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

Abstract

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.

New viral‐genetic mapping uncovers an enrichment of corticotropin‐releasing hormone‐expressing neuronal inputs to the nucleus accumbens from stress‐related brain regions

“…Notably, retrogradely‐labeled CRH + cells tended to reside in the posterior BLA, as 40% of all CRH‐expressing cells in this region projected to the NAc. Notably, both CRH (Itoi et al, ) and CRH mRNA (Kono et al, ) have been observed in mice and specifically in transgenic mice (CRH‐Cre and a Venus reporter), using tools that are completely independent from those employed here.…”

Stress-related synaptic plasticity in the hypothalamus

“…The adoption of new tools that enable specific activation of identified cell populations will be extremely beneficial for gain ing a better understanding of the interactions between cotransmitters in the PVN. In this vein, recent studies have described new mouse models that allow for both the identification and the genetic targeting of specific subsets of PNCs 99,113,114 . One important limitation that must be addressed is the dearth of information about the activity of PNCs and CRHproducing neurons in vivo, particularly in response to stress exposure; developing and implementing approaches that allow for the use of electrophysiology and/or imaging techniques in awake, behaving animals will be a considerable advance in help ing to make causal links between changes in synaptic function or plasticity and the output of stress circuitry.…”

Chronic Unpredictable Mild Stress Induces Loss of GABA Inhibition in Corticotrophin-Releasing Hormone-Expressing Neurons through NKCC1 Upregulation

“…Visualizing CRH-expressing neurons through immunohistochemical analysis is difficult without prior administration of compounds that inhibit axonal transport to promote the somatic accumulation of neuropeptides [4]. In transgenic mice, PVN-CRH neurons can be tagged by specifically expressing green fluorescent protein driven by the endogenous Crh promoter [5-7]. However, many studies use established rat stress models to study HPA axis activity.…”