Pituitary Adenylate Cyclase-Activating Polypeptide-27 (PACAP-27) in the Thalamic Paraventricular Nucleus Is Stimulated by Ethanol Drinking

Abstract

Background: The paraventricular nucleus of the thalamus (PVT) is a limbic brain structure that affects ethanol (EtOH) drinking, but the neurochemicals transcribed in this nucleus that may participate in this behavior have yet to be fully characterized. The neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), is known to be transcribed in other limbic areas and to be involved in many of the same behaviors as the PVT itself, possibly including EtOH drinking. It exists in 2 isoforms, PACAP-38 and PACAP-27, with the former expressed at higher levels in most brain regions. The purpose of this study was to characterize PACAP in the PVT and to assess its response to EtOH drinking.

Methods: First, EtOH-naïve, Sprague Dawley rats were examined using quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry, to characterize PACAP mRNA and peptide throughout the rostrocaudal axis of the PVT. Next, EtOH-naïve, vGLUT2-GFP transgenic mice were examined using immunohistochemistry, to identify the neurochemical phenotype of the PACAPergic cells in the PVT. Finally, Long Evans rats were trained to drink 20% EtOH under the intermittent-access paradigm and then examined with PCR and immunohistochemistry, to determine the effects of EtOH on endogenous PACAP in the PVT.

Results: Gene expression of PACAP was detected across the entire PVT, denser in the posterior than the anterior portion of this nucleus. The protein isoform, PACAP-27, was present in a high percentage of cell bodies in the PVT, again particularly in the posterior portion, while PACAP-38 was instead dense in fibers. All PACAP-27⁺ cells colabeled with glutamate, which itself was identified in the majority of PVT cells. EtOH drinking led to an increase in PACAP gene expression and in levels of PACAP-27 in individual cells of the PVT.

Conclusions: This study characterizes the PVT neuropeptide, PACAP, and its understudied protein isoform, PACAP-27, and demonstrates that it is involved in pharmacologically relevant EtOH drinking. This indicates that PACAP-27 should be further investigated for its possible role in EtOH drinking.

Keywords: Anterior Paraventricular Nucleus of the Thalamus; Intermittent Access; PACAP-38; Pituitary Adenylate Cyclase-Activating Polypeptide; Posterior Paraventricular Nucleus of the Thalamus.

Fig 1.

Gene expression of pituitary adenylate cyclase-activating polypeptide (PACAP) in the paraventricular nucleus of the thalamus (PVT) of Sprague-Dawley rats (N = 5), as assessed using quantitative real-time PCR (Experiment 1). Bars labeled with the same letter are not significantly different from each other. Values are mean ± SEM. Abbreviations: a/mPVT, anterior/middle paraventricular nucleus of the thalamus; m/pPVT, middle/posterior paraventricular nucleus of the thalamus.

Fig 2.

Cells expressing pituitary adenylate cyclase-activating polypeptide (PACAP) protein and 4’,6-diamidino-2-phenylindole (DAPI) in the paraventricular nucleus of the thalamus (PVT) of Sprague-Dawley rats (N = 5), as assessed using immunofluorescent histochemistry (Experiment 2). A. Quantification of cells expressing PACAP-27 and PACAP-38 as a percentage of cells expressing DAPI in the anterior PVT (aPVT), middle PVT (mPVT), and posterior PVT (pPVT). Bars labeled with the same letter are not significantly different from each other. Values are mean ± SEM. B. Confocal photomicrographs showing PACAP-27 (red), PACAP-38 (green), and DAPI (blue) in the aPVT, mPVT, and pPVT. Inset is a higher magnification of the image marked with a white square. Images on the far right are single-channel photomicrographs of the triple-labeled image of the posterior PVT. Scale bars = 100 μm in main image and 30 μm in inset.

Fig 3.

Cells expressing pituitary adenylate cyclase-activating polypeptide (PACAP) protein in the paraventricular nucleus of the thalamus (PVT) of Sprague-Dawley rats (N = 5), as assessed using immunohistochemistry with an antibody that targets both PACAP isoforms (Experiment 2). A. Quantification of cells expressing PACAP in the anterior PVT (aPVT), middle PVT (mPVT), and posterior PVT (pPVT). Bars labeled with the same letter are not significantly different from each other. Values are mean ± SEM. B. Photomicrograph showing PACAP in the mPVT. Inset is a higher magnification of the image marked with a white square. Scale bars = 100 μm in main image and 30 μm in inset.

Fig 4.

Confocal photomicrograph showing cells expressing pituitary adenylate cyclase-activating polypeptide-27 (PACAP-27, red) protein, vesicular-glutamate-transporter 2 (vGLUT2, green), and 4’,6-diamidino-2-phenylindole (DAPI, blue) in the paraventricular nucleus of the thalamus (PVT) of male vGLUT2-GFP transgenic mice (Experiment 3). Inset is a higher magnification of the image marked with a white square. Scale bars = 100 μm in main image and 30 μm in inset.

Fig 5.

Gene expression of pituitary adenylate cyclase-activating polypeptide (PACAP) in the paraventricular nucleus of the thalamus (PVT) of male Long-Evans rats, trained to drink 20% ethanol under the intermittent-access two-bottle-choice paradigm or maintained on water and chow only (n = 8/group), as assessed using quantitative real-time PCR (Experiment 4). Bars labeled with the same letter are not significantly different from each other. Values are mean ± SEM. Abbreviations: a/mPVT, anterior/middle paraventricular nucleus of the thalamus; m/pPVT, middle/posterior paraventricular nucleus of the thalamus.

Fig 6.

Pituitary adenylate cyclase-activating polypeptide (PACAP) protein in the paraventricular nucleus of the thalamus (PVT) of male Long-Evans rats, trained to drink 20% ethanol or maintained on water and chow only (n = 6/group), as assessed using immunofluorescent histochemistry (Experiment 5). A. Density of immunofluorescence for PACAP-27 and PACAP-38 in the PVT. B. Total immunofluorescence for PACAP-27 in individual cells in the PVT. Bars labeled with the same letter are not significantly different from each other. Values are mean ± SEM. C. Confocal photomicrographs showing cells expressing PACAP-27 (red), PACAP-38 (green), and 4’,6-diamidino-2-phenylindole (DAPI, blue) in the mPVT of ethanol and water drinkers. Scale bar = 100 μm.