[Show abstract][Hide abstract] ABSTRACT: Electromagnetic frequencies up to a few terahertz (THz) can yield real-time and noninvasive measurements on biological matter. Unfortunately, strong absorption in aqueous solutions and low spatial resolution return difficult free-space investigations. A new approach based on integrated THz circuits was used. The authors designed and fabricated a BioMEMS (Biological MicroElectro-Mechanical System) compatible with microfluidic circulation and electromagnetic propagation. It is dedicated to the ex vivo detection of nitric oxide synthase (NOS) activity, which is involved in neurodegenerative phenomena.
The biological model was a leech's central nervous system. After its injury, the production of NO was observed and measured in the far-THz spectral domain. The nerve cord was put inside a BioMEMS realized in polydimethylsiloxane (PDMS) sealed on a glass wafer. Glass is a good material for supporting high-frequency integrated waveguides such as coplanar waveguides (CPWs). Measurements were performed with vectorial network analyser (VNA).
The transmission parameter in the frequency range of 0.14-0.22 THz was measured through CPWs located just below the microchannel containing the injured leech nerve cord. The lesion caused a decreased transmission coefficient due to the NOS activity. L-NAME was injected inside the microchannel and it was verified that it inhibits this activity.
It was demonstrated that THz spectroscopy can detect a biochemical event, such as NOS activity around an injured leech's nerve cord, in real time. Future studies will be dedicated to quantitative measurements of the reaction products using the sophisticated management of several drugs allowed with microfluidic microsystems.
Medical science monitor: international medical journal of experimental and clinical research 10/2009; 15(9):MT121-5. · 1.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mass spectrometry (MS) has become an essential tool for the detection, identification, and characterization of the molecular components of biological processes, such as those responsible for the dynamic properties of the nervous system. Generally, the application of these powerful techniques requires the destruction of the specimen under study, but recent technological advances have made it possible to apply the matrix-assisted laser desorption/ionization (MALDI) MS technique directly to tissue sections. The major advantage of direct MALDI analysis is that it enables the acquisition of local molecular expression profiles, while maintaining the topographic integrity of the tissue and avoiding time-consuming extraction, purification, and separation steps, which have the potential for introducing artifacts. With automation and the ability to display complex spectral data using imaging software, it is now possible to create multiple 2D maps of selected biomolecules in register with tissue sections, a method now known as MALDI Imaging, or MSI (for Mass Spectrometry Imaging). This creates, for example, an opportunity to correlate functional states, determined a priori with live recording or imaging, with the corresponding molecular maps obtained at the time the tissue is frozen and analyzed with MSI. We review the increasing application of MALDI Imaging to the analysis of molecular distributions of proteins and peptides in nervous tissues of both vertebrates and invertebrates, focusing in particular on recent studies of neurodegenerative diseases and early efforts to implement assays of neuronal development.
[Show abstract][Hide abstract] ABSTRACT: A common technique for the long-term storage of tissues in hospitals and clinical laboratories is preservation in formalin-fixed paraffin-embedded (FFPE) blocks. Such tissues stored for more than five years have not been useful for proteomic studies focused on biomarker discovery. Recently, MS-based proteomic analyses of FFPE showed positive results on blocks stored for less than 2 days. However, most samples are stored for more than one year, and thus our objective was to establish a novel strategy using as a model system 6-hydroxydopamine (6-OHDA) treated rat brain tissues stored in FFPE blocks for more than 9 years. We examined MALDI tissue profiling combining the use of automatic spotting of the MALDI matrix with in situ tissue enzymatic digestion. On adjacent sections, the identification of compounds is carried out by tissue digestion followed by nanoLC/MS-MS analysis. The combination of these approaches provides MALDI direct analysis, MALDI/MS imaging, as well as the localization of a large number of proteins. This method is validated since the analyses confirmed that ubiquitin, trans-elongation factor 1, hexokinase, and the Neurofilament M are down-regulated as previously shown in human or Parkinson animal models. In contrast, peroxidoredoxin 6, F1 ATPase, and alpha-enolase are up-regulated. In addition, we uncovered three novel putative biomarkers, the trans-elongation factor 1 (eEF1) and the collapsin response mediator 1 and 2 from protein libraries. Finally, we validate the CRMP-2 protein using immunocytochemistry and MALDI imaging based on the different ions from trypsic digestion of the protein. The access to archived FFPE tissue using MALDI profiling and imaging opens a whole new area in clinical studies and biomarker discovery from hospital biopsy libraries.
