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.     

Substance P and substance K in the rat hypothalamus following monosodium glutamate lesions of the arcuate nucleus.

Adult rats treated neonatally with monosodium glutamate (MSG) exhibit lesions in the arcuate nucleus of the hypothalamus. Following MSG lesioning, dopamine content in median eminence/arcuate nucleus (ME/AN) tissue extracts declined by 60-70%. Substance P (SP) content as determined by radioimmunoassay was significantly decreased in the paraventricular nucleus (PVN) (531 +/- 30 pg, mean +/- SEM) compared to controls (871 +/- 110 pg) but was unchanged in ME/AN extracts. Substance K (SK) content decreased to 257 +/- 20 pg in the PVN of lesioned animals compared to controls (367 +/- 31 pg) and the ME/AN content of SK was also significantly decreased (236 +/- 36 pg compared to control levels of 619 +/- 65 pg). The CRF-41 content of the PVN and ME/AN was unchanged by MSG lesioning, indicating that these areas are not affected by MSG. The partial depletion of SP and SK in the PVN following MSG treatment provides evidence that at least some of the neurokinin content of the PVN may originate in cell bodies of the arcuate nucleus. However, the lack of response of ME/AN SP to MSG treatment may suggest that the arcuate nucleus is not the major source of SP in the median eminence.     

The luteinizing hormone-releasing hormone (LH-RH) neuronal networks of the guinea pig brain. II. The regulation on gonadotropin secretion and the origin of terminals in the median eminence.

Cell bodies synthesizing LH-RH are located throughout the central nervous system including the hypothalamic arcuate nucleus, the medial preoptic area and medial septal nucleus. The contribution of each of these cell groups to the LH-RH terminals in the median eminence was assessed by immunocytochemistry following placement of radiofrequency lesions in male guinea pigs. Lesions in the arcuate nucleus resulted in an almost complete absence of LH-RH fibers in the median eminence. Lesions in the medial preoptic area or suprachiasmatic nucleus produced a decrease in the amount of immunoreactivity throughout the median eminence; but there was only a small decrease in the numbers of labeled fibers, which was localized to the internal zone of the median eminence and the external zone on the ventral and lateral surfaces of the infundibular stalk. Lesions of the mammillary bodies, medial amygdaloid nucleus, septal nucleus and fornix had no effect. The effects of these lesions on pituitary gonadotropin secretion was also assessed. Only lesions in the arcuate nucleus diminished plasma concentrations of luteinizing hormone (LH) and testosterone in the male guinea pigs. Similary only arcuate lesions prevented the postcastration rise in plasma LH in ovariectomized female guinea pigs. These data strongly suggest that the arcuate nucleus is the major but not the sole source of LH-RH terminals in the median eminence, and these are responsible for the neural regulation of tonic gonadotropin secretion.     

Estrous Cycle Variations in Gonadotropin-Releasing Hormone, Substance P and Beta-Endorphin Contents in the Median Eminence, the Arcuate Nucleus and the Medial Preoptic Nucleus in the Rat: A Detailed Analysis of Proestrus Changes

The concentrations of gonadotropin-releasing hormone, substance P and β-endorphin were measured in the median eminence, the arcuate nucleus and the medial preoptic nucleus of 4-day cycling female rats. Very well marked estrous cycle-related fluctuations were registered for these neuropeptides in these areas. The largest variations in concentrations of peptide levels were observed in the median eminence. Substance P concentration was highest throughout the day of proestrus as compared to the three others days of the cycle. At the time of the preovulatory luteinizing hormone surge, on the afternoon of proestrus, there was a marked increase in gonadotropin-releasing hormone concentration and a marked decrease in β-endorphin concentration.     

