Differentially expressed genes in the rat cochlear nucleus.

The cochlear nucleus is the first central pathway involved in the processing of peripheral auditory activity. The anterior ventral cochlear nucleus (AVCN), posterior ventral cochlear nucleus (PVCN) and dorsal cochlear nucleus (DCN) each contain predominant populations of neurons that have been well characterized regarding their morphological and electrophysiological properties. Little is known, however, of the underlying genetic factors that contribute to these properties and the initial steps in auditory processing. Serial analysis of gene expression (SAGE), supported by microarray experiments, was performed on each subdivision of the rat cochlear nucleus to identify genes that may sub-serve specialized roles in the central auditory system. Pair-wise comparisons between SAGE libraries from the AVCN, PVCN and DCN were correlated with microarray experiments to identify individual transcripts with significant differential expression. Twelve highly correlated genes were identified representing cytoskeletal, vesicular, metabolic and g-protein regulating proteins. Among these were Rgs4 which showed higher expression in the DCN, Sst and Cyp11b1 with very high expression in the AVCN and Calb2 with preferential expression in the PVCN. The differential expression of these genes was validated with real-time reverse transcriptase-polymerase chain reaction. These experiments provide a basis for understanding normal auditory processing on a molecular level and a template for investigating changes that may occur in the cochlear nucleus with hearing loss, the generation and percept of tinnitus, and central auditory processing disorders.

Comparative metabolism of benzo[a]pyrene and drugs in human liver.

The oxidative metabolism of antipyrine, hexobarbital, coumarin, zoxazolamine, 7-ethoxycoumarin, and the chemical carcinogen benzo[a]pyrene (BP) was studied in 32 adult human livers obtained at autopsy. When enzyme activity for one substrate was plotted against enzyme activity for a second substrate for each of the 32 livers, statistically significant correlations were found between the rates of metabolism of BP and the rates of metabolism of each of the other five drug substrates. The degree of correlation was dependent upon the substrate pair that was studied. Highly significant statistical correlations (p less than 0.001) for monooxygenase activities among the different livers were observed for BP with antipyrine (r = 0.85), antipyrine with zoxazolamine (r = 0.82), antipyrine with hexobarbital (r = 0.79), zoxazolamine with 7-ethoxycoumarin (r = 0.75), antipyrine with coumarin ( r= 0.72), zoxazolamine with coumarin (r = 0.72), BP with hexobarbital (r = 0.72), hexobarbital with coumarin (r = 0.71), BP with zoxazolamine (r = 0.69), hexobarbital with zoxazolamine (r = 0.64), coumarin with 7-ethoxycoumarin (r = 0.61), and BP with coumarin (r = 0.57). Less significant correlations were obtained for BP with 7-ethoxycoumarin (r = 0.35; p = 0.05). It is not known whether the relationships between the metabolism of the several substrates described here for autopsy livers would also occur with fresh livers. The lack of a perfect correlation for any of the substrate pairs suggests the presence in human liver of multiple monooxygenase enzyme systems for the metabolism of benzo[a]pyrene and the five other substrates studied, as well as heterogeneity in their distribution among the 32 livers that were examined. The approach described in the present report may have significance in the study of the comparative metabolism of drugs, chemical carcinogens, and other environmental pollutants by human tissues and may help us find predictor drugs that will be useful for evaluating the drug- and carcinogen-metabolizing capacity of different individuals in the human population.

Motoneurons dorsolateral to the central canal innervate perineal muscles in the Mongolian gerbil.

