, eGFP mRNA was observed in the PVN and the SON, and eGFP fluorescence was seen in the PVN and the SON, and also in the posterior pituitary, indicating transport of transgene protein down MNC axons to storage in nerve terminals. The expression of the AVP-eGFP transgene and eGFP fluorescence in the PVN and the SON was markedly increased after dehydration and chronic salt-loading. On the other hand, AVP-containing parvocellular neurosecretory cells in the PVN that are involved in the activation of the hypothalamic-pituitary adrenal axis exhibit robust AVP-eGFP fluorescence after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide. In the median eminence, the internal and external layer showed strong fluorescence for eGFP after osmotic stimuli and stressful conditions, respectively, again indicating appropriate transport of transgene traslation products. Brain slices and acutely-dissociated MNCs and axon terminals also exhibited strong fluorescence, as observed under fluorescence microscopy. The AVP-eGFP transgenic animals are thus unique and provide a useful tool to study AVP-secreting cells in vivo for electrophysiology, imaging analysis such as intracellular Ca²⁺ imaging, organ culture and in vivo monitoring of dynamic change in AVP secretion.     

Light and electron microscopic immunocytochemistry of β-endorphin/β-LPH-like immunoreactive neurons in the arcuate nucleus and surrounding areas of the rat hypothalamus

β-Endorphin/β-LPH-like immunoreactive neurons in the hypothalamic arcuate nucleus and its surrounding areas were visualized by light and electron microscopic immunocytochemistry. Immunoreactive processes were found in the vicinity of the pia mater, in the lateral part of the external layer of the median eminence and near the lateral wall of the third ventricle. Neuronal perikarya contained immunoreactive dense granules as well as developed cell organellae. They received neuronal inputs from other neurons through axoplasmic and axodendritic synapses. Immunoreactive neuronal processes containing dense granules and mitochondria were found as preterminal elements on non-immunoreactive neuronal soma and dendrites. Immunoreactive processes also make intimate contact with capillaries in the arcuate nucleus near the median eminence.     

GRF immunoreactive neurons in the paraventricular nucleus of the rat: an immunohistochemical study with monoclonal and polyclonal antibodies

Our study demonstrates a complex GRF neuronal system within the rat hypothalamus. Using both high affinity polyclonal and high specificity monoclonal antibodies to rat (r) GRF, we have substantiated evidence for immunoreactive GRF (GRF-i) perikarya in the parvocellular portion of the paraventricular nucleus. Other hypothalamic areas containing rGRF-positive perikarya include the lateral arcuate nucleus, lateral hypothalamus, perifornical area and dorsomedial nucleus. GRF-i neuronal terminals were seen in the external zone of the median eminence, more rostrally in the periventricular nucleus, and near the suprachiasmatic nucleus and more caudally in the dorsomedial nucleus and ventral premammillary nucleus.     

Release of vasopressin from supraoptic neurons within the median eminence in vivo. A combined microdialysis and push-pull perfusion study in the rat

The present study was designed to investigate whether or not arginine vasopressin (AVP) is released from magnocellular neurons within the median eminence (ME) in vivo. Urethane-anesthetized adult male Wistar rats were equipped with a microdialysis probe aimed at the supraoptic (SON) or paraventricular nucleus (PVN), a push-pull perfusion probe resting in the ME, and a blood microdialysis probe within the jugular vein. Dialysis of the SON (but not the PVN) with Ringer's solution containing 56 mmol l⁻¹ K⁺ resulted in an increase in AVP release within the ME (to 492 ± 192% of release during basal conditions,P < 0.05) and into blood (to 138 ± 9%,P < 0.01) whereby the release probably occurred from axonal swellings and nerve terminals of supraoptic neurons which project through the internal zone of the ME to the posterior pituitary. The calculated amount of AVP released into the extracellular fluid of the ME was high enough (approximately 1 pg/μ1) to hypothesize that the neuropeptide could enter the portal blood capillaries in physiologically relevant concentrations. Taken together, the present study indicates that activation of magnocellular neurons is accompanied by release of AVP within the median eminence. We assume that AVP released in this way might mediate a communication between the hypothalamic-neurohypophysial system and the hypothalamic-pituitary-adrenal axis in response to selected stressful stimuli.     

