Transgenic Mice Expressing Green Fluorescent Protein under the Control of the Corticotropin-Releasing Hormone Promoter

Abstract

CRH is widely expressed in the brain and is of broad functional relevance to a number of physiological processes, including stress response, parturition, immune response, and ingestive behavior. To delineate further the organization of the central CRH network, we generated mice expressing green fluorescent protein (GFP) under the control of the CRH promoter, using bacterial artificial chromosome technology. Here we validate CRH-GFP transgene expression within specific brain regions and confirm the distribution of central GFP-producing cells to faithfully recapitulate that of CRH-expressing cells. Furthermore, we confirm the functional integrity of a population of GFP-producing cells by demonstrating their apposite responsiveness to nutritional status. We anticipate that this transgenic model will lend itself as a highly tractable tool for the investigation of CRH expression and function in discrete brain regions.

A transgenic mouse line expressing green fluorescent protein under the control of the corticotropin-releasing hormone promoter (CRH-GFP) was generated to facilitate the investigation of endogenous CRH expression and function.

CRH is a 41-amino acid peptide that is widely expressed in the brain and a select number of peripheral sites (1,2,3,4,5). The pluripotent effects of CRH in the human and rodent are mediated via activation of G protein-coupled CRH₁ and CRH₂ receptors which are expressed both centrally and peripherally (6,7,8).

Secretion of CRH from a subpopulation of neurons in the paraventricular nucleus of the hypothalamus (PVH) into the hypophyseal portal system leads to the release of ACTH from the anterior pituitary. In this hormonal capacity, CRH is a principle regulatory component of the hypothalamic-pituitary-adrenal axis with a recognized involvement in mediating physiological responses to stress and infection (1). In addition to these roles, CRH has been implicated in the modulation of energy homeostasis. Early support for CRH’s effect on ingestive behavior was provided through pharmacological studies demonstrating that intracerebroventricular administration of CRH reduced food intake in rodents (9,10). In normal rats, refeeding after food deprivation significantly increases the expression of c-fos, a marker for neuronal activation, and CRH within the PVH (11,12). Consistently, both CRH knockout mice and CRH₂ receptor knockout mice exhibit impaired refeeding after food deprivation (13,14). CRH has also been shown to mediate part of the anorectic effect of leptin, a critical signal of peripheral energy store status (15,16).

Historically, visualizing central sites of endogenous CRH expression through immunohistochemistry has proved difficult without prior brain administration of compounds that inhibit axonal transport and promote the somatic accumulation of neuropeptides. To improve on previous technology and thus facilitate investigation of CRH neuronal circuitry, we generated CRH-GFP bacterial artificial chromosome (BAC) transgenic mice expressing Tau-topaz green fluorescent protein (GFP) under the transcriptional control of the CRH promoter, thereby enhancing visualization of discrete CRH-expressing cells. Characterization of this model confirms CRH-neuron-specific expression of the CRH-GFP transgene in multiple brain regions and the functional validity of PVH GFP-expressing cells in a physiologically relevant assessment of CRH-neuron activity.

Materials and Methods

Generation of CRH-GFP transgenic mice expressing GFP under the transcriptional control of the CRH promoter

CRH-GFP BAC transgenic mice expressing Tau-topaz GFP under the transcriptional control of the CRH promoter were generated using BAC transgenic technology (17). The GFP transgene was introduced into the ATG site of the CRH BAC (BAC ID no. 397J12) by homologous recombination (supplemental Fig. S1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). The GFP transgene included a Tau-GFP fusion protein, followed by a poly(A) signal. Tau, A bovine microtubule binding protein, was used to increase axonal labeling by GFP to facilitate studies of neuronal connectivity. BAC filters (BAC mouse II) were obtained from Genome Systems (St. Louis, MO). The CRH-GFP construct was cloned into the shuttle vector PSV1 for the BAC modification. The PSV1 vector has a temperature-sensitive origin of replication, a copy of the RecA gene (which introduces recombination in a recombination-deficient Escherichia coli host), and tetracycline resistance gene (Tet+). The shuttle vector has 0.6 kb upstream and 0.5 kb downstream arms of CRH sequence flanking the GFP transgene. The shuttle vector was transformed into a DH10B E. coli host harboring the CRH BAC. After two homologous recombination events, the modified CRH BAC was selected based on temperature and antibiotic sensitivity. The homologous recombination of the modified BAC was confirmed through Southern blot analysis. The modified BAC DNA construct was microinjected into the pronucleus of fertilized oocytes from a CBA/C57BL/6 F1 mouse strain to generate four transgenic founder lines using the Rockefeller University transgenic facility. Founder animals were mated with C57BL/6J (Jackson Laboratory, Bar Harbor, ME) mice to generate F1 progeny.

