Sex steroid modulation of neurokinin B gene expression in the arcuate nucleus of adult male rats

Human menopause is associated with hypertrophy and increased gene expression of neurokinin (NKB) neurons in the infundibular (arcuate) nucleus of the hypothalamus. We have hypothesized that these changes are secondary to gonadal failure. In the present study, we determined that orchidectomy resulted in an increase in the mean profile area and the number of neurons expressing NKB mRNA in the rat arcuate nucleus. No changes were seen when orchidectomy was combined with testosterone or estradiol replacement. These findings support our hypothesis and demonstrate that gonadal steroids modulate NKB neurons in the arcuate nucleus of adult male rats.     

Corticotropin-releasing hormone-synthesizing neurons of the human hypothalamus receive neuropeptide Y-immunoreactive innervation from neurons residing primarily outside the infundibular nucleus

Immunohistochemical single- and double-labeling studies were performed on the hypothalami of postmortem human brains to elucidate the distribution of corticotropin-releasing hormone (CRH)-immunoreactive (IR) neuronal elements and their interaction with the neuropeptide Y (NPY)-ergic neuronal system. The great majority of CRH-IR perikarya were found in the paraventricular nucleus (PVN), whereas a considerable number of CRH-IR neurons were also observed in the periventricular and infundibular nuclei. The dorsomedial nucleus and the perifornical region contained only scattered CRH-IR neurons. Dense CRH-IR fiber networks were found throughout the hypothalamus. However, the medial preoptic, the dorsolateral part of the supraoptic, the suprachiasmatic, the ventromedial, and the different mammillary nuclei showed a relative paucity of fibers. The terminal fields of NPY-IR axons overlapped the distribution of CRH-IR neurons in the hypothalamus. NPY-IR axon varicosities were juxtaposed to both dendrites and perikarya of the majority of CRH-IR neurons residing in the paraventricular, periventricular, and infundibular nuclei. These neurons were frequently contacted by multiple NPY axons that either formed baskets around their perikarya or completely ensheathed the emanating CRH dendrites. Because NPY and agouti-related protein (AGRP) are co-contained in neurons of the human infundibular nucleus, we used AGRP as a marker of NPY fibers originating exclusively from the infundibular nucleus. Only a small proportion of CRH neurons in the PVN was contacted by AGRP-IR axon varicosities, suggesting that NPY-IR innervation of CRH neurons in the PVN derive mainly from regions outside the infundibular nucleus. The present morphological findings support the view that NPY regulates the CRH system of the human hypothalamus and therefore at least some of the effects of NPY on metabolic, autonomic, and endocrine functions may be mediated through CRH.     

A vasopressin and oxytocin containing nucleus in the pig hypothalamus that shows neuronal changes during puberty

A vasopressin and oxytocin containing nucleus is described for the first time in the pig hypothalamus. It is located near the third ventricle, just dorsal to the suprachiasmatic nucleus, and consists of magnocellular neurons, similar to those of the supraoptic nucleus and paraventricular nucleus. Morphometric analysis of neuronal number, size, density, and volume was performed at four different ages: 1 day, 7 weeks, 16 weeks, and 30 weeks postnatally. No sex difference in these parameters was observed. In this period the volume of the nucleus increased gradually from 6.6 x 10(-3) to 54.2 x 10(-3) mm3. One day after birth 1,215 +/- 191 (mean +/- SEM) neurons were present in the vasopressin and oxytocin containing nucleus, followed by a decrease to 771 +/- 80 neurons at 7 weeks and 697 +/- 116 at 16 weeks. Between 16 and 30 weeks (puberty) there was a dramatic increase in neuron number up to 1,765 +/- 214 neurons. This increase in the number of vasopressin and oxytocin containing neurons in the pig hypothalamus is much later in development than has ever been reported so far.     