The Mongolian gerbil provides a model in which sexually dimorphic areas in the hypothalamus are correlated with sociosexual behaviors such as scent marking and male copulatory behavior. To extend this model, investigations were conducted to determine whether sexually dimorphic areas existed in the spinal cord that could be relevant to male sexual behavior. The focus of these investigations was the perineal muscles associated with the penis. Therefore, this research identified the spinal motoneurons that innervate the bulbocavernosus, levator ani, anal sphincter, and ischiocavernosus muscles of Mongolian gerbils. The motoneuron pool that innervates the bulbocavernosus, levator ani, and anal sphincter was designated the spinal nucleus of the bulbocavernosus (SNB), as for other species of rodents. The motoneuron pool innervating the ischiocavernosus was identified as the dorsolateral nucleus, again, to be consistent with the designation for other rodents. The motoneurons of the gerbil SNB were distributed dorsolateral to the central canal in the lumbosacral transition zone of the spinal column. These motoneurons are located in the region classically defined as area X of the spinal cord. The number of SNB motoneurons was sexually dimorphic, with male gerbils having about five times as many SNB motoneurons as do female gerbils. The size of SNB motoneurons was also sexually dimorphic. The SNB motoneurons of males were 1.5 times larger than the SNB motoneurons of females. The effects of adult castration on the male SNB were also studied. After castration, the size, but not the number, of SNB motoneurons in males was significantly decreased. This decrease was prevented by testosterone treatment. The percentage of calcitonin gene-related peptide (CGRP)-immunoreactive SNB motoneurons was also affected by adult castration. The percentage of CGRP-immunoreactive motoneurons was significantly decreased after adult castration. Again, this decrease was reversed by testosterone treatment. These findings suggest that the SNB of gerbils is sexually dimorphic and is sensitive to circulating levels of gonadal steroids. The unique placement of the SNB motoneurons suggests that an alternative laminar organizational scheme may be necessary for Mongolian gerbil.

Developmentally regulated expression of calcitonin gene-related peptide in the superior olive.

During postnatal development, the expression of calcitonin gene-related peptide (CGRP) and its mRNA was investigated in the superior olivary complex of the hamster in order to better understand its role in the development of the efferent olivocochlear (OC) pathway. Although both the peptide and its mRNA were expressed at birth in a few periolivary cells, neither CGRP mRNA nor any immunoreactivity could be detected in the lateral superior olive until after postnatal day (P) 5. By P9, CGRP expression had significantly increased and was mostly contained within the lateral superior olive. Between P7 and P18, there appears to be a transient increase in the transcript expression both in periolivary regions and in the lateral superior olive. Notably, both peptide and mRNA expression decreased precipitously throughout the superior olive after P18. In comparison, although both the facial and trigeminal motor nuclei had significant CGRP expression at birth, the facial motor nucleus demonstrated a decrease in the level of CGRP expression between P1 and P6, while the trigeminal motor nucleus reached a maximal level of expression around P18. If CGRP expression is related to synaptogenesis in OC neurons, as has been suggested for certain motor neurons, then we predict that the ephemeral increases in transcript expression in OC neurons are related to synaptogenetic mechanisms in the cochlear periphery. Importantly, the time course for CGRP expression in lateral OC neurons indicates that their OC terminals in the cochlear periphery may not begin forming synapses until near the end of the 1st postnatal week.

Isolation and characterization of a novel oligodendrocyte-specific protein.

Myelin plays a critical role in nervous system function and alterations in myelin-specific proteins cause a variety of neurologic disorders. We isolated a novel cDNA from the CNS that shares little nucleotide sequence homology with previously reported genes but appears to encode a protein related to peripheral myelin protein-22 (PMP-22) based on its amino acid sequence, predicted structure, and cellular localization. PMP-22 is important in peripheral myelination and Schwann cell proliferation, and mutations in its gene cause diseases of peripheral nerves. The isolated cDNA is 1.8 kb in length with an open reading frame of 621 bp. Northern blot analysis detected hybridization of labeled cDNA with a single 2.1-kb transcript only in the CNS. In situ hybridization revealed expression of this cDNA in oligodendrocytes of brain and spinal cord as well as in oligodendrocyte-enriched cultures; therefore we have named it oligodendrocyte-specific protein (OSP) cDNA. An OSP-specific polyclonal antibody reacted with a single 22-kd protein present in CNS myelin and oligodendrocytes. Developmental expression of OSP mRNA in the spinal cord was similar to that of the mRNA for a major myelin protein, proteolipid protein (PLP), and similar to PMP-22 in peripheral nerves. Since OSP is localized to oligodendrocytes and myelin, has a similar structure with PMP-22, and has a developmental pattern of expression like other myelin proteins, it probably has an important role in CNS myelinogenesis.