Animal husbandry and tissue preparation

CRH-GFP heterozygous and wild-type mice used were of a CBA/C57BL/6J genetic background and were generated by breeding within the population. Genotypes were determined by PCR amplification of tail-derived genomic DNA using primers specific to the GFP sequence (forward, 5′-CCG AGG ATC CTA CCA TGG TGA GCA AGG GCG A and reverse, 5′-CAG CTT GTG CCC CAG GAT GT). All mice had ad libitum access to water and food (unless stated otherwise) and were housed in a temperature-controlled room (21.5–22.5 C) with a standard 12-h light, 12-h dark cycle.

To collect tissue for histology, mice were deeply anesthetized with isoflurane gas and perfused transcardially with 20–30 ml diethyl pyrocarbonate (Sigma, St. Louis, MO)-treated 0.9% saline followed by 40–50 ml of 10% neutral buffered formalin (Sigma). Brains were extracted, postfixed in 10% neutral buffered formalin for 4 h, and immersed in 20% sucrose in diethyl pyrocarbonate-treated PBS for 24–48 h at 4 C. Brains were sectioned at 25 μm on a freezing sliding microtome and collected in five equal series. One series of tissue was counterstained with thionin and used as a reference to determine nuclear boundaries for subsequent analysis. The remaining series were stored at either −20 C in an antifreeze solution (18) for in situ hybridization histochemistry (ISHH) experiments or at 4 C in 0.02% sodium azide in PBS for single- and dual-immunohistochemistry (IHC) experiments. Data obtained with ISHH and IHC were intended to provide relative cell counts, not accurate determination of absolute cell number. Data displayed in figures were generated using an Axioskop II with Axiovision (Zeiss, Thornwood, NY), Photoshop (Adobe, San Jose, CA), and/or Canvas (ACD Systems, Victoria, British Columbia, Canada) software. All procedures were in accordance with the guidelines set forth by the Rockefeller University Laboratory Animal Research Center (New York, NY).

ISHH

Adult male C57BL6J mice (Jackson Laboratory) weighing 25–35 g were used to map the distribution of CRH mRNA in the wild-type brain (n = 4) using ISHH methods previously reported (18,19,20) (supplementary methods). The CRH riboprobe used corresponds to 770 bp of the rat CRH sequence starting 82 bp 3′ of the start codon (21) and was 96% identical with the corresponding mouse sequence. The riboprobe was generated by in vitro transcription with T7 polymerase (Ambion, Austin, TX) in the presence of ³⁵S-labeled uridine 5-triphosphate, according to the manufacturer’s protocol.

IHC

Single- and dual-label IHC was performed to evaluate single-labeled expression and coexpression of GFP and CRH in adult male CRH-GFP and wild-type mice (n = 4 per genotype). To identify single-labeled CRH-immunoreactive (IR) neurons and dual-labeled CRH-IR and GFP-IR neurons, mice were pretreated with 40 μg of colchicine (Sigma) in 10 μl of 0.9% saline into the lateral ventricle under isoflurane gas anesthesia. This procedure was performed to enhance the detection of CRH-IR in cell bodies. Thirty-six to 48 h later, mice were perfused and tissue was prepared as described above. Standard methods for IHC were used (20,22,23) (supplementary methods). Detection of GFP-IR and CRH-IR was performed using primary antisera [GFP mouse, 1:10,000, Chemicon International (Temecula, CA) and CRH rabbit, 1:200, Phoenix Pharmaceuticals (Burlingame, CA)].

Single-labeled cells were assessed throughout the brain and were counted as labeled if the stain conformed to the outline of a cell. General estimates of the abundance of GFP-IR labeled neurons in the brain were recorded in tabular format. A schematic representation of the distribution of GFP-IR neurons was also performed at 12 rostrocaudal levels of the brain.

