Bertalan Dudas

Evidence that 5-HT2A receptors in the hypothalamic paraventricular nucleus mediate neuroendocrine responses to (-)DOI.

The present study determined whether the serotonin2A (5-HT2A) receptors in the hypothalamic paraventricular nucleus mediate the neuroendocrine responses to a peripheral injection of the 5-HT2A/2Creceptor agonist (−)DOI [(−)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane]. The 5-HT2A receptor antagonist MDL100,907 ((±)-α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol), the 5-HT2C receptor antagonist SB-242084 (6-chloro-5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-indoline), or vehicle were microinjected bilaterally through a chronically implanted double-barreled cannula into the hypothalamic paraventricular nucleus 15 min before a peripheral injection of (−)DOI in conscious rats. (−)DOI significantly elevated plasma levels of oxytocin, prolactin, ACTH, corticosterone, and renin. Neither the 5-HT2A receptor antagonist nor the 5-HT2Creceptor antagonist, injected alone, altered the basal levels of these hormones. MDL100,907 (0.748, 7.48, and 18.7 nmol) dose dependently inhibited the (−)DOI-induced increase in all of the hormones except corticosterone. In contrast, SB-242084 (10 nmol) did not inhibit (−)DOI-increased hormone levels. To confirm the presence of 5-HT2A receptors in the hypothalamic paraventricular nucleus, 5-HT2A receptors were mapped using immunohistochemistry. Densely labeled magnocellular neurons were observed throughout the anterior and posterior magnocellular subdivisions of the hypothalamic paraventricular nucleus. Moderately to densely labeled cells were also observed in parvicellular regions. Thus, it is likely that 5-HT2A receptors are present on neuroendocrine cells in the hypothalamic paraventricular nucleus. These data provide the first direct evidence that neuroendocrine responses to a peripheral injection of (−)DOI are predominantly mediated by activation of 5-HT2A receptors in the hypothalamic paraventricular nucleus.

Three‐Dimensional Representation of the Neurotransmitter Systems of the Human Hypothalamus: Inputs of the Gonadotrophin Hormone‐Releasing Hormone Neuronal System

The gonadotrophin‐releasing hormone (GnRH) represents the final common pathway of a neuronal network that integrates multiple external and internal factors to control fertility. Among the many inputs GnRH neurones receive, oestrogens play the most important role. In females, oestrogen, in addition to the negative feedback, also exhibits a positive feedback influence upon the activity and output of GnRH neurones to generate the preovulatory luteinising hormone surge and ovulation. Until recently, the belief has been that the GnRH neurones do not contain oestrogen receptors and that the action of oestrogen upon GnRH neurones is indirect, involving several, oestrogen‐sensitive neurotransmitter and neuromodulator systems that trans‐synaptically regulate the activity of the GnRH neurones. Although this concept still holds for humans, recent studies indicate that oestrogen receptor‐beta is expressed in GnRH neurones of the rat. This review provides three dimensional stereoscopic images of GnRH‐immunoreactive (IR) and some peptidergic (neuropeptide Y‐, substance P‐, β‐endorphin‐, leu‐enkaphalin‐, corticotrophin hormone‐releasing‐ and galanin‐IR) and catecholaminergic neurones and the communication of these potential oestrogen‐sensitive neuronal systems with GnRH neurones in the human hypothalamus. Because the post‐mortem human tissue does not allow the electron microscopic identification of synapses on GnRH neurones, the data presented here are based on light microscopic immunocytochemical experiments using high magnification with oil immersion, semithin sections or confocal microscopy.

Topography and associations of luteinizing hormone–releasing hormone and neuropeptide Y–immunoreactive neuronal systems in the human diencephalon

