Sex-related differences in endogenous pituitary adenylate cyclase-activating polypeptide (PACAP) in the thalamic paraventricular nucleus: Implications for addiction neuroscience

Abstract

Males and females exhibit differences in motivated and affective behavior; however, the neural substrates underlying these differences remain poorly understood. In the paraventricular nucleus of the thalamus (PVT), sex-related differences in neuronal activity have been identified in response to motivated behavior tasks and affective challenges. Within the PVT, the neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), is highly expressed and is also involved in motivated and affective behavior. The purpose of this study was to compare the expression of PACAP mRNA and peptide in the PVT of males and females. Analysis with quantitative real-time PCR in mice revealed that females had significantly higher levels of PACAP mRNA than males in the whole PVT, but no differences in the neuropeptides enkephalin or corticotropin releasing factor (CRF) in this brain region. While in rats, females demonstrated a trend for greater gene expression than males in the anterior/middle and middle/posterior PVT, they again showed no differences in enkephalin or CRF. Analysis with immunofluorescent histochemistry revealed that female mice had significantly more PACAP-containing cells than males as a function of area throughout the PVT, and that female rats had significantly more PACAP-27 and PACAP-38-containing cells than males, both as a percentage of total cells and as a function of PVT area. For PACAP-27, this specifically occurred in the anterior PVT, and for PACAP-38, it occurred throughout the anterior, middle, and posterior PVT. These results suggest that sex-related differences in PVT PACAP may underly some of the established sex-related differences in motivated and affective behavior.

1. Introduction

Both clinically and preclinically, males and females have been found to exhibit differences in motivated and affective behavior [1–3]. The neural substrates underlying these sex-related differences, however, are poorly understood. The paraventricular nucleus of the thalamus (PVT) is one brain region where sex-related differences have been identified in response to motivated behavior tasks and affective challenges, through examination of immediate early genes in the PVT [4–6] and of electrophysiological activity in PVT projections to the bed nucleus of the stria terminalis (BNST) [7]. Recently, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP), which is known to be involved in motivated and affective behavior [8, 9], was found to be densely expressed in the PVT of both mice and rats [10, 11]. Importantly, however, PACAP was examined only in males. While sex-related differences in neuronal activity in the PVT have been identified, possible sex-related differences in neuropeptide expression in the PVT remain to be determined.

The PVT is a midline brain structure that has been implicated in affective behavior [12], as well as the intake of a number of drugs of abuse, including morphine, amphetamine, cocaine, nicotine, tetrahydrocannabinol, and ethanol [13, 14]. Positioned as a major node in the limbic system, the PVT receives projections from regions that include the hypothalamus, prefrontal cortex, and hippocampus, and innervates regions such as the nucleus accumbens, BNST, and central nucleus of the amygdala [15, 16]. While it uses glutamate as its primary classical neurotransmitter [10, 17, 18], the PVT also densely expresses numerous behavior-related neuropeptides, including not only PACAP, but also enkephalin and corticotropin releasing factor (CRF), among others [10, 11, 19, 20]. Primarily due to its nuclear configurations, the PVT is sometimes examined as two subregions, the anterior PVT and the posterior PVT [15, 21, 22]. Although these subregions share similarities in their anatomical connections, the anterior and posterior PVT also demonstrate subtle differences that may account for their hypothesized differences in function. For example, the anterior PVT has denser projections to the suprachiasmatic nucleus and dorsomedial nucleus accumbens shell [23], and has been proposed to be more highly involved than the posterior PVT in arousal, reward-seeking, and positive emotional valence [12, 23, 24]. In contrast, the posterior PVT, which more heavily innervates the ventromedial nucleus accumbens shell, dorsolateral BNST, and central amygdala [15, 23, 25, 26], has been hypothesized to be more involved in aversive motivation and negative emotional valence [12, 23, 24]. In cases where a third subregion, the middle PVT, has been differentiated, it is often found to act as a transition between the anterior and posterior PVT, in terms of gene expression and efferent projections [10, 23, 27]. Beyond sex-related differences in neuropeptide expression in the PVT, it also remains to be determined if there are sex-related differences in neuropeptide expression across the antero-posterior axis of the PVT.

