Characterisation and origins of melanin-concentrating hormone immunoreactive fibres of the posterior lobe of the pituitary and median eminence during lactation in the Long-Evans rat.

Although the melanin-concentrating hormone (MCH) and its coding mRNA are predominantly found in the tuberal hypothalamus, there is detectable synthesis of MCH in the preoptic hypothalamus exclusively in lactating dams, suggesting a participation of MCH in the alterations that take place after parturition. Also implicated in the dam physiology is oxytocin, a neurohormone released from the posterior pituitary that is necessary for milk ejection. Because the projection fields from oxytocin-immunoreactive (-IR) neurones and the mediobasal preoptic hypothalamus overlap and MCH-IR neurones are found in proximity to oxytocin neurones, we investigated the spatial relationship between MCH and oxytocin fibres. Accordingly, we employed multiple immunohistochemistry labelling for MCH and oxytocin for light and electron microscopy techniques, in addition to i.v. tracer injection combined with in situ hybridisation to identify MCH neurones that project to neurosecretory areas. As described for other strains, lactating Long-Evans dams also display immunoreactivity for MCH in the preoptic hypothalamus on days 12 and 19 of lactation. The appearance of these neurones is contemporaneous with an increase in MCH-IR fibres in both the internal layer of the median eminence and the posterior pituitary. In both regions, MCH- and oxytocin-IR fibres were found in great proximity, although there was no evidence for synaptic interaction between these two populations at the ultrastructural level. The tracer injection revealed that only mediobasal preoptic MCH neurones project to the posterior pituitary, suggesting a neuroendocrine-modulatory role for this population. When taken together, the results obtained in the present study indicate that neuroplasticity events at the mediobasal preoptic hypothalamus that occur during late lactation may be part of a neuroendocrinology control loop involving both MCH and oxytocin.

Effects of acute restraint and unpredictable chronic mild stress on brain corticotrophin releasing factor mRNA in the elevated T-maze.

Corticotrophin releasing factor (CRF) modulates stress/anxiety-related responses. Previous studies showed that exposure to acute restraint and unpredictable chronic mild stress (UCMS) facilitates elevated T-maze (ETM) avoidance responses, an anxiogenic-like effect. This study verified the role of CRF in the modulation of ETM avoidance and escape reactions, in unstressed rats and in animals exposed to acute restraint or to UCMS, by quantifying CRF mRNA concentrations in stress/anxiety-related brain regions, through semiquantitative in situ hybridization. Results showed that stress exposure altered CRF mRNA in regions related to the modulation of stress/anxiety: the cingulate cortex, the hippocampus, the paraventricular and dorsomedial hypothalamus, the medial and central amygdalas, the dorsal region of the dorsal raphe (dDR) and the ventrolateral periaqueductal gray. A regression analysis showed that the anxiogenic-like effects observed in acute restraint animals were particularly associated to increases in CRF mRNA in the paraventricular hypothalamus, medial and central amygdalas and dDR. On the other hand, anxiogenic-like effects observed after UCMS exposure are associated to increases in CRF mRNA in the medial and central amygdalas, in the BNST and in the ventrolateral periaqueductal grey. This observation suggests important differences in the neurocircuitry that mediates responses to acute and chronic stress exposure. CRF mRNA in regions traditionally related to the modulation of panic reactions (the dorsal periaqueductal grey and the lateral wings of the dorsal raphe) were not observed, what might explain the absence of panicogenic-like effects of stress exposure. These results contribute to a better understanding of the role played by CRF in stress/anxiety-related responses.

Anatomical Organization of Urocortin 3-Synthesizing Neurons and Immunoreactive Terminals in the Central Nervous System of Non-Human Primates [Sapajus spp.].

Urocortin 3 (UCN3) is a neuropeptide member of the corticotropin-releasing factor (CRF) peptide family that acts as a selective endogenous ligand for the CRF, subtype 2 (CRF2) receptor. Immunohistochemistry and in situ hybridization data from rodents revealed UCN3-containing neurons in discrete regions of the central nervous system (CNS), such as the medial preoptic nucleus, the rostral perifornical area (PFA), the medial nucleus of the amygdala and the superior paraolivary nucleus. UCN3-immunoreactive (UCN3-ir) terminals are distributed throughout regions that mostly overlap with regions of CRF2 messenger RNA (mRNA) expression. Currently, no similar mapping exists for non-human primates. To better understand the role of this neuropeptide, we aimed to study the UCN3 distribution in the brains of New World monkeys of the Sapajus genus. To this end, we analyzed the gene and peptide sequences in these animals and performed immunohistochemistry and in situ hybridization to identify UCN3 synthesis sites and to determine the distribution of UCN3-ir terminals. The sequencing of the Sapajus spp. UCN3-coding gene revealed 88% and 65% identity to the human and rat counterparts, respectively. Additionally, using a probe generated from monkey cDNA and an antiserum raised against human UCN3, we found that labeled cells are mainly located in the hypothalamic and limbic regions. UCN3-ir axons and terminals are primarily distributed in the ventromedial hypothalamic nucleus (VMH) and the lateral septal nucleus (LS). Our results demonstrate that UCN3-producing neurons in the CNS of monkeys are phylogenetically conserved compared to those of the rodent brain, that the distribution of fibers agrees with the distribution of CRF2 in other primates and that there is anatomical evidence for the participation of UCN3 in neuroendocrine control in primates.