Neuropeptide Y (NPY) potentiates the effect of luteinizing hormone–releasing hormone (LHRH) on luteinizing hormone secretion in several species, including human. In addition to the pituitary sites, the interactions of the NPY and LHRH systems may involve diencephalic loci. However, the morphologic basis of this putative communication has not yet been elucidated in the human brain. To discover interaction sites, the distribution and connections of LHRH and NPY‐immunoreactive (IR) neuronal elements in the human hypothalamus were investigated by means of light microscopic single‐ and double‐label immunocytochemistry. NPY‐IR perikarya and fibers were found to be widely distributed in the ventral diencephalon, with high densities in the preopticoseptal, periventricular, and tuberal regions. Small neuronal cell groups were infiltrated with a dense network of varicose NPY‐IR fibers in the lateral preoptic area. The LHRH‐IR perikarya were located mainly in the preopticoseptal region, diagonal band of Broca, lamina terminalis, and periventricular and infundibular nuclei. A few LHRH‐IR neurons and fibers were scattered in the mamillary region. The overlap between the NPY and LHRH systems was apparent in the periventricular, paraventricular, and infundibular nuclei. Double‐labeling immunohistochemistry showed NPY‐IR axon varicosities in contact with LHRH‐IR perikarya and main dendrites. The putative innervation of LHRH neurons by NPY‐IR fibers was also seen in 1‐μm‐thick plastic sections and with confocal laser scanning microscope, thus further supporting the functional impact of NPY‐IR terminals on LHRH‐IR neurons. The present findings suggest that the hypophysiotropic LHRH‐synthesizing neurons may be innervated by intrahypothalamic NPY‐IR fibers. Confirmation by ultrastructural analysis would demonstrate that the LHRH system in the human hypothalamus is regulated by NPY, as has been demonstrated in nonhuman species. J. Comp. Neurol. 427:593–603, 2000.

Oral and subcutaneous administration of the glycosaminoglycan C3 attenuates Aβ(25–35)-induced abnormal tau protein immunoreactivity in rat brain

High molecular weight glycosaminoglycans (GAG) and proteoglycans (PG) affect pathological changes of the brain in Alzheimers disease (AD). PG stimulate the processing and aggregation of amyloid-beta (Abeta), protect the protein from proteolysis, and increase the formation of neurofibrillary tangles by inducing the hyperphosphorylation of tau protein. These effects may be competitively inhibited by GAG. We have studied the effects of orally (by gavage) and subcutaneously (s.c.) administered low molecular weight heparin, C3 (4-10 oligosaccharides; MW = 2.1 kDa; USP value = 12 U/mg), on abnormal tau-2 protein immunoreactivity in the rat hippocampus following a single, unilateral intra-amygdaloid administration of Abeta(25-35). Oral administration of C3 (25 mg/kg; once daily) was initiated 3 days prior to Abeta(25-35) administration, and was continued daily for an additional 14 days. S.c. administration of C3 (2.5 mg/kg, twice daily), was started 3 days prior to, and was continued for 32 days after, Abeta(25-35) administration. Animal brains were subsequently processed for tau-2, ChAT-immunoreactivity, choline acetyltransferase (ChAT) activity and acetylcholinesterase (AChE) activity. Both oral and s.c. administration of C3 attenuated Abeta(25-35) induced appearance of tau-2-immunoreactive (IR) perikarya in the ipsilateral hippocampus (P < 0.05). Hippocampal cholinergic enzyme activity in C3 treated animals was not significantly different from control animals. The present findings suggest that C3 might be used successfully to prevent abnormal tau protein formation in chronic neurologic diseases, such as AD. Moreover, our data demonstrate that the mechanism of this effect does not appear to influence the cholinergic system of the brain.

The blood–brain barrier accessibility of a heparin-derived oligosaccharides C3

Although heparin-derived oligosaccharide(s) (HDO) have been clinically used for the management of neurological disorders, such as stroke and Alzheimers disease (AD), very little information on the mechanism of their therapeutic action is known. To test the hypothesis that HDO may pass through the blood-brain barrier (BBB) to mediate their effects, a pharmacodynamic (PD) model was developed and the presence of HDO in the cerebrospinal fluid (CSF) was used as a BBB accessibility index. Rats were treated with an ultralow molecular weight (MW) heparin fragment C3 via the intravenous or subcutaneous routes at 5-10 mg/kg. At varying periods, the plasma, CSF, and brain samples were collected, and functional anti-factor Xa activities were measured to quantitate the CSF/plasma ratios (CPR) and the brain uptake. C3 showed CPR of 1.7% and 0.8% after intravenous and subcutaneous injections, respectively. These findings were verified by intravenous administration of tritium-labeled C3 followed by detection of the radioactivity in the CSF and brain homogenates. These data suggest that ultralow MW HDO may pass through the BBB.