One of the most highly-expressed neuropeptides in the PVT, PACAP [10], appears to be differentially expressed across the antero-posterior PVT. In male rats, both gene expression and peptide levels have been found to be higher in the posterior PVT compared to the anterior PVT [11]. The gene for PACAP, Adcyap1, is independently processed into two peptide isoforms, PACAP-27 and PACAP-38 [28–30]. While these isoforms can both bind to any of three receptors, the PACAP type 1 (PAC1) receptor, vasoactive intestinal polypeptide type 1 (VPAC1) receptor, and VIP type 2 (VPAC2) receptor [31–33], they show different affinities for these receptors. For example, PACAP-38 binds to the PAC1 receptor with greater affinity than PACAP-27, but PACAP-27 binds to the VIP1 receptor with higher affinity than PACAP-38 [34]. Although PACAP-38 is more densely expressed throughout most of the brain [35, 36], we have previously demonstrated in male rats that it is PACAP-27 that is more densely expressed than PACAP-38 in the PVT [11], and PACAP-27 is more densely expressed in the posterior than the anterior half of the PVT. Like the PVT, PACAP is involved in aspects of motivated and affective behavior, including the intake of ethanol and cocaine [8, 37], and although much of this work has been conducted in males, there is some indication that females may be more sensitive to stimulation by PACAP-27 [8]. Notably, however, studies to date, which have not examined the PVT, have demonstrated no significant sex-related differences in the expression or levels of PACAP in the brain [38–40]. Thus, while there are sex-related differences in motivated and affective behavior, and both the PVT and PACAP are involved in these behaviors, and the PVT has demonstrated sex-related differences in relation to these behaviors, whether or not there are sex-related differences in PACAP in the PVT remains unknown.

The goal of this study was to determine and characterize sexual dimorphism in the expression of PACAP in the PVT. We examined both gene and peptide expression of PACAP in the PVT in both male and female mice and rats. Given the greater sensitivity of females to stimulation by PACAP, we hypothesized that males would demonstrate higher expression of PACAP compared to females. With both PACAP and the PVT being involved in addictive and affective behaviors, our findings begin to shed light on the neurobiological bases of sex-related differences in behavior.

2. Materials and methods

2.1. Subjects

Adult male and female C57BL/6J mice (n = 19 males, 18 females; cohorts 1 and 3: males and females both 3 − 6 months old, cohort 2: 6 months old; bred in-house from mice originally purchased from Jackson Laboratory, Bar Harbor, ME, USA) and male and female Sprague-Dawley rats (n = 10 males, 11 females; all 2 months old; Charles River Laboratories International, Inc., Malvern, PA, USA) and male and female Long-Evans rats (n = 9/group; all 4 months old; Charles River Laboratories International, Inc., Malvern, PA, USA) were housed in an AAALAC accredited facility, on a 12-hour reversed light/dark cycle (lights off at 0600 h for mice and 0900 h for rats). Mice were group housed, while rats were individually housed. All animals received ad libitum chow and water throughout the study. Experiments were approved by the Institutional Animal Care and Use Committee of Drexel University College of Medicine and complied with the ARRIVE guidelines, carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised 1978). Data on PACAP mRNA and peptide isoforms in the male Sprague-Dawley rats were previously reported [11].