Double-labeled cells were assessed and quantified at four levels of the bed nucleus of the stria terminalis (BST; 0.38, 0.26, 0.14, 0.02 mm from bregma), four levels of the PVH (−0.58, −0.70, −0.82, −0.94 mm from bregma), four levels of the lateral hypothalamic area (LHA; −1.46, −1.58, −1.70, −1.82 mm from bregma), one level of area A11 (−2.30 mm from bregma), and two levels of Barrington’s nucleus (−5.40, −5.52 mm from bregma). Double-labeling was performed using fluorescent visualization with the appropriate fluorophores and filters and was recorded if GFP-IR and CRH-IR labeled the same neuron.

Dual-label ISHH and IHC

To confirm exclusive expression of GFP in CRH mRNA containing neurons, concurrent visualization of CRH mRNA and GFP-IR was performed by combining IHC with ISHH. Coexpression in the BST, PVH, LHA, area A11, and Barrington’s nucleus in CRH-GFP mice was examined (for specific levels of each region assessed, see section above). Brain tissue from adult male CRH-GFP mice (n = 4) was first processed for ³⁵S-labeled CRH using ISHH and then IHC for GFP as detailed above (supplementary methods). Double-labeled cells were recorded if GFP-IR cell bodies contained overlying black grains (³⁵S-CRH labeling) that were in a quantity greater than 3 × background (background, grains in 100 μm² area in the internal capsule) and conformed to the shape of the GFP-IR cell bodies.

Functional assessment of GFP-producing cells during after postfast refeeding

Individually housed CRH-GFP mice were either fasted for 18 h and refed for 4 h or fasted for 18 h (n = 4 per condition). Mice were anesthetized and transcardially perfused 4–6 h after the onset of the dark cycle. Brain tissue was processed for single- and dual-labeled IHC as described above. Analysis of single-label GFP-IR and c-fos immunoreactivity (FOS-IR; using rabbit anti-c-fos primary antibody, 1:10,000; Calbiochem International, La Jolla, CA) and dual-label GFP-IR and FOS-IR was performed using fluorescent visualization with the appropriate fluorophores and filters.

Results

Generation of CRH-GFP transgenic mice expressing GFP under the transcriptional control of the CRH promoter

Four transgenic founder lines of CRH-GFP mice were generated, and all genotypes were born at the expected Mendelian frequency and were viable, fertile, and appeared grossly normal upon physical inspection. The transgenic line with highest GFP expression was used for further characterization.

Distribution of CRH mRNA in the wild-type mouse brain

To characterize endogenous CRH mRNA expression in the wild-type mouse brain, ISHH was performed using a ³⁵S-labeled CRH riboprobe specific to the endogenous gene. CRH mRNA was evident in discrete brain regions throughout the neuraxis (Table 1 and supplemental Fig. S1), as expected based on previous reports (2,24).

Table 1.

Comparative distribution of CRH mRNA and GFP-IR

Brain region	CRH mRNA (Wild- type)	GFP-IR (CRH-GFP)
Olfactory tubercle	d	d
Cortex	c	SC
Piriform cortex	a	a
Shell of nucleus accumbens	d	d
Lateral septum	SC	d
Substantia innominata	d	c
Interstitial nucleus of the posterior limb of the anterior commissure	d	SC
BST	b	b
Ventral pallidum	d/-	d/-
Preoptic area	c	d
Central nucleus of the amygdala	b	c
Basomedial nucleus of the amygdala	c	c
PVH
Parvocellular	a	b
Magnocellular	d	d/-
Zona incerta	c	c
Hippocampus	SC	SC
LHA	b	b
Dorsomedial hypothalamus	d	d
A11	d	d
Medial part of the medial geniculate	c	c
Periaqueductal gray	c	c
Deep mesencephalic nucleus	c	c
Raphe magnus	d	d
Pontine reticular nucleus	d	d
Peduncular pontine tegmental nucleus	c	c
Inferior colliculus	c	c
Lateral parabrachial nucleus	c	c
Lateral dorsal tegmental nucleus	c	c
Barrington’s nucleus	a	a
Reticulotegmental nucleus of the pons	c	c
Medial vestibular nucleus	c	c
Gigantocellular reticular nucleus, alpha	d	d
Inferior olive	a	a

Comparative qualitative distribution of ³⁵S-labeled CRH riboprobe in the wild-type mouse brain and GFP-IR in the CRH-GFPmouse brain (n = 4 for each genotype). SC, Scattered cells. 

