Graphology for the diagnosis of suicide attempts: a blind proof of principle controlled study.

To evaluate the ability of two graphologists and two practising internists not trained in graphology to differentiate letters written by subjects who have attempted to commit suicide by self-poisoning and healthy volunteers, we performed a maximal blind controlled study vs. healthy volunteers. Forty fully recovered patients who had attempted to commit suicide and 40 healthy volunteers wrote and signed a short letter or story not related to the parasuicide or their mental health status. The evaluators classified the 80 letters as 'suicide' or 'no suicide' in an intention-to-treat analysis. Letters expressing sadness were subsequently excluded for a per-protocol analysis. Correct diagnosis of suicide and of healthy controls was made in, respectively, 32 of 40 and 33 of 40 letters by the graphologists and in 27 of 40 and 34 of 40 letters by the internists. After the exclusion of 12 letters expressing sadness, the sensitivity, specificity, positive predictive value and negative predictive value were, respectively, 73, 88, 81 and 82% for the graphologists and 53, 89, 80 and 71% for the internists. Both classified the letters with significantly more effectiveness than chance (p < 0.001) with no statistically significant difference between the two groups of evaluators. We concluded that graphological analysis is able to differentiate letters written by patients who attempt suicide from those written by healthy controls. This technique shows an acceptable degree of accuracy and could therefore become an additional discharge or decision-making tool in Psychiatry or Internal Medicine.

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.

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.

Expression of the human insulin gene in the gastric G cells of transgenic mice.

The goal of this study was to engineer gastrin-producing G cells of the gastric antrum to produce insulin. A pGas-Ins chimeric gene in which the gastrin promoter drives expression of the human insulin gene was constructed and was validated by transient transfection of GH4 and AGS cells. RT-PCR analysis and sequencing revealed three forms of differentially spliced insulin mRNA in GH4 cells transiently transfected by pGas-Ins. Gas-Ins transgenic mice were generated utilizing this chimeric gene. Northern blot analysis, in situ hybridization, and immunohistochemistry demonstrated expression of the human insulin gene specifically in antral G cells. Northern blot analysis demonstrated that the shortest of the insulin mRNA three forms is predominantly expressed in stomach tissue. RT-PCR analysis also showed expression of the transgene in colon, pancreas, and brain tissues that was undetectable by northern analysis. We conclude that gastrin promoter can be used for targeting expression of human insulin to antral G cells and that antral G cells can express human insulin. Further refining of the chimeric gene design is required to enhance expression.

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.

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.

Topographic distribution of nicotinic acetylcholine receptors in the cristae of a turtle.

The neurochemical basis of cholinergic efferent modulation of afferent function in the vestibular periphery remains incompletely understood; however, there is cellular, biochemical and molecular biological evidence for both muscarinic and nicotinic acetylcholine (ACh) receptors (nAChRs) in this system. This study examined the topographic distribution of alpha-bungarotoxin (alpha-BTX) nAChRs in the cristae of a turtle species. Cristae were perfusion-fixed, cut at 20 micrometer on a cryostat and incubated with alpha-BTX or polyclonal antibodies raised against Torpedo nAChR. Light microscopy showed abundant specific labeling of nAChR in the central zone of each hemicrista on the calyx-bearing afferents surrounding type I hair cells and on the base of the type II hair cells. Within the peripheral zone, dense labeling of type II hair cells near the torus and sparse or no label was observed on type II hair cells near the planum. The alpha-BTX binding showed a similar pattern within the cristae. The similarity between the topographic distribution of alpha-BTX binding nAChR and of efferent inhibition of afferents supports the notion that the inhibitory effect of afferents is mediated by nAChR.

Partial cDNAs encoding G-protein alpha subunits in the rat vestibular periphery.

To begin understanding what G-proteins are involved in signal transduction in the vestibular periphery, the expression of Galpha subunits in rat primary afferent neurons (Scarpa's ganglia) and end-organs was studied. Reverse transcription-polymerase chain reaction (RT-PCR) with degenerate primers corresponding to two conserved regions of the Galpha protein coding sequence produced partial cDNAs encoding two distinct forms of Galpha(s) subunit (Galpha(s2) and anove) Galpha(s2) subunit,GenBank accession number AF1841510); and two forms of Galpha(i2) subunits. A novel truncated form of Galpha(i2) (designated Galpha(i2(vest)),Gen Bank accession number AF189020) was detected in the vestibular periphery. Galpha(i2(vest)) was also expressed in rat cerebellum and heart. The possible role of the identified Galpha protein cDNAs in the function of the vestibular periphery is discussed.