The centrally projecting Edinger-Westphal nucleus--I: Efferents in the rat brain.

The oculomotor accessory nucleus, often referred to as the Edinger-Westphal nucleus [EW], was first identified in the 17th century. Although its most well known function is the control of pupil diameter, some controversy has arisen regarding the exact location of these preganglionic neurons. Currently, the EW is thought to consist of two different parts. The first part [termed the preganglionic EW-EWpg], which controls lens accommodation, choroidal blood flow and pupillary constriction, primarily consists of cholinergic cells that project to the ciliary ganglion. The second part [termed the centrally projecting EW-EWcp], which is involved in non-ocular functions such as feeding behavior, stress responses, addiction and pain, consists of peptidergic neurons that project to the brainstem, the spinal cord and prosencephalic regions. However, in the literature, we found few reports related to either ascending or descending projections from the EWcp that are compatible with its currently described functions. Therefore, the objective of the present study was to systematically investigate the ascending and descending projections of the EW in the rat brain. We injected the anterograde tracer biotinylated dextran amine into the EW or the retrograde tracer cholera toxin subunit B into multiple EW targets as controls. Additionally, we investigated the potential EW-mediated innervation of neuronal populations with known neurochemical signatures, such as melanin-concentrating hormone in the lateral hypothalamic area [LHA] and corticotropin-releasing factor in the central nucleus of the amygdala [CeM]. We observed anterogradely labeled fibers in the LHA, the reuniens thalamic nucleus, the oval part of the bed nucleus of the stria terminalis, the medial part of the central nucleus of the amygdala, and the zona incerta. We confirmed our EW-LHA and EW-CeM connections using retrograde tracers. We also observed moderate EW-mediated innervation of the paraventricular nucleus of the hypothalamus and the posterior hypothalamus. Our findings provide anatomical bases for previously unrecognized roles of the EW in the modulation of several physiologic systems.

Kinin B1 receptor gene ablation affects hypothalamic CART productionb.

A role for the kinin B1 receptor in energy-homeostatic processes was implicated in previous studies; notably, the studies where kinin B1 receptor knockout mice (B1-/-) were shown to have impaired adiposity, impaired leptin and insulin production, lower feed efficiency, protection from liver steatosis and diet-induced obesity when fed a high fat diet (HFD). In particular, in a model where the B1 receptor is expressed exclusively in the adipose tissue, it rescues the plasma insulin concentration and the weight gain seen in wild type mice. Taking into consideration that leptin participates in the formation of hypothalamic nuclei, which modulate energy expenditure, and feeding behavior, we hypothesized that these brain regions could also be altered in B1-/- mice. We observed for the first time a difference in the gene expression pattern of cocaine and amphetamine related transcript (CART) in the (lateral hypothalamic area (LHA) resulting from the deletion of the kinin B1 receptor gene. The correlation between CART expression in the LHA and the thwarting of diet-induced obesity corroborates independent correlations between CART and obesity. Furthermore, it seems to indicate that the mechanism underlying the 'lean' phenotype of B1-/- mice does not stem solely from changes in peripheral tissues but may also receive contributions from changes in the hypothalamic machinery involved in energy homeostasis processes.

Hypothalamic melanin-concentrating hormone projections to the septo-hippocampal complex in the rat.