^a, Very high expression; 

^b, High expression; 

^c, Moderate expression; 

^d, Modest expression; */-, Mild expression; SC, Scattered cells. 

Anatomical distribution of GFP-IR in the CRH-GFP mouse brain

The distribution pattern of GFP-IR cells in CRH-GFP mice was highly consistent with CRH mRNA expression in wild-type mice (Table 1 and Fig. 1). Of note, fewer GFP-IR neurons were identified in the cortex of CRH-GFP mice compared with CRH mRNA expression in wild-type mice. Minor differences in the degree of expression were also found in brain regions in which modest and scattered GFP-IR cells were observed in CRH-GFP mice compared with CRH mRNA expression in wild-type mice, as illustrated in Table 1. Importantly, GFP-IR in CRH-GFP mice was not identified in any brain region in which endogenous CRH mRNA was not expressed in wild-type mice.

Figure 1.

Schematic representation of GFP-IR in CRH-GFP mouse brain. A series of representative rostrocaudally aligned schematic transverse sections (A–L) depicting the location of the neurons labeled for GFP-IR (black circles) in the CRH-GFP mouse brain. Each black circle represents a single GFP-IR neuron. 3V, Third ventricle; 4V, fourth ventricle; 7n, facial nerve; II/III, cerebral cortex, layer 2 and 3; V/VI, cerebral cortex, layer 5 and 6; ac, anterior commissure; AcbSh, shell part of accumbens nucleus; Aq, aqueduct; AP, area postrema; Bar, Barrington’s nucleus; BMA, anterior part of basomedial amygloid nucleus; BSTLD, lateral division of bed nucleus of stria terminalis, dorsal part; BSTLV, lateral division of bed nucleus of stria terminalis, ventral part; CA, hippocampus; CC, central canal; CeA, central nucleus of the amygdala; Cg, cingulate cortex; cp, cerebral peduncle, basal part; CPu, caudate putamen; D3V, dorsal third ventricle; DG, dentate gyrus; DMD, dorsal part of the dorsomedial nucleus of the hypothalamus; DpMe, deep mesencephalic nucleus; f, fornix; fmi, forceps minor of the corpus callosum; fr, fasciculus retroflexus; GiA, gigantocellular reticular nucleus, α ic, internal capsule; IC, inferior colliculus; IL, infralimbic cortex; IO, inferior olive; IPAC, interstitial nucleus of the posterior limb of the anterior commissure; LDTg, laterodorsal tegmental nucleus; LRt, lateral reticular nucleus; LPB, lateral parabrachial nucleus; LS, lateral septal nucleus; LV, lateral ventricle; MeA, medial amygdaloid nucleus; MGM, medial geniculate nucleus; ml, medial lemniscus; MM, medial mammillary nucleus, medial part; Mo, motor cortex; MPO, medial preoptic area; MS, medial septal nucleus; mt, mammillothalamic tract; MVe, medial vestibular nucleus; RMg, raphe magnus nucleus; RgTg, reticulotegmental nucleus of the pons; PAG, pariaqueductal gray; PaLM, lateral magnocellular pat of the paraventricular nucleus of hypothalamus; Pir, piriform cortex; PnO, pontine reticular nucleus, oral part; PPTg, pedunculopotine tegmental nucleus; RMg, raphe magnus nucleus; SI, substantia innominata; SNc, substantia nigra, compact part; SNr, substatia nigra, reticular part; sol, nucleus of solitary tract; SS, somatosensory cortex; st, stria terminalis; Tu, olfactory tubercle; VP, ventral pallidum; XSCP, decussation of the superior cerebellar peduncle; ZI, zona incerta. Scale bar (L), 2 mm.

Colocalization of GFP-IR and CRH-IR/CRH mRNA in CRH-GFP mice

Dual-labeling was performed to characterize expression of the GFP transgene specifically in CRH-containing neurons of CRH-GFP mice. We assessed two regions with dense CRH expression (PVH and Barrington’s nucleus), two regions with high expression (BST and LHA) and one region with modest CRH expression (area A11). The counts of single- and dual-labeled neurons were made in CRH-GFP mice using CRH-IR to identify endogenous CRH protein-containing cells and GFP-IR to label cells expressing the GFP transgene. GFP-IR neurons consistently coexpressed CRH-IR in these regions. Specifically, 91% of GFP-IR neurons in the BST, 99% in the PVH, 93% in the LHA, 99% in A11, and 98% in Barrington’s nucleus coexpressed CRH-IR (Fig. 2A-J”).