Monosynaptic Pathway Between the Arcuate Nucleus Expressing Glial Type II Iodothyronine 5'-Deiodinase mRNA and the Median Eminence-Projective TRH Cells of the Rat Paraventricular Nucleus

Recent evidence suggests that the thyroid regulation of thyrotropin-releasing hormone (TRH)-containing neurons in the paraventricular nucleus of the hypothalamus involves the activation of other hypothalamic neural circuits. For example, the arcuate nucleus and not the paraventricular nucleus contains the highest enzyme activity of 5′-deiodinase type II, an enzyme that is pivotal for the local synthesis of T3. This experiment was undertaken to demonstrate whether a monosynaptic pathway exists between the arcuate nucleus and those TRH cells of the paraventricular nucleus that are neuroendocrine, i.e. project to the external layer of the median eminence. A specific cRNA probe derived from the coding region of deiodinase type II was used for the in situ hybridization histochemistry which was combined with immunocytochemistry for a specific marker of glial cells, glial fibrillary acidic protein (GFAP). The hybridization signals were present within the hypothalamus in the arcuate nucleus–median eminence region and in the periventricular area. The periventricular labeling was localized to the ependymal layer of the third ventricle and no hybridization product was detected in the paraventricular nucleus and other hypothalamic nuclei adjacent to the third ventricle. Within the median eminence, numerous cells containing the hybridization product were located in the internal layer adjacent to the floor of the third ventricle and in the external layer adjacent to the surface of the brain. In the dorso- and ventromedial regions of the arcuate nucleus, deiodinase type II mRNA-containing cells were also detected. Numerous type II deiodinase mRNA-containing cells in the median eminence and arcuate nucleus were also found to be immunopositive for GFAP. The abundance of arcuate cells expressing the hybridization product was lower than those in the periventricular region or in the median eminence. The anterograde tracer, Phaseolus vulgaris leucoagglutinin, was injected into the medial parts of the arcuate nucleus where the in situ hybridization experiment detected deiodinase type II mRNA. Simultaneously with the anterograde tracing, the retrograde tracer, Fluoro-Gold, was injected into either the median eminence or the general circulation. Light and electron microscopic double and triple immunolabeling experiments on vibratome sections of colchicine-pretreated animals revealed that arcuate fibers innervate TRH cells within the parvicellular region of the paraventricular nucleus. Populations of these TRH cells receiving afferents from the arcuate nucleus were also retrogradely labelled from either the median eminence or the general circulation indicating their direct role in the regulation of thyrotropin secretion from the anterior pituitary. The majority of arcuate nucleus efferents on TRH cells were found to establish symmetrical synaptic connections. The present results provided direct evidence of a monosynaptic pathway between the hypothalamic site of local thyroid hormone production, the arcuate nucleus, and neuroendocrine TRH cells in the paraventricular nucleus. This signalling modality may play an important role in thyroid feedback on TRH cells. Since the arcuate nucleus is involved in the regulation of central mechanisms controlling diverse homeostatic functions, including reproduction and feeding, the pathway described in this study may also carry integrated signals related to reproduction and ingestion to TRH-producing cells.     

Astrocytes in the arcuate nucleus and median eminence that take up a fluorescent dye from the circulation express leptin receptors and neuropeptide Y Y1 receptors

Following systemic injection, several different dyes and markers are found to accumulate rapidly in cells in the arcuate nucleus and median eminence, and the capillaries in this region appear specialised for exchange of molecules. The present study used hydroxystilbamidine (FluoroGold equivalent) to identify cells that take up molecules from the circulation in these regions; 2-6 h following injection, uptake was seen in the external and intermediate zones of the median eminence and the adjacent ventral part of the arcuate nucleus, but not in other regions of the hypothalamus. The labelled cells were small; double-labelling experiments revealed that they expressed glial fibrillary acid protein (GFAP), but not NeuN, Agouti-related protein (AgRP) or beta-endorphin. They had the morphology of astrocytes and were readily distinguished from tanycytes by staining for vimentin. Many of these labelled astrocytes also expressed leptin receptors and neuropeptide Y Y1 receptors. The surrounding neurons that expressed these receptors did not take up this dye. This demonstrates that astrocytes take up molecules from the circulation in the median eminence and adjacent arcuate nucleus, and may have a significant signalling role in regulation of food intake.     

Electrophysiologic evidence for connections between the supraoptic and the arcuate/ventromedial hypothalamic nuclei in the rat

Extracellular action potentials were recorded from 48 single units located in the hypothalamic arcuate and ventromedial nuclei. Fifteen percent of the cells were identified as projecting to the median eminence and some of these cells may have belonged to the tuberoinfundibular dopaminergic systems. Responses of all cells to stimulation of the ipsilateral supraoptic nucleus were recorded; 17% of ventromedial nucleus neurons were antidromically identified as projecting to the supraoptic nucleus. None of the latter cells was also identified as projecting to the median eminence. Three of six identified tuberoinfundibular and eight unidentified ventromedial nucleus cells were found to be excited by stimulation of the supraoptic nucleus. One arcuate cell identified as projecting to the median eminence was nonresponsive to supraoptic stimulation. Orthodromic inhibitory responses were recorded from 17% of all cells recorded but no inhibitory responses were recorded from cells identified as projecting to the median eminence. We suggest that these results may provide some neurophysiologic explanations for the observed interrelationships between oxytocin and prolactin secretion, and between vasopressin and growth hormone secretion.     

