Interleukin-6 receptor α is co-localised with melanin-concentrating hormone in human and mouse hypothalamus.

Interleukin (IL)-6 deficient mice develop mature-onset obesity. Furthermore, i.c.v. administration of IL-6 increases energy expenditure, suggesting that IL-6 centrally regulates energy homeostasis. To investigate whether it would be possible for IL-6 to directly influence the energy homeostasis via hypothalamic regulation in humans and rodents, we mapped the distribution of the ligand binding IL-6 receptor α (IL-6Rα) in this brain region. In the human hypothalamus, IL-6Rα-immunoreactivity was detected in perikarya and first-order dendrites of neurones. The IL-6Rα-immunoreactive (-IR) neurones were observed posterior to the level of the interventricular foramen. There, IL-6Rα-IR neurones were located in the lateral hypothalamic, perifornical, dorsal and posterior hypothalamic areas, the hypothalamic dorsomedial nucleus and in the zona incerta. In the caudal part of the hypothalamus, the density of the IL-6Rα-IR neurones gradually increased. Double-labelling immunofluorescent studies demonstrated that IL-6Rα immunoreactivity was localised in the same neurones as the orexigenic neuropeptide, melanin-concentrating hormone (MCH). By contrast, IL-6Rα-immunoreactivity was not observed in the orexin B-IR neurones. To determine whether the observed expression of IL-6Rα is evolutionary conserved, we studied the co-localisation of IL-6Rα with MCH and orexin in the mouse hypothalamus, where IL-6Rα-immunoreactivity was present in numerous MCH-IR and orexin-IR neurones. Our data demonstrate that the MCH neurones of the human hypothalamus, as well as the MCH and orexin neurones of the mouse hypothalamus, contain IL-6Rα. This opens up the possibility that IL-6 influences the energy balance through the MCH neurones in humans, and both MCH and orexin neurones in mice.

Co-localisation of kisspeptin with galanin or neurokinin B in afferents to mouse GnRH neurones.

The gonadotrophin-releasing hormone (GnRH) secreting neurones, which form the final common pathway for the central regulation of reproduction, are directly targeted by kisspeptin (KP) via the G protein-coupled receptor, GPR54. In these multiple labelling studies, we used ovariectomised mice treated with 17ß-oestradiol (OVX + E(2)) or vehicle (OVX + oil) to determine: (i) the ultrastructural characteristics of KP-immunoreactive (IR) afferents to GnRH neurones; (ii) their galanin or neurokinin B (NKB) content; and (iii) the co-expression of galanin or NKB with KP in the two major subpopulations of KP neurones located in the rostral periventricular area of the third ventricle (RP3V) and the arcuate nucleus (Arc). Electron microscopic investigation of the neuronal juxtapositions revealed axosomatic and axodendritic synapses; these showed symmetrical or asymmetrical characteristics, suggesting a phenotypic diversity of KP afferents. Heterogeneity of afferents was also demonstrated by differential co-expression of neuropeptides; in OVX + E(2) mice, KP afferents to GnRH neurones showed galanin-immunoreactivity with an incidence of 22.50 ± 2.41% and NKB-immunoreactivity with an incidence of 5.61 ± 2.57%. In OVX + oil animals, galanin-immunoreactivity in the KP afferents showed a major reduction, appearing in only 5.78 ± 1.57%. Analysis for co-localisation of galanin or NKB with KP was extended to the perikaryal level in animal models, which showed the highest KP incidence; these were OVX + E(2) females for the RP3V and OVX + oil females for the ARC. In the RP3V of colchicine-treated OVX + E(2) animals, 87.84 ± 2.65% of KP-IR neurones were galanin positive. In the Arc of the colchicine-treated OVX + oil animals, galanin immunoreactivity was detected in only 12.50 ± 1.92% of the KP expressing neurones. By contrast, the incidence of co-localisation with NKB in the Arc of those animals was 98.09 ± 1.30%. In situ hybridisation histochemistry of sections from OVX + E(2) animals identified galanin message in more than a third of the KP neurones in the RP3V (38.67 ± 11.57%) and in the Arc (42.50 ± 12.52%). These data suggest that GnRH neurones are innervated by chemically heterogeneous KP cell populations, with a small proportion deriving from the Arc group. The presence of galanin within KP axons innervating GnRH neurones and the oestrogen-dependent regulation of that presence add a new dimension to the roles played by galanin in the central regulation of reproduction.