Peptide GEGLSS-Like Immunoreactivity in the Rat Central Nervous System

A rabbit antiserum was raised against the N-terminal fragment peptide, GEGLSS (Gly-Glu-Gly-Leu-Ser-Ser) of bovine neuropeptide AF (NPAF, A18Famide). NPAF is an octadecapeptide isolated from the bovine brain together with neuropeptide FF (NPFF). GEGLSS-like immunoreactivity was localized with immunofluorescence technique in colchicine-treated rats in neuronal cell bodies of the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. A few neurons were also observed in the retrochiasmatic part of the SON. GEGLSS-like immunoreactivity was also localized to nerve terminals of the posterior pituitary. No GEGLSS-ir neuronal cell bodies were observed in the medial hypothalamus, in an area that contains NPFF-ir neurons. GEGLSS immunoreactivity was also seen in the fibers and terminals of nucleus of the solitary tract. We injected a retrograde tracer, fluorogold, to the posterior pituitary gland and visualized GEGLSS-ir neuronal cell bodies double-labeled with the tracer in SON, PVN, and SOR. The pituitary stalk transsection totally abolished the GEGLSS-ir structures from the posterior pituitary. Our results suggest that GEGLSS immunoreactivity in the rat brain has a more limited distribution than NPFF immunoreactivity. GEGLSS immunoreactivity was partially colocalized with arginine-vasopressin and oxytocin in neuronal cell bodies in the SON and PVN. Considering the fact that the known rat NPFF-NPAF precursor does not contain GEGLSS structure, the detected GEGLSS immunoreactivity may be derived from a previously unknown precursor.     

Localization of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) in the Hypothalamus-Pituitary System in Rats: Light and Electron Microscopic Immunocytochemical Studies

The localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the hypothalamus-pituitary system in rats was examined in light and electron microscopic immunocytochemistry using a specific antiserum to synthetic PACAP 1–38 (R0831). In light microscopic study, intensely PACAP-immunostained perikarya were observed in the supraoptic and paraventricular magnocellular nucleus in the hypothalamus. In the median eminence, many immunoreactive nerve fibers were observed in the internal layer, but a few immunoreactive terminals were noticed in the external layer. In the pituitary gland, numerous immunoreactive nerve fibers were observed in the posterior lobe. In the intermediate lobe, moderately immunostained cells were observed, but in the anterior lobe no immunostained cells were noticed. In electron microscopic study, PACAP-immunoreactivity was examined by avidin-biotin peroxidase complex method. In the perikarya of the supraoptic and paraventricular magnocellular nucleus, DAB-reaction products were distributed diffusely in the cytoplasmic matrix, frequently attaching to the rough-surfaced endoplasmic reticulum. In the nerve terminals of the posterior lobe, reaction products were observed among the secretory granules, but sometimes upon them. In the cells of the intermediate lobe, reaction products were also distributed in the cytoplasmic matrix.     

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.     

Immunocytochemical distribution of catecholamine-synthesizing neurons in the hypothalamus and pituitary gland of pigs: Tyrosine hydroxylase and dopamine-β-hydroxylase