Interconnection between orexigenic neuropeptide Y- and anorexigenic alpha-melanocyte stimulating hormone-synthesizing neuronal systems of the human hypothalamus

Peripheral feeding-related hormones such as leptin, insulin, and ghrelin exert their main central effects through neuropeptide Y- (NPY) synthesizing and alpha-melanocyte-stimulating hormone- (alpha-MSH) synthesizing neurons of the hypothalamic arcuate nucleus. In rodents, recent reports have described an asymmetric signaling between these neuron populations by showing that while NPY influences alpha-MSH-synthesizing neurons, the melanocortin-receptor agonist Melanotan II (MTII) does not modulate the electrophysiological properties of NPY neurons. The functional neuroanatomy of the relationship between these cell populations is unknown in humans. The aim of the current study was to analyze the putative relationship of the orexigenic NPY and anorexigenic alpha-MSH systems in the infundibular nucleus of the human hypothalamus, the analogue of the rodent arcuate nucleus. Double-labeling fluorescent immunocytochemistry for NPY and alpha-MSH was performed on postmortem sections of the human hypothalamus. The sections were analyzed by confocal laser microscopy. Both NPY- and alpha-MSH-immunoreactive (IR) neurons were embedded in dense, intermingling networks of NPY- and alpha-MSH-IR axons in the human infundibular nucleus. NPY-IR varicosities were observed in juxtaposition to all alpha-MSH-IR neurons. The mean number of NPY-IR axon varicosities on the surface of an alpha-MSH-IR neuron was approximately six. The majority of NPY-IR neurons were also contacted by alpha-MSH-IR varicosities, although, the number of such contacts was lower (two alpha-MSH-IR varicosities per NPY neuron). In summary, the present data demonstrate that these two antagonistic, feeding-related neuronal systems are interconnected in the infundibular nucleus, and the neuronal wiring possesses an asymmetric character in the human hypothalamus.     

Hemorrhage activates proopiomelanocortin neurons in the rat hypothalamus

Severe blood loss lowers arterial pressure through a central mechanism that is thought to include opioid neurons. In this study, we investigated whether hemorrhage activates proopiomelanocortin (POMC) neurons by measuring Fos immunoreactivity and POMC mRNA levels in the medial basal hypothalamus. Hemorrhage (2.2 ml/100 g body weight over 20 min) increased the number of Fos immunoreactive neurons throughout the rostral-caudal extent of the arcuate nucleus, the retrochiasmatic area and the peri-arcuate region lateral to the arcuate nucleus where POMC neurons are located. Double label immunohistochemistry revealed that hemorrhage increased Fos expression by beta-endorphin immunoreactive neurons significantly. The proportion of beta-endorphin immunoreactive neurons that expressed Fos immunoreactivity increased approximately four-fold, from 11.7+/-1.4% in sham-operated control animals to 42.0+/-5.2% in hemorrhaged animals. Hemorrhage also increased POMC mRNA levels in the medial basal hypothalamus significantly, consistent with the hypothesis that blood loss activates POMC neurons. To test whether activation of arcuate neurons contributes to the fall in arterial pressure evoked by hemorrhage, we inhibited neuronal activity in the caudal arcuate nucleus by microinjecting the local anesthetic lidocaine (2%; 0.1 or 0.3 microl) bilaterally 2 min before hemorrhage was initiated. Lidocaine injection inhibited hemorrhagic hypotension and bradycardia significantly although it did not influence arterial pressure or heart rate in non-hemorrhaged rats. These results demonstrate that hemorrhage activates POMC neurons and provide evidence that activation of neurons in the arcuate nucleus plays an important role in the hemodynamic response to hemorrhage.     