Journal of Proteome Research 04/2008; 7(3):969-78. DOI:10.1021/pr070464x · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: With new emerging mass spectrometry technologies, it can now be demonstrated that direct tissue analysis is feasible using matrix-assisted laser desorption/ionization (MALDI) sources. A major advantage of direct MALDI analysis is to avoid time-consuming extraction, purification or separation steps, which have the potential for producing artifacts. Direct MALDI analysis of tissue sections enables the acquisition of cellular expression profiles while maintaining the cellular and molecular integrity. With automation and the ability to reconstruct complex spectral data using imaging software, it is now possible to produce multiplex imaging maps of selected bio-molecules within tissue sections. Thus, direct MALDI spectral data obtained from tissue sections can be converted into imaging maps, a method now known as MALDI-imaging. MALDI-imaging combines the power of mass spectrometry, namely exquisite sensitivity and unequivocal structural information, within an intact and unaltered morphological context. Critical improvements to increase image resolution are presented in this manuscript e.g., solvent treatment, new solid ionic matrices, gold sputtering, nickel support or laser focalization. One of the most important developments is the ability to carry out either direct MALDI analysis or MALDI imaging on paraffin tissue sections, thus opening the path to an archival "gold-mine" of existing pathology samples to proteomic analysis. These developments provide new avenues for biomarker hunting and diagnostic follow-up in the clinical setting. Further developments in MALDI-imaging of specific targets provide an added dimension, as validated disease-marker-gene RNA transcripts can be analyzed along with their translation by targeting their specific protein products or metabolites. Disease/health states will thus be closely molecularly monitored at protein and nucleic acids levels, with a single technique. Taken together, MALDI imaging will become a key tool for pathology proteomic studies.
Current pharmaceutical design 02/2007; 13(32):3317-24. DOI:10.2174/138161207782360672 · 3.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of maternal 50% food restriction (FR) during the last week of gestation and/or lactation on pituitary-gonadal axis (at birth and weaning), on circulating levels of leptin (at weaning), and on the onset of puberty have been determined in rats at birth and at weaning. Maternal FR during pregnancy has no effect at term on the litter size, on the basal level of testosterone in male pups, and on the drastic surge of circulating testosterone that occurs 2 h after birth. At weaning, similar retardation of body growth is observed in male and female pups from mothers exposed to FR. This undernutrition induces the most drastic effects when it is performed during both gestation and lactation or during lactation alone. Drastic retardation of testicle growth with reduction of cross-sectional area and intratubular lumen of the seminiferous tubules is observed in male pups from mothers exposed to undernutrition during both gestation and lactation or during lactation alone. Maternal FR during the perinatal period reduces circulating levels of FSH in male pups without affecting LH and testosterone concentrations. Maternal FR does not affect circulating levels of LH, estradiol, and progesterone in female pups. Female pups from mothers exposed to FR during both gestation and lactation show a significant increase of plasma FSH as well as a drastic retardation of ovarian growth. The follicular population was also altered. The number of antral follicles of small size (vesicular follicles) was increased, although the number of antral follicles of large size (graafian follicles) was reduced. Maternal FR occurring during both late gestation and lactation (male and female pups), during lactation alone (male and female pups), or during late gestation (female pups) induces a drastic reduction of plasma leptin and fat mass in pups at weaning. The onset of puberty is delayed in pups of both sexes from mothers exposed to FR during lactation and during both gestation and lactation. In conclusion, these data demonstrate that a perinatal growth retardation induced by maternal FR has long-term consequences on both size and histology of the genitals, on plasma gonadotropins and leptin levels, on fat stores at weaning, and on the onset of puberty.
