Type 1 corticotropin-releasing factor receptor expression reported in BAC transgenic mice: implications for reconciling ligand-receptor mismatch in the central corticotropin-releasing factor system

Abstract

In addition to its established role in initiating the endocrine arm of the stress response, corticotropin-releasing factor (CRF) can act in the brain to modulate neural pathways that effect coordinated physiological and behavioral adjustments to stress. Although CRF is expressed in a set of interconnected limbic and autonomic cell groups implicated as primary sites of stress-related peptide action, most of these are lacking or impoverished in CRF receptor (CRFR) expression. Understanding the distribution of functional receptor expression has been hindered by the low resolution of ligand binding approaches and the lack of specific antisera, which have supported immunolocalizations at odds with analyses at the mRNA level. We have generated a transgenic mouse that shows expression of the principal, or type 1, CRFR (CRFR1). This mouse expresses GFP in a cellular distribution that largely mimics that of CRFR1 mRNA and is extensively colocalized with it in individual neurons. GFP-labeled cells display indices of activation (Fos induction) in response to central CRF injection. At the cellular level, GFP labeling marks somatic and proximal dendritic morphology with high resolution and is also localized to axonal projections of at least some labeled cell groups. This includes a presence in synaptic inputs to central autonomic structures such as the central amygdalar nucleus, which is implicated as a stress-related site of CRF action, but lacks cellular CRFR1 expression. These findings validate a new tool for pursuing the role of central CRFR signaling in stress adaptation and suggest means by which the pervasive ligand-receptor mismatch in this system may be reconciled.

Figure 1

Transgene design. A BAC (rp23-4B21) that contains most, if not all of the genomic locus of CRFR1, including the coding region, promoter and enhancer elements was used to construct the transgene (A). The first exon of CRFR1, which includes the translation start, was replaced with sequences encoding GFP and a translation stop signal such that only GFP would be transcribed from the modified, transgenic, CRFR1 locus (B).

Figure 2

Morphological detail revealed by CRFR1-driven GFP expression. Immunoperoxidase staining for GFP shows the extent of neuronal labeling provided by the CRFR1-GFP mouse in the lateral hypothalamic area (A) and pontine reticular formation (B). Cell bodies, primary dendrites, dendritic branching and varicose (presumably axonal) processes are all clearly labeled. The degree of cellular and axonal labeling exhibits regional variation. In the dentate gyrus (C), neurons in the hilar region are strongly labeled, while staining of granule cells (gr) is weaker and sporadic. A dense plexus of fine varicosities occupies the outer two-thirds of the molecular layer (mol). This is not seen in the inner third, which receives commissural and associational input from hilar neurons. In cerebellar cortex (D) labeling of small cells in the granule cell layer (gr) and punctate (axonal) elements in the molecular layer (mol) contrast with a distinct lack of staining of Purkinje cells (pcl). The latter have been reported as sites of CRFR1 expression in some histochemical studies. Other abbreviations: fx, fornix, hf, hippocampal fissure. Scale bars: 25 μm.

Figure 3

Comparison of transgenic GFP and CRFR1 mRNA expression. Adjacent sections through two levels of the forebrain in the CRFR1-GFP animal showing immunoperoxidase localization of GFP (A, C) and hybridization histochemical demonstration of CRFR1 mRNA (B, D). The two markers are consistent in revealing major cellular sites of expression in isocortex (Iso), piriform cortex (Pir), hippocampus (Hipp), medial septum (MS), globus palliidus (GP), reticular nucleus of the thalamus (RT, medial n of the amygdala (MeA) and arcuate nucleus of the hypothalamus (ArH). Other abbreviations: CeA, Central n. of the amygdala; CPu, caudoputamen; LA, lateral n. of the amygdala; LS, lateral septum. VMH, ventromedial hypothalamic n.; BLA, basolateral n. of the amygdala; Scale bar: 100 μM.

