Diversity of Reporter Expression Patterns in Transgenic Mouse Lines Targeting Corticotropin-Releasing Hormone-Expressing Neurons

Abstract

Transgenic mice, including lines targeting corticotropin-releasing factor (CRF or CRH), have been extensively employed to study stress neurobiology. These powerful tools are poised to revolutionize our understanding of the localization and connectivity of CRH-expressing neurons, and the crucial roles of CRH in normal and pathological conditions. Accurate interpretation of studies using cell type-specific transgenic mice vitally depends on congruence between expression of the endogenous peptide and reporter. If reporter expression does not faithfully reproduce native gene expression, then effects of manipulating unintentionally targeted cells may be misattributed. Here, we studied CRH and reporter expression patterns in 3 adult transgenic mice: Crh-IRES-Cre;Ai14 (tdTomato mouse), Crfp3.0CreGFP, and Crh-GFP BAC. We employed the CRH antiserum generated by Vale after validating its specificity using CRH-null mice. We focused the analyses on stress-salient regions, including hypothalamus, amygdala, bed nucleus of the stria terminalis, and hippocampus. Expression patterns of endogenous CRH were consistent among wild-type and transgenic mice. In tdTomato mice, most CRH-expressing neurons coexpressed the reporter, yet the reporter identified a few non-CRH-expressing pyramidal-like cells in hippocampal CA1 and CA3. In Crfp3.0CreGFP mice, coexpression of CRH and the reporter was found in central amygdala and, less commonly, in other evaluated regions. In Crh-GFP BAC mice, the large majority of neurons expressed either CRH or reporter, with little overlap. These data highlight significant diversity in concordant expression of reporter and endogenous CRH among 3 available transgenic mice. These findings should be instrumental in interpreting important scientific findings emerging from the use of these potent neurobiological tools.

Figure 1.

CRH-ir neurons in adult C57BL/6J mouse (WT) vs CRH-null mouse (KO). A and B, In the hypothalamus, CRH-ir neurons are apparent in the parvocellular subregion of the PVN in WT mice, but no signal is detected in KO mice. C and D, Abundant CRH-ir neurons with a bipolar shape (arrows) are evident in layers II and III of the neocortex (motor area) in WT mice but not in KO mice. Scale bars, 100 μm.

Figure 2.

The expression of tdTomato reporter in the PVN and median eminence in the Crh-IRES-Cre;Ai14 tdTomato mouse. A, In the PVN, the vast majority of reporter-expressing neurons (tdTomato) in the parvocellular subregion coexpress endogenous CRH. B, In the median eminence, parvocellular CRH-expressing neurosecretory neuron axons terminated within the external layer. A similar pattern was apparent for tdTomato-expressing terminals. Scale bars, 50 μm.

Figure 3.

Expression patterns of the GFP reporter and endogenous CRH in the PVN of the Crf.p3.0CreGFP mouse. A, Dual-labeling ICC for CRH (red) and the GFP reporter (green). Two distinct populations of neurons were visualized in the hypothalamus: CRH-ir neurons were located in the dorsomedial parvocellular division, whereas GFP reporter-expressing neurons tended to reside more laterally. Boxed areas in A3 were magnified. B, In the median eminence, a partial overlap of CRH-expressing terminals and GFP reporter-expressing terminals was observed in external layer of this structure. Scale bars, 50 μm.

Figure 4.

Expression patterns of the GFP reporter and endogenous CRH in the hypothalamus in the BAC transgenic mouse. A, A group of GFP reporter-expressing neurons was detected at the anterior level of the hypothalamus, at which CRH-expressing terminals were abundant, but no CRH-ir parvocellular cell bodies; 200 μm posterior to this level, CRH-ir parvocellular cells in the anterior PVN were apparent. However, no GFP reporter-expressing neurons were detected. B, In accordance with the termination of CRH-expressing cell in naïve mouse and rat, CRH-ir terminals were apparent in the external layer of the median eminence. In contrast, the GFP reporter signal was visible in the inner layer of the structure. Scale bars, 50 μm.

Figure 5.