Catecholamine neurons in the squirrel monkey hypothalamus

Summary Catecholamine neurons were found in the periventricular nucleus, arcuate nucleus, and a region around the mammillothalamic tract in the hypothalamus of the squirrel monkey. These neurons did not fluoresce in control or monoamine-oxidase-inhibited animals, and exhibited fluorescence only after intraventricular injections of alpha-methyl-norepinephrine. Dense accumulations of Catecholamine varicosities appeared in the periventricular nucleus, external contact zone of the median eminence, and perivascular region of the median eminence; the arcuate nucleus contained no more than a moderate density of Catecholamine varicosities. The median eminence fluorescence had a regional character, with greatest intensity in the caudal portion.     

The distribution of enkephalin in the mediobasal hypothalamus of the mouse brain: effects of neonatal administration of MSG

The immunocytochemical distribution of the pentapeptides, methionine- and leucine-enkephalin (Enk) were examined in the mediobasal hypothalamus of control mice and mice with a neurotoxic lesion of the arcuate nucleus due to neonatal treatment of monosodium glutamate (MSG). In control mice immunoreactive Enk cells and fibers were present in the arcuate nucleus. Enk fibers were found in the subependymal, internal and external layers of the median eminence.At 60 days of age, mice receiving neonatal injections of MSG displayed symptoms of neuroendocrine deficiencies. Histochemical methods revealed a loss of Enk immunoreactivity from the arcuate nucleus and all layers of the median eminence. These results suggest that the lack of Enk cells and fibers in the mediobasal hypothalamus of MSG-treated animals is partially responsible for the endocrine dysfunctions exhibited by these animals.     

Ultrastructural morphometric analysis of somatostatin-like immunoreactive neurones in the rat central nervous system after labelling with colloidal gold

Nerve terminals of the rat median eminence, arcuate nucleus and spinal cord were examined in the electron microscope after post-embedding, colloidal gold labelling of immunoreactivity to somatostatin. Strong immunostaining was thus obtained together with adequate morphological preservation. Reactive boutons showed clusters of gold particles essentially confined to dense-cored vesicles. In the median eminence, the positive varicosities made up more than half of all terminals and averaged 735 nm in diameter. Those in the arcuate nucleus and spinal cord were much less numerous and generally smaller (575 nm). The labelled vesicles had mean external diameters of 109, 95 and 79 nm in the median eminence, arcuate nucleus and spinal cord, respectively. Calculations of the likely amounts of somatostatin within the vesicles of the median eminence and arcuate nucleus yielded values of 0.7 and 1.4 mM, corresponding to 190 and 230 molecules of the peptide, respectively. These data support a neurotransmitter or modulator role for somatostatin in the central nervous system.     

Somatostatin neurons and their projections in dog diencephalon.

Immunocytochemical localization of the tetradecapeptide somatostatin was performed in dog brain using the unlabeled antibody enzyme method. A large population of immunoreactive neurons was seen in the periventricular areas of the preoptic area and anterior hypothalamus. This field of neurons extended into the paraventricular nucleus, arcuate nucleus and tuberal areas surrounding the ventromedial nuclei. Fibers from the periventricular somatostatin cells projected into the median eminence, the third ventricle, the pars nervosa of the hypophysis, the organum vasculosum of the lamina terminalis, the medial preoptic area and the interstitial nucleus of the stria terminalis. The tuberal cells projected to the ventromedial nucleus and the cells of the arcuate nucleus terminated within the arcuate nucleus as well as within the contact zone of the median eminence. These findings suggest that somatostatin can exert hormonal effects via the vasculature or the cerebrospinal fluid, or transmitter and/or neuromodulatory effects via contacts with other neurons.     