Biology of Reproduction 03/2003; 68(2):390-400. DOI:10.1095/biolreprod.102.003269 · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It has become apparent that galanin as well as proopiomelanocortin-derived peptides, such as beta-endorphin, play an important role in the hypothalamic circuitry that regulates neuroendocrine functions and appetite behavior. We have recently shown that GalR1 and GalR2 galanin receptor mRNAs are expressed in proopiomelanocortin neurons of the arcuate nucleus, suggesting a direct modulatory action of galanin on the proopiomelanocortin neuronal system. In the present study, we investigated the effect of galanin on beta-endorphin release and proopiomelanocortin mRNA expression from male rat mediobasal hypothalamic fragments incubated ex vivo. Galanin induced a decrease of spontaneous beta-endorphin release within the first 30-60 min of incubation and this effect was blocked by the galanin receptor antagonist galantide. Co-incubation of galanin with FK-506 (tacrolimus), a calcineurin inhibitor, suppressed the inhibitory effect of galanin on beta-endorphin release, suggesting that calcineurin is involved in the galanin-evoked decrease in beta-endorphin release. Measurement of beta-endorphin levels in the tissues at the end of the incubation period (120 min) revealed that galanin caused a two-fold increase of beta-endorphin peptide concentration in the mediobasal hypothalamic tissues. Concurrently, galanin induced an increase in the mean density of silver grains overlying proopiomelanocortin neurons after 60 min of incubation, an effect antagonized by galantide. Finally, reverse transcription-polymerase chain reaction analysis revealed that the mRNAs for the three galanin receptor subtypes (i.e. GalR1, GalR2, and GalR3) were expressed in the incubated mediobasal hypothalamic fragments. Taken as a whole, our results indicate that galanin plays a modulatory role on proopiomelanocortin neurons and this interrelation contributes to the elucidation of the neural circuitry that controls, among others, gonadotropin-releasing hormone function.
[Show abstract][Hide abstract] ABSTRACT: Recent studies from our laboratory suggested that the vascular endothelium of the median eminence was involved via nitric oxide secretion in the modulation of GnRH release during the estrous cycle. To further investigate that issue, we studied the variations of endothelial nitric oxide synthase protein and mRNA in the median eminence of female rats killed at different time points of the day and/or of the estrous cycle. Endothelial nitric oxide synthase protein levels were measured by Western blot, and endothelial nitric oxide synthase mRNA analysis was performed with semiquantitative RT-PCR (for each time point, n = 4). The results revealed that endothelial nitric oxide synthase synthesis varied markedly across the estrous cycle. Indeed, endothelial nitric oxide synthase protein (n = 20) and mRNA (n = 16) levels increase significantly on 0800 h and 1600 h proestrus compared with 1400 h diestrus II. In a second step, quantification analysis were made in median eminence obtained from ovariectomized and ovariectomized, E2 benzoate primed rat. The results show a significant increase in expression of endothelial nitric oxide synthase protein as well as endothelial nitric oxide synthase mRNA in ovx-E2 primed rat median eminence. Concurrently, the levels of the cav-1 protein, a specific endogenous inhibitor of endothelial nitric oxide synthase, were measured in median eminence during estrous cycle and in ME from ovx and ovx-E2 primed rats. A significant decrease of median eminence cav-1 was noted on 1600 h proestrus and in ovx-E2 primed rats when compared with 1400 h diestrus II and ovx, respectively. Altogether, these results strongly suggest that high NO release from median eminence observed on proestrus may be due to an increase of endothelial nitric oxide synthase expression and a decrease of the cav-1 protein levels. These findings demonstrate that E2 is able to modulate endothelial nitric oxide synthase and cav-1 expression both during the estrous cycle and in experimental conditions and consequently reinforce the idea that nitric oxide acting on GnRH release, is essentially endothelial in origin. These results may also imply that variations of endothelial nitric oxide synthase expression are essential for the pulsatile/cyclic nitric oxide median eminence release observed in a previous study.