Expression of BDNF and TrkB mRNAs in the crista neurosensory epithelium and vestibular ganglia following ototoxic damage.

Following ototoxic lesion with the aminoglycoside gentamicin, the vestibular neurosensory epithelia undergo degeneration and then limited spontaneous regeneration. The spatio-temporal expression of brain-derived neurotrophic factor (BDNF) and of its high affinity receptor (trkB) mRNA was investigated in the vestibular end organs and ganglia of chinchillas following gentamicin ototoxicity. In the vestibular ganglia of untreated chinchillas, the level of expression of BDNF mRNA is low. At 1 and 2 weeks after intraotic treatment with gentamicin, BDNF mRNA levels in the vestibular ganglia were elevated significantly compared to untreated chinchillas and chinchillas 4 weeks after treatment. At 4 weeks after gentamicin treatment, BDNF mRNA levels were at intact levels of expression. In the crista ampullaris, high levels of BDNF transcripts were found in the untreated chinchillas. At 1 and 2 weeks after treatment, when only supporting cells are present in the crista, BDNF mRNA was undetectable. Four weeks after aminoglycoside treatment BDNF mRNA was present in the epithelium but at lower levels than in the intact epithelium. In contrast to its ligand, high levels of trkB mRNA hybridization were present in the vestibular ganglia of untreated chinchillas and trkB mRNA levels did not change following gentamicin treatment. In the vestibular epithelia, trkB mRNA was not detected either in the intact epithelium or after gentamicin ototoxicity. These data suggest that BDNF may be involved in the maintenance of the vestibular ganglia and contribute to neurite outgrowth to new and repaired hair cells following ototoxic damage.

The passerine hippocampus is a site of high aromatase: inter- and intraspecies comparisons.

The vertebrate hippocampus (HP) plays a critical role in the organization of memory. Estrogens alter synaptic morphology and function in the mammalian HP and may potentiate memory performance. Previous studies suggest that the songbird HP itself is a site of significant aromatase expression, intimating that local estrogen synthesis may provide a source of this steroid to estrogen-sensitive neural circuits. To explore the potential role of local estrogen synthesis on HP structure and function, we have characterized aromatase message and activity in the zebra finch HP. Toward this end we have compared (a) HP aromatase mRNA with that at other neural loci, (b) HP aromatase activity between adults of both sexes, and (c) HP and hypothalamic preoptic area (HPOA) aromatase activity among songbirds and nonsongbirds. Finally we asked whether aromatase activity was intrinsic to the HP by maintaining it in culture, isolated from the rest of the telencephalon. The HP of every songbird studied expresses aromatase, with comparable levels across sexes. Notably, aromatase activity was found at higher levels in the songbird HP than in the HPOA. In both nonsongbird species investigated, however, HP aromatase activity was undetectable under identical assay conditions. Additionally, the developing songbird HP continues to express aromatase when cultured in isolation from the rest of the telencephalon. The data suggest that HP aromatase is characteristic of passeriformes and, as in the HPOA, may represent a mechanism whereby estrogen is made available to neural circuits. Passerines may prove invaluable animal models for investigations of the estrogenic modulation of HP structure and function.

Estrogen-induced alteration of mu-opioid receptor immunoreactivity in the medial preoptic nucleus and medial amygdala.

The mu-opioid receptor (mu-OR), like most G-protein-coupled receptors, is rapidly internalized after agonist binding. Although opioid peptides induce internalization in vivo, there are no studies that demonstrate mu-OR internalization in response to natural stimuli. In this study, we used laser-scanning microscopy to demonstrate that estrogen treatment induces the translocation of mu-OR immunoreactivity (mu-ORi) from the membrane to an internal location in steroid-sensitive cell groups of the limbic system and hypothalamus. Estrogen-induced internalization was prevented by the opioid antagonist naltrexone, suggesting that translocation was largely dependent on release of endogenous agonists. Estrogen treatment also altered the pattern of mu-ORi at the bright-field light microscopic level. In the absence of stimulation, the majority of immunoreactivity is diffuse, with few definable mu-OR+ cell bodies or processes. After stimulation, the density of distinct processes filled with mu-ORi was significantly increased. We interpreted the increase in the number of mu-OR+ processes as indicating increased levels of internalization. Using this increase in the density of mu-OR+ fibers, we showed that treatment of ovariectomized rats with estradiol benzoate induced a rapid and reversible increase in the number of fibers. Significant internalization was noted within 30 min and lasted for >24 hr after estrogen treatment in the medial preoptic nucleus, the principal part of the bed nucleus, and the posterodorsal medial amygdala. Naltrexone prevented the increase of mu-OR+ processes. These data imply that estrogen treatment stimulates the release of endogenous opioids that activate mu-OR in the limbic system and hypothalamus providing a "neurochemical signature" of steroid activation of these circuits.

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.

Infusion of CCK-A receptor mRNA antisense oligodeoxynucleotides inhibits lordosis behavior.

Cholecystokinin (CCK) acting on discrete receptors in the limbic-hypothalamic circuit modulates lordosis behavior. Neurons in the medial preoptic nucleus (MPN) express CCK-A subtype receptor mRNA, and site-specific infusions of CCK facilitate lordosis, suggesting that CCK-A receptor activation positively modulates lordosis. In the present study, we demonstrated CCK binding in the central portion of the MPN (MPNc) and the disruption of lordosis behavior by reducing the expression of CCK-A receptors in this nucleus. Antisense oligodeoxynucleotides (ODN) specific for CCK-A receptor mRNA were infused into the MPN of ovariectomized female rats. The expression of estrogen-induced lordosis behavior was blocked in animals receiving infusions of antisense ODN into the MPN (LQ = 10.0 +/- 1.0) compared to animals receiving infusions of nonsense ODN (containing the same nucleotide bases in a random order; LQ = 92.5 +/- 7.5). In vitro, AR42J pancreatic acinar carcinoma cells treated with antisense ODN had lower levels of CCK-A and CCK-B subtype receptor binding than nonsense ODN treated cells. In vivo, however, infusions of CCK-A mRNA antisense ODN did not alter CCK-B receptor binding levels. These results suggest that CCK, acting via CCK-A receptors in the MPNc, is critical for the display of lordosis behavior.

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.

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.

TRPM-2 expression and tunel staining in neurodegenerative diseases: studies in wobbler and rd mice.

Neuronal apoptosis has been described during development but little is known about whether apoptosis plays a role in neurodegenerative disease. Neurodegenerative cell death can be difficult to study because it is often a slow process and it is limited to only a few cells among many nondying cells. We used molecular methods to study cell death in the spinal cords of wobbler mice, a model of motoneuron disease, and compared it to retinas of rd mice, a model of retinitis pigmentosa, where it is known that photoreceptors die by apoptosis. Increased levels of mRNA of testosterone-repressed prostate message 2 (TRPM-2) were found in motoneurons of wobbler mice and the retinas of rd mice. In motoneurons, TRPM-2 mRNA colocalized with increased expression of the message for growth-associated protein (GAP-43). In rd retinas, TRPM-2 mRNA was localized to ganglion cells of the inner retina known to survive the disease. These suggest that TRPM-2 expression is associated with cell membrane remodeling in surviving cells associated with synaptic reorganization or change in afferent input. In situ labeling of fragmented DNA (TUNEL staining) identified dying photoreceptors in the rd mouse. In the wobbler spinal cords dying motoneurons were not labeled. These data suggest that the process of neurodegenerative motoneuron cell death in wobbler mice is different from the apoptotic process of rd photoreceptors.

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.

Expression of brain-derived neurotrophic factor and its receptor mRNA in the vestibuloauditory system of the bullfrog.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin which has been suggested to play a crucial role in the development and maintenance of the inner ear. In the present study, we investigated the expression of mRNAs of BDNF and its high-affinity receptor trkB in the vestibuloauditory system of the adult bullfrog. In situ hybridization was performed using riboprobes transcribed from Xenopus BDNF and trkB cDNA clones. BDNF mRNA was expressed in the sensory epithelia of the ampullary cristae, utricular and saccular maculae, lagena, and amphibian and basilar papillae. Strong hybridization for BDNF mRNA was also found in neuron somata of the vestibuloauditory nuclear complex. trkB mRNA was detected in the sensory epithelia of all vestibular and auditory endorgans. High levels of both BDNF and trkB mRNAs were found in vestibuloauditory ganglion cells. These results support the hypothesis that BDNF participates in the maintenance of vestibuloauditory neurons and may be important for the trophic regulation of vestibular and auditory sensory epithelia in this animal model.

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.