This study describes the distribution of catecholaminergic neurons in the hypothalamus and the pituitary gland of the domestic pig, Sus scrofa, an animal that is widely used as an experimental model of human physiology in addition to its worldwide agricultural importance. Hypothalamic catecholamine neurons were identified by immunocytochemical staining for the presence of the catecholamine synthesizing enzymes, tyrosine hydroxylase and dopamine-β-hydroxylase. Tyrosine hydroxylase-immunoreactive perikarya were observed in the periventricular region throughout the extent of the third ventricle, the anterior and retrochiasmatic divisions of the supraoptic nucleus, the suprachiasmatic nucleus, the ventral and dorsolateral regions of the paraventricular nucleus and adjacent dorsal hypothalamus, the ventrolateral arcuate nucleus, and the posterior hypothalamus. Perikarya ranged from parvicellular (10–15 μm) to magnocellular (25–50 μm) and were of multiple shapes (rounded, fusiform, triangular, or multipolar) and generally had two to five processes with branched arborization. No dopamine-β-hydroxylase immunoreactive perikarya were observed within the hypothalamus or in the adjacent basal forebrain structures. Both tyrosine hydroxylase- and dopamine-β-hydroxylase-immunoreactive fibers and punctate varicosities were observed throughout areas containing tyrosine hydroxylase perikarya, but dopamine-β-hydroxylase immunoreactivity was very sparse within the median eminence. Within the pituitary gland, only tyrosine hydroxylase fibers, and not dopamine-β-hydroxylase immunoreactive fibers, were located throughout the neurohypophyseal tract and within the posterior pituitary in both pars intermedia and pars nervosa regions. Generally, the location and patterns of both catecholamine-synthesizing enzymes were similar to those reported for other mammalian species except for the absence of the A15 dorsal group and the very sparse dopamine-β-hydroxylase immunoreactive fibers and varicosities in the median eminence in the pig. These findings provide an initial framework for elucidating behavioral and neuroendocrine species differences with regard to catecholamine neurotransmitters. © 1996 Wiley-Liss, Inc.     

Increased Expression of Nitric Oxide Synthase in Hypothalamic Neuronal Regeneration

This investigation deals with the histochemical and scanning electron microscopic (SEM) correlates that depict regeneration of the neurohypophyseal system that may be nitric oxide dependent following hypophysectomy in the rodent hypothalamus. NOS histochemistry and correlative SEM were employed to establish the rates of regrowth and appearance of NOS-positive supraoptic (SON) and paraventricular (PVN) neurites and their cell bodies following hypophysectomy. NOS activity increased significantly in SON and PVN neuronal perikarya and regenerating axons by 2 weeks. NOS-positive neurites were observed to regrow into the adjacent median eminence and insinuate into the lumen of the third cerebral ventricle. By 4 weeks posthypophysectomy, NOS staining of SON and PVN neurons and their regrown neurites had returned to normal control levels. Despite this fact, large complexes of apparent magnocellular neurites remained upon the floor of the third cerebral ventricle as observed with SEM. These observations support the hypothesis that NO may play a fundamental role in the process of regeneration, plasticity, and retargeting of SON and PVN axons following injury.     

Neuronal expression of fos protein in the forebrain of diabetic rats

We sought to identify the areas that have altered neuronal activity within the hypothalamus of diabetic rats by mapping neuronal expression of c-fos protein (Fos) and Fos-related antigens. After a standard PAP immunocytochemical protocol, Fos-like immunoreactivity was observed in the paraventricular nucleus (PVN), supraoptic nucleus (SON), median preoptic area (MnPO), anterior hypothalamus (AH) and posterior hypothalamus (PH) of control (vehicle; n=6) and diabetic rats (Sprague-Dawley rats injected with STZ 65 mg/kg/ip 4 weeks prior to the experiment; n=6). Blood glucose levels were significantly elevated in the diabetic group (370+/-8 mg/dl) compared to control group (104+/-3 mg/dl). Diabetic rats had a significantly higher number of Fos-positive cells in PVN (2.5x), SON (7x) and MnPO (2x) compared to the control rats. However, diabetic rats had significantly fewer Fos-positive cells in the AH (0.3x) and no difference was observed in the PH between the diabetic and control rats. Despite the elevated number of Fos-positive cells in the diabetic rats, dehydration (water withdrawal for 24 h) or hypertonic challenge (1.5 ml of 0.1 M NaCl i.p. injection) produced a further increase in the number of Fos-positive cells in the PVN, SON and MnPO. Dehydration did not alter the number of Fos-positive cells in the AH or PH, but hypertonic challenge produced a significant increase in the Fos-positive cells in both the AH and PH of diabetic rats. This study demonstrates that: (1) there is increased basal neuronal activity in the PVN, SON and MnPO, a decrease in neuronal activity in the AH and no change in neuronal activity in the PH as indicated by Fos staining in diabetic rats; and (2) dehydration or hypertonic challenge produces a further increase in the number of Fos-positive cells in the PVN, SON, and MnPO which is comparable to control rats. These data support the conclusion that vasopressin producing neurons in the PVN and SON and autonomic areas within the lamina terminalis and hypothalamus are activated during diabetes and may contribute to the elevated levels of vasopressin and autonomic dysfunction during diabetes.     