[Show abstract][Hide abstract] ABSTRACT: The role of nitric oxide (NO) from vascular endothelium in the control of GnRH release at the median eminence (ME) level is well established. Interactions between NPY receptor/endothelium/nitric oxide are clearly demonstrated. While several studies implicate NPY Y1 receptor in the control of GnRH/LH at the time of the preovulatory LH surge, our results also demonstrate the importance of NPY Y2 receptor in the control of GnRH release via endothelial NO. We conclude that NPY may be one of the elements implicated in the generation of the spontaneous NO/GnRH via Y2 receptor located on endothelium.
[Show abstract][Hide abstract] ABSTRACT: The purpose of the present study was to determine whether TGF beta, a cytokine secreted by hypothalamic astrocytes, was able to regulate POMC neurons in the arcuate nucleus. In a first set of experiments, mediobasal hypothalamic fragments were exposed to TGF beta(1), and the relative POMC mRNA expression was assessed by in situ hybridization using a radiolabeled POMC riboprobe. The results showed that 4 x 10(-10) M TGF beta(1) was efficient in decreasing significantly the amounts of POMC mRNA (P < 0.01). Interestingly, the decrease of relative POMC mRNA levels was higher in the rostral than in the caudal parts of the arcuate nucleus. In a second set of experiments, we examined the occurrence of TGF beta receptors expression in arcuate POMC neurons. Dual labeling in situ hybridization and in situ hybridization, coupled to immunohistochemical labeling, were performed to examine mRNA expression of the type I serine-threonine kinase receptor for TGF beta and the presence of type II receptor for TGF beta, respectively, in POMC neurons. The results indicated that TGF beta receptor I mRNA and TGF beta receptor II protein were expressed in numerous POMC neurons. Regional analysis revealed that the highest proportion of POMC neurons expressing TGF beta receptors was located in the rostral part of the arcuate nucleus. Using dual labeling immunohistochemistry, we also found that Smad2/3 immunoreactivity, a TGF beta(1) downstream signaling molecule, was present in the cytoplasm and nucleus of some POMC (beta-endorphin) neurons. We next examined whether the number of POMC neurons expressing TGF beta-RI mRNA was affected by sex steroids. Quantification of the number of POMC neurons expressing TGF beta receptor I mRNA in ovariectomized, ovariectomized E2-treated, and ovariectomized E2 plus progesterone-treated animals revealed that estrogen treatment decreased the expression of TGF beta receptor I mRNA in POMC neurons located in the rostral half of the arcuate nucleus, an effect reversed by progesterone in a subset of the most rostral cells. Taken together, these data reveal that TGF beta(1) may directly modulate the activity of POMC neurons through the activation of TGF beta receptors. Therefore, the present study provides additional evidence for the involvement of TGF beta(1) in the regulation of neuroendocrine functions and supports the existence of a glial-to-neurons communication within the arcuate nucleus.
[Show abstract][Hide abstract] ABSTRACT: The purpose of the present study was to determine whether TGF, a cytokine secreted by hypothalamic astrocytes, was able to regulate POMC neurons in the arcuate nucleus. In a first set of experiments, mediobasal hypothalamic fragments were exposed to TGF1, and the relative POMC mRNA expres- sion was assessed by in situ hybridization using a radiola- beled POMC riboprobe. The results showed that 4 1010 M TGF1 was efficient in decreasing significantly the amounts of POMC mRNA (P < 0.01). Interestingly, the decrease of relative POMC mRNA levels was higher in the rostral than in the caudal parts of the arcuate nucleus. In a second set of exper- iments, we examined the occurrence of TGF receptors expression in arcuate POMC neurons. Dual labeling in situ hybridization and in situ hybridization, coupled to immuno- histochemical labeling, were performed to examine mRNA expression of the type I serine-threonine kinase receptor for TGF and the presence