Estradiol down-regulates RF-amide-related peptide (RFRP) expression in the mouse hypothalamus.

In most mammals, RF-amide-related peptides are synthesized in the dorsomedial hypothalamic nucleus and regulate reproduction via inhibiting GnRH neurons and, possibly, adenohypophyseal gonadotrophs. In the present study, we investigated the possibility that RFRP-synthesizing neurons are involved in estrogen feedback signaling to the reproductive axis in mice. First, we used quantitative in situ hybridization and compared the expression of prepro-RFRP mRNA of ovariectomized mice, with and without 17ß-estradiol (E2) replacement. Subcutaneous administration of E2 via silastic capsules for 4 d significantly down-regulated prepro-RFRP mRNA expression. The underlying receptor mechanism was investigated with immunohistochemistry. In ovariectomized mice, low levels of nuclear estrogen receptor (ER)-α immunoreactivity were detectable in 18.7 ± 3.8% of RFRP neurons. The majority of RFRP neurons showed no ER-α signal, and RFRP neurons did not exhibit ER-ß immunoreactivity. Results of these studies indicate that RFRP is a negatively estradiol-regulated neurotransmitter/neuromodulator in mice. The estrogenic down-regulation of RFRP expression may contribute to estrogen feedback to the reproductive axis. The issue of whether E2 regulates RFRP neurons directly or indirectly remains open given that ER-α immunoreactivity is present only at low levels in a subset of these cells.

Blockade of central nicotine acetylcholine receptor signaling attenuate ghrelin-induced food intake in rodents.

Here we sought to determine whether ghrelin's central effects on food intake can be interrupted by nicotine acetylcholine receptor (nAChR) blockade. Ghrelin regulates mesolimbic dopamine neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens, partly via cholinergic VTA afferents originating in the laterodorsal tegmental area (LDTg). Given that these cholinergic projections to the VTA have been implicated in natural as well as drug-induced reinforcement, we sought to investigate the role of cholinergic signaling in ghrelin-induced food intake as well as fasting-induced food intake, for which endogenous ghrelin has been implicated. We found that i.p. treatment with the non-selective centrally active nAChR antagonist, mecamylamine decreased fasting-induced food intake in both mice and rats. Moreover, central administration of mecamylamine decreased fasting-induced food intake in rats. I.c.v. ghrelin-induced food intake was suppressed by mecamylamine i.p. but not by hexamethonium i.p., a peripheral nAChR antagonist. Furthermore, mecamylamine i.p. blocked food intake following ghrelin injection into the VTA. Expression of the ghrelin receptor, the growth hormone secretagogue receptor 1A, was found to co-localize with choline acetyltransferase, a marker of cholinergic neurons, in the LDTg. Finally, mecamylamine treatment i.p. decreased the ability of palatable food to condition a place preference. These data suggest that ghrelin-induced food intake is partly mediated via nAChRs and that nicotinic blockade decreases the rewarding properties of food.

The kisspeptin system of the human hypothalamus: sexual dimorphism and relationship with gonadotropin-releasing hormone and neurokinin B neurons.