Differential regulation of alpha-calcitonin gene-related peptide and preprocholecystokinin messenger RNA expression in alpha-motoneurons: effects of testosterone and inactivity induced factors.

alpha-Calcitonin gene-related peptide expression in alpha-motoneurons is regulated by spinal cord transection, axotomy and testosterone, but to date there are no studies which examine the regulation of cholecystokinin expression in motoneurons. In the present study, we compared the regulation of preprocholecystokinin and alpha-calcitonin gene-related peptide messenger RNA levels in motoneurons of the spinal nucleus of the bulbocavernosus. Previously, we demonstrated that manipulations which decrease activity in target muscles of the spinal nucleus of the bulbocavernosus motoneurons increase alpha-calcitonin gene-related peptide message and peptide levels in spinal nucleus of the bulbocavernosus motoneurons. This muscle-nerve interaction is mediated by a soluble factor which is increased by castration. We now report that decreasing plasma testosterone levels decreased preprocholecystokinin messenger RNA levels. Testosterone replacement at the time of castration restored preprocholecystokinin messenger RNA levels to intact values. Injections of crude extracts prepared from denervated bulbocavernosus/levator ani into the homologous muscles of gonadally intact rats increased the levels of alpha-calcitonin gene-related peptide messenger RNA in spinal nucleus of the bulbocavernosus motoneurons. The levels of preprocholecystokinin messenger RNA did not differ in rats injected with denervated bulbocavernosus/levator ani extract or buffer, both of which were significantly higher than in intact, untreated rats. The results of the present experiments imply that levels of preprocholecystokinin and alpha-calcitonin gene-related peptide messenger ribonucleic acid are differentially regulated in spinal nucleus of the bulbocavernosus motoneurons.

Expression and distribution of mu opioid receptors in the inner ear of the rat.

Opioid peptides have demonstrated modulatory effects on the vestibular afferent discharge and are putative vestibular efferent neuromodulators. The distribution of their receptors in the mammalian vestibular epithelia is not known. We used reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, Western blots and immunohistochemistry to study the expression of mu opioid receptor (MOR) in the Scarpa's ganglia and cristae ampullares of rats. MOR transcript was only detected in the somata of the vestibular afferent neurons. MOR-like immunoreactivity was observed in the somata of vestibular afferents and in nerve terminals in the cristae ampullares epithelia both in the center and peripheral regions. Double labeling of cristae sections with the MOR1 antibody in combination with antibodies against calretinin (a marker for vestibular afferents terminating in calices) and peripherin (a marker for afferents terminating in boutons), respectively showed that MOR1 immunoreactivity was in calyx, dimorphic and bouton vestibular afferents. MOR immunoreactivity was not detected in vestibular efferent fibers identified with choline acetyltransferase immunohistochemistry. These results indicate that MOR may mediate effects of vestibular efferents on afferents.

Anabolic-androgenic steroid induced alterations in choline acetyltransferase messenger RNA levels of spinal cord motoneurons in the male rat.

The effect of chronic supraphysiological doses of anabolic-androgenic steroids, such as those illegally used by recreational, amateur and professional athletes to increase muscle mass and strength, on motoneurons has not been established. The choline acetyltransferase activity levels of perineal muscles in the male rat are modulated by plasma testosterone levels. These muscles are innervated by the sexually dimorphic motoneurons of the spinal nucleus of the bulbocavernosus. Since the primary source of choline acetyltransferase in muscle is motoneuronal, testosterone may modulate perineal muscle choline acetyltransferase activity by regulating choline acetyltransferase messenger RNA levels in motoneurons. The purpose of this study was to determine if choline acetyltransferase messenger RNA levels in cervical and lumbar spinal motoneurons are affected by chronic (four weeks) changes of plasma testosterone levels in the adult male rat. Using in situ hybridization, choline acetyltransferase messenger RNA levels were analysed in four motor columns: the spinal nucleus of the bulbocavernosus, the retrodorsal lateral nucleus of the lumbar spinal cord, and the lateral motor columns of the cervical and lumbar spinal cords. Chronic exposure to supraphysiological levels of testosterone (five- to ten-times physiologic levels) significantly increased choline acetyltransferase messenger RNA in all four motor columns. Subsequent to castration, choline acetyltransferase messenger RNA levels decreased in motoneurons of the spinal nucleus of the bulbocavernosus and the retrodorsal lateral nucleus. This observation suggests that the decrease in choline acetyltransferase activity levels of muscles innervated by spinal nucleus of the bulbocavernosus motoneurons may be due to changes in choline acetyltransferase protein levels. Indeed, testosterone replacement therapy of castrated males prevented the decline of choline acetyltransferase messenger RNA levels in motoneurons. The results of this study demonstrate that anabolic-androgenic steroids can affect the levels of specific messenger RNAs in motoneuron populations throughout the spinal cord suggesting that motoneuronal characteristics are modulated by circulating anabolic-androgenic steroid levels regardless of the purported "androgen sensitivity" of the specific neuromuscular system.