Über die neuronale Hypertrophie der Pars subventricularis nuclei infundibularis bei Hypophysentumoren

Summary For the first time, the hypertrophy of nerve cells of the basal and basolateral regions of the infundibular nucleus is reported in four male patients who suffered from pituitary neoplasms (two chromophobe adenomas, two intrasellar craniopharyngiomas). In each patient, the pituitary gland was completely destroyed. This resulted in marked degeneration of the testicles. Each patient showed remarkably large neurons in an area first described as the subventricular nucleus by Sheehan and Kovács (1966) which we designate as the subventricular part of the infundibular (=arcuate) nucleus. Using an automatic image analyzer (Quatimet 720), the neuronal hypertrophy could be quantitatively confirmed and statistically secured when compared to normal controls. The hypertrophic neurons are marked by a variegated morphology, cytoplasmic vacuoles and a numerical increase of peripherally located Nissl bodies. Especially remarkable is the high frequency of so-called nuclear spheroids, that are regarded as cytoplasmic invaginations into the nucleus. Based on quantitative analytical findings and the distribution of the nuclear spheroids, the subventricular part represents a semilumar band of cells in the basal and basolateral region of the arcuate nucleus and is, as a special neuronal group, associated with certain functional states. In each case, a lack of gonadotropins and, thereby, of gonadal steroids is to be surmised. Since the arcuate nucleus represents the major part of the hypophyseotrophic region, this neuronal hypertrophy appears to indicate increased neuroendocrine activity based on a deficient negative feed-back. The subventricular part of the infundibular nucleus is an example of cerebral architectonics, discovered by pathological findings.

Mit Unterstützung des Sonderforschungsbereichs 33 der Deutschen Forschungsgemeinschaft und der Alexander von Humboldt-Stiftung     

CNS connections with the median raphe nucleus: retrograde tracing with WGA-apoHRP-Gold complex in the rat

In this work we examined the neuronal input to one of the serotoninergic centers in the brain, median raphe nucleus (MR). Special consideration is given to projections of the hypothalamus. To describe the afferents to MR, a retrograde transport technique was used after microinjection of WGA-apoHRP-Gold complex under pressure and subsequent gold-silver intensification on formaldehyde-fixed rat brain sections. Optimal conditions were obtained when the coordinates of the injection site were A +/- 1.5, L +/- 0.15, and H +/- 2.7 according to Paxinos and Watson (The Rat Brain in Stereotaxic Coordinates. New York: Academic Press, '82). Results obtained under these conditions show a heterogeneous distribution of labeled neurons throughout the brain, including a large proportion (+/- 65%) of hypothalamic neurons. Extra-hypothalamic neurons projecting to MR were from the prefrontal cortex, lateral and medial habenular nuclei, the pontine area of the central grey, interpeduncular nucleus, dorsal raphe nucleus, oculomotor and trochlear nuclei, dorsal and laterodorsal tegmental nuclei, parabrachial nuclei, and lateral and interpositus cerebellar nuclei. Hypothalamic neurons connected to MR were found to be from medial and lateral preoptic areas, lateral hypothalamus, dorsomedian nucleus, the perifornical area, and the complex of mammillary bodies. Many other discrete regions contained different densities of labeled perikarya: the medial preoptic nucleus, paraventricular nucleus, retrochiasmatic area, arcuate nucleus, lateral magnocellular nucleus, and the posterior area. The MR appears as an integrative center receiving many neuroanatomically and functionally heterogeneous inputs from the whole brain.     

Distribution of the vestibular neurons projecting to the oculomotor and trochlear nuclei in rabbits

Horseradish peroxidase and Fast Blue were injected into the oculomotor and trochlear nuclei of rabbits so as to study the distribution of vestibular neurons that project to these nuclei. After the oculomotor nucleus was injected, labelled neurons were found in the superior, medial, and descending vestibular nuclei as well as in cell group Y. In the superior nucleus, most of the neurons (510 +/- 46) were ipsilateral to the injection, although contralaterally labelled neurons were also observed (104 +/- 19) more peripherally. In cell group Y, 186 +/- 24 contralaterally labelled neurons were observed, whereas hardly any (8 +/- 3) were found on the ipsilateral side. The largest group of labelled neurons (811 +/- 65) constituted a neuronal band located contralaterally in the medial nucleus and rostral part of the descending nucleus. This band rostromedially continued with the caudal part of the group of internuclear neurons of the abducens nucleus. Only 190 +/- 31 neurons were labelled in the medial and descending nucleus ipsilateral to the injected oculomotor nucleus. After injection of the trochlear nucleus, labelled neurons were found in the ipsilateral superior nucleus and contralateral medial and descending nuclei: a few labelled cells were also observed in the ipsilateral medial and descending nuclei as well as in the contralateral cell group Y.     

