Chronic pain enhances excitability of corticotropin-releasing factor-expressing neurons in the oval part of the bed nucleus of the stria terminalis.

We previously reported that enhanced corticotropin-releasing factor (CRF) signaling in the bed nucleus of the stria terminalis (BNST) caused the aversive responses during acute pain and suppressed the brain reward system during chronic pain. However, it remains to be examined whether chronic pain alters the excitability of CRF neurons in the BNST. In this study we investigated the chronic pain-induced changes in excitability of CRF-expressing neurons in the oval part of the BNST (ovBNSTCRF neurons) by whole-cell patch-clamp electrophysiology. CRF-Cre; Ai14 mice were used to visualize CRF neurons by tdTomato. Electrophysiological recordings from brain slices prepared from a mouse model of neuropathic pain revealed that rheobase and firing threshold were significantly decreased in the chronic pain group compared with the sham-operated control group. Firing rate of the chronic pain group was higher than that of the control group. These data indicate that chronic pain elevated neuronal excitability of ovBNSTCRF neurons.

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.

An intact pituitary vasopressin system is critical for building a robust circadian clock in the suprachiasmatic nucleus.

The circadian clock is a biological timekeeping system that oscillates with a circa-24-h period, reset by environmental timing cues, especially light, to the 24-h day-night cycle. In mammals, a "central" clock in the hypothalamic suprachiasmatic nucleus (SCN) synchronizes "peripheral" clocks throughout the body to regulate behavior, metabolism, and physiology. A key feature of the clock's oscillation is resistance to abrupt perturbations, but the mechanisms underlying such robustness are not well understood. Here, we probe clock robustness to unexpected photic perturbation by measuring the speed of reentrainment of the murine locomotor rhythm after an abrupt advance of the light-dark cycle. Using an intersectional genetic approach, we implicate a critical role for arginine vasopressin pathways, both central within the SCN and peripheral from the anterior pituitary.

Differential CRH expression level determines efficiency of Cre- and Flp-dependent recombination.

Corticotropin-releasing hormone expressing (CRH+) neurons are distributed throughout the brain and play a crucial role in shaping the stress responses. Mouse models expressing site-specific recombinases (SSRs) or reporter genes are important tools providing genetic access to defined cell types and have been widely used to address CRH+ neurons and connected brain circuits. Here, we investigated a recently generated CRH-FlpO driver line expanding the CRH system-related tool box. We directly compared it to a previously established and widely used CRH-Cre line with respect to the FlpO expression pattern and recombination efficiency. In the brain, FlpO mRNA distribution fully recapitulates the expression pattern of endogenous Crh. Combining both Crh locus driven SSRs driver lines with appropriate reporters revealed an overall coherence of respective spatial patterns of reporter gene activation validating CRH-FlpO mice as a valuable tool complementing existing CRH-Cre and reporter lines. However, a substantially lower number of reporter-expressing neurons was discerned in CRH-FlpO mice. Using an additional CRH reporter mouse line (CRH-Venus) and a mouse line allowing for conversion of Cre into FlpO activity (CAG-LSL-FlpO) in combination with intersectional and subtractive mouse genetic approaches, we were able to demonstrate that the reduced number of tdTomato reporter expressing CRH+ neurons can be ascribed to the lower recombination efficiency of FlpO compared to Cre recombinase. This discrepancy particularly manifests under conditions of low CRH expression and can be overcome by utilizing homozygous CRH-FlpO mice. These findings have direct experimental implications which have to be carefully considered when targeting CRH+ neurons using CRH-FlpO mice. However, the lower FlpO-dependent recombination efficiency also entails advantages as it provides a broader dynamic range of expression allowing for the visualization of cells showing stress-induced CRH expression which is not detectable in highly sensitive CRH-Cre mice as Cre-mediated recombination has largely been completed in all cells generally possessing the capacity to express CRH. These findings underscore the importance of a comprehensive evaluation of novel SSR driver lines prior to their application.

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.

Cre-dependent ACR2-expressing reporter mouse strain for efficient long-lasting inhibition of neuronal activity.

Optogenetics is a powerful tool for manipulating neuronal activity by light illumination with high temporal and spatial resolution. Anion-channelrhodopsins (ACRs) are light-gated anion channels that allow researchers to efficiently inhibit neuronal activity. A blue light-sensitive ACR2 has recently been used in several in vivo studies; however, the reporter mouse strain expressing ACR2 has not yet been reported. Here, we generated a new reporter mouse strain, LSL-ACR2, in which ACR2 is expressed under the control of Cre recombinase. We crossed this strain with a noradrenergic neuron-specific driver mouse (NAT-Cre) to generate NAT-ACR2 mice. We confirmed Cre-dependent expression and function of ACR2 in the targeted neurons by immunohistochemistry and electrophysiological recordings in vitro, and confirmed physiological function using an in vivo behavioral experiment. Our results show that the LSL-ACR2 mouse strain can be applied for optogenetic inhibition of targeted neurons, particularly for long-lasting continuous inhibition, upon crossing with Cre-driver mouse strains. The LSL-ACR2 strain can be used to prepare transgenic mice with homogenous expression of ACR2 in targeted neurons with a high penetration ratio, good reproducibility, and no tissue invasion.