of type II receptor for TGF, respec- tively, in POMC neurons. The results indicated that TGF receptor I mRNA and TGF receptor II protein were ex- pressed in numerous POMC neurons. Regional analysis re- vealed that the highest proportion of POMC neurons express- ing TGF receptors was located in the rostral part of the arcuate nucleus. Using dual labeling immunohistochemistry, we also found that Smad2/3 immunoreactivity, a TGF1 down- stream signaling molecule, was present in the cytoplasm and nucleus of some POMC (-endorphin) neurons. We next ex- amined whether the number of POMC neurons expressing TGF-RI mRNA was affected by sex steroids. Quantification of the number of POMC neurons expressing TGF receptor I mRNA in ovariectomized, ovariectomized E2-treated, and ovariectomized E2 plus progesterone-treated animals re- vealed that estrogen treatment decreased the expression of TGF receptor I mRNA in POMC neurons located in the ros- tral half of the arcuate nucleus, an effect reversed by proges- terone in a subset of the most rostral cells. Taken together, these data reveal that TGF1 may directly modulate the ac- tivity of POMC neurons through the activation of TGF re- ceptors. Therefore, the present study provides additional ev- idence for the involvement of TGF1 in the regulation of neuroendocrine functions and supports the existence of a gli- al-to-neurons communication within the arcuate nucleus. (Endocrinology 142: 4055- 4065, 2001)
[Show abstract][Hide abstract] ABSTRACT: The involvement of nitric oxide (NO) as a gaseous neurotransmitter in the hypothalamic control of pituitary LH secretion has been demonstrated. NO, as a diffusible signaling gas, has the ability to control and synchronize the activity of the neighboring cells. NO is secreted at the median eminence (ME), the common termination field for the antehypophysiotropic neurons, under the stimulation of other signaling substances. At the ME, NO stimulates GnRH release from neuroendocrine terminals. The present studies were undertaken to determine whether NO is secreted spontaneously from ME fragments ex vivo and whether its secretion is correlated to GnRH release. To accomplish this, female rats were killed at different time points of the day and/or of the estrous cycle. The spontaneous NO release was monitored in real time, with an amperometric probe, during 4 periods of 30 min, from individual ME fragments (for each time point, n = 4). GnRH levels were measured in parallel for each incubation-period by RIA. The results revealed that NO was released in a pulsatile manner from female ME fragments and, unambiguously, that the amplitude of NO secretion varied markedly across the estrous cycle. Indeed, though the NO pulse period (32 +/- 1 min, n = 36) and duration (21 +/- 2 min, n = 36) did not vary significantly across the estrous cycle, the amplitude of this secretion pulse was significantly higher on proestrus (Pro; 39 +/- 3 nM, n = 20), compared with diestrus (16 +/- 1 nM, n = 8) or estrus (23 +/- 3 nM, n = 8, P < 0.05). The GnRH levels in the incubation medium were positively correlated to NO secretion across the estrous cycle (r = 0.86, P < 0.003, n = 9), confirming that NO and GnRH release are coupled. Furthermore, 5 x 10(-7) M L-N(5)-(1-iminoethyl)ornithine (L-NIO), a NO synthase inhibitor, succeeded in inhibiting the strong NO-GnRH secretory coupling and GnRH release on PRO: Because at this concentration, L-NIO selectively inhibits endothelial NO synthase, the results further demonstrate that the major source of NO involved in GnRH release at the ME is endothelial in origin. Additionally, the induction of a massive NO/GnRH release in 15-day ovariectomized rat treated with estradiol benzoate strongly suggested that estradiol is participating in the stimulation of NO release activity between diestrus II and PRO: The present study is the first demonstrating that ME can spontaneously release NO and that NO's rhythm of secretion varies markedly across the estrous cycle. This pulsatile/cyclic ME NO release may constitute the synchronizing link to anatomically scattered GnRH neurons.