Protection against inflammatory neurodegeneration and glial cell death by 7β-hydroxy epiandrosterone, a novel neurosteroid

It has been demonstrated that neuroprotective effects of dehydroepiandrosterone (DHEA) may be mediated by its 7alpha- and 7beta-hydroxy derivatives. Epiandrosterone is also converted to 7beta-hydroxy epiandrosterone (7beta-OH EPIA) in numerous tissues. The aim of the present study was to establish whether treatment with 7beta-hydroxy epiandrosterone has a neuroprotective effect in animal models of Alzheimers disease (AD) lesions. Intra-amygdaloid administration of amyloid beta [Abeta(25-35)] increased the number of tau-positive cells in the ipsilateral hippocampus. Intracerebroventricular administration of ethylcholine aziridinium (AF64A) caused cholinergic damage in the septum, and glial lesions in the lateral septal nucleus and in the lateral zones of the hippocampus. These effects were almost completely prevented when animals were treated subcutaneously (b.i.d.) for 10 days with 0.1 mg/kg 7beta-hydroxy epiandrosterone. These findings indicate that 7beta-hydroxy epiandrosterone has powerful cytoprotective effects suggesting that (a) this neurosteroid may have therapeutic potential in various neurodegenerative conditions such as Alzheimers disease, and (b) 7beta-hydroxy steroids may constitute a novel class of endogenous neuroprotective agents.

Morphology and distribution of neurons expressing serotonin 5-HT1A receptors in the rat hypothalamus and the surrounding diencephalic and telencephalic areas.

Disorders of serotonergic neurotransmission are involved in disturbances of numerous hypothalamic functions including circadian rhythm, mood, neuroendocrine functions, sleep and feeding. Among the serotonin receptors currently recognized, 5-HT(1A) receptors have received considerable attention due to their importance in the etiology of mood disorders. While previous studies have shown the presence of 5-HT(1A) receptors in several regions of the rat brain, there is no detailed map of the cellular distribution of 5-HT(1A) receptors in the rat diencephalon. In order to characterize the distribution and morphology of the neurons containing 5-HT(1A) receptors in the diencephalon and the adjacent telencephalic areas, single label immunohistochemistry was utilized. Large, multipolar, 5-HT(1A)-immunoreactive (IR) neurons were mainly detected in the magnocellular preoptic nucleus and in the nucleus of diagonal band of Broca, while the supraoptic nucleus contained mainly fusiform neurons. Medium-sized 5-HT(1A)-IR neurons with triangular or round-shaped somata were widely distributed in the diencephalon, populating the zona incerta, lateral hypothalamic area, anterior hypothalamic nucleus, substantia innominata, dorsomedial and premamillary nuclei, paraventricular nucleus and bed nucleus of stria terminalis. The present study provides schematic mapping of 5-HT(1A)-IR neurons in the rat diencephalon. In addition, the morphology of the detected 5-HT(1A)-IR neural elements is also described. Since rat is a widely used laboratory animal in pharmacological models of altered serotoninergic neurotransmission, detailed mapping of 5-HT(1A)-IR structures is pivotal for the neurochemical characterization of the neurons containing 5-HT(1A) receptors.

Associations between the human growth hormone-releasing hormone- and neuropeptide-Y-immunoreactive systems in the human diencephalon: A possible morphological substrate of the impact of stress on growth

Previous studies revealed that stress is a pivotal factor in the regulation of growth. Psychological harassment may result in psychosocial dwarfism with delayed puberty, short stature and depression. Growth hormone (GH) secretion is suppressed by stress, possibly via the attenuation of growth hormone-releasing hormone (GHRH) secretion. However, the morphological substrate of this phenomenon has not been elucidated yet. Since neuropeptide Y (NPY) levels in the plasma is increased by administration of various stressors, the common consensus is that NPY plays a crucial role in the stress response. In the present study, we examined the putative juxtapositions between the NPY- and GHRH-immunoreactive (IR) systems in the human hypothalamus using double-label immunohistochemistry. Our findings revealed that the majority of the GHRH-IR perikarya formed intimate associations with NPY-IR fiber varicosities. The majority of these juxtapositions were found in the infundibular nucleus/median eminence where NPY-IR fiber varicosities often covered a significant surface area of the GHRH neurons. Since the juxtapositions between the GHRH-IR perikarya and NPY-IR fiber varicosities may be functional synapses, they may represent the morphological substrate of stress-suppressed GH secretion. The large number of contacting elements indicates that NPY plays a pivotal role in GH release, and may be considered as a major factor in the attenuation of growth by stress in humans.