Hippocampal stimulation produces neuronal death in the immature brain.

We re-examined the proposed resistance of the immature brain to seizure-induced damage. In awake, freely moving rat pups, intermittent perforant path stimulation produced selective hippocampal cell loss and reduction in paired-pulse inhibition. During 16 h of stimulation, animals showed frequent wet dog shakes and hind-limb scratching movements but no convulsive motor activity. In situ end-labelling performed 2 h after the end of stimulation showed an intense band of positively-labelled eosinophilic cells with condensed profiles bilaterally in the dentate granule cell layer of stimulated animals. Control animals showed no in situ end-labelling positivity in the dentate gyrus. These cells were not observed 24 h later, suggestive of rapidly scavenged apoptotic cells. One day after the end of stimulation, many necrotic interneurons with eosinophilic cytoplasm and pyknotic nuclei were observed in the hilus of the stimulated dentate gyrus in all rats tested. Hippocampal pyramidal cells in CA1, CA3 and subiculum showed bilateral damage greater on the side of stimulation, and prepiriform cortex sustained bilateral symmetrical lesions. One month after perforant path stimulation, Cresyl Violet staining showed the number of large hilar interneurons (>15 microm) was reduced on the stimulated side (54.1 +/- 12.2) compared to the non-stimulated side (100.5 +/- 10.2 cells, P<0.01). Immunohistochemical analysis showed significant losses in somatostatin (8.5 +/- 1.6 stimulated side, 22.8 +/- 3.8 unstimulated side, P<0.05) and neuropeptide Y (12.8 +/- 3.2 stimulated side, 17.0 +/- 4.1 unstimulated side, P<0.05) immunoreactive cells in the stimulated hilus but no loss of parvalbumin-immunoreactive cells. Significant reductions in paired-pulse inhibition were found after stimulation but there was some return of inhibition by one month. These combined data demonstrate that the immature brain can incur damage as a result of prolonged seizure-like hippocampal activity mimicking status epilepticus in immature rats. The hippocampal damage produced by perforant path stimulation is associated with the immediate loss of physiological inhibition suggesting important modification of excitatory control in an extremely epileptogenic region of the brain.

Steroid regulation of calcitonin gene-related peptide mRNA expression in motoneurons of the spinal nucleus of the bulbocavernosus.

Motoneurons express calcitonin gene-related peptide (CGRP). Previous studies have shown that CGRP immunoreactivity is regulated by testosterone in the androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB). In this research the effect of plasma levels of testosterone on the expression of alpha CGRP mRNA in the SNB motoneurons of adult male rats was studied with in situ hybridization. The number of motoneurons expressing alpha CGRP mRNA and the level of alpha CGRP mRNA expression was significantly higher in the SNB of castrated male rats than in the SNB of gonadally intact rats. Using a 5x background labeling criterion in castrated rats 88.1 +/- 4.5% while in intact rats 75.3 +/- 6.4% of SNB motoneurons expressed alpha CGRP mRNA. Testosterone replacement at the time of castration prevented the effect of castration on the expression of alpha CGRP mRNA in SNB motoneurons. In castrated rats, the increase in the number of SNB cells expressing CGRP was the result of increased steady state levels of alpha CGRP mRNA in all SNB neurons.

The role of target muscles in the expression of calcitonin gene-related peptide mRNA in the spinal nucleus of the bulbocavernosus.