Figure 4

Finer grained comparison of GFP and receptor mRNA distributions. Adjoining sections through the olfactory bulb (A, B), temporal cortex (C, D) and ventral midbrain (E, F) prepared for immunoperoxidase localization of GFP (left column) and CRFR1 mRNA (right). The distribution and relative strength of labeling for the two markers in similar in most layers of the olfactory bulb, isocortex, the substantia nigra (SNc, SNr) and ventral tegmental area (VTA). Transgenic GFP expression labels granule cells in the olfactory bulb much more decisively than in situ hybridization. In contrast, relatively strong secondary focus of CRFR1 mRNA expression in layer 2/3 of isocortex (B) is not matched by comparable GFP labeling (A). Other abbreviations: cp, cerebral penuncle; epl, external plexiform layer, gl, glomerular layer, gr, granule cell layer; mi, mitral cell layer; SNc, substantia nigra, compact part; SNr, substantia nigra, reticular part. Scale bars: 100 μm,

Figure 5

Characterization of CRFR1-GFP expression in the paraventricular nucleus (PVH) and pituitary. A-C: Confocal microscopic images through the PVH of the CRFR1-GFP mouse showing immunofluorescence staining for GFP, CRF and merged channels. The substantial population of GFP-stained cells does not overlap appreciably with the CRF-expressing contingent. D-F: Sections though the pituitary co- stained for GFP (A) and ACTH (B) reveal substantial congruence in the anterior lobe (al; white cells in merged image, C), but that only a subset of melanotropes in intermediate lobe (il) are GFP-positive. Scale bar: 50 μM.

Figure 6

GFP expression in and around the locus coeruleus (LC). Confocal images of a section through the LC showing concurrent immunoflourescence labeling for GFP (A), CRF (B), tyrosine hydroxylase (TH; C) and merged channels (D). GFP-stained cell bodies are numerous in adjoining regions, but none co-stain for TH, a marker of LC neurons. Punctate, presumably axonal, elements are labeled for both GFP and CRF within the LC, defining potential substrates for interactions between peptidergic terminals and presynaptic CRFR1 receptors in this cell group. Scale Bar: 100 μM.

Figure 7

Overlay of transgenic GFP with molecular and functional indices of CRFR1 expression. Left column: Concurrent dual labeling for GFP (brown) and CRFR1 mRNA (black grains) showing extensive overlap between the two markers in the dentate nucleus of the cerebellum (DN; panel A), the globus pallidus (GP; B) and gracile nucleus (Gr; C). Arrowheads mark examples of doubly-labeled neurons. Note the lack of expression of either marker in the nucleus of the solitary tract (NTS; C), a central autonomic cell group identified as a site of CRF action. Right column: Dual immunoperoxidase staining for GFP (brown cytoplasm) and the inducible activation marker, Fos (black nuclei), in sections from CRFR1-GFP mice that received an icv injection of CRF (1μg), 2 hr before perfusion. Nearly all Fos-positive cells in the red nucleus (RN; D) and reticular part of the substania nigra (SNr; E) are GFP positive. The central nucleus of the amygdala (CeA; F), another central autonomic cell group, shows a strong activational response, but little or no GFP expression. Some cells immediately adjoining the CeA (arrowhead) are double labeled. Scale bar: 25 μm.

Figure 8

Some central autonomic cell groups harbor axonal and dendritic, but not somatic, expression of CRFR1-GFP. Top two rows: Sections through the oval nucleus of the BTS (BSTov; top) and CeA (second row) co-stained for transgenic GFP (A, D) and endogenous CRF (B, E) expression; merged images are shown at the right (C, F). Both cell groups are surrounded by GFP-labeled perikarya, but contain very few within their borders. Both are invested with GFP-positive processes, which intermingle with a denser CRF-immunoreactive innervation. G-I: Dual staining for GFP and Map2, a dendritic marker, reveals that some of the coarser elements in the CeA are identifiable as CRFR1-expressing dendrites (arrow) while many GFP fibers in the CeA are Map2 negative, indicating they are axonal (arrowheads). J-L: Higher power confocal micrography reveals a fine GFP positive fiber (J) containing puncta positive for SV2 (K, arrow, blue), a marker of presynaptic terminals, adjacent to puncta containing CRF (K, magenta, arrowhead). The arrow in the merged micrograph (I) points to an example of a presynaptic terminal from a CRFR1 expressing neuron that might respond to locally released CRF (arrowhead). Scale Bars: A-F, 50 μM; G-I, 2 μM; J-L, 1 μm.

Figure 9

Fine structure of GFP labeling in the central nucleus of the amygdala. Electron micrographs showing preembedding immunoperoxidase localization of transgenic GFP expression in dendrites and axon terminals in the central nucleus. A, B: We frequently observed smaller caliber dendritic processes (D*) that were commonly encircled by unlabeled axon terminals (open arrows), some of which formed (mainly asymmetric) synaptic contacts with the GFP+ dendrite. In addition, GFP-stained axon terminals (AT*) were also found apposed (curved arrows) to one or more unlabeled dendrites, with some contacts displaying clear synaptic specializations (D). Scale bars: 1 μm in A and C; 0.5μm in B and D.