A Transgenic Rat for Investigating the Anatomy and Function of Corticotrophin Releasing Factor Circuits

Abstract

Corticotrophin-releasing factor (CRF) is a 41 amino acid neuropeptide that coordinates adaptive responses to stress. CRF projections from neurons in the central nucleus of the amygdala (CeA) to the brainstem are of particular interest for their role in motivated behavior. To directly examine the anatomy and function of CRF neurons, we generated a BAC transgenic Crh-Cre rat in which bacterial Cre recombinase is expressed from the Crh promoter. Using Cre-dependent reporters, we found that Cre expressing neurons in these rats are immunoreactive for CRF and are clustered in the lateral CeA (CeL) and the oval nucleus of the BNST. We detected major projections from CeA CRF neurons to parabrachial nuclei and the locus coeruleus, dorsal and ventral BNST, and more minor projections to lateral portions of the substantia nigra, ventral tegmental area, and lateral hypothalamus. Optogenetic stimulation of CeA CRF neurons evoked GABA-ergic responses in 11% of non-CRF neurons in the medial CeA (CeM) and 44% of non-CRF neurons in the CeL. Chemogenetic stimulation of CeA CRF neurons induced Fos in a similar proportion of non-CRF CeM neurons but a smaller proportion of non-CRF CeL neurons. The CRF1 receptor antagonist R121919 reduced this Fos induction by two-thirds in these regions. These results indicate that CeL CRF neurons provide both local inhibitory GABA and excitatory CRF signals to other CeA neurons, and demonstrate the value of the Crh-Cre rat as a tool for studying circuit function and physiology of CRF neurons.

Keywords: CRF; Cre recombinase; Fos; R121919; central amygdala; channelrhodopsin-2; designer receptors exclusively activated by designer drugs; transgenic rat models.

Figure 1

Crh-Cre rats express Cre recombinase activity in the CeL and dlBNST. (A,B) Bigenic progeny of a Crh-Cre X DsRed2/GFP cross display robust GFP labeling in the CeL and dlBNST. Scale bars, 200 μm. (C,D) Cre-dependent mCherry expression in Cre-expressing neurons of the CeL and dlBNST. Scale bars, 100 μm in panel (C); 200 μm in panel (D). (E) Cre-dependent eYFP co-localizes with CRF immunoreactivity in the CeL. Scale bar, 200 μm. (F) Rendered isosurface analysis demonstrates co-localization of CRF immunoreactivity within CeL neurons that also express Cre-dependent eYFP. Arrows point to an example of eYFP and CRF in the same neuron. AC, anterior commissure; AMY, amygdala; BLA, basolateral amygdala; CeC, capsular central amygdala; CeM, medial central amygdala; cst, commissural stria terminalis; Hip, Hippocampus; IC, internal capsule; LV, lateral ventricle; Str, Striatum; Th, Thalamus.

Figure 2

Coexpression of other neuropeptides in CeL CRF neurons. (A) A large percentage of CRF neurons identified by expression of Cre-dependent mCherry are immunoreactive for dynorphin and somatostatin, while few express enkephalin or PKCδ. Scale bars, 100 μm. Medial is to the left. (B) Quantification of mCherry expression with neuropeptide immunoreactivity; CRF n = 3 rats, 12 amygdala sections/rat; dynorphin n = 4 rats, 6 amygdala sections/rat; somatostatin n = 4 rats, 6 amygdala sections/rat; enkephalin n = 4 rats, 6 amygdala sections/rat; PKCδ, n = 6 rats, 10–12 amygdala sections/rat.

Figure 3

CeL CRF neurons project strongly to brainstem nuclei. (A) After injection of AAV-hSyn-DIO-mCherry into the CeL, mCherry expressing fibers were detected in the lateral and medial parabrachial nuclei (Bregma −9.0). Scale bar, 500 μm. (B) mCherry expressing fibers were also detected in the medial parabrachial nucleus just lateral to the locus coeruleus (Bregma -9.6). Red, mCherry; Green, Tyrosine Hydroxylase. Scale bar, 500 μm. (C,D) High-magnification examples of mCherry fibers from the CeL and noradrenergic LC neurons. CeL fibers appear to run orthogonally to noradrenergic dendrites extending laterally from the LC core into the medial parabrachial nucleus. Scale bars, 100 μm in panel (C); 20 μm in panel (D). MPBN, medial parabrachial nucleus; LPBN, lateral parabrachial nucleus; LC, locus coeruleus.