[Show abstract][Hide abstract] ABSTRACT: The present investigation concerns 80-90-day-old female rats born from morphine-exposed mothers (2x10 mg/kg per day from day 11-18 of gestation) or saline-treated ones (controls). The former showed reduced size and activity of the adrenals at birth. At adult stage, they present: (1) higher increase of plasma adrenocorticotrophic hormone level on proestrus; (2) significant rise of plasma corticosterone level on diestrus morning and estrus evening; (3) adrenal atrophy which was significant only on diestrus and estrus morning; (4) more corticosterone binding sites of type I (mineralocorticoid receptors) on proestrus morning in the hippocampus; (5) more corticosterone binding sites of type II (glucocorticoid receptors) in the hippocampus on proestrus morning and in the hypothalamus on estrus morning. In both experimental groups, B(max) for hypothalamic mineralocorticoid receptors were drastically higher on estrus morning than on the other stages of the estrous cycle. The activity of the pituitary-gonadal axis is poorly affected by prenatal morphine-exposition. In both experimental groups drastic and comparable surges of both plasma progesterone and luteinizing hormone were observed during proestrus. Nevertheless morphine-exposed females showed higher levels of plasma estradiol on diestrus morning but lower levels on metestrus morning. In conclusion, prenatal exposition to morphine has long-term effects mainly on pituitary-adrenal axis as well as on binding sites for corticosterone in the hypothalamus and the hippocampus which are dependent on the estrous cycle stages in adult females.
Brain Research 06/2001; 902(1):66-73. DOI:10.1016/S0006-8993(01)02339-3 · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of the polypeptide hormone prolactin (PRL) in the development and regulation of benign prostate hyperplasia (BPH) and also in prostate cancer are not very well characterized. This study examines the action of PRL, either alone or in association with androgens [testosterone (T) or dihydrotestosterone (DHT)], in the rat prostate gland. The effects of PRL and androgens were investigated after 30 and 60 days in control, castrated, castrated with a substitutive implant of T or DHT, and sham-operated Wistar rats. To enhance PRL release, we induced hyperprolactinemia by administering chronic injections of sulpiride (40 mg. kg(-1). day(-1)). Chronic hyperprolactinemia induces enlargement and inflammation of the lateral rat prostate without any histological changes on ventral and dorsal lobes. We also demonstrate that hyperprolactinemia induces Bcl-2 overexpression in the lateral rat prostate and that this could inhibit the level of apoptosis. The in vivo model established here is a useful in vivo approach for studying the hormonal regulation of normal and pathological prostate development.
AJP Endocrinology and Metabolism 02/2001; 280(1):E120-9. · 3.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide (NO) as well as beta-endorphin are involved in the neuroendocrine control of gonadotropin-releasing hormone (GnRH) secretion. Recently, morphological and microdialysis experiments have suggested that beta-endorphin may exert an inhibitory influence on NO release in the preoptic area of rat hypothalamus. The present study determines if the mu opioid receptor mRNA is expressed in neuronal NO synthase (nNOS)-immunopositive neurons and if this expression varies among the regions of the basal forebrain being examined. We found, through the use of immunohistochemical and in situ hybridization techniques, that the mu opioid receptor mRNA is expressed in a representative subpopulation of nNOS-immunoreactive neurons in the rat preoptic area. Interestingly, the mu opioid receptor mRNA/nNOS-immunoreactive coexpression is predominant in the rostral and median preoptic area, containing most of GnRH cell bodies. These results strongly suggest that beta-endorphin, via an action through mu opioid receptors, may directly participate in the regulation of NO production in the preoptic area. Our results strengthen the hypothesis that beta-endorphin may participate in GnRH neuronal modulation at the cell body level by regulating NO release from the interneurons of the preoptic area that express nNOS.
Molecular Brain Research 09/2000; 80(1):46-52. DOI:10.1016/S0169-328X(00)00118-2 · 2.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study was designed to determine whether transforming growth factor (TGF)beta and/or activin participate in the regulation of the gonadotropin releasing hormone (GnRH) neuroendocrine axis in vivo. Single-label in situ hybridization histochemistry was used to determine the anatomical distribution of a TGFbeta and activin type I receptor (B1) mRNA, in the adult female rat hypothalamic areas that are known to be important sites for the regulation of reproduction. Dual-label in situ hybridization histochemistry was performed to determine whether B1 mRNA was expressed in GnRH neurones. The results of these studies revealed an extensive distribution of B1 mRNA in the hypothalamic regions, including diagonal bands of Broca, preoptic area, arcuate nucleus and median eminence. In the median eminence, B1 mRNA was detected in tanycytes and in the endothelial cells of the pituitary portal blood capillaries. Dual-label in situ hybridization histochemistry showed that 31+/-5% of GnRH neurones expressed B1 mRNA, thus providing evidence that TGFbeta and/or activin can act directly on GnRH neurones to modulate their activity. Taken together, these data provide morphological arguments in favour of a participation of TGFbeta and/or activin in the regulation of reproduction at the hypothalamic level.