The gonadotropin-releasing hormone containing ventral hypothalamic tract in the fetal rhesus monkey (Macaca mulatta)

A well-defined, gonadotropin-releasing hormone (GnRH)-containing fiber pathway, the ventral hypothalamic tract (VHT), is described by immunostaining in fetal rhesus macaques (109-156 days gestation). The VHT arises above the lateral aspects of the optic chiasm near the supraoptic nucleus, and courses ventromedially close to the ventral hypothalamic surface to terminate in the infundibulum and zona externa of the median eminence. It is formed by the confluence of GnRH-immunopositive (GnRH+) axons from local neurons, from a few GnRH+ cells in the inferior thalamic peduncle, and probably from more anterior neurons in the septum and preoptic area. Bipolar GnRH+ neurons contributing directly to the VHT are grouped at its origin dorsolateral to the optic chiasm, dorsal and medial to the optic tracts, at the infundibular lip, and within the pathway between. At the infundibular lip, GnRH+ perikarya are generally lateral or ventral to the infundibular (arcuate) nucleus, and are rarely within the nucleus itself. Cell bodies here are sometimes tripolar, but GnRH+ intercellular contacts are seldom seen. A few VHT fibers extend to the ventral surface of the brain just beneath the pia mater. Abundant capillaries in the subarachnoid space suggest a possible route for delivery of GnRH to the adenohypophysis in early gestation, before maturation of the hypophysial portal system occurs. Posterior to the infundibulum, a few VHT fibers are joined by descending periventricular fibers forming a dense fiber band beneath the premammillary recess of the third ventricle. Totals of GnRH+ cell bodies in the prosencephalon of the fetal rhesus macaque are estimated to be 5,600 in females (n = 2) and 2,600 in males (n = 3). More than 60% of VHT neurons are located in the medial basal hypothalamus, and the majority of basal hypothalamic GnRH+ neurons (86%) are associated with the VHT. Furthermore, reports of the autonomy of the medial basal hypothalamic-hypophysial unit in control of gonadotropin secretion suggest that the VHT may be the most important GnRH system involved in primate reproduction. It is clear that fetal material may offer the best model to study the GnRH neuronal system in primates.     

Importance of endogenous nitric oxide synthase in the rat hypothalamus and amygdala in mediating the response to capsaicin

Although capsaicin has been shown to activate certain neuronal groups in the hypothalamus and amygdala, the neurotransmitters involved and the exact mechanism of action are not clearly understood at present. The aim of this study was to examine the hypothesis that the effect of capsaicin in the rat hypothalamus and amygdala primarily involves direct activation of the endogenous nitric oxide synthase (NOS) neurons responsible for the synthesis of nitric oxide (NO). Subcutaneous capsaicin injection in male rats, compared with vehicle, caused a significant increase in Fos expression in the paraventricular nucleus (PVN), supraoptic nucleus (SON), and medial and cortical amygdala. The expression of nicotinamide adenine dinucleotide phosphate diaphorase, a histochemical marker for NOS, was also increased in these brain areas in addition to the periventricular and lateral hypothalamic area and central amygdaloid nucleus. Also, capsaicin significantly increased the expression of neuronal NOS messenger RNA and protein in the PVN, SON, and medial amygdala as demonstrated by in situ hybridization and immunohistochemistry, respectively. A higher proportion of the NOS neurons in the PVN, periventricular region, SON and amygdala showed Fos expression in response to capsaicin than vehicle injection. There was little, if any, Fos activation in the NOS-positive neurons in the lateral hypothalamic area. The capsaicin-induced activation of the hypothalamic PVN and SON neurons and the medial amygdaloid nucleus was attenuated in the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) -pretreated animals in comparison with the inactive enantiomer D-NAME. These observations indicate that activation of the endogenous NOS system and production of NO constitute a major pathway through which capsaicin exerts its effect within the hypothalamus and amygdala.     