Figure 2.

Colocalization of GFP-IR and CRH-IR (A–J“) and GFP-IR and ³⁵S-labeled CRH (K–T) in CRH-GFP mouse brain. A–E, Merged photomicrographs of representative regions (BSTLD, PVH, LHA, A11, and Barrington’s nucleus, respectively) expressing GFP-IR (green fluorescence) and CRH-IR (red fluorescence). F–J, Higher-power magnification of boxed area in A–E, respectively, and indicate neurons expressing GFP-IR. F’–J’, Neurons express CRH-IR. F”–J“, Merged images of GFP-IR and CRH-IR. Arrows indicate colocalization of GFP-IR and CRH-IR. Scale bar (A), 300 μm applies to A; scale bar (E), 300 μm applies to B–E; scale bar (J”), 25 μm, applies to all other micrographs. K–O, Photomicrographs of representative regions (BST, PVH, LHA, A11, and Barrington’s nucleus, respectively) expressing GFP-IR (brown stain) and ³⁵S-labeled CRH (cluster of black grains). P–T, Higher-power magnification of boxed area (K–O), respectively, and illustrate neurons coexpressing GFP-IR and ³⁵S-labeled CRH. Scale bar (O), 300 μm applies to K–O; scale bar (T), 25 μm, applies to P–T. For abbreviation definition, see legend to Fig. 1.

GFP-IR also consistently colocalized with CRH-containing neurons identified using ISHH. A ³⁵S-labeled CRH riboprobe was used to label endogenous CRH mRNA-expressing cells and GFP-IR used to identify cells expressing the GFP transgene. Counts of single- and dual-labeled cells were again made in the BST, PVH, LHA, A11, and Barrington’s nucleus, regions representing varying levels of CRH mRNA abundance. Using these methods, 89% of neurons identified as GFP-IR in the BST, 90% in the PVH, 90% in the LHA, 90% in A11, and 98% in Barrington’s nucleus coexpressed ³⁵S-labeled CRH (Fig. 2, K–T).

Together these findings suggest that CRH-GFP mice may be used as a tool to investigate the anatomical and physiological role of CRH neurons in the BST, PVH, LHA, A11, and Barrington’s nucleus.

GFP-producing cells of the PVH are functionally reactive to postfast refeeding

Endogenous hypothalamic, and in particular PVH, CRH neurons are activated upon refeeding after food deprivation (11,12). To characterize the functional integrity of cells expressing GFP, we assessed neuronal activation patterns of GFP-positive cells within the PVH of postfast refed CRH-GFP mice. Specifically, transgenic animals were fasted for 18 h before 4 h of ad libitum refeeding. Consistent with previous reports (11,12), refeeding significantly increased the number of FOS-IR neurons in the PVH at all levels of the PVH analyzed compared with fasted mice (Table 2). Furthermore, dual-labeling for GFP-IR and FOS-IR indicated that 76% of GFP-expressing cells within the PVH were activated by refeeding (Table 2 and Fig. 3). This demonstrates that PVH neurons expressing GFP are physiologically responsive and confirms the functional veracity of the GFP-CRH model.

Table 2.

FOS-IR and GFP-IR colocalization within the PVH of fasted and postfast refed CRH-GFP transgenic mice

	PVH (level from bregma)
	−1.40			−1.60			−1.80			−1.88
	GFP-IR	FOS-IR + GFP-IR	Percent	GFP-IR	FOS-IR + GFP-IR	Percent	GFP-IR	FOS-IR + GFP-IR	Percent	GFP-IR	FOS-IR + GFP-IR	Percent
Fasted	9	1	6	28	2	5	26	1	4	13	1	4
Refed	14	9	65a	30	23	77a	28	21	76a	12	11	90a

FOS-IR and GFP-IR colocalization within the PVH of fasted and postfast refed CRH-GFP transgenic mice. The mean number of total GFP-IR cells and FOS-IR + GFP-IR cells were determined at four levels of the PVH in the brains of fasted (control; n = 4) and postfast refed CRH-GFP mice (n = 4). The mean percentage of GFP-IR cells expressing fos at each level was then calculated. The number of FOS-IR + GFP-IR cells was significantly increased by postfast refeeding, compared with fasted animals, at all four levels. 