Journal of Neuroendocrinology 08/2000; 12(7):665-70. · 3.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous studies have shown that galanin-containing fibers make synaptic contacts with POMC neurons in the arcuate nucleus. However, the ability of POMC neurons to express galanin receptors has never been assessed. The present study was designed to investigate whether POMC neurons express galanin receptor messenger RNA (mRNA) and whether testosterone could modulate galanin receptor gene expression. A dual-labeling in situ hybridization histochemistry, using 35S-labeled (galanin receptors GalR1 or GalR2) and digoxigenin-labeled (POMC) riboprobes, was performed on brain sections from intact, castrated, and testosterone-replaced adult male rats. For analysis, the arcuate nucleus was divided into four rostro-caudal areas. The results revealed that both GalR1 and GalR2 mRNAs were expressed in POMC neurons. Most POMC neurons expressing galanin receptor mRNAs were found in the rostral parts of the nucleus. Castration reduced the labeling density of galanin receptor mRNAs in POMC neurons, and testosterone prevented the effects of castration in all rostro-caudal subdivisions of the arcuate nucleus. Taken together, these data indicate that galanin can directly modulate the activity of POMC neurons, via an action on GalR1 or GalR2 receptors, particularly in the rostral-arcuate nucleus. In addition, testosterone can modulate the expression of GalR1 and GalR2. Because POMC neurons located in the rostral part of the nucleus are known to project preferentially to the preoptic area, POMC neurons expressing the galanin receptor genes may play an important role in the regulation of the GnRH neuroendocrine axis.
[Show abstract][Hide abstract] ABSTRACT: We have shown previously at the ultrastructural level that morphological changes occur in the external zone of the median eminence allowing certain GnRH nerve terminals to contact the pericapillary space on the day of proestrus. The present study was designed to determine whether the intrinsic determinant of neuronal outgrowth, growth-associated protein-43 (GAP-43), was expressed in GnRH neurons of adult female rats, and whether its expression varied throughout the estrous cycle. To accomplish this, we perfusion-fixed groups of adult female rats at 0800 and 1600 h on diestrous day 2 (diestrous II), at 0800 h and 1600 h on proestrus, and at 0800 and 1600 h on estrus (n = 4 rats/group) and used double labeling in situ hybridization and quantification to compare the levels of GAP-43 messenger RNA (mRNA) in cells coexpressing GnRH mRNA. GnRH mRNA was detected with an antisense complementary RNA (cRNA) probe labeled with the hapten digoxigenin, whereas the GAP-43 cRNA probe was labeled with 35S and detected by autoradiography. In addition, GAP-43 protein was identified with immunohistochemistry in the median eminence. The results show that many GnRH neurons expressed GAP-43 mRNA and that GAP-43 protein was present in many GnRH axon terminals in the outer layer of the median eminence. The number of GnRH neurons expressing GAP-43 mRNA was significantly higher on proestrus (64 +/- 5%) than on diestrous II (40 +/- 2%; P < 0.001) or on estrus (45 +/- 8%; P < 0.05), and the GAP-43 mRNA levels in GnRH neurons also varied as a function of time of death during the estrous cycle. The GAP-43 mRNA levels in GnRH neurons were higher on proestrus and estrus than on diestrous II (P < 0.05). These data show that 1) GAP-43 is expressed in adult GnRH neurons; 2) GAP-43 mRNA expression in GnRH neurons fluctuates during the estrous cycle; and 3) GAP-43 mRNA content in GnRH neurons is highest on the day of proestrus, before and during the onset of the LH surge. These observations suggest that the increased GAP-43 mRNA expression in GnRH neurons on the day of proestrus could promote the outgrowth of GnRH axon terminals to establish direct neurovascular contacts in the external zone of the median eminence and thus facilitate GnRH release into the pituitary portal blood.