Figure 4

CeL CRF neurons provide inputs to other limbic brain structures. (A,B) A dense bundle of mCherry expressing fibers from the CeL were observed in the dorsolateral and ventral BNST. (A) Rostrally, fibers clustered mainly in the oval nucleus of the dorsal bed nucleus and in the subcommisural zone of the ventral bed nucleus (Bregma −0.12). (B) Caudally, dense fibers of the stria terminalis were present in the dorsal region (Bregma −0.6). Scale bars, 200 μm. (C) Less dense projections were detected ventral and lateral to the ventral BNST in the substantia innominata and the ventral pallidum (Bregma −0.12). Scale bar, 200 μm. (D) Fibers were detected throughout the lateral hypothalamus (Bregma -4.20) within the nigrostriatal bundle. Scale bar, 200 μm. (E) Fibers also projected dorsomedially into the caudal dorsal raphe nucleus and ventrolateral periaqueductal gray (Bregma −7.7). TPH, tryptophan hydroxylase. Scale bar, 200 μm. (F–I) Some fibers projected as far as the nucleus tractus solitarius where they came in close contact to noradrenergic processes and cell bodies in the most caudal regions. Bregma (−12.9) – (−14.0). TH, tyrosine hydroxylase. Scale bars, 200 μm in panel (F); 200 μm in panel (G); 100 μm in panel (H); 100 μm in panel (I). ac, anterior commissure; ic, internal capsule; Str, striatum; st, stria terminalis; VP, ventral pallidum; SIB, substantia innominata; cp, cerebral peduncle; pLH, posterior lateral hypothalamus; Aq, central aqueduct; vlPAG, ventrolateral periaqueductal gray; 4V, fourth ventricle.

Figure 5

CeL CRF projections to the substantia nigra and VTA. (A) Representative example of CeL CRF fibers in the rostral VTA and substantia nigra pars compacta (SNc; Bregma −5.0). Scale bar, 500 μm. (B) CeL CRF fibers were observed projecting through the SNc, but not contacting the VTA slightly more caudally (Bregma -5.5). Scale bar, 500 μm. (C) CeL CRF fibers were present at the most caudal aspects of the VTA and SNc (Bregma −6.1). Scale bar, 500 μm. (D) CeL CRF fibers course through the most dorsolateral region of the SNc. Scale bar, 100 μm. (E) Low density collaterals were present in the rostral VTA surrounding dopamine neurons. Scale bar, 100 μm. Green, tyrosine hydroxylase.

Figure 6

ChR2 stimulation of CRF CeL terminals evokes IPSCs in a subset of CeA neurons. (A) Coronal section through the CeA with superimposed reference frame to identify the location of recorded neurons across slices. (B) Example of live CeL neurons (Scale bar, 10 μm) and fibers (Scale bar, 50 μM) expressing ChR2-eYFP. (C) Examples of IPSCs evoked after stimulation of ChR2 in CRF CeL inputs, which were blocked by picrotoxin. (D) Picrotoxin also blocked spontaneous IPSCs. (E) Diagram showing distribution of recorded cells relative to the cluster of CRF cell bodies and dendrites in the CeL (dotted blue circle). Filled symbols represent neurons with IPSC responses; open symbols are neurons without evoked IPSC responses. One CeL neuron with an IPSC response was found outside the cluster of CRF cell bodies and dendrites but within the confines of the CeL. (F) Similar magnitude of evoked IPSCs in CeL and CeM neurons and at two different LED intensities. BLA, basolateral amygdala; CeL, lateral central amygdala; CeM, medial central amygdala; LA, lateral amygdala.

Figure 7

CeL CRF neurons activate non-CRF neurons in the CeL and CeM. (A) Representative overlay images of Fos immunoreactivity and mCherry fluorescence in control, hM4Di, and hM3Dq-expressing CeL neurons. Scale bar, 100 μm. Insets show high-magnification examples of mCherry expressing neurons immunostained for Fos. (B) Percentage of mCherry+ neurons co-expressing Fos following administration of CNO. (C,D) Fos induction in non-CRF neurons of the CeL (C) and the CeM (D) after administration of CNO. ^****p < 0.001 compared with Control or hM4Di, n = 4–5 rats, 10 sections/rat, Tukey's multiple comparisons test. (E) Representative overlay images of Fos immunoreactivity and native mCherry fluorescence in hM3Dq-expressing cells from vehicle- or R121919-treated rats. Scale bar, 100 μm. (F) Percentage of Fos+ neurons in the CeL and CeM after administration of R121919. (G) Total neuron counts per amygdala section are equivalent between groups. (H) Percentage of hM3Dq neurons expressing Fos after administration of CNO is equivalent between groups. ^***p < 0.001, ^*p < 0.05, n = 4–5 rats, 10–12 sections/rat, Tukey's multiple comparisons test.

Figure 8

Sagittal rat brain schematic of CeL CRF neuron projections. BNST, bed nucleus of the stria terminalis; CeL, lateral central amygdala; DRN, dorsal raphe nuclei; LC, locus coeruleus; LH, lateral hypothalamus; NTS, nucleus tractus solitarius; PBN, parabrachial nucleus; SIB, Substantia innominata; SNc, substantia nigra pars compacta; vlPAG, ventrolateral periaqueductal gray; VP, ventral pallidum.