Kisspeptin signaling via the kisspeptin receptor G-protein-coupled receptor-54 plays a fundamental role in the onset of puberty and the regulation of mammalian reproduction. In this immunocytochemical study we addressed the (i) topography, (ii) sexual dimorphism, (iii) relationship to gonadotropin-releasing hormone (GnRH) neurons and (iv) neurokinin B content of kisspeptin-immunoreactive hypothalamic neurons in human autopsy samples. In females, kisspeptin-immunoreactive axons formed a dense periventricular plexus and profusely innervated capillary vessels in the infundibular stalk. Most immunolabeled somata occurred in the infundibular nucleus. Many cells were also embedded in the periventricular fiber plexus. Rostrally, they formed a prominent periventricular cell mass (magnocellular paraventricular nucleus). Robust sex differences were noticed in that fibers and somata were significantly less numerous in male individuals. In dual-immunolabeled specimens, fine kisspeptin-immunoreactive axon varicosities formed axo-somatic, axo-dendritic and axo-axonal contacts with GnRH neurons. Dual-immunofluorescent studies established that 77% of kisspeptin-immunoreactive cells in the infundibular nucleus synthesize the tachykinin peptide neurokinin B, which is known to play crucial role in human fertility; 56 and 17% of kisspeptin fibers in the infundibular and periventricular nuclei, respectively, contained neurokinin B immunoreactivity. Site-specific co-localization patterns implied that kisspeptin neurons in the infundibular nucleus and elsewhere contributed differentially to these plexuses. This study describes the distribution and robust sexual dimorphism of kisspeptin-immunoreactive elements in human hypothalami, reveals neuronal contacts between kisspeptin-immunoreactive fibers and GnRH cells, and demonstrates co-synthesis of kisspeptins and neurokinin B in the infundibular nucleus. The neuroanatomical information will contribute to our understanding of central mechanisms whereby kisspeptins regulate human fertility.

Evidence for suprachiasmatic vasopressin neurones innervating kisspeptin neurones in the rostral periventricular area of the mouse brain: regulation by oestrogen.

In rodents, a circadian signal from the suprachiasmatic nucleus (SCN) is essential for the pro-oestrous surge of gonadotrophin-releasing hormone (GnRH), which, in turn, induces luteinising hormone (LH) surge and ovulation. We hypothesised that kisspeptin (KP) neurones in the anteroventral periventricular and periventricular preoptic nuclei (AVPV/PeN) form part of the communication pathway between the SCN and GnRH neurones. In anterograde track tracing studies, we first identified vasopressin (VP)-containing axons of SCN origin in apposition to KP-immunoreactive (IR) neurones. Studies to quantify this input relied on the observation that VP-synthesising neurones in the SCN differ from other VP systems in their lack of galanin expression. In ovariectomised mice, 30.79 +/- 1.63% of KP-IR perikarya and proximal dendrites within the AVPV/PeN received galanin-negative VP-IR varicosities. Oestrogen-treatment significantly increased the number of KP-IR neurones, with their percentage apposed by galanin-negative VP-IR varicosities (46.95 +/- 1.88%) and the number of VP-IR appositions on individual KP-IR neurones. At the ultrastructural level, the VP-IR terminals formed symmetric synapses with KP-IR neurones, which was in accordance with the morphology of inhibitory synapses established by SCN neurones. By contrast to VP, vasoactive intestinal polypeptide (VIP), which is synthesised by a distinct subset of SCN neurones, occurred only rarely in axons apposed to KP-IR neurones. Altogether, our results are consistent with the hypothesis that KP neurones located in the mouse AVPV/PeN receive circadian information from the SCN via a vasopressinergic monosynaptic pathway, which is enhanced by oestrogen.

Oestrogen receptor alpha and beta immunoreactive cells in the suprachiasmatic nucleus of mice: distribution, sex differences and regulation by gonadal hormones.

Oestrogen regulates various aspects of circadian rhythm physiology. The presence of oestrogen receptors within the suprachiasmatic nucleus (SCN), the principal circadian oscillator, indicates that some actions of oestrogen on circadian functions may be exerted at that site. The present study analysed sex differences, topographic distribution, and neurochemical phenotype of neurones expressing the alpha and beta subtypes of oestrogen receptors (ERalpha and ERbeta) in the mouse SCN. We found that relatively few neurones in the SCN are immunoreactive (IR) for ERalpha (approximately 4.5% in females and 3% in males), but five- to six-fold more SCN neurones express ERbeta. ER-IR neurones are primarily in the shell subdivision of the nucleus and show differences between the sexes, significantly greater numbers being found in females. Treatment of male or female gonadectomised mice with oestradiol benzoate for 24 h substantially reduced the number of ERbeta-IR neurones, but not ERalpha-IR neurones. Double-labelling immunocytochemical experiments to characterise the phenotype of the oestrogen-receptive neurones showed the presence of the calcium-binding proteins calretinin or calbindin D28K in approximately 12% and 10%, respectively, of ERalpha-IR neurones. A higher proportion (approximately 38%) of ERbeta-IR neurones contains calbindin D28K; a few (approximately 2%) express calretinin or vasopressin. These double-labelled cells appear primarily in the shell subdivision of the SCN. Neither vasoactive intestinal polypeptide- nor gastrin releasing peptide-immunoreactivity was observed in ER-IR neurones. These data indicate that the primary target cells for oestrogen are in the shell subdivision of the nucleus. The sexually differentiated expression and distribution of ERalpha and ERbeta in various cell populations of the SCN suggest multiple modes of oestrogen signalling within this nucleus, which may modulate circadian functions.