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.

Sex differences in pain-induced modulation of corticotropin-releasing hormone neurons in the dorsolateral part of the stria terminalis in mice.

We earlier reported female-biased, sex-specific involvement of the dorsolateral bed nucleus of the stria terminalis (dl BST) in the formalin-induced pain response in rats. The present study investigated pain effects on mice behaviors. Because the dl BST is densely populated with corticotropin-releasing hormone (CRH) neurons, we examined sex differences in these parameters for the dl BST CRH neurons in male and female mice of a mouse line for which the CRH gene promoter (corticotropin-releasing factor [CRF]-Venus ΔNeo) controls the expression of the modified yellow fluorescent protein (Venus). Approximately 92% of Venus-positive cells in the dl BST were also CRH mRNA-positive, irrespective of sex. Therefore, the cells identified using Venus fluorescence were regarded as CRH neurons. A female-biased sex difference was observed in pain-induced behaviors during the interphase (5-15 min after formalin injection) but not during the later phase (phase 2, 15-60 min) in wild-type mice. In CRF-Venus ΔNeo mice, a female-biased difference was observed in either the earlier phase (phase 1, 0-5 min) or the interphase, but not in phase 2. Patch-clamp recordings taken using an acute BST slice obtained from a CRF-Venus ΔNeo mouse after formalin injection showed miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs). Remarkably, the mEPSCs frequency was higher in the Venus-expressing cells of formalin-injected female mice than in vehicle-treated female mice. Male mice showed no increase in mEPSC frequency by formalin injection. Formalin injection had no effect on mEPSC or mIPSC amplitudes in either sex. Pain-induced changes in mEPSC frequency in putative CRH neurons were phase-dependent. Results show that excitatory synaptic inputs to BST CRH neurons are temporally enhanced along with behavioral sex differences in pain response, suggesting that pain signals alter the BST CRH neurons excitability in a sex-dependent manner.

Distinctive Regulation of Emotional Behaviors and Fear-Related Gene Expression Responses in Two Extended Amygdala Subnuclei With Similar Molecular Profiles.

The central nucleus of the amygdala (CeA) and the lateral division of the bed nucleus of the stria terminalis (BNST) are the two major nuclei of the central extended amygdala that plays essential roles in threat processing, responsible for emotional states such as fear and anxiety. While some studies suggested functional differences between these nuclei, others showed anatomical and neurochemical similarities. Despite their complex subnuclear organization, subnuclei-specific functional impact on behavior and their underlying molecular profiles remain obscure. We here constitutively inhibited neurotransmission of protein kinase C-δ-positive (PKCδ+) neurons-a major cell type of the lateral subdivision of the CeA (CeL) and the oval nucleus of the BNST (BNSTov)-and found striking subnuclei-specific effects on fear- and anxiety-related behaviors, respectively. To obtain molecular clues for this dissociation, we conducted RNA sequencing in subnuclei-targeted micropunch samples. The CeL and the BNSTov displayed similar gene expression profiles at the basal level; however, both displayed differential gene expression when animals were exposed to fear-related stimuli, with a more robust expression change in the CeL. These findings provide novel insights into the molecular makeup and differential engagement of distinct subnuclei of the extended amygdala, critical for regulation of threat processing.

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.

Variation of pro-vasopressin processing in parvocellular and magnocellular neurons in the paraventricular nucleus of the hypothalamus: Evidence from the vasopressin-related glycopeptide copeptin.

Arginine vasopressin (AVP) is synthesized in parvocellular- and magnocellular neuroendocrine neurons in the paraventricular nucleus (PVN) of the hypothalamus. Whereas magnocellular AVP neurons project primarily to the posterior pituitary, parvocellular AVP neurons project to the median eminence (ME) and to extrahypothalamic areas. The AVP gene encodes pre-pro-AVP that comprises the signal peptide, AVP, neurophysin (NPII), and a copeptin glycopeptide. In the present study, we used an N-terminal copeptin antiserum to examine copeptin expression in magnocellular and parvocellular neurons in the hypothalamus in the mouse, rat, and macaque monkey. Although magnocellular NPII-expressing neurons exhibited strong N-terminal copeptin immunoreactivity in all three species, a great majority (~90%) of parvocellular neurons that expressed NPII was devoid of copeptin immunoreactivity in the mouse, and in approximately half (~53%) of them in the rat, whereas in monkey hypothalamus, virtually all NPII-immunoreactive parvocellular neurons contained strong copeptin immunoreactivity. Immunoelectron microscopy in the mouse clearly showed copeptin-immunoreactivity co-localized with NPII-immunoreactivity in neurosecretory vesicles in the internal layer of the ME and posterior pituitary, but not in the external layer of the ME. Intracerebroventricular administration of a prohormone convertase inhibitor, hexa-d-arginine amide resulted in a marked reduction of copeptin-immunoreactivity in the NPII-immunoreactive magnocellular PVN neurons in the mouse, suggesting that low protease activity and incomplete processing of pro-AVP could explain the disproportionally low levels of N-terminal copeptin expression in rodent AVP (NPII)-expressing parvocellular neurons. Physiologic and phylogenetic aspects of copeptin expression among neuroendocrine neurons require further exploration.