Immunocytochemical distribution of [Met]enkephalin-Arg-Gly-Leu immunoreactivity in the rat diencephalon

Recently, [Met]Enkephalin-Arg-Gly-Leu (MEAGL) was isolated from bovine adrenal glands, and it was found to be derived exclusively from proenkephalin. Therefore, we investigated the distribution of MEAGL-like immunoreactive neuronal perikarya and fibers in the rat diencephalon pretreated with colchicine by PAP immunocytochemistry. In the thalamus MEAGL immunoreactive neuronal perikarya were distributed in the paraventricular nucleus and the ventral part of the lateral geniculate nucleus. Immunoreactive fibers were found in the paraventricular, paracentral, anteroventral, reuniens and rhomboid nuclei. In addition, immunoreactive fibers were also noted in the anterior pretectal nucleus. In the hypothalamus, immunoreactive neuronal perikarya were observed in the medial preoptic area, anterior and lateral hypothalamic nuclei, perifornical region, parvocellular and postero-magnocellular regions of paraventricular nucleus, ventromedial nucleus, dorsomedial nucleus, arcuate nucleus, premammillary, medial mammillary and lateral mammillary nuclei. The distribution of immunoreactive fibers was similar to that of neuronal perikarya. However, immunoreactive fibers were also observed in the supraoptic and suprachiasmatic nuclei where no immunoreactive neuronal perikarya were detected. Numerous immunoreactive fibers were detected in the external layer of the median eminence, but there were few in the internal layer. The similarity and difference in the distribution between MEAGL and other proenkephalin peptides such as [Met]enkephalin were also discussed.     

Neurotensin in the rat median eminence: the possible sources of neurotensin-like fibers and varicosities in the external layer

The possible sources of neurotensin-like immunoreactive axons in the median eminence were studied after several experimental surgical approaches including unilateral lateral retrochiasmatic area transection, midsagittal knife cut through the median eminence, complete surgical isolation of the medial basal hypothalamus and bilateral paraventricular nucleus lesions. Both immunohistochemical and radioimmunoassay data demonstrate that neurotensin-containing neuronal located in the hypothalamic arcuate nuclei represent the main source of neurotensin occurring in the external zone of the median eminence of the rat: (1) neither the complete isolation of the medial basal hypothalamus nor the transection of the major neuronal input channel to the median eminence in the lateral retrochiasmatic area altered neurotensin-like immunoreactivity in the median eminence; (2) bilateral lesioning of the paraventricular nucleus resulted in insignificant changes of neurotensin level in the median eminence; and (3) two days after lesioning the median eminence an increased amount of retrogradely accumulated neurotensin-like immunoreactivity was found in several perikarya of the arcuate nuclei due to the blockage of axonal transport in the transected fibers. Retrograde accumulation of neurotensin-like material in other cells scattered in the anterior hypothalamus (in the paraventricular, paraventricular and anterior hypothalamic nuclei) indicates that in addition to the arcuate neurons these neurons may also participate in the neurotensin innervation of the median eminence.