Novel aspects of glutamatergic signalling in the neuroendocrine system.

L-glutamate, the main excitatory neurotransmitter, influences virtually all neurones of the neuroendocrine hypothalamus via synaptic mechanisms. Vesicular glutamate transporters (VGLUT1-3), which selectively accumulate L-glutamate into synaptic vesicles, provide markers with which to visualise glutamatergic neurones in histological preparations; excitatory neurones in the endocrine hypothalamus synthesise the VGLUT2 isoform. Results of recent dual-label in situ hybridisation studies indicate that glutamatergic neurones in the preoptic area and the hypothalamic paraventricular, supraoptic and periventricular nuclei include parvocellular and magnocellular neurosecretory neurones which secrete peptide neurohormones into the bloodstream to regulate endocrine functions. Neurosecretory terminals of GnRH, TRH, CRF-, somatostatin-, oxytocin- and vasopressin-secreting neurones contain VGLUT2 immunoreactivity, suggesting the co-release of glutamate with hypophysiotrophic peptides. The presence of VGLUT2 also indicates glutamate secretion from non-neuronal endocrine cells, including gonadotrophs and thyrotrophs of the anterior pituitary. Results of in vitro studies show that ionotropic glutamate receptor analogues can elicit hormone secretion at neuroendocrine/endocrine release sites. Structural constituents of the median eminence, adenohypophysis and neurohypophysis contain elements of glutamatergic transmission, including glutamate receptors and enzymes of the glutamate/glutamine cycle. The synthesis of VGLUT2 exhibits robust up-regulation in response to certain endocrine challenges, indicating that altered glutamatergic signalling may represent an important adaptive mechanism. This review article discusses the newly emerged non-synaptic role of glutamate in neuroendocrine and endocrine communication.

Gonadotropin-releasing hormone neurons express estrogen receptor-beta.

CONTEXT: Recent identification of the second estrogen receptor (ER) isoform (ER-beta) within GnRH neurons of the rodent brain has generated much enthusiasm in the field of neuroendocrine research by questioning the dogma that GnRH cells do not directly sense changes in circulating estrogens. OBJECTIVE: To address the issue of whether GnRH neurons of the human hypothalamus also contain ER-beta, we have performed dual-label immunocytochemical studies. DESIGN: Tissue sections were prepared from autopsy samples of male human individuals (n = 8; age < 50 yr), with sudden causes of death. Technical efforts were made to minimize postmortem interval (<24 h), optimize tissue fixation (use of a mixture of 2% paraformaldehyde and 4% acrolein for four tissue samples), and sensitize the immunocytochemical detection of ER-beta (application of silver-intensified nickel-diaminobenzidine chromogen). MAIN OUTCOME MEASURE: Distribution and percent ratio of GnRH neurons that also contained ER-beta immunoreactivity were analyzed under the light microscope. RESULTS: With acrolein in tissue fixative, nuclear ER-beta immunoreactivity was observed in 10.8-28.0% of GnRH neurons of the four different individuals. ER-beta-containing GnRH neurons were widely distributed in the hypothalamus, without showing a noticeable preference in regional location. CONCLUSIONS: The demonstration of ER-beta and the previous lack of detection of ER-alpha in human GnRH cells indicate that estrogens may exert direct actions upon GnRH neurons exclusively through ER-beta. In the light of differing ligand-binding characteristics of ER-beta from those of ER-alpha, this discovery offers a potential new approach to influence estrogen feedback to GnRH neurons through ER-beta-selective receptor ligands.