Sex differences in the distribution of androgen receptors in the human hypothalamus

The present study reports for the first time the distribution of androgen receptor immunoreactivity (AR-ir) in the human hypothalamus of ten human subjects (five men and five women) ranging in age between 20 years and 39 years using the antibody PG21. Prolonged postmortem delay (72:00 hours) or fixation time (100 days) did not influence the AR-ir. In men, intense nuclear AR-ir was found in neurons of the horizontal limb of the diagonal band of Broca, in neurons of the lateromamillary nucleus (LMN), and in the medial mamillary nucleus (MMN). An intermediate nuclear staining was found in the diagonal band of Broca, sexually dimorphic nucleus of the preoptic area, paraventricular nucleus, suprachiasmatic nucleus, ventromedial nucleus, and infundibular nucleus, whereas weaker labeling was found in the bed nucleus of the stria terminalis, medial preoptic area, dorsal and ventral zones of the periventricular nucleus, supraoptic nucleus, and nucleus basalis of Meynert. In most brain areas, women revealed less staining than men. In the LMN and the MMN, a strong sex difference was found. Cytoplasmic labeling was observed in neurons of both sexes, although women showed a higher variability in the intensity of such staining. However, no sex differences in AR-ir were observed in the bed nucleus of the stria terminalis, the nucleus basalis of Meynert, or the islands of Calleja. Species differences and similarities of the AR-ir distribution are discussed. The present results suggest the participation of androgens in the regulation of various hypothalamic processes that are sexually dimorphic.     

Neurons expressing neuropeptide Y mRNA in the infundibular hypothalamus of Japanese quail are activated by fasting and co-express agouti-related protein mRNA

The neural mechanisms involved in the compensatory hyperphagia exhibited by many vertebrate species after a fast are not fully understood but, in mammals, appear to involve nutritionally-sensitive neurons that co-express neuropeptide Y (NPY) and agouti-related protein (AGRP) in the infundibular hypothalamus. We investigated whether these neurons have been evolutionarily conserved in a non-mammalian vertebrate, the Japanese quail. Birds exhibited compensatory hyperphagia 1 h after return of food following a 24-h fast. We addressed a potential regulatory role for NPY, first, by using in situ hybridisation (ISH) to map NPY gene expression in the hypothalamus. This revealed a strong signal in the infundibular nucleus (IN). Secondly, we quantified NPY gene expression in 24-h fasted birds compared to ad libitum fed controls using two independent methods. In whole hypothalamus, measured by ribonuclease protection assay, NPY mRNA increased 1.5-fold in fasted birds. A similar, 1.7-fold, increase was observed specifically in the IN when analysed by ISH. No differences in NPY expression between fed and fasted birds were observed in other brain regions. To determine whether NPY neurons in the avian IN co-express AGRP, we cloned a fragment of the quail AGRP gene and used it to localise AGRP mRNA by ISH. The gene was expressed exclusively in the hypothalamus, specifically in the IN, where its distribution matched that of NPY. Double-label ISH revealed that the majority of NPY neurons in the IN co-express AGRP mRNA. Collectively, these data indicate that this cell type has been neuroanatomically and functionally conserved during vertebrate evolution.     