Identification of substances which regulate activity of corticotropin-releasing factor-producing neurons in the paraventricular nucleus of the hypothalamus.

The stress response is a physiological system for adapting to various internal and external stimuli. Corticotropin-releasing factor-producing neurons in the paraventricular nucleus of the hypothalamus (PVN-CRF neurons) are known to play an important role in the stress response as initiators of the hypothalamic-pituitary-adrenal axis. However, the mechanism by which activity of PVN-CRF neurons is regulated by other neurons and bioactive substances remains unclear. Here, we developed a screening method using calcium imaging to identify how physiological substances directly affect the activity of PVN-CRF neurons. We used acute brain slices expressing a genetically encoded calcium indicator in PVN-CRF neurons using CRF-Cre recombinase mice and an adeno-associated viral vector under Cre control. PVN-CRF neurons were divided into ventral and dorsal portions. Bath application of candidate substances revealed 12 substances that increased and 3 that decreased intracellular calcium concentrations. Among these substances, angiotensin II and histamine mainly increased calcium in the ventral portion of the PVN-CRF neurons via AT1 and H1 receptors, respectively. Conversely, carbachol mainly increased calcium in the dorsal portion of the PVN-CRF neurons via both nicotinic and muscarinic acetylcholine receptors. Our method provides a precise and reliable means of evaluating the effect of a substance on PVN-CRF neuronal activity.

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.

Organizing activity of Fgf8 on the anterior telencephalon.

The anterior part of the embryonic telencephalon gives rise to several brain regions that are important for animal behavior, including the frontal cortex (FC) and the olfactory bulb. The FC plays an important role in decision-making behaviors, such as social and cognitive behavior, and the olfactory bulb is involved in olfaction. Here, we show the organizing activity of fibroblast growth factor 8 (Fgf8) in the regionalization of the anterior telencephalon, specifically the FC and the olfactory bulb. Misexpression of Fgf8 in the most anterior part of the mouse telencephalon at embryonic day 11.5 (E11.5) by ex utero electroporation resulted in a lateral shift of dorsal FC subdivision markers and a lateral expansion of the dorsomedial part of the FC, the future anterior cingulate and prelimbic cortex. Fgf8-transfected brains had lacked ventral FC, including the future orbital cortex, which was replaced by the expanded olfactory bulb. The olfactory region occupied a larger area of the FC when transfection efficiency of Fgf8 was higher. These results suggest that Fgf8 regulates the proportions of the FC and olfactory bulb in the anterior telencephalon and has a medializing effect on the formation of FC subdivisions.

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.

A novel GABA-mediated corticotropin-releasing hormone secretory mechanism in the median eminence.

Corticotropin-releasing hormone (CRH), which is synthesized in the paraventricular nucleus (PVN) of the hypothalamus, plays an important role in the endocrine stress response. The excitability of CRH neurons is regulated by γ-aminobutyric acid (GABA)-containing neurons projecting to the PVN. We investigated the role of GABA in the regulation of CRH release. The release of CRH was impaired, accumulating in the cell bodies of CRH neurons in heterozygous GAD67-GFP (green fluorescent protein) knock-in mice (GAD67(+/GFP)), which exhibited decreased GABA content. The GABAA receptor (GABAAR) and the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), but not the K(+)-Cl(-) cotransporter (KCC2), were expressed in the terminals of the CRH neurons at the median eminence (ME). In contrast, CRH neuronal somata were enriched with KCC2 but not with NKCC1. Thus, intracellular Cl(-) concentrations ([Cl(-)]i) may be increased at the terminals of CRH neurons compared with concentrations in the cell body. Moreover, GABAergic terminals projecting from the arcuate nucleus were present in close proximity to CRH-positive nerve terminals. Furthermore, a GABAAR agonist increased the intracellular calcium (Ca(2+)) levels in the CRH neuron terminals but decreased the Ca(2+) levels in their somata. In addition, the increases in Ca(2+) concentrations were prevented by an NKCC1 inhibitor. We propose a novel mechanism by which the excitatory action of GABA maintains a steady-state CRH release from axon terminals in the ME.

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.