Glutamatergic innervation of the hypothalamic median eminence and posterior pituitary of the rat.

Recent studies have localized the glutamatergic cell marker type-2 vesicular glutamate transporter (VGLUT2) to distinct peptidergic neurosecretory systems that regulate hypophysial functions in rats. The present studies were aimed to map the neuronal sources of VGLUT2 in the median eminence and the posterior pituitary, the main terminal fields of hypothalamic neurosecretory neurons. Neurons innervating these regions were identified by the uptake of the retrograde tract-tracer Fluoro-Gold (FG) from the systemic circulation, whereas glutamatergic perikarya of the hypothalamus were visualized via the radioisotopic in situ hybridization detection of VGLUT2 mRNA. The results of dual-labeling studies established that the majority of neurons accumulating FG and also expressing VGLUT2 mRNA were located within the paraventricular, periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area. In contrast, only few FG-accumulating cells exhibited VGLUT2 mRNA signal in the arcuate nucleus. Dual-label immunofluorescent studies of the median eminence and posterior pituitary to determine the subcellular location of VGLUT2, revealed the association of VGLUT2 immunoreactivity with SV2 protein, a marker for small clear vesicles in neurosecretory endings. Electron microscopic studies using pre-embedding colloidal gold labeling confirmed the localization of VGLUT2 in small clear synaptic vesicles. These data suggest that neurosecretory neurons located mainly within the paraventricular, anterior periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area secrete glutamate into the fenestrated vessels of the median eminence and posterior pituitary. The functional aspects of the putative neuropeptide/glutamate co-release from neuroendocrine terminals remain to be elucidated.

Direct targeting of hippocampal neurons for apoptosis by glucocorticoids is reversible by mineralocorticoid receptor activation.

An important question arising from previous observations in vivo is whether glucocorticoids can directly influence neuronal survival in the hippocampus. To this end, a primary postnatal hippocampal culture system containing mature neurons and expressing both glucocorticoid (GR) and mineralocorticoid (MR) receptors was developed. Results show that the GR agonist dexamethasone (DEX) targets neurons (microtubule-associated protein 2-positive cells) for death through apoptosis. GR-mediated cell death was counteracted by the MR agonist aldosterone (ALDO). Antagonism of MR with spironolactone ([7alpha-(acetylthio)-3-oxo-17alpha-pregn-4-ene-21 carbolactone] (SPIRO)) causes a dose-dependent increase in neuronal apoptosis in the absence of DEX, indicating that nanomolar levels of corticosterone present in the culture medium, which are sufficient to activate MR, can mask the apoptotic response to DEX. Indeed, both SPIRO and another MR antagonist, oxprenoate potassium ((7alpha,17alpha)-17-hydroxy-3-oxo-7-propylpregn-4-ene-21-carboxylic acid, potassium salt (RU28318)), accentuated DEX-induced apoptosis. These results demonstrate that GRs can act directly to induce hippocampal neuronal death and that demonstration of their full apoptotic potency depends on abolition of survival-promoting actions mediated by MR.

Estrogen receptor-beta immunoreactivity in luteinizing hormone-releasing hormone neurons of the rat brain.

Feedback regulation of luteinizing hormone-releasing hormone (LHRH) neurons by estradiol plays important roles in the neuroendocrine control of reproduction. Recently, we found that the majority of LHRH neurons in the rat contain estrogen receptor-beta (ER-beta) mRNA, whereas, they seemed to lack ER-alpha mRNA expression. In addition, we observed nuclear uptake of (125)I-estrogen by a subset of these cells. These data suggest that ER-beta is the chief receptor isoform mediating direct estrogen effects upon LHRH neurons. To verify the translation of ER-beta protein within LHRH cells, the present studies applied dual-label immunocytochemistry (ICC) to free-floating sections obtained from the preoptic area of rats. The improved ICC method using the silver-gold intensification of nickel-diaminobenzidine chromogen, enabled the observation of nuclear ER-beta-immunoreactivity in the majority of LHRH cells. The incidence of ER-beta expression was similarly high in LHRH neurons of ovariectomized female (87.8 +/- 2.3%, mean +/- SEM), estradiol-primed female (74.9 +/- 3.2%) and intact male (85.0 +/- 4.7%) rats. The presence of ER-beta mRNA, ER-beta immunoreactivity and (125)I-estrogen binding sites in LHRH neurons of the rat provide strong support for the notion that these cells are directly regulated by estradiol, through ER-beta. The gene targets and molecular mechanisms of this regulation remain unknown.