Previous studies have shown that calcitonin gene-related peptide (CGRP) mRNA steady-state levels and CGRP immunoreactivity in motoneurons of the spinal nucleus of the bulbocavernosus (SNB) are inversely related to the gonadal steroid environment in male rats. Since both the SNB motoneurons and their target muscles, the bulbocavernosus and the levator ani, are steroid sensitive, gonadal steroids may act at either site to regulate CGRP expression. In the present study, we tested the hypothesis that gonadal steroids influence CGRP expression in SNB motoneurons through their effects on the bulbocavernosus and levator ani muscles. We determined the levels of alpha-CGRP mRNA and immunoreactive CGRP in SNB motoneurons of adult male rats following injection of the bulbocavernosus with muscle extracts from bulbocavernosus/levator ani of castrated rats, paralysis of the bulbocavernosus or pudendal nerve cuts. Following injection of the bulbocavernosus/levator ani with extracts from castrated rats, the level of CGRP expression and the number of SNB motoneurons with alpha-CGRP message were increased. These studies suggest that the bulbocavernosus/levator ani muscles from castrated rats produce a factor that increases levels of CGRP. Injections of extract prepared from the bulbocavernosus and levator ani muscles of gonadally intact rats did not change the expression of alpha-CGRP mRNA in the SNB. Paralysis of the bulbocavernosus/levator ani with a local anesthetic increased the number of SNB motoneurons expressing alpha-CGRP mRNA and CGRP immunoreactive neurons. To determine whether nerve damage accounted for the observed effects following injection of anesthetic, the pudendal nerves were cut bilaterally.(ABSTRACT TRUNCATED AT 250 WORDS)

alpha-CGRP mRNA levels in motoneurons innervating specific rat muscles.

Along with acetylcholine, motoneurons express several neuromodulatory peptides. The most extensively studied of these peptides is calcitonin gene-related peptide (CGRP). CGRP modulates the biochemical, physiological and metabolic properties of skeletal muscle primarily through activation of membrane receptors. Virtually all motor pool contain motoneurons that are immunoreactive for CGRP. The purpose of this study was to determine the proportions of motoneurons that express alpha-CGRP in motor pools innervating muscles with different motor unit compositions. These include the soleus, extensor digitorum longus, tensor fascia latae and the diaphragm muscles as well as the spinal nucleus of the bulbocavernosus. The spinal nucleus of the bulbocavernosus provides innervation to the bulbocavernosus/levator ani muscle complex and the external anal sphincter muscle. The spinal nucleus of the bulbocavernosus contained the greatest proportion of alpha-CGRP mRNA-positive motoneurons, followed in descending rank order by the tensor fascia latae, the extensor digitorum longus, the soleus and the diaphragm motor pools. In addition, significant differences between motor pools were observed in the mean relative alpha-CGRP mRNA level among those motoneurons expressing alpha-CGRP. The highest mean relative alpha-CGRP mRNA level was observed in soleus and the extensor digitorum longus motor pools; followed in descending rank order by the tensor fascia latae, the diaphragm and the spinal nucleus of the bulbocavernosus. We have previously shown that muscle contractile inactivity increases the number of motoneurons that express alpha-CGRP and in the relative mRNA levels. The results of the present study suggest that the proportion of motoneurons within a motor pool that express alpha-CGRP may be closely related to the contractile activity (i.e. activation history) of the target muscle.

Expression of the AMPA-selective receptor subunits in the vestibular nuclei of the chinchilla.

The distribution of the AMPA type glutamate receptor has been investigated throughout the central nervous system; however, no detailed description of its distribution is available in the vestibular nuclei. In the present study, in situ hybridization histochemistry and immunohistochemistry were used to localize the messenger RNAs and proteins of the AMPA-selective receptor subunits GluR1, GluR2, GluR3 and GluR4 in the vestibular nuclei of the chinchilla. Immunohistochemistry with subunits specific antisera showed differential distribution of the subunits in the vestibular nuclei. GluR2/3 antiserum labeled the most neurons, suggesting that many if not all vestibular neurons receive glutamatergic input. GluR1-positive neurons were fewer than GluR2/3 immunoreactive neurons and GluR4 immunoreactivity was found in the fewest number of neurons. GluR1 and GluR4 immunoreactivity was also found in astrocyte-like structures. In situ hybridization with 35S-labeled complementary RNA probes confirmed the distribution of the AMPA receptor subunits obtained by immunohistochemistry. Quantitative analysis of the levels of hybridization showed a high degree of diversity in the levels of expression of the GluR2 subunit mRNA, with the highest levels of expression in the giant Deiter's cells of the lateral vestibular nuclei and the lowest levels in the small neurons throughout the vestibular nuclei. The subunit compositions of the AMPA receptors determine their physiological properties. Differential distribution and levels of expression of the receptor subunits in the vestibular nuclei may be related to the characteristics of information processing through the vestibular system.

Radiation hybrid mapping of 11 alpha and beta nicotinic acetylcholine receptor genes in Rattus norvegicus.