2.2. Quantitative real-time PCR (qRT-PCR)

To determine differences between males and females in levels of PACAP mRNA in the PVT, qRT-PCR was used. Male and female C57BL/6J mice (run in two cohorts (cohorts 1 and 2): n = 5/group and n = 6/group) were sacrificed during their dark cycle and their whole PVT was dissected out to examine mRNA levels of PACAP, as well as enkephalin and CRF for comparison. Whole PVT was needed from the mice, to obtain a sufficiently high mRNA yield for analysis in our laboratory. Immediately after sacrifice, the brain was placed in a matrix slicing guide, with the ventral surface facing up. Two coronal cuts were made, at the middle optic chiasm and 1.0 mm caudal to this, yielding one brain slice (Bregma −0.75 mm to −1.75 mm) [41]. This slice was then placed under a microscope on a petri dish filled with ice, and the whole PVT was dissected, as an inverted isosceles triangle below the third ventricle, approximately 0.3 mm wide at the base. Male and female Sprague-Dawley rats (n = 5 males, 6 females) and male and female Long-Evans rats (n = 9/group) were also sacrificed during their dark cycle and their anterior/middle PVT and middle/posterior PVT were dissected out to examine mRNA levels of PACAP, as well as enkephalin and CRF for comparison. Immediately after sacrifice, the brain was placed in a matrix slicing guide set on ice, with the ventral surface facing up. Three coronal cuts were made, at the middle optic chiasm (Bregma −1.5 mm) and going 2.0 mm caudal to this (Bregma −2.5 mm and −3.5 mm), yielding two slices (anterior/middle PVT: Bregma −1.5 mm to −2.5 mm; middle/posterior PVT: Bregma −2.5 mm to −3.5 mm) [42]. These slices were then placed under a microscope on a petri dish filled with ice, and the anterior/middle PVT and middle/posterior PVT were dissected as inverted isosceles triangles directly ventral to the dorsal third ventricle, approximately 0.8 mm wide at the base. Brain sections were stored at −20 °C in RNAlater (Qiagen Inc., Valenia, CA, USA) until extraction of RNA.

For both mouse and rat tissue, total RNA from each brain sample was extracted using an RNeasy Mini Kit (Qiagen Inc.) and DNA was removed using RNase-free DNase 1 (Qiagen Inc.). The yield was quantified with a NanoDrop Lite spectrophotometer (Thermo Electron North America LLC, Madison Wisconsin) with A260/A280 ratios between 1.80 and 2.27, indicating high purity. The cDNA was reverse transcribed using SuperScript^® VILO^™ Master Mix (Invitrogen, Grand Island, NY, USA) in a SimpliAmp^™ Thermal Cycler (Applied Biosystems, Waltham, MA, USA), using 1 μg of RNA from each sample. The qRT-PCR used a SYBR Green PCR core reagents kit (Applied Biosystems, Grand Island, NY, USA), with 12.5 ng of cDNA template in a 25 μl reaction volume in MicroAmp^® Fast Optical 96-Well Reaction Plates (Applied Biosystems). A StepOnePlus Real-Time PCR System (Applied Biosystems) was used to carry out the reaction, which was run under the conditions of 2 min at 50 °C (primer annealing), 10 min at 95 °C (polymerase activation and sequence extension), and 40 cycles of 15 s at 95 °C (denaturation) plus 1 min at 60 °C (annealing and extension). Each sample was run in triplicate, and each run included a no-template control. Target gene expression was quantified using the relative quantification method (ΔΔC_T), with cyclophilin-A as the housekeeping gene. Primers were designed with the NCBI Primer design tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) [43], and purchased from Invitrogen at ThermoFisher Scientific (Grand Island, NY, USA) (Table 1). Those that were newly-designed for this study (mouse cyclophilin-A, PACAP, enkephalin, and CRF) were first validated with tissue from the target region, using serial dilutions to test for primer efficiency and examining the melt curve for primer specificity.

Table 1.

Primer sequences and concentrations used for quantitative real-time polymerase chain reaction in Experiments 1 and 2.

Primer	Sequence	Concentration
Cyclophilin-A Mouse	5′-ATTCATGTGCCAGGGTGGTG-3′ (forward) 5′-TGCCAGGACCTGTATGCTTT-3′ (reverse)	200 nM
PACAP Mouse	5′-GACCAGAAGACGAGGCTTACG-3′ (forward) 5′-GTCCGCTGGATAGTAAAGGGC-3′ (reverse)	200 nM
Enkephalin Mouse	5′-GCCGCTTTACACTTGCCTTC-3′ (forward) 5′-CTