Dual action of serotonin on local excitatory and inhibitory neural circuits regulating the corticotropin-releasing factor neurons in the paraventricular nucleus of the hypothalamus.

Serotonergic neurons originating from the raphe nuclei have been proposed to regulate corticotropin-releasing factor (CRF) neurons in the paraventricular nucleus of the hypothalamus (PVH). Since glutamate- and γ-aminobutyric acid (GABA)-containing neurons, constituting the hypothalamic local circuits, innervate PVH CRF neurons, we examined whether they mediate the actions of serotonin (5-hydroxytryptamine [5-HT]) on CRF neurons. Spontaneous excitatory postsynaptic currents (sEPSCs) or spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in PVH CRF neurons, under whole cell patch-clamp, using the CRF-modified yellow fluorescent protein (Venus) ΔNeo mouse. Serotonin elicited an increase in the frequency of sEPSCs in 77% of the cells and a decrease in the frequency of sIPSCs in 71% of the cells, tested in normal medium. Neither the amplitude nor decay time of sEPSC and sIPSC was affected, thus the site(s) of action of serotonin may be presynaptic. In the presence of tetrodotoxin (TTX), serotonin had no significant effects on either parameter of sEPSC or sIPSC, indicating that the effects of serotonin are action potential-dependent, and that the presynaptic interneurons are largely intact within the slice; distant neurons may exist, though, since some 20%-30% of neurons did not respond to serotonin without TTX. We next examined through what receptor subtype(s) serotonin exerts its effects on presynaptic interneurons. DOI (5-HT2A/2C agonist) mimicked the action of serotonin on the sIPSCs, and the serotonin-induced decrease in sIPSC frequency was inhibited by a selective 5-HT2C antagonist RS102221. 8-OH-DPAT (5-HT1A/7 agonist) mimicked the action of serotonin on the sEPSCs, and the serotonin-induced increase in sEPSC frequency was inhibited by a selective 5-HT7 antagonist SB269970. Thus, serotonin showed a dual action on PVH CRF neurons, by upregulating glutamatergic- and downregulating GABAergic interneurons; the former may partly be mediated by 5-HT7 receptors, whereas the latter by 5-HT2C receptors. The CRF-Venus ΔNeo mouse was useful for the electrophysiological examination.

Vasopressin Expressed in Hypothalamic CRF Neurons Causes Impaired Water Diuresis in Secondary Adrenal Insufficiency.

Patients with secondary adrenal insufficiency can present with impaired free water excretion and hyponatremia, which is due to the enhanced secretion of vasopressin (AVP) despite increased total body water. AVP is produced in magnocellular neurons in the paraventricular nucleus of the hypothalamus (PVH) and supraoptic nucleus and in parvocellular corticotropin-releasing factor (CRF) neurons in the PVH. This study aimed to elucidate whether magnocellular AVP neurons or parvocellular CRF neurons coexpressing AVP are responsible for the pathogenesis of hyponatremia in secondary adrenal insufficiency. The number of CRF neurons expressing copeptin, an AVP gene product, was significantly higher in adrenalectomized AVP-floxed mice (AVPfl/fl) than in sham-operated controls. Adrenalectomized AVPfl/fl mice supplemented with aldosterone showed impaired water diuresis under ad libitum access to water or after acute water loading. They became hyponatremic after acute water loading, and it was revealed under such conditions that aquaporin-2 (AQP2) protein levels were increased in the kidney. Furthermore, translocation of AQP2 to the apical membrane was markedly enhanced in renal collecting duct epithelial cells. Remarkably, all these abnormalities observed in the mouse model for secondary adrenal insufficiency were ameliorated in CRF-AVP-/- mice that lacked AVP in CRF neurons. Our study demonstrates that CRF neurons in the PVH are responsible for the pathogenesis of impaired water excretion in secondary adrenal insufficiency.

Congenital Hypothyroidism and Brain Development: Association With Other Psychiatric Disorders.