Acetylcholine is the main neurotransmitter of the vestibular efferents and a wide variety of muscarinic and nicotinic acetylcholine receptors are expressed in the vestibular periphery. To date, 11 nicotinic subunits (alpha and beta) have been reported in mammals. Previously, our group [Brain Res. 778 (1997) 409] reported that these nicotinic acetylcholine receptor alpha and beta subunits were differentially expressed in the vestibular periphery of the rat. To begin an understanding of the molecular genetics of these vestibular efferents, this study examined the chromosomal locations of these nicotinic acetylcholine receptor genes in the rat (Rattus norvegicus). Using radiation hybrid mapping and a rat radiation hybrid map server (www.rgd.mcw.edu/RHMAP SERVER/), we determined the chromosomal position for each of these genes. The alpha2-7, alpha9, alpha10, and beta2-4 nicotinic subunits mapped to the following chromosomes: alpha2, chr. 15; alpha3, chr. 8; alpha4, chr. 3; alpha5, chr. 8; alpha6, chr. 16; alpha7, chr. 1; alpha9, chr. 14; alpha10, chr. 7; beta2, chr. 2; beta3, chr. 16; and beta4, chr. 8. With the location for each of these nicotinic subunits known, it is now possible to develop consomic and/or congenic strains of rats that can be used to study the functional genomics of each of these subunits.

The localization of sensory nerve fibers and receptor binding sites for sensory neuropeptides in canine mesenteric lymph nodes.

Previous work has established that the central nervous system can modulate the immune response. Direct routes through which this regulation may occur are the sympathetic and sensory innervation of lymphoid organs. We investigated the innervation of canine mesenteric lymph nodes using immunohistochemistry and the expression of binding sites for sensory neuropeptides using quantitative receptor autoradiography. The sympathetic innervation of lymph nodes was examined by immunohistochemical methods using an antiserum directed against tyrosine hydroxylase (TOH), the rate limiting enzyme in catecholamine synthesis. TOH-containing fibers were associated with 90% of the blood vessels (arteries, veins, arterioles and venules) in the hilus, medullary and internodular regions of lymph nodes and in trabeculae with no obvious relationship to blood vessels. The sensory innervation of lymph nodes was investigated using antisera directed against the putative sensory neurotransmitters calcitonin gene-related peptide (CGRP) and substance P (SP). CGRP- and SP-containing fibers were detected in the hilus, the medullary region, and the internodular region of lymph nodes usually in association with arterioles and venules. About 50% of the arterioles and venules exhibited a CGRP innervation and a smaller fraction (5-10%) were innervated by SP-containing fibers. Few if any TOH, CGRP, and SP nerve fibers were detected in the germinal centers of lymph nodes. Using quantitative receptor autoradiography we studied the distribution of receptor binding sites for the sensory neuropeptides CGRP, SP, substance K (SK), vasoactive intestinal peptide (VIP), somatostatin (SOM), and bombesin. Specific CGRP binding sites were expressed throughout lymph nodes by trabeculae, arterioles, venules and 25% of the germinal centers. SP receptor binding sites were localized to arterioles and venules in the T cell regions and 25-30% of the germinal centers. VIP binding sites were localized to the internodular and T cell regions, to medullary cords, and to 10-20% of germinal centers. SK, SOM, and bombesin binding sites were not detected in the lymph nodes, although receptor binding sites for these peptides were detected with high specific/nonspecific binding ratios in other canine peripheral tissues. Taken together with previous results these findings suggest that the sympathetic and sensory innervation of mesenteric lymph nodes appears to be involved with the regulation of their blood and lymph flow. The neuropeptide receptor binding sites in lymph node germinal centers may be expressed by lymphocytes upon activation by antigens.(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of calcitonin gene-related peptide and its receptors in a striated muscle.

The distribution of calcitonin gene-related peptide (CGRP) and its binding sites in the bulbocavernosus, a striated muscle, are reported. We used immunohistochemistry and [125I]CGRP autoradiography. The pattern of [125I]CGRP binding was restricted to a discrete band that coincides with the distribution of end-plates in this muscle as determined by CGRP immunohistochemistry and acetylcholinesterase staining. CGRP has been shown to increase the level of acetylcholine receptor (AChR) alpha-subunit mRNA. The role of CGRP as the endogenous factor by which motoneurons regulate the expression of junctional AChR is discussed.