Neuroprotective Properties of Glycosaminoglycans: Potential Treatment for Neurodegenerative Disorders

Previous studies suggest that proteoglycans and glycosaminoglycans (GAGs) may play an important role in the pathogenesis and/or alleviation of neurodegenerative disorders, including Alzheimer’s disease (AD). Proteoglycans increase the formation of neurofibrillary tangles, and stimulate the aggregation of β-amyloid (Aβ). This effect, on the other hand, is believed to be competitively inhibited by certain GAGs. Over the past few years, we have examined the neuroprotective properties of Neuroparin (C3), a low-molecular-weight GAG (approx. 2.1 kDa), in animal models of lesions characteristic of AD. Neuroparin is composed of 4–10 oligosaccharides, and it is derived from heparin involving depolymerization of heparin by gamma irradiation. In our experiments, Neuroparin protected against cholinergic lesions induced by intracerebroventricular injection of a specific cholinotoxin, AF64A, in rats. Administration of Neuroparin attenuated AF64A-stimulated, low-affinity nerve growth factor receptor-immunoreactive axonal varicosities in the rat septum, and increased arborization of hippocampal CA1 neurons. Neuroparin also reduced the septal caspase 3 immunoreactivity induced by AF64A treatment. Moreover, Neuroparin reduced tau 2 immunoreactivity in the rat hippocampus, stimulated by intra-amygdaloid injection of Aβ25–35. These findings are in good agreement with our previous data indicating a neuroprotective role of GAGs. These results, plus others, all suggest that Neuroparin may possess neuroprotective properties against many of the characteristic neural lesions in AD. Since our pharmacokinetic studies revealed that Neuroparin is capable of crossing the blood-brain barrier, Neuroparin may, conceivably, open an entirely new avenue in the treatment of neurodegenerative disorders. Phase I studies have been completed, and have proven to be extremely supportive in that regard.

Bi-directional associations between galanin and luteinizing hormone-releasing hormone neuronal systems in the human diencephalon.

Evidence suggests that galanin plays an important role in the regulation of reproduction in the rat. Galanin is colocalized with luteinizing hormone (LH)-releasing hormone (LHRH) in a subset of LHRH neurons in female rats and galanin-immunoreactive (galanin-IR) nerve terminals innervate LHRH neurons. Recent studies indicate that galanin may control gonadal functions in rats at two different levels: (i) via direct modulation of pituitary LH secretion and/or (ii) indirectly via the regulation of the hypothalamic LHRH release. However, the morphological substrate of any similar modulation is not known in human. In the present series of experiments we first mapped the galanin-IR and LHRH-IR neural elements in human brain, utilizing single label immunohistochemistry. Then, following the superimposition of the maps of these systems, the overlapping sites were identified with double labeling immunocytochemistry and examined in order to verify the putative juxtapositions between galanin-IR and LHRH-IR structures. LHRH and galanin immunoreactivity were detected mainly in the medial basal hypothalamus, in the medial preoptic area and along the diagonal band of Broca. Careful examination of the IR elements in the overlapping areas revealed close, bi-directional contacts between galanin-IR and LHRH-IR structures, which have been verified in semithin plastic sections. These galanin-LHRH and LHRH-galanin juxtapositions were most numerous in the medial preoptic area and in the infundibulum/median eminence of the human diencephalon. In conclusion, the present study is the first to reveal bi-directional juxtapositions between galanin- and LHRH-IR neural elements in the human diencephalon. These galanin-LHRH and LHRH-galanin contacts may be functional synapses, and they may be the morphological substrate of the galanin-controlled gonadal functions in humans.