Isabelle Boutelet

Melanocortin‐3 Receptor mRNA Expression in Pro‐Opiomelanocortin Neurones of the Rat Arcuate Nucleus

The melanocortins α‐ and γ‐melanocyte‐stimulating hormones (α‐ and γ‐MSH) derive from the pro‐opiomelanocortin (POMC) precursor. Melanocortins exert a wide range of biological activities in the brain through activation of at least three distinct melanocortin receptor (MC‐R) subtypes. In order to determine whether POMC neurones can modulate their own activity, we looked for the possible expression of the MC3‐R gene in POMC‐positive cell bodies in the rat hypothalamus. In situ hybridization experiments revealed that the density of MC3‐R mRNA is particularly high in the arcuate nucleus which contains the main population of POMC neurones in the brain. The occurrence of MC3‐R mRNA in POMC‐positive cell bodies was demonstrated using a double‐labelling in situ hybridization technique. The proportion of POMC neurones expressing MC3‐R mRNA was significantly higher in the most rostral (43.5%) than in the most posterior part of the arcuate nucleus (8.2%). These results indicate that melanocortins likely exert a direct regulatory feedback on POMC neurones through activation of MC3‐R receptors. Our data also suggest that MC3‐R may be involved in the neuroendocrine responses induced by centrally administered melanocortins.

Pituitary adenylate cyclase-activating polypeptide inhibits food intake in mice through activation of the hypothalamic melanocortin system.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and the proopiomelanocortin (POMC)-derived peptide, α-melanocyte-stimulating hormone (α-MSH), exert anorexigenic activities. While α-MSH is known to inhibit food intake and stimulate catabolism via activation of the central melanocortin-receptor MC4-R, little is known regarding the mechanism by which PACAP inhibits food consumption. We have recently found that, in the arcuate nucleus of the hypothalamus, a high proportion of POMC neurons express PACAP receptors. This observation led us to investigate whether PACAP may inhibit food intake through a POMC-dependent mechanism. In mice deprived of food for 18 h, intracerebroventricular administration of PACAP significantly reduced food intake after 30 min, and this effect was reversed by the PACAP antagonist PACAP6-38. In contrast, vasoactive intestinal polypeptide did not affect feeding behavior. Pretreatment with the MC3-R/MC4-R antagonist SHU9119 significantly reduced the effect of PACAP on food consumption. Central administration of PACAP induced c-Fos mRNA expression and increased the proportion of POMC neuron-expressing c-Fos mRNA in the arcuate nucleus. Furthermore, PACAP provoked an increase in POMC and MC4-R mRNA expression in the hypothalamus, while MC3-R mRNA level was not affected. POMC mRNA level in the arcuate nucleus of PACAP-specific receptor (PAC1-R) knock-out mice was reduced as compared with wild-type animals. Finally, i.c.v. injection of PACAP provoked a significant increase in plasma glucose level. Altogether, these results indicate that PACAP, acting through PAC1-R, may inhibit food intake via a melanocortin-dependent pathway. These data also suggest a central action of PACAP in the control of glucose metabolism.

Expression of Melanocortin MC3 and MC4 Receptor mRNAs by Neuropeptide Y Neurons in the Rat Arcuate Nucleus

Neuropeptide Y (NPY) and α-melanocyte-stimulating hormone (α-MSH), two neuropeptides that are synthesized in neurons of the arcuate nucleus of the hypothalamus, exert opposite actions on food intake and body weight. NPY is orexigenic and decreases energy expenditure whereas α-MSH reduces food consumption and stimulates catabolism. α-MSH is an endogenous ligand for the central melanocortin receptors, MC3-R and MC4-R. In order to determine whether α-MSH may act directly on NPY neurons in the arcuate nucleus, we have investigated the possible occurrence of MC3-R and MC4-R mRNA in NPY-expressing cell bodies in the rat hypothalamus. Double-labeling in situ hybridization histochemistry using 35S-labeled (MC3-R or MC4-R) and digoxigenin-labeled (NPY) riboprobes revealed that 38 ± 1% of the NPY mRNA-positive perikarya expressed MC3-R mRNA while only 9 ± 2% of the NPY-producing neurons contained MC4-R mRNA. The proportions of NPY neurons that express MC3-R mRNA or MC4-R mRNA were not significatively different in the anterior and posterior aspects of the arcuate nucleus. The present study shows that a large proportion of NPY neurons in the rat hypothalamus express MC3-R mRNA while a much lower number of NPY neurons express MC4-R mRNA, suggesting that melanocortins may directly modulate the activity of the hypothalamic NPY system, mainly through activation of MC3-R. These data provide additional evidence for the complex interactions between the stimulatory (NPY) and inhibitory (α-MSH) pathways controlling feeding behavior and energy homeostasis.

