High-resolution characterization of a PACAP-EGFP transgenic mouse model for mapping PACAP-expressing neurons.

Pituitary adenylate cyclase-activating polypeptide (PACAP, gene name Adcyap1) regulates a wide variety of neurological and physiological functions, including metabolism and cognition, and plays roles in of multiple forms of stress. Because of its preferential expression in nerve fibers, it has often been difficult to trace and identify the endogenous sources of the peptide in specific populations of neurons. Here, we introduce a transgenic mouse line that harbors in its genome a bacterial artificial chromosome containing an enhanced green fluorescent protein (EGFP) expression cassette inserted upstream of the PACAP ATG translation initiation codon. Analysis of expression in brain sections of these mice using a GFP antibody reveals EGFP expression in distinct neuronal perikarya and dendritic arbors in several major brain regions previously reported to express PACAP from using a variety of approaches, including radioimmunoassay, in situ hybridization, and immunohistochemistry with and without colchicine. EGFP expression in neuronal perikarya was modulated in a manner similar to PACAP gene expression in motor neurons after peripheral axotomy in the ipsilateral facial motor nucleus in the brainstem, providing an example in which the transgene undergoes proper regulation in vivo. These mice and the high-resolution map obtained are expected to be useful in understanding the anatomical patterns of PACAP expression and its plasticity in the mouse. J. Comp. Neurol. 524:3827-3848, 2016. © 2016 Wiley Periodicals, Inc.

Evidence for limited D1 and D2 receptor coexpression and colocalization within the dorsal striatum of the neonatal mouse.

The striatum is the major input nucleus of the basal ganglia involved in reward processing, goal-directed behaviors, habit learning, and motor control. The striatum projects to the basal ganglia output nuclei via the "direct" and "indirect" pathways, which can be distinguished by their projection fields and their opposing effects on behavior. In adult animals, the functional opposition is modulated by the differential actions of D1 and D2 dopamine receptors (D1R, D2R), the expression of which is largely separated between these pathways. To determine whether a similar degree of separation exists earlier in development, we used dual-label immunohistochemistry to map dorsal-striatal D1R and D2R expression at the promoter level in postnatal day 0 (PD0) Drd1a-tdTomato/Drd2-GFP BAC transgenic mice, and at the receptor level by costaining for native D1R and D2R in wildtype (WT) PD0 animals. To assess for potential molecular interactions between D1R and D2R we also employed a recently developed proximity-ligation assay (PLA). Limited coexpression and colocalization of the D1R and D2R proteins was found in clusters of neurons endemic to the "patch" compartment as identified by costaining with tyrosine hydroxylase, but not outside these clusters. Moreover, in contrast to our recent findings where we failed to detect a D1R-D2R PLA signal in the adult striatum, in PD0 striatum we did identify a clear PLA signal for this pair of receptors. This colocalization at close proximity points to a possible role for D1R/D2R-mediated crosstalk in early striatal ontogeny.

Neuroanatomical characterization of a growth hormone secretagogue receptor-green fluorescent protein reporter mouse.

Growth hormone secretagogue receptor (GHSR) 1a is the only molecularly identified receptor for ghrelin, mediating ghrelin-related effects on eating, body weight, and blood glucose control, among others. The expression pattern of GHSR within the brain has been assessed previously by several neuroanatomical techniques. However, inherent limitations to these techniques and the lack of reliable anti-GHSR antibodies and reporter rodent models that identify GHSR-containing neurons have prevented a more comprehensive functional characterization of ghrelin-responsive neurons. Here we have systematically characterized the brain expression of an enhanced green fluorescence protein (eGFP) transgene controlled by the Ghsr promoter in a recently reported GHSR reporter mouse. Expression of eGFP in coronal brain sections was compared with GHSR mRNA expression detected in the same sections by in situ hybridization histochemistry. eGFP immunoreactivity was detected in several areas, including the prefrontal cortex, insular cortex, olfactory bulb, amygdala, and hippocampus, which showed no or low GHSR mRNA expression. In contrast, eGFP expression was low in several midbrain regions and in several hypothalamic nuclei, particularly the arcuate nucleus, where robust GHSR mRNA expression has been well-characterized. eGFP expression in several brainstem nuclei showed high to moderate degrees of colocalization with GHSR mRNA labeling. Further quantitative PCR and electrophysiological analyses of eGFP-labeled hippocampal cells confirmed faithful expression of eGFP within GHSR-containing, ghrelin-responsive neurons. In summary, the GHSR-eGFP reporter mouse model may be a useful tool for studying GHSR function, particularly within the brainstem and hippocampus; however, it underrepresents GHSR expression in nuclei within the hypothalamus and midbrain.