Monosodium glutamate lesions in rat hypothalamus studied by immunohistochemistry for gonadotropin releasing hormone, neurotensin, tyrosine hydroxylase, and glutamic acid decarboxylase and by autoradiography for [3H] estradiol

Adult male and female rats treated neonatally with monosodium glutamate (MSG) exhibit lesions in the arcuate nucleus of the hypothalamus. Immunohistochemical analysis of the distribution of tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD), neurotensin (NT) and gonadotropin-releasing hormone (GnRH) reveals substantial destructions of tuberoinfundibular dopamine and NT systems accompanied by a marked reduction of immunoreactivity in the median eminence. GAD immunoreactivity in the arcuate nucleus and median eminence is greatly reduced, while GnRH containing structures in the mediobasal hypothalamus are not noticeably affected. Evaluation of autoradiograms after intravenously administered [3H] estradiol in the ventral hypothalamus indicate an almost complete loss of target neurons in the arcuate nucleus but not in the nearby ventromedial nucleus. The results suggest that: (a) NT- and dopamine-containing neurons of the arcuate nucleus project to the median eminence via tuberoinfundibular NT and dopaminergic pathways; (b) GABA in the median eminence originates to a major extent from neurons of the arcuate nucleus through a tuberoinfundibular GABAergic system; (c) GnRH is produced in the rat outside the arcuate nucleus; (d) the MSG-induced lesion in the basal tuberal region abolishes or strongly diminishes estradiol target neurons in the arcuate nucleus.     

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.     

Intrahypothalamic terminals of stress conducting fibers

Surgical stress did not elevate plasma corticosterone level in rats with bilateral surgical transection of the lateral retrochiasmatic area (RCAL). After RCAL transections light and electron microscopic terminal degeneration was observed in both the external and the internal layers of the median eminence as well as in the arcuate and ventromedial nuclei. Incisions lateral to the medial forebrain bundle did not prevent the stress-induced rise in plasma corticosterone level and were not followed by degeneration in the median eminence. Transection of the medial forebrain bundle rostral or caudal to the RCAL caused degeneration in both the median eminence and the arcuate nucleus.     

The effect of parenteral glutamate treatment on the localization of neurotransmitters in the mediobasal hypothalamus.

The localization of cholinergic, aminergic and amino acid-ergic neurones in the mediobasal hypothalamus has been studied in normal rat brain and in brains where neurones in nucleus arcuatus were destroyed by repeated administration of 2 mg/g body weight monosodium glutamate to newborn animals. In normal animals acetylcholinesterase staining, choline acetyltransferase and aromatic L-amino acid decarboxylase were concentrated in the median eminence and the arcuate nucleus. Glutamate decarboxylase was concentrated at the boundary between the ventromedial and the arcuate nuclei, with lower activity in the arcuate nucleus and very low activity in the median eminence. Nucleus arcuatus contained an intermediate level of high affinity glutamate uptake. In the lesioned animals, there were significant decreases in choline acetyltransferase, acetylcholinesterase staining and glutamate decarboxylase in the median eminence, whereas choline acetyltransferase activity and acetylcholinesterase staining, but not glutamate decarboxylase activity, were decreased in nucleus arcuatus. Aromatic L-amino acid decarboxylase was unchanged in all regions studied. The high affinity uptakes of glutamate, dopamine and noradrenaline, and the endogenous amino acid levels were also unchanged in the treated animals. The results indicate the existence of acetylcholine- and GABA-containing elements in the tuberoinfundibular tract. They further indicate that the dopamine cells in the arcuate nucleus are less sensitive to the toxic effect of glutamate than other cell types, possibly because they contain less glutamate receptors.     

Monosodium glutamate lesions in rat hypothalamus studied by immunohistochemistry for gonadotropin releasing hormone, neurotensin, tyrosine hydroxylase, and glutamic acid decar☐ylase and by autoradiography for [H]estradiol