^aP < 0.001; Student’s t-test. 

Figure 3.

Refeeding substantially activates GFP neurons in CRH-GFP mice. A, Merged photomicrographs of FOS-IR (green fluorescence), used as a marker of neuronal activation, and GFP-IR (red fluorescence) in the PVH of CRH-GFP mice after 18 h food deprivation proceeded by a 4-h bout of refeeding. B, Higher-power magnification of PVH FOS-IR neurons. B’, The same section as in B but illustrating GFP-IR. B“, The merged photomicrograph of B and B’ illustrating coexpression of FOS-IR and GFP-IR in a representative sample of PVH neurons. Arrows indicate colocalization. Scale bar (A), 100 μm applies to A; scale bar (B”), 25 μm, applies to B–B“. For abbreviation definition, see legend to Fig. 1.

Discussion

Here we describe the generation and validation of BAC transgenic mice expressing GFP under the transcriptional control of the CRH promoter, a new tool that may be used to characterize CRH function and connectivity within the brain. CRH-GFP mice are viable and fertile and appear grossly normal on physical inspection. This indicates that although the modified BAC contains sequences from neighboring genes that could be overexpressed, the BAC targeting method yielded mice exhibiting an overtly normal phenotype.

Expression of the GFP transgene in CRH neurons was confirmed using ISHH and IHC in a selection of brain regions varying in degree of CRH mRNA abundance. The CRH riboprobe used for analyses does not detect the mRNA product of the modified allele, and furthermore, the GFP transgene would not be identified by the CRH antibody, which detects endogenous CRH protein expression. Therefore, the results suggest that the flanking sequences used in this BAC are sufficient to confer cell type specific expression of the CRH gene.

In CRH-GFP mice, abundant GFP-IR was limited exclusively to regions demonstrating CRH-IR and ³⁵S-labeled CRH neurons. Further dual-histochemical labeling was performed to directly examine coexpression in a selection of brain regions, the BST, PVH, LHA, A11, and Barrington’s nucleus. Extensive coexpression was observed in these nuclei. Whereas we cannot discount that minor inconsistencies observed in the coexpression of GFP-IR and endogenous CRH may be due to ectopic expression of the GFP transgene, it is more likely that these small differences are associated with methodological factors related to dual-histochemical labeling analysis. The consistent coexpression of GFP-IR and ³⁵S-labeled CRH or CRH-IR identified in the BST, PVH, LHA, A11, and Barrington’s nucleus indicate that these mice may be used as a tool to investigate the anatomical and physiological function of CRH neurons in these regions. Despite faithful recapitulation of CRH expression within most brain regions, qualitative analysis of CRH-GFP transgene expression using GFP-IR within the cortex indicated an underrepresentation of cells compared with ³⁵S-CRH-labeled cells in the same region of wild-type mice. This may be due to methodological factors associated with the sensitivity of the histochemical labels used (i.e. IHC for GFP and ISHH for CRH) or to the absence of a critical regulatory region specific to cortical CRH neurons within the CRH BAC. Until this is resolved, caution should be used with using the CRH-GFP transgenic line to study cortical CRH neurons.

Lastly, to further validate the CRH-GFP mouse line, we sought to confirm the functional integrity of GFP-expressing cells. The PVH is a critical brain region involved in regulating energy balance and the parvocellular division contains the highest density of mouse hypothalamic CRH (and GFP) neurons (e.g. Ref 2). We demonstrate that these cells are functionally responsive to a physiologically salient stimulus, postfast refeeding. This observation is consistent with previous reports of PVH CRH-neuron activation under similar conditions in the rat (11,12) and confirms the functional integrity of the CRH-GFP line.

In summary, CRH-synthesizing neurons are distributed widely in the mammalian neuraxis and are implicated in the regulation of numerous physiological processes. To further understand CRH circuitry and function, we generated a CRH-GFP transgenic mouse line that affords greatly refined neuroanatomical resolution (without the need for prior colchicine administration). Characterization of this reporter line confirms faithful recapitulation of central CRH expression and preserved functionality in the regions assessed. We suggest that the integrity and tractability of this CRH-GFP line will prove to be of significant value to CRH-related research.