Gastric electrical stimulation modulates hypothalamic corticotropin-releasing factor–producing neurons during post-operative ileus in rat

High-frequency/low-energy gastric electrical stimulation (GES) is an efficient therapy to treat gastric emptying-related disorders but its mechanism of action remains poorly understood. We aimed to assess the effects of high-frequency/low-energy GES on corticotropin-releasing factor (CRF)-producing neurons in the paraventricular nucleus of the hypothalamus (PVN), which are involved in gastric ileus induced by laparotomy. Two electrodes were implanted in the rat gastric antrum during laparotomy, then stimulation (amplitude: 2 mA; pulse duration 330 micros; frequency: 2 Hz; 1 min ON/2 min OFF) or sham stimulation (control group) were applied. Using immunohistochemistry, the number of c-Fos protein-expressing neurons (c-Fos protein-immunoreactive cells, Fos-IR) was quantified in the PVN after 1 h of stimulation. The number of neurons expressing simultaneously c-Fos protein and CRF mRNA was measured by means of immunocytochemistry combined with in situ hybridization. Finally, c-Fos and CRF mRNA levels in the hypothalamus were determined by in situ hybridization or quantitative reverse transcriptase-polymerase chain reaction. Fos-IR in the PVN was significantly decreased 1 h after GES (P<0.05) but was not affected by sub-diaphragmatic vagotomy. The number of neurons containing c-Fos protein and CRF mRNA was lower in the GES group compared with the control group (P<0.05). In addition, c-Fos and CRF mRNA levels in the PVN were significantly decreased by GES (P<or=0.05). It is concluded that acute GES reduces the number of CRF-producing neurons and decreases CRF expression in the PVN during post-operative gastric ileus.

Pituitary adenylate cyclase-activating polypeptide directly modulates the activity of proopiomelanocortin neurons in the rat arcuate nucleus.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and the proopiomelanocortin (POMC)-derived peptide alpha-melanocyte-stimulating hormone (alpha-MSH) both regulate multiple neuroendocrine functions and feeding behavior. Two subtypes of PACAP receptor mRNAs, pituitary adenylate cyclase-activating polypeptide-specific receptor (PAC1-R) and pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide mutual receptor (VPAC2-R), are actively expressed in the arcuate nucleus of the hypothalamus, where POMC cell bodies are located. This observation led us to investigate the possible regulatory action of PACAP on rat POMC neurons. Double-labeling in situ hybridization histochemistry revealed that approximately 50% of POMC-producing neurons express PAC1-R and/or VPAC2-R mRNAs. The proportion of POMC neurons that also contain PAC1-R mRNA was homogeneous along the rostro-caudal axis of the arcuate nucleus while POMC-positive cell bodies expressing the VPAC2-R subtype were more abundant in the rostral region. Incubation of mediobasal hypothalamic explants with PACAP (10(-7) M; 30 min) increased POMC mRNA expression, and this effect was blocked by PACAP6-38 (10(-6) M). In contrast, incubation with vasoactive intestinal polypeptide (10(-7) M) did not affect POMC mRNA level. Incubation of hypothalamic fragments with PACAP (10(-7) M) caused a significant increase in alpha-MSH content in the tissue and in the incubation medium. Altogether, the present results reveal that exogenous PACAP, acting probably through PAC1-R, regulates the activity of POMC neurons in the rat hypothalamus. These data suggest that the effects of PACAP on the gonadotropin-releasing hormone neuroendocrine axis and the regulation of feeding behavior may be mediated, at least in part, through modulation of POMC neurons.

Expression of PACAP receptor mRNAs by neuropeptide Y neurons in the rat arcuate nucleus.

Abstract:  Neuropeptide Y (NPY) and pituitary adenylate cyclase‐activating polypeptide (PACAP) exert opposite actions in energy homeostasis: NPY is a potent orexigenic peptide whereas PACAP reduces food intake. PAC1‐R and VPAC2‐R mRNAs are actively expressed in the arcuate nucleus of the hypothalamus which contains a prominent population of NPY neurons. By using a double‐labeling in situ hybridization technique, we now show that a significant proportion of NPY neurons express PAC1‐R or VPAC2‐R mRNA. This observation indicates that PACAP may regulate the activity of NPY neurons, suggesting that the inhibitory effect of PACAP on food intake may be mediated, at least in part, through modulation of NPY neurotransmission.

Prolyl endopeptidase mRNA expression in the central nervous system during rat development.

Prolyl endopeptidase (PEP) is a serine protease that cleaves small peptides at the carboxyl side of L-proline. PEP has been reported to have important functions in the brain being implicated in learning and memory processes, psychological disorders and neurodegenerative diseases. Several PEP substrates have been shown to play a role during brain development and this observation led us to investigate the expression of PEP mRNA in the rat brain and spinal cord, from embryo to adult stages. In situ hybridization revealed that PEP mRNA is expressed early, from embryonic day 15, notably in germinative areas including the neocortical, hippocampal, pallidal, thalamic, anterior hypothalamic, tectal, cerebellar, pontine and medullary neuroepithelia. PEP mRNA was also found in the differentiating fields of the olfactory bulb, the orbital and cingulate cortex, the hippocampal formation, the cortical plate and the subventricular zone of the cortex. Quantitative RT-PCR analysis in various brain areas and the spinal cord showed that PEP mRNA levels are more abundant during the perinatal stages, coinciding with a period of neuronal migration and differentiation. From then on, PEP mRNA expression decreased, reaching its lowest levels at adulthood. Overall, the present data support the possibility that PEP exerts specific functions related to neurodevelopment besides those proposed to date.

pH Control of dehydrogenase systems

Abstract The pH dependence of the forward and backward reactions catalysed by various dehydrogenases, especially alcohol dehydrogenase, were established under standardized conditions. Except for malate dehydrogenase, the pH-dependences of both directions are very different from each other and the distance between the two optimal pH values is about 2 pH units. Some information about protonation of enzyme-substrate complexes is deduced. The design of a chemical oscillator is shown to illustrate how dehydrogenase associations can be used and how they can be controlled by pH.