Thyroid hormones play an important role in brain development, and thyroid hormone insufficiency during the perinatal period results in severe developmental delays. Perinatal thyroid hormone deficiency is clinically known as congenital hypothyroidism, which is caused by dysgenesis of the thyroid gland or low iodine intake. If the disorder is not diagnosed or not treated early, the neuronal architecture is perturbed by thyroid hormone insufficiency, and neuropathological findings, such as abnormal synapse formation, defects in neuronal migration, and impairment of myelination, are observed in the brains of such patients. Furthermore, the expression of psychiatric disorder-related molecules, especially parvalbumin, is significantly decreased by thyroid hormone insufficiency during the perinatal period. Animal experiments using hypothyroidism models display decreased parvalbumin expression and abnormal brain architecture, and these experimental results show reproducibility and stability. These basic studies reinforce the results of epidemiological studies, suggesting the relevance of thyroid dysfunction in psychiatric disorders. In this review, we discuss the disruption of brain function associated with congenital hypothyroidism from the perspective of basic and clinical research.

Downstream projection of Barrington's nucleus to the spinal cord in mice.

Barrington's nucleus (Bar), which controls micturition behavior through downstream projections to the spinal cord, contains two types of projection neurons, BarCRH and BarESR1, that have different functions and target different spinal circuitry. Both types of neurons project to the L6-S1 spinal intermediolateral (IML) nucleus, whereas BarESR1 neurons also project to the dorsal commissural nucleus (DCN). To obtain more information about the spinal circuits targeted by Bar, we used patch-clamp recording in spinal slices from adult mice in combination with optogenetic stimulation of Bar terminals. Recording of opto-evoked excitatory postsynaptic currents (oEPSCs) in 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate (DiI)-labeled lumbosacral preganglionic neurons (LS-PGNs) revealed that both Bar neuronal populations make strong glutamatergic monosynaptic connections with LS-PGNs, whereas BarESR1 neurons also elicited smaller-amplitude glutamatergic polysynaptic oEPSCs or polysynaptic opto-evoked inhibitory postsynaptic currents (oIPSCs) in some LS-PGNs. Optical stimulation of BarCRH and BarESR1 terminals also elicited monosynaptic oEPSCs and polysynaptic oIPSCs in sacral DCN neurons, some of which must include interneurons projecting to either the IML or ventral horn. Application of capsaicin increased opto-evoked firing during repetitive stimulation of Bar terminals through the modulation of spontaneous postsynaptic currents in LS-PGNs. In conclusion, our experiments have provided insights into the synaptic mechanisms underlying the integration of inputs from Bar to autonomic circuitry in the lumbosacral spinal cord that may control micturition.NEW & NOTEWORTHY Photostimulation of BarCRH or BarESR1 axons in the adult mouse spinal cord elicits excitatory or inhibitory postsynaptic responses in multiple cell types related to the autonomic nervous system including preganglionic neurons (PGNs) in the lumbosacral intermediolateral nucleus and interneurons in the lumbosacral dorsal commissure nucleus. Integration of excitatory inputs from Bar and from visceral primary afferents in PGNs may be important in the regulation of micturition behavior.

Thyroid hormone insufficiency alters the expression of psychiatric disorder-related molecules in the hypothyroid mouse brain during the early postnatal period.

The functional role of thyroid hormone (TH) in the cortex and hippocampus of mouse during neuronal development was investigated in this study. TH insufficiency showed a decrease in the expression of parvalbumin (PV) in the cortex and hippocampus of pups at postnatal day (PD) 14, while treatment with thyroxine from PD 0 to PD 14 ameliorated the PV loss. On the other hand, treatment with antithyroid agents in adulthood did not result in a decrease in the expression of PV in these areas. These results indicate the existence of a critical period of TH action during the early postnatal period. A decrease in MeCP2-positive neuronal nuclei was also observed in the cortical layers II-IV of the cerebral cortex. The brains were then stained with CUX1, a marker for cortical layers II-IV. In comparison with normal mice, CUX1 signals were decreased in the somatosensory cortex of the hypothyroid mice, and the total thickness of cortical layers II-IV of the mice was lower than that of normal mice. These results suggest that TH insufficiency during the perinatal period strongly and broadly affects neuronal development.

Cholinergic modulation of CRH and non-CRH neurons in Barrington's nucleus of the mouse.

Corticotropin-releasing hormone (CRH) is expressed in Barrington's nucleus (BarN), which plays an essential role in the regulation of micturition. To control the neural activities of BarN, glutamatergic and GABAergic inputs from multiple sources have been demonstrated; however, it is not clear how modulatory neurotransmitters affect the activity of BarN neurons. We have employed knock-in mice, CRH-expressing neurons of which are labeled with a modified yellow fluorescent protein (Venus). Using whole cell patch-clamp recordings, we examined the responses of Venus-expressing (putative CRH-expressing) neurons in BarN (BarCRH), as well as non-CRH-expressing neurons (BarCRH-negative), following bath application of cholinergic agonists. According to the present study, the activity of BarCRH neurons could be modulated by dual cholinergic mechanisms. First, they are inhibited by a muscarinic receptor-mediated mechanism, most likely through the M2 subclass of muscarinic receptors. Second, BarCRH neurons are excited by a nicotinic receptor-mediated mechanism. BarCRH-negative neurons also responded to cholinergic agents. Choline transporter-immunoreactive nerve terminals were observed in close proximity to the neurites, as well as the somata of BarCRH. The present results suggest that BarN neurons are capable of responding to cholinergic input.NEW & NOTEWORTHY This study investigates the effects of bath-applied cholinergic agonists on Barrington's nucleus (BarN) neurons in vitro. They were either excitatory, through nicotinic receptors, or inhibitory, through muscarinic receptors. Putative corticotropin-releasing hormone (CRH)-expressing neurons in BarN, as well as putative non-CRH-expressing neurons, responded to cholinergic agonists.

Correction to: Female-biased sexual dimorphism of corticotropin-releasing factor neurons in the bed nucleus of the stria terminalis.

Following publication of the original article [1], we noticed a number of errors.

Female-biased sexual dimorphism of corticotropin-releasing factor neurons in the bed nucleus of the stria terminalis.

BACKGROUND: The bed nucleus of the stria terminalis (BNST) contains the highest density of corticotropin-releasing factor (CRF)-producing neurons in the brain. CRF-immunoreactive neurons show a female-biased sexual dimorphism in the dorsolateral BNST in the rat. Since CRF neurons cannot be immunostained clearly with available CRF antibodies in the mouse, we used a mouse line, in which modified yellow fluorescent protein (Venus) was inserted to the CRF gene, and the Neo cassette was removed, to examine the morphological characteristics of CRF neurons in the dorsolateral BNST. Developmental changes of CRF neurons were examined from postnatal stages to adulthood. Gonadectomy (GDX) was carried out in adult male and female mice to examine the effects of sex steroids on the number of CRF neurons in the dorsolateral BNST. METHODS: The number of Venus-expressing neurons, stained by immunofluorescence, was compared between male and female mice over the course of development. GDX was carried out in adult mice. Immunohistochemistry, in combination with Nissl staining, was carried out, and the effects of sex or gonadal steroids were examined by estimating the number of Venus-expressing neurons, as well as the total number of neurons or glial cells, in each BNST subnucleus, using a stereological method. RESULTS: Most Venus-expressing neurons co-expressed Crf mRNA in the dorsolateral BNST. They constitute a group of neurons without calbindin immunoreactivity, which makes a contrast to the principal nucleus of the BNST that is characterized by calbindin immunostaining. In the dorsolateral BNST, the number of Venus-expressing neurons increased across developmental stages until adulthood. Sexual difference in the number of Venus-expressing neurons was not evident by postnatal day 5. In adulthood, however, there was a significant female predominance in the number of Venus expressing neurons in two subnuclei of the dorsolateral BNST, i.e., the oval nucleus of the BNST (ovBNST) and the anterolateral BNST (alBNST). The number of Venus-expressing neurons was smaller significantly in ovariectomized females compared with proestrous females in either ovBNST or alBNST, and greater significantly in orchiectomized males compared with gonadally intact males in ovBNST. The total number of neurons was also greater significantly in females than in males in ovBNST and alBNST, but it was not affected by GDX. CONCLUSION: Venus-expressing CRF neurons showed female-biased sexual dimorphism in ovBNST and alBNST of the mouse. Expression of Venus in these subnuclei was controlled by gonadal steroids.

Genome-Wide Analysis of Glucocorticoid-Responsive Transcripts in the Hypothalamic Paraventricular Region of Male Rats.

Glucocorticoids (GCs) are essential for stress adaptation, acting centrally and in the periphery. Corticotropin-releasing factor (CRF), a major regulator of adrenal GC synthesis, is produced in the paraventricular nucleus of the hypothalamus (PVH), which contains multiple neuroendocrine and preautonomic neurons. GCs may be involved in diverse regulatory mechanisms in the PVH, but the target genes of GCs are largely unexplored except for the CRF gene (Crh), a well-known target for GC negative feedback. Using a genome-wide RNA-sequencing analysis, we identified transcripts that changed in response to either high-dose corticosterone (Cort) exposure for 12 days (12-day high Cort), corticoid deprivation for 7 days (7-day ADX), or acute Cort administration. Among others, canonical GC target genes were upregulated prominently by 12-day high Cort. Crh was upregulated or downregulated most prominently by either 7-day ADX or 12-day high Cort, emphasizing the recognized feedback effects of GC on the hypothalamic-pituitary-adrenal (HPA) axis. Concomitant changes in vasopressin and apelin receptor gene expression are likely to contribute to HPA repression. In keeping with the pleotropic cellular actions of GCs, 7-day ADX downregulated numerous genes of a broad functional spectrum. The transcriptome response signature differed markedly between acute Cort injection and 12-day high Cort. Remarkably, six immediate early genes were upregulated 1 hour after Cort injection, which was confirmed by quantitative reverse transcription PCR and semiquantitative in situ hybridization. This study may provide a useful database for studying the regulatory mechanisms of GC-dependent gene expression and repression in the PVH.

Distribution of corticotropin-releasing factor neurons in the mouse brain: a study using corticotropin-releasing factor-modified yellow fluorescent protein knock-in mouse.

We examined the morphological features of corticotropin-releasing factor (CRF) neurons in a mouse line in which modified yellow fluorescent protein (Venus) was expressed under the CRF promoter. We previously generated the CRF-Venus knock-in mouse, in which Venus is inserted into the CRF gene locus by homologous recombination. In the present study, the neomycin phosphotransferase gene (Neo), driven by the pgk-1 promoter, was deleted from the CRF-Venus mouse genome, and a CRF-Venus∆Neo mouse was generated. Venus expression is much more prominent in the CRF-Venus∆Neo mouse when compared to the CRF-Venus mouse. In addition, most Venus-expressing neurons co-express CRF mRNA. Venus-expressing neurons constitute a discrete population of neuroendocrine neurons in the paraventricular nucleus of the hypothalamus (PVH) that project to the median eminence. Venus-expressing neurons were also found in brain regions outside the neuroendocrine PVH, including the olfactory bulb, the piriform cortex (Pir), the extended amygdala, the hippocampus, the neocortices, Barrington's nucleus, the midbrain/pontine dorsal tegmentum, the periaqueductal gray, and the inferior olivary nucleus (IO). Venus-expressing perikarya co-expressing CRF mRNA could be observed clearly even in regions where CRF-immunoreactive perikarya could hardly be identified. We demonstrated that the CRF neurons contain glutamate in the Pir and IO, while they contain gamma-aminobutyric acid in the neocortex, the bed nucleus of the stria terminalis, the hippocampus, and the amygdala. A population of CRF neurons was demonstrated to be cholinergic in the midbrain tegmentum. The CRF-Venus∆Neo mouse may be useful for studying the structural and functional properties of CRF neurons in the mouse brain.

Visualization of corticotropin-releasing factor neurons by fluorescent proteins in the mouse brain and characterization of labeled neurons in the paraventricular nucleus of the hypothalamus.

Corticotropin-releasing factor (CRF) is the key regulator of the hypothalamic-pituitary-adrenal axis. CRF neurons cannot be distinguished morphologically from other neuroendocrine neurons in the paraventricular nucleus of the hypothalamus (PVH) without immunostaining. Thus, we generated a knock-in mouse that expresses modified yellow fluorescent protein (Venus) in CRF neurons (CRF-Venus), and yet its expression is driven by the CRF promoter and responds to changes in the interior milieu. In CRF-Venus, Venus-expressing neurons were distributed in brain regions harboring CRF neurons, including the PVH. The majority of Venus-expressing neurons overlapped with CRF-expressing neurons in the PVH, but many neurons expressed only Venus or CRF in a physiological glucocorticoid condition. After glucocorticoid deprivation, however, Venus expression intensified, and most Venus neurons coexpressed CRF. Conversely, Venus expression was suppressed by excess glucocorticoids. Expression of copeptin, a peptide encoded within the vasopressin gene, was induced in PVH-Venus neurons by glucocorticoid deprivation and suppressed by glucocorticoid administration. Thus, Venus neurons recapitulated glucocorticoid-dependent vasopressin expression in PVH-CRF neurons. Noradrenaline increased the frequency of glutamate-dependent excitatory postsynaptic currents recorded from Venus-expressing neurons in the voltage clamp mode. In addition, the CRF-iCre knock-in mouse was crossed with a CAG-CAT-EGFP reporter mouse to yield the Tg(CAG-CAT-EGFP/wt);CRF(iCre/wt) (EGFP/CRF-iCre) mouse, in which enhanced green fluorescent protein (EGFP) is driven by the CAG promoter. EGFP was expressed more constitutively in the PVH of EGFP/CRF-iCre mice. Thus, CRF-Venus may have an advantage for monitoring dynamic changes in CRF neurons and CRF networks in different glucocorticoid states.

Amelioration of improper differentiation of somatostatin-positive interneurons by triiodothyronine in a growth-retarded hypothyroid mouse strain.

Thyroid hormone (TH) plays an important role in brain development, and TH deficiency during pregnancy or early postnatal periods leads to neurological disorders such as cretinism. Hypothyroidism reduces the number of parvalbumin (PV)-positive interneurons in the neocortex and hippocampus. Here we used a mouse strain (growth-retarded; grt) that shows growth retardation and hypothyroidism to examine whether somatostatin (Sst)-positive interneurons that are generated from the same pool of neural progenitor cells as PV-positive cells are also altered by TH deficiency. The number of PV-positive interneurons was significantly decreased in the neocortex and hippocampus of grt mice as compared with normal control mice. In contrast to the decrease in the number of PV neurons, the number of Sst-positive interneurons in grt mice was increased in the stratum oriens of the hippocampus and the hilus of the dentate gyrus, although their number was unchanged in the neocortex. These changes were reversed by triiodothyronine administration from postnatal day (PD) 0 to 20. TH supplementation that was initiated after PD21 did not, however, affect the number of PV- or Sst-positive cells. These results suggest that during the first three postnatal weeks, TH may be critical for the generation of subpopulations of interneurons.

Targeting of locus ceruleus noradrenergic neurons expressing human interleukin-2 receptor α-subunit in transgenic mice by a recombinant immunotoxin anti-Tac(Fv)-PE38: a study for exploring noradrenergic influence upon anxiety-like and depression-like behaviors.

The noradrenergic (NA) neurons in the locus ceruleus (LC) were ablated with a high degree of selectivity by immunotoxin-mediated neuronal targeting. Transgenic mice were used in which the human interleukin-2 receptor-α subunit (hIL-2Rα; Tac) is expressed under the promoter of dopamine ß-hydroxylase. The recombinant immunotoxin, which is composed of the Fv fragment of an anti-hIL-2Rα monoclonal antibody fused to a truncated form of Pseudomonas exotoxin [anti-Tac(Fv)-PE38], was injected bilaterally into the LC of the mouse. As a result, the LC-NA neurons disappeared almost completely, and tissue noradrenaline was depleted in brain regions that receive NA inputs from the LC. The decrement of tissue noradrenaline content was more profound compared with that in mice treated with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), a neurotoxin capable of ablating axons originating from the LC-NA neurons. Mice treated with either the immunotoxin or DSP-4 presented increased anxiety-like behaviors; in contrast, only the immunotoxin-treated mice, and not the DSP-4-treated mice, showed increased depression-like behavior. The immunotoxin-mediated neuronal targeting may provide a means for further unraveling the links between the LC and pathological manifestations of neurological disorders.

Quantitative analysis of thyroid-stimulating hormone messenger RNA and heterogeneous nuclear RNA in hypothyroid rats.

Thyroid-stimulating hormone (TSH) stimulates the synthesis and release of thyroid hormones including triiodothyronine (T3) and thyroxine (T4). Semiquantitative analyses using northern blot and in situ hybridization suggested that TSH gene transcription is upregulated under conditions of hypothyroidism. However, no quantitative analysis of TSH gene expression using real-time polymerase chain reaction (PCR) has been reported. In this study, we quantitated the TSHbeta messenger ribonucleic acid (mRNA) level as well as the TSHbeta heterogeneous nuclear ribonucleic acid (hnRNA) level in the anterior pituitary of hypothyroid rats, by real-time PCR using the LightCycler system. The hnRNA is the primary deoxyribonucleic acid (DNA) transcript, which reflects the transcription rate more reliably than the mRNA because of its short half-life. In the anterior pituitary of rats with methimazol-induced chronic hypothyroidism, both mRNA and hnRNA expression of TSHbeta were upregulated fourfold relative to normal rats (n=4). Our method provides a rapid and accurate measure of gene transcription. In the present report, we described a technique for accurate measurement of TSHbeta hnRNA level.

Topographic analysis of cell proliferation in the hippocampus of the adult mouse.

The dentate gyrus of the hippocampus generates new neurons throughout life. The distribution of neural progenitor cells in the hippocampus along the anteroposterior axis is, however, not known. To determine whether mitotic activity differs along the anteroposterior axis, we examined mitotic activity in serial hippocampal sections of two experimental groups: normal bred (control) and exercised groups. In both groups, the number of 5-bromo-2'-deoxyuridine-positive cells was markedly decreased in the caudal dentate gyrus; exercised groups also had mitotic activity of about 1.5 times that of control groups. Mitotic activity tended to increase in the suprapyramidal blade of the middle dentate gyrus of exercised groups. These results indicate that the adult hippocampus has regional differences in mitotic activity.

Impaired neurogenesis in the growth-retarded mouse is reversed by T3 treatment.

Triiodothyronine (T3) regulates neuronal development, and T3 deficiency impairs the formation of the central nervous system during childhood. Here, we used the hypothyroidal growth-retarded (grt) mouse to investigate whether the generation of new neurons is affected by T3 deficiency. Cell proliferation in the hippocampus, as measured by 5-bromo-2'-deoxyuridine (BrdU) incorporation, was markedly decreased in the grt mouse while the number of BrdU-positive cells in T3-treated grt mice was equivalent to that of wild type mice. The cellular differentiation rate did not differ among the experimental groups. These results suggest that T3 plays an important role during neurogenesis.

Differential effects of forced swim-stress on the corticotropin-releasing hormone and vasopressin gene transcription in the parvocellular division of the paraventricular nucleus of rat hypothalamus.

Corticotropin-releasing hormone (CRH) and vasopressin (AVP) colocalize in the parvocellular division of the paraventricular nucleus of the hypothalamus (PVN). We examined the effect of forced swim-stress on the CRH and AVP primary transcript (hnRNA) levels in the rat PVN by semi-quantitative in situ hybridization. CRH hnRNA increased markedly following 10-min swim-stress and returned to the basal level by 2 h. AVP hnRNA in the parvocellular division of the PVN, where AVP colocalizes with CRH, did not change significantly immediately after the swim-stress, but it did increase significantly 2 h after the stress. Pretreatment with dexamethasone abolished the increases in CRH and AVP hnRNA levels after the swim-stress. The present results demonstrate the differential effects of forced swim-stress on the CRH and AVP gene transcription in the parvocellular PVN, confirming the diverse response of the dual peptide-containing system in the face of acute stressful events.