Detection of estrogen receptor-beta messenger ribonucleic acid and 125I-estrogen binding sites in luteinizing hormone-releasing hormone neurons of the rat brain.

Luteinizing hormone-releasing hormone (LHRH) neurons of the forebrain play a pivotal role in the neuroendocrine control of reproduction. Although serum estrogen levels influence many aspects of LHRH neuronal activity in the female, earlier studies were unable to detect estrogen receptors (ERs) within LHRH neurons, thus shaping a consensus view that the effects of estradiol on the LHRH neuronal system are mediated by interneurons and/or the glial matrix. The present studies used dual-label in situ hybridization histochemistry (ISHH) and combined LHRH-immunocytochemistry/125I-estrogen binding to readdress the estrogen-receptivity of LHRH neurons in the female rat. In ISHH experiments we found that the majority of LHRH neurons exhibited hybridization signal for the "beta" form of ER (ER-beta). The degree of colocalization was similar in topographically distinct populations of LHRH neurons and was not significantly altered by estradiol (67.2+/-1.8% in ovariectomized and 73.8+/-4.2% in ovariectomized and estradiol-treated rats). In contrast, the mRNA encoding the classical ER-alpha could not be detected within LHRH neurons. In addition, in vivo binding studies using 125I-estrogen revealed a subset of LHRH-immunoreactive neurons (8.8%) which accumulated the radioligand thus providing evidence for the translation of ER protein(s) within these cells. The findings that most LHRH neurons in the female rat express ER-beta mRNA and at least some are capable of binding 125I-estrogen challenge the current opinion that estrogen does not exert direct effects upon the LHRH neuronal system.

Hypophysiotropic thyrotropin-releasing hormone-synthesizing neurons in the human hypothalamus are innervated by neuropeptide Y, agouti-related protein, and alpha-melanocyte-stimulating hormone.

We recently demonstrated that three arcuate nucleus-derived peptides, neuropeptide Y (NPY), agouti-related protein (AGRP), and alphaMSH, are contained in axon terminals that heavily innervate hypophysiotropic TRH neurons in the rat brain and may contribute to the altered set-point of the hypothalamo-pituitary-thyroid axis during fasting. To determine whether a similar regulatory system exists in human brain, we performed a series of immunohistochemical studies using antisera against NPY, AGRP, alphaMSH, and TRH in adult hypothalami obtained within 15 h of death. Numerous small to medium-sized, fusiform and multipolar NPY-, AGRP-, and alphaMSH-immunoreactive (-IR) cells were widely distributed throughout the rostro-caudal extent of the infundibular (arcuate) nucleus. A similar distribution pattern was found for NPY- and AGRP-IR neurons in the arcuate nucleus, whereas alphaMSH-IR cells appeared to form a separate cell population. By double labeling fluorescent immunohistochemistry, 82% of NPY neurons cocontained AGRP, and 87% of AGRP neurons coexpressed NPY. No colocalization was found between alphaMSH- and AGRP-IR neurons. NPY-, AGRP-, and alphaMSH-containing axons densely innervated the hypothalamic paraventricular nucleus and were found in close juxtaposition to TRH-synthesizing cell bodies and dendrites. These studies demonstrate that in man, the NPY-, AGRP-, and alphaMSH-IR neuronal systems in the infundibular and paraventricular nuclei are highly reminiscent of that observed in the rat and may similarly be involved in regulating the hypothalamo-pituitary-thyroid axis in the human brain.

C5a receptor expression by TGW neuroblastoma cells.

