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.

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.

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.