Regulation of corticotropin releasing hormone receptor (CRH-R) in the rat anterior pituitary as assessed by radioimmunoassay.

Since the cloning of corticotropin releasing hormone receptor type 1 (CRH-R1), an essential component of the hypothalamo-pituitary-adrenal (HPA) axis, numerous studies have been conducted to monitor its changes in transcription levels under various conditions. However, the precise dynamics at the protein levels are yet to be elucidated. In the present study we aimed at establishing an RIA system for CRH-R1 protein, with an antiserum against the C-terminal fragment of human/rat CRH-R1. The generated antiserum showed a moderate cross-reactivity with CRH-R2. We examined the in vivo effect of adrenalectomy (ADX) on immunoreactive CRH-R (irCRH-R) levels in the rat AP, and the in vitro profile of irCRH-R levels in cultured rat AP cells after administration of CRH. The irCRH-R in the AP membrane of intact rats was 51.8 +/- 6.8 fmol/mg protein, which is comparable to those reported in binding studies. ADX elicited a significant decrease of irCRH-R to approximately 50% of the control level one day after ADX, which returned to the baseline level the following day. Addition of CRH to cultured AP cells resulted in a significant decrease of irCRH-R in the membrane fraction to 18% of the control level at 4 h, and it returned rapidly to 70% at 8 h. These experiments together with our previous study implicate that irCRH-R makes a different profile, with an earlier recovery than that of mRNA. Although this system cannot precisely discriminate between CRH-R1 and CRH-R2, our findings may serve to demonstrate differing CRH receptor regulations at the synthesis level and at the protein level in the rat AP.

Stimulatory effects of prostaglandin E2 on neurogenesis in the dentate gyrus of the adult rat.

Neurogenesis in the dentate gyrus of adult rodents is elicited by transient global ischemia. Cyclooxygenase (COX) -2, a rate-limiting enzyme for prostanoid synthesis, is also induced by ischemia. We recently found that the administration of a non-selective COX inhibitor to ischemic animals suppressed cell proliferation in the subgranular zone (SGZ) at the dentate gyrus of the hippocampus. To clarify whether prostaglandin E2 (PGE2) synthesis by COX's is involved in neurogenesis, sulprostone, an analogue of PGE2, was injected into the rat hippocampus. Sulprostone injection increased the number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells in the SGZ. BrdU-positive cells also expressed polysialylated isoforms of neural cell adhesion molecule and neuronal nuclear antigen. These results suggest that PGE2 plays an important role in the proliferation of cells in the SGZ.

Basic properties and annual changes of follicle-stimulating hormone receptors in the testis of horseshoe bats, Rhinolophus ferrumequinum.

The unique reproductive patterns, delayed fertilization in females, and asynchrony between spermatogenesis and mating behavior in males are well documented in bats living in temperate latitudes. The present study was undertaken to examine follicle-stimulating hormone (FSH) receptors in the testis of bats, Rhinolophus ferrumequinum, during the annual reproductive cycle. Male bats were captured at natural roosting sites and testicular preparations were subjected to a radioligand binding assay for FSH receptors. The weight of paired testes increased considerably in the spermatogenic period and decreased from the mating to hibernation periods. Meiotic division in the testis was observed in the spermatogenic period but not the mating period. Serum testosterone concentrations increased in the spermatogenic period and rapidly decreased in the mating period. The binding of FSH was specific for mammalian FSHs and detected primarily in the testis. Scatchard plot analyses of the binding of FSH to bat testicular preparations showed straight lines, suggesting the presence of a single class of binding sites. The affinities (equilibrium association constant) of FSH receptors were consistent throughout the annual reproductive cycle. The specific binding per unit weight of testis and total binding in the paired testes were highest in the mating period and in the spermatogenic period, respectively, among reproductive periods. The accumulation of cyclic adenosine 3', 5'-monophosphate to FSH stimulation was higher in the spermatogenic period than in the hibernation period. These findings suggest that testicular function of bats is associated with seasonal changes in the number of binding sites, while the number per target cell and the activation of adenylate cyclase led by FSH-receptor complex considerably decreases in the hibernation period.