We recently reported that not only lymphoid cells, but cells of neuronal origin may harbor C5a receptors (C5aR) as suggested by results of RT-PCR testing and that an apoptotic pathway is associated with the C5aR. To determine whether C5aR is expressed as an integral membrane protein, we generated mono- and polyclonal anti-C5aR antibodies. Flow cytometry showed a low-level expression of C5aR in TGW neuroblastoma cells. Epitope mapping suggested that a conformation change in C5aR occurs when exposed to C5a. Although an aphysiologically high concentration of C5a is necessary for inducing a transient increase in the intracellular Ca2+ level, TGW cells do employ the signal transduction pathway associated with C5aR, suggesting that these cells may serve as putative model for C5aR-expressing neurons.

Peptidergic innervation of human cerebral blood vessels and saccular aneurysms.

Peptidergic innervation of the human cerebral vasculature has not yet been described in detail and its role in the maintenance of cerebral autoregulation still needs to be established. Similarly, few data exist on the innervation of vascular malformations. The aim of this study was to clarify the peptidergic innervation patterns of human cerebral arteries of various sizes, and, for the first time, that of saccular aneurysms. Light microscopic study of whole-mount preparations of human cerebral arteries and aneurysm sacs resected either during tumor removal or after neck-clipping were carried out by means of silver-intensified light microscopic immunocytochemistry visualizing neuropeptide-Y, calcitonin gene-related peptide and substance P immunoreactivity. Systematic morphological investigations confirmed the presence of longitudinal fiber bundles on the adventitia and a network-like deeper peptidergic system at the adventitia-media border, while in smaller pial and intraparenchymal vessels, only sparse longitudinal immunopositive axons could be detected. The innervation pattern was totally absent in the wall of saccular aneurysms with the complete disappearance of peptidergic nerve fibers in some areas. To the best of our knowledge neither the disappearance of this network on small pial and intraparenchymal vessels, nor the absence of an innervation pattern in saccular aneurysms have been described before. Nonhomogeneous peptidergic innervation of the human cerebral vascular tree might be one of the factors responsible for the distinct autoregulatory properties of the capacitance and resistance vessels. Malfunction of this vasoregulatory system might lead to the impairment of autoregulation during pathological conditions such as subarachnoid hemorrhage.

Estrogen receptor immunoreactivity is present in the majority of central histaminergic neurons: evidence for a new neuroendocrine pathway associated with luteinizing hormone-releasing hormone-synthesizing neurons in rats and humans.

The central regulation of the preovulatory LH surge requires a complex sequence of interactions between neuronal systems that impinge on LH-releasing hormone (LHRH)-synthesizing neurons. The reported absence of estrogen receptors (ERs) in LHRH neurons indicates that estrogen-receptive neurons that are afferent to LHRH neurons are involved in mediating the effects of this steroid. We now present evidence indicating that central histaminergic neurons, exclusively located in the tuberomammillary complex of the caudal diencephalon, serve as an important relay in this system. Evaluation of this system revealed that 76% of histamine-synthesising neurons display ERalpha-immunoreactivity in their nucleus; furthermore histaminergic axons exhibit axo-dendritic and axo-somatic appositions onto LHRH neurons in both the rodent and the human brain. Our in vivo studies show that the intracerebroventricular administration of the histamine-1 (H1) receptor antagonist, mepyramine, but not the H2 receptor antagonist, ranitidine, can block the LH surge in ovariectomized estrogen-treated rats. These data are consistent with the hypothesis that the positive feedback effect of estrogen in the induction of the LH surge involves estrogen-receptive histamine-containing neurons in the tuberomammillary nucleus that relay the steroid signal to LHRH neurons via H1 receptors.

Glycosylation of microtubule-associated protein tau in Alzheimer's disease brain.