Melanin-concentrating hormone (MCH) and neuropeptide glutamic acid-isoleucine (NEI) are expressed in neurons that are located mainly in the hypothalamus and project widely throughout the rat central nervous system. One of the main targets of melanin-concentrating hormone is the hippocampal formation, although the exact origin of the projections is unknown. By using injections of the retrograde tracer True Blue into the hippocampus, together with immunohistochemical analysis, we observed retrogradely labeled melanin-concentrating hormone-containing neurons in the lateral hypothalamic area, incerto-hypothalamic area, perifornical area, the periventricular nucleus of the hypothalamus, and in the internuclear area (between the dorsomedial and ventromedial nuclei of the hypothalamus), as well as a few retrogradely labeled and melanin-concentrating hormone-immunoreactive cells in the supramammillary nucleus. The afferents from the lateral hypothalamic area were confirmed using injection of the anterograde tracer biotinylated dextran amine, which enabled us to use histochemical analysis in order to visualize fibers and terminals in the hippocampal formation. In the medial septal nucleus, we found cholinergic neurons that are also putatively innervated by melanin-concentrating hormone immunoreactive fibers and project to the hippocampal formation. Finally, using two different protocols for immunoperoxidase, we were able to show GABAergic basket cells presumably innervated by melanin-concentrating hormone-immunoreactive fibers in the hippocampal formation. On the basis of the data collected herein, we hypothesize that the MCH/NEI projections from hypothalamic nuclei participate in spatial memory and learning through direct and indirect pathways. These pathways would enable the animal to organize its exploratory behavior during foraging.

mRNA expression of corticotropin-releasing factor and urocortin 1 after restraint and foot shock together with alprazolam administration.

Corticotropin-releasing factor (CRF) is expressed in the paraventricular nucleus of the hypothalamus (PVN), and act centrally to provoke stress-like autonomic and behavioral responses. Urocortins 1-3 are additional ligands to the CRF receptors 1 and 2. Ucn 1 neurons are primarily concentrated in the Edinger-Westphal (EW) nucleus and also have been associated with stress responses. It is also known that UCN 1 respond in different ways depending on the stressor presented. Benzodiazepines can act via the CRF peptidergic system and chronic administration of alprazolam does not interfere with CRF mRNA expression in the PVN, but significantly increase Ucn 1 mRNA expression in the EW. The aim of our study was to investigate the relationship between different stressor stimuli, foot shock (FS) and restraint (R), and the mRNA expression of CRF and Ucn 1 in the PVN and EW using alprazolam (A). We employed fos activation and in situ hybridization. Restraint group presented increased fos-ir and CRF mRNA expression in the PVN compared to FS group. The stress responses of R group were prevented by A. In the EW, fos-ir was higher in the FS group than in the R group, whereas Ucn 1 mRNA expression was higher in the R group than in the FS group. Alprazolam significantly increased fos-ir and Ucn 1 mRNA expression in both groups. Our results show that PVN and EW respond in different ways to the same stressors. Furthermore, EW of stressed animals replies in a complementary way comparing to PVN with the use of Alprazolam.

Expression profile and distribution of Efhc1 gene transcript during rodent brain development.

One of the putative causative genes for juvenile myoclonic epilepsy (JME) is EFHC1. We report here the expression profile and distribution of Efhc1 messenger RNA (mRNA) during mouse and rat brain development. Real-time polymerase chain reaction revealed that there is no difference in the expression of Efhc1 mRNA between right and left hemispheres in both species. In addition, the highest levels of Efhc1 mRNA were found at intra-uterine stages in mouse and in adulthood in rat. In common, there was a progressive decrease in Efhc1 expression from 1-day-old neonates to 14-day-old animals in both species. In situ hybridization studies showed that rat and mouse Efhc1 mRNAs are expressed in ependymal cells of ventricle walls. Our findings suggest that Efhc1 expression is more important during initial phases of brain development and that at this stage it could be involved in key developmental mechanisms underlying JME.

Leptin fragments induce Fos immunoreactivity in rat hypothalamus.

Leptin presents an important role in energy balance and neuroendocrine control in mammals. In an attempt to identify regions of the leptin molecule responsible for its bioactivity, we have synthesized six peptides based on the protein three-dimensional structure. Fragments were synthesized by the solid-phase methodology, purified by reverse-phase high-performance liquid chromatography (RP-HPLC), and characterized by liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS). They were injected intravenously and their ability to induce Fos immunoreactivity (Fos-ir) in rat hypothalamus was compared with that of the recombinant human leptin and saline. Fragment Ac-[Ser117]Lep116-140-NH2 (V) induced Fos-ir in hypothalamic nuclei that express leptin receptor long form. No similar ability was observed for the other five fragments. To investigate whether Fos-ir was induced in the same neuronal group activated by leptin, we proceeded with a dual-label immunohistochemistry for cocaine- and amphetamine-regulated transcript (CART), a neuropeptide related to leptin action in rat hypothalamus. We found that Ac-[Ser117]Lep116-140-NH2 (V) differentially activates CART neurons through the rostrocaudal extension of the arcuate nucleus. These results suggest that this fragment acts in the same group of neurons that mediate leptin response. This approach may offer the basis for the development of leptin-related compounds, having potential application in human or veterinary medicine.