Adult male and female rats treated neonatally with monosodium glutamate (MSG) exhibit lesions in the arcuate nucleus of the hypothalamus. Immunohistochemical analysis of the distribution of tyrosine hydroxylase (TH), glutamic acid decar☐ylase (GAD), neurotensin (NT) and gonadotropin-releasing hormone (GnRH) reveals substantial destructions of tuberoinfundibular dopamine and NT systems accompanied by a marked reduction of immunoreactivity in the median eminence. GAD immunoreactivity in the arcuate nucleus and median eminence is greatly reduced, while GnRH containing structures in the mediobasal hypothalamus are not noticeably affected. Evaluation of autoradiograms after intravenously administered [³H]estradiol in the ventral hypothalamus indicate an almost complete loss of target neurons in the arcuate nucleus but not in the nearby ventromedial nucleus. The results suggest that: (a) NT- and dopamine-containing neurons of the arcuate nucleus project to the median eminence via tuberoinfundibular NT and dopaminergic pathways; (b) GABA in the median eminence originates to a major extent from neurons of the arcuate nucleus through a tuberoinfundibular GABAergic system; (c) GnRH is produced in the rat outside the arcuate nucleus; (d) the MSG-induced lesion in the basal tuberal region abolishes or strongly diminishes estradiol target neurons in the arcuate nucleus.     

Effect of electrical stimulation of the arcuate nucleus on neurochemical estimates of tuberoinfundibular and tuberohypophysial dopaminergic neuronal activities

The activity of nigrostriatal dopaminergic neurons has been estimated biochemically by measuring the rates of dopamine (DA) synthesis (accumulation of dihydroxyphenylalanine (DOPA) after NSD 1015) and turnover (decline of DA concentrations after alpha-methyltyrosine) in the striatum. It has been assumed that the activities of tuberoinfundibular dopaminergic (TIDA) and tuberohypophysial dopaminergic (THDA) neurons can also be estimated by making the same measurements in the terminals of these neurons in the median eminence and the posterior pituitary, respectively. In the present study, this assumption was tested directly by measuring the rates of DA synthesis and turnover in the median eminence and posterior pituitary following electrical stimulation of TIDS and THDA cell bodies in the arcuate nucleus. Electrical stimulation of the arcuate nucleus increased the rate of DOPA accumulation and the alpha-methyltyrosine-induced decline of DA concentrations in the median eminence and in the neural and intermediate lobes of the posterior pituitary. gamma-Butyrolactone (GBL), an anesthetic that selectively inhibits DA impulse flow, reduced the rates of DA synthesis and turnover in the median eminence. GBL also increased prolactin secretion which is tonically inhibited by DA released from TIDA neurons. Serum prolactin levels were significantly decreased by arcuate nucleus stimulation in GBL-anesthetized rats. These results indicate that the rates of DA synthesis and turnover within the median eminence and posterior pituitary reflect the activities of TIDA and THDA neurons, respectively.     

Distribution of immunoreactive substance P neurons in the hypothalamus and pituitary of the rhesus monkey

The distribution of immunoreactive substance P (IR-SP) neurons was examined in the hypothalamus and pituitary gland of the rhesus monkey by using the peroxidase-antiperoxidase immunocytochemical technique. Immunoreactive SP cell bodies were observed in the arcuate nucleus, in the region lateral to the arcuate nucleus, and in the median eminence (ME). Immunoreactive SP cells were also seen in the periventricular area of the dorsal tuberal region. A rich network of SP fibers was concentrated in the arcuate region, and the fiber stain was particularly dense in the external zone of the median eminence and in the external layer of the infundibular stalk. Also, substance P fibers were seen in the internal layer of the pituitary stalk and in the neural lobe of the pituitary gland. Outside the hypothalamus a dense network of IR-SP fibers was observed in the globus pallidus.     

Diffusional barrier around the hypothalamic arcuate nucleus in the rat

The contour lines of horseradish peroxidase injection sites in the ventrobasal hypothalamus were distorted by the border between arcuate and ventromedial nuclei as well as between arcuate nucleus and median eminence. The dense array of tanycyte processes is assumed to isolate the arcuate nucleus from the neighboring territories by establishing a diffusional barrier surface.     

Distributions of periventricular projections of the paraventricular nucleus to the median eminence and arcuate nucleus

Neuronal projections from the periventricular subnucleus of the hypothalamic paraventricular nucleus to the median eminence and the arcuate nucleus were investigated in the rat by the anterograde tract-tracer, Phaseolus vulgaris leucoagglutinin. The vast majority of labeled fibers coursed ventrally along the third ventricle and distributed in the external layer of the median eminence bilaterally, with ipsilateral predominance moving caudalwards. Periventricular fibers also terminated in the arcuate nucleus, but this innervation was exclusively ipsilateral.