Selective effects of kainic acid on diencephalic neurons

Injections of kainic acid (KA) into the lateral hypothalamus (LH) produce neuronal loss in this region without apparent damage to medial forebrain bundle fibers passing through the area. Cellular destruction is not limited to the LH; the neuronal loss in the thalamic reticular nucleus, the subthalamic nucleus and zona incerta is more extensive than that in the LH. Since all of the nuclei of ventral thalamic origin except the ventral lateral geniculate nucleus (VLGN) were destroyed by LH injections of KA, we sought to determine whether this nucleus also is sensitive to KA. Injections directly into the VLGN produce total neuronal loss here as well as in the thalamic reticular nucleus, zona incerta and subthalamic nucleus. Other areas showing cell loss are the dorsal LGN, medial geniculate, lateral portion of the ventrobasal complex and midline thalamic nuclei. Injections of KA into medial hypothalamus adjacent to the suprachiasmatic nucleus produced no neuronal degeneration. In addition, no neuronal loss was noted in medial hypothalamic nuclei lying adjacent to areas of LH in which KA was injected. Therefore, the sensitivity of diencephalic nuclei appears to range from highly sensitive regions such as derivatives of ventral thalamus and midline thalamic nuclei to regions of moderate sensitivity such as the LH, geniculate nuclei and ventrobasal thalamic nucleus, to regions resistant to KA toxicity such as the suprachiasmatic nucleus and other nuclei and areas of medial hypothalamus.     

Neuroprotective effects of prostaglandin A1 in animal models of focal ischemia

The distribution of motor and proprioceptive neurons was investigated in the trigeminal nervous system of wild-type and Brn-3a knockout mice at embryonic day 18.5 and postnatal day 0. We found that the trigeminal motor nucleus (Mo5) contained abundant motoneurons in wild-type (mean number +/- SD per section = 128 +/- 22, range = 93-167) and knockout (mean number +/- SD per section = 121 +/- 23, range = 75-158) mice and that the cell size of Mo5 neurons was similar between these mice (wild-type, mean +/- SD = 165 +/- 59 microm2, range = 65-326 microm2; knockout, mean +/- SD = 167 +/- 59 microm2, range = 71-327 microm2). Mo5 neurons were immunoreactive for calcitonin gene-related peptide and such immunoreactive neurons were abundant in both wild-type and mutant mice. In the mesencephalic tract nucleus (Mes5) of wild-type mice, many proprioceptors (mean number +/- SD per section = 56 +/- 19, range = 27-85) that contained parvalbumin immunoreactivity were also observed. In knockout mice, however, Mes5 neurons could not be detected. The area of brainstems which normally contained the Mes5 was devoid of parvalbumin-immunoreactive proprioceptors. The present study suggests that Brn-3a is required for the development of proprioceptors but not motoneurons in the trigeminal nervous system.     

mu-opioid receptor mRNA expression in identified hypothalamic neurons

It has been known for a number of years that mu-opioid receptor agonists (e.g., morphine, beta-endorphin, and enkephalin) inhibit luteinizing hormone (LH), vasopressin (VP), and oxytocin (OT) release and stimulate prolactin secretion in rodents and primates by an action at the level of the brain. Also, electrophysiological studies have established that hypothalamic neurons, including gonadotropin-releasing hormone (GnRH), VP, OT, beta-endorphin, and dopamine neurons, are responsive to mu-receptor activation. Although mu-receptor expression has been demonstrated in the hypothalamus, there have been few studies localizing these receptors in neurosecretory neurons. Therefore, we sought to document mu-opioid receptor mRNA expression in immunocytochemically identified hypothalamic neurons. The brains from both female and male guinea pigs were examined by using in situ hybridization and immunocytochemistry. The studies revealed that mu-receptor mRNA was expressed in different diencephalic regions including the preoptic area, the bed nuclei stria terminalis, the paraventricular nucleus thalamus, and the anterior hypothalamus, as well as the supraoptic (SON), paraventricular (PVH), ventromedial, dorsomedial, and arcuate nuclei of the hypothalamus. Importantly, mu-opioid receptors were expressed in subpopulations of GnRH neurons (33.25 +/- 4.6% and 33.6 +/- 3.7% in females and males, respectively), dopamine neurons (51.7 +/- 5.8% to 75.0 +/- 2.6%, depending on neuronal location), beta-endorphin neurons (68.3.0 +/- 4.4%), and VP neurons (41-70%, depending on neuronal location). Because mu-opioid receptors couple via G-proteins to activate inwardly rectifying potassium channels and to inhibit calcium channels, the presence of these receptors is likely to play a major role in directly controlling the excitability of hypothalamic neurons.