In the neurofibrillary pathology of Alzheimer's disease (AD), neurofibrillary tangles (NFTs) contain paired helical filaments (PHFs) as their major fibrous component. Abnormally hyperphosphorylated, microtubule-associated protein tau is the major protein subunit of PHFs. A recent in vitro study showed that PHF tangles from AD brains are highly glycosylated, whereas no glycan is detected in normal tau. Deglycosylation of PHF tangles converts them into bundles of straight filaments and restores their accessibility to microtubules. We showed that PHF tangles from AD brain tissue were associated with specific glycan molecules by double immunostaining with peroxidase and alkaline phosphatase labeling. Intracellular tangles and dystrophic neurites in a neuritic plaque with abnormally hyperphosphorylated tau, detected with the monoclonal antibodies AT-8 and anti-tau-2, were also positive with lectin Galanthus nivalis agglutinin (GNA) which recognizes both the N- and O-glycosidically linked saccharides. Colocalization was not seen in the extracellular tangles and amyloid deposition, suggesting that the glycosylation of tau might be associated with the early phase of insoluble NFT formation. Thus, although abnormal phosphorylation might promote aggregation of tau and inhibition of the assembly of microtubules, glycosylation mediated by a GNA-positive glycan appears to be responsible for the formation of the PHF structures in vivo.

Complement C5a anaphylatoxin fragment causes apoptosis in TGW neuroblastoma cells.

Human neuroblastoma TGW cells express a C5a anaphylatoxin receptor-like molecule termed neuronal C5a receptor. A C5a-receptor fragment peptide (termed PR226-multiple antigenic peptide) can induce rapid apoptosis in TGW cells via neuronal C5a receptor-associated signal transduction pathways. In order to analyse role of activated complement system in neurodegeneration, TGW cells were exposed to an oligomer form of a C5a fragment (amino acids: 37-53) peptide termed PL37-multiple antigenic peptide. Upon treatment with PL37-multiple antigenic peptide, an increased nuclear c-fos expression was shown within 30 min. DNA fragmentation, a hallmark of apoptosis, was noted within 4 h. Extracellular administration of 100 nM PL37-multiple antigenic peptide evoked inward calcium current pulses. At higher doses (0.5 microM-1 microM), PL37-multiple antigenic peptide evoked higher current pulses, followed by an irreversible, high inward current. To exert its apoptotic effect, PL37-multiple antigenic peptide utilizes a pertussis toxin-sensitive signal transduction pathway associated with the neuronal C5a receptor. Activation of the complement system and therefore release of C5a has already been reported in Alzheimer's disease. In addition, the presence of the Kunitz-type proteinase inhibitors indicates an impaired protease function and a possible abnormal fragmentation of C5a anaphylatoxin. Our data suggest that neurons expressing neuronal C5a receptor are more vulnerable to the apoptosis associated with the neuronal C5a receptor and the possibility that abnormal activation of C5a receptor and C5a anaphylatoxin fragments might be involved in the pathogenesis of Alzheimer's disease.

A neuronal C5a receptor and an associated apoptotic signal transduction pathway.

1. We report the first experimental evidence of a neuronal C5a receptor (nC5aR) in human cells of neuronal origin. Expression of nC5aR mRNA was demonstrated by the reverse transcriptase-polymerase chain reaction (RT-PCR) in TGW human neuroblastoma cells. 2. Expression of a functional C5aR was supported by the finding that C5a evoked a transient increase in the intracellular calcium level as measured by flow cytometry (FACS). 3. To analyse the function of the nC5aR, an antisense peptide fragment of the C5aR was used. Previous data showed that a C5aR fragment (a peptide termed PR226) has C5aR agonist and antagonist effects in U-937 cells depending on the concentration of the peptide. We found that a multiple antigenic peptide (MAP) form of the same peptide (termed PR226-MAP) induced rapid elevation of nuclear c-fos immunoreactivity and resulted in DNA fragmentation, a characteristic sign of apoptosis, in TGW cells. 4. Early electrophysiological events characteristic of apoptosis were also detected: intermittent calcium current pulses were recorded within 1-2 min of peptide administration. C5a pretreatment delayed the onset of this calcium influx. 5. We also demonstrated that the apoptotic pathway is linked to nC5aR via pertussis toxin-sensitive G-proteins. 6. Although the function of C5a and its receptor on neurons is unknown, these results suggest that an abnormal activation of this signal transduction pathway can result in apoptosis and, subsequently, in neurodegeneration.