Regulation of cholecystokinin receptors in the ventromedial nucleus of the hypothalamus: sex steroid hormone effects.

Quantitative autoradiography was used to analyze cholecystokinin receptor (CCK-R) binding in the ventromedial hypothalamic nucleus (VMH) of gonadectomized rats treated with estrogen or testosterone. In the rostral VMH, sex steroids did not affect binding levels. In the caudal VMH, in both gonadectomized male and female rats, the levels of CCK-R binding were decreased 24 h after injection of 50 micrograms of estrogen benzoate but were not changed after injection of 300 micrograms of testosterone propionate. We hypothesize that these changes in CCK-R binding in the VMH reflect ligand-induced down-regulation that result from an estrogen-facilitation of stimulated CCK release in the VMH.

The role of apoptosis in sexual differentiation of the rat sexually dimorphic nucleus of the preoptic area.

The sexually dimorphic nucleus of the preoptic area (SDN-POA) in the rat hypothalamus is larger in volume in males than in females due to a larger number of cells in the nucleus. Although the SDN-POA, and its development, have been extensively studied, the actual mechanism of its sexual differentiation has not been established. The results of previous studies have not supported a role for gonadal steroids in the regulation of neurogenesis or the determination of the migratory pathway perinatally. In this study, the role of cell death in the development of the sexual dimorphism in the SDN-POA was investigated using in situ end-labeling to visualize fragmented DNA in apoptotic cells. In the experiments described here, the incidence of apoptosis was determined in part of the SDN-POA, the central division of the medial preoptic nucleus (MPNc), over the first 13 days postnatally in male and female rats. There was a sex difference in the incidence of apoptosis in the MPNc between postnatal days 7 and 10; the incidence was higher in females. The role of testosterone (T) in regulating the incidence of apoptosis in the developing MPNc was examined in neonatally castrated males following T or vehicle injection. Testosterone had a profound inhibitory effect on the incidence of apoptosis between days 6 and 10. In a control region within the lateral preoptic area, there was no sex difference in the incidence of apoptosis, nor was there an effect of T. Thus, the data indicate that the regulation of apoptosis by T is one mechanism involved in the sexual differentiation of the SDN-POA.

Adenylyl cyclase isoforms in the vestibular periphery of the rat.

The expression of adenylyl cyclase (AC) isoforms in the adult rat vestibular periphery was investigated using reverse transcription polymerase chain reaction (RT-PCR). AC II, IV and V mRNAs were expressed in both Scarpa's ganglion and vestibular end organs. In addition, in the vestibular end organs, an AC mRNA not previously reported in the rat was identified. The cloned sequence (GenBank accession no. AF184150) represented 95 amino acids with 100% similarity to the human AC VII and 94% to the bovine AC VII. AC VII mRNA also was found in the cerebellum but was undetectable in heart, kidney, liver and Scarpa's ganglion.

Calcitonin gene-related peptide-like immunoreactivity in spinal motoneurons of the male mouse is affected by castration and genotype.

Calcitonin gene-related peptide (CGRP) is found in motoneurons of the mammalian spinal cord, including motoneurons of the androgen-dependent spinal nucleus of the bulbocavernosus (SNB) of the mouse. Motoneurons of the SNB innervate the bulbocavernosus (BC), a striated muscle involved in penile reflexes. CGRP is though to be a trophic factor produced by motoneurons to regulate the expression of the acetylcholine receptor at the neuromuscular junction. In rats, the number of SNB motoneurons containing CGRP is increased by gonadal steroids. This regulation appears to rely on an activity-dependent factor produced by the BC muscle. The purpose of the present study was to examine, using immunohistochemistry, the steroid dependence of CGRP in the SNB of male house mice. Genotypic differences in the steroid regulation of CGRP immunoreactivity were examined in three strains of mice that differ in their behavioral sensitivity to castration. The results demonstrate that castration reduces the number of CGRP-positive SNB motoneurons in mice. The magnitude of the change in CGRP in response to castration and the length of time required following castration to alter CGRP were dependent on genotype. Interestingly, the effect of castration in mice, to reduce the number of CGRP-immunoreactive SNB motoneurons, is opposite in direction from the increase in CGRP SNB motoneurons observed in rats observed following castration. These experiments suggest that androgens may alter neuromuscular junction function of mouse SNB by regulating the production of CGRP in a species-specific, genotypically dependent fashion.