Diversity of reporter-expressing neurons in the central amygdala of transgenic mice. A, In the adult C57BL/6J mouse, CRH-expressing neurons resided primarily in the central nucleus of the amygdala, where ir cell bodies (inset) and a dense mesh of CRH-ir fibers/terminals were apparent. Scale bar, 100 μm. B, The distribution pattern of CRH expression (green) in the central amygdala was largely recapitulated in the Crh-IRES-Cre;Ai14 tdTomato mouse. The vast majority of tdTomato reporter-expressing neurons coexpressed the native peptide. Scale bar, 50 μm. C, In the Crfp3.0CreGFP mouse, both native CRH (red) and reporter-expressing neurons (green) were apparent. Images shown are within the posterior central amygdala. Confocal high magnification and thin serial sections (0.2–0.5 μm of thickness) revealed a partial overlap of native CRH and reporter for both cell bodies and fibers. A magnification of the boxed area in the middle panel is shown on the right, scanned at 0.5 μm of virtual sections. This method enabled visualization of clear colocalization (arrowheads) of CRH and reporter. Arrows denote lack of overlap. Scale bars, 50 μm (middle) and 20 μm (right). D, In the Crh-BAC transgenic mouse, sections of the central amygdala that harbored most CRH-ir soma and fibers had few reporter-expressing neurons (left). GFP reporter-expressing neurons were apparent in the posterior level of central amygdala, yet the signal did not overlap with CRH-ir neurons (right). Scale bars, 60 μm (left) and 30 μm (right).

Figure 6.

Expression patterns of native CRH and of reporters in the BNST. A, CRH-ir neurons and fibers in the anterior (A1) and posterior (A2) BNST of adult C57BL/6J mice. Cell bodies (inset in A1) of CRH-ir neurons were apparent in the dorsolateral subdivision of anterior BNST, whereas dense networks of ir axon terminals (inset in A2) were found in the posterior region. ac, anterior commissure. Scale bars, 100 μm (A1) and 200 μm (A2). B, In the anterior BNST, the distribution pattern of CRH expression in naïve mouse was recapitulated in the Crh-IRES-Cre;Ai14 tdTomato mouse. Most reporter-expressing neurons in the dorsolateral subdivision coexpressed the native peptide (arrowheads). Scale bars, 50 μm (left) and 20 μm (right). C, In the posterior BNST, a group of reporter-expressing neurons was observed in the Crfp3.0CreGFP mouse, with a limited coexpression (arrowheads) of native peptide. Arrows point reporter expression only. Scale bars, 50 μm (left) and 20 μm (right). Boxed areas in B and C were magnified to show the colocalization. BNST sections were not available for the CRH-BAC mouse.

Figure 7.

Patterns of CRH- and reporter-expressing neuronal distribution in the hippocampus of naïve and 3 transgenic mice. A, The distribution and structure of CRH-ir neurons in the hippocampus of adult C57BL/6J mouse. Boxed areas in the top panel were magnified in the insets. CRH-expressing neurons (arrows) in CA1 and CA3 pyramidal cell layers appear eccentric, bipolar, and possess a network of terminals surrounding the unlabeled pyramidal cells. Additionally, a heterogeneous population of elongated and multipolar interneuronal-like cells expressing CRH are visible. Scale bars, 500 μm (top) and 32 μm (middle and bottom). B, In the Crh-IRES-Cre;Ai14 tdTomato mouse, the large majority of CRH-ir neurons coexpress the tdTomato reporter (see more detailed analysis in Figure 8). Arrowheads point the colocalization. SO, stratum oriens; SP, stratum pyramidale; SLM, stratum lacunosum-moleculare. Scale bar, 50 μm. C, In the Crfp3.0CreGFP mouse, reporter-expressing neurons were sparse in area CA1 as well as in area CA3 (data not shown). Arrows point to CRH-ir neurons. Scale bar, 50 μm. D, In the Crh-BAC transgenic mouse, both CRH-ir neurons/fibers and GFP reporter-expressing neurons/fibers were clearly apparent. However, most GFP-positive cells appeared pyramidal in structure, and there was no overlap with CRH-ir neurons. Scale bar, 30 μm.

Figure 8.

Concordant identities of CRH-expressing hippocampal neurons in naïve mice and of expressing hippocampal neurons in the tdTomato mouse. A, In the Crh-IRES-Cre;Ai14 tdTomato mouse, interneuron-like reporter-expressing neurons coexpressed CRH (arrowheads) in the pyramidal cell layer of area CA1 (as well as in CA3). However, those with soma shape and dendritic processes typical of pyramidal cell were devoid of CRH expression (arrows). B, A subset of CRH-ir neurons (brown) in the pyramidal cell layer of area CA1 of naïve C57BL/6J mice coexpressed PV (blue granular deposits). C, In the Crh-IRES-Cre;Ai14 tdTomato mouse, a coexpression (arrowhead) of PV (green), and the reporter was apparent in similar cells. D, In the dentate gyrus of adult C57BL/6J mice, a robust population of CRH-ir neurons (brown) coexpressed calretinin (blue). E, In the Crh-IRES-Cre;Ai14 tdTomato mouse, the colocalization (arrowheads) of calretinin (green) and the reporter is visible in the same cell population. Scale bars: 50 μm (A), 25 μm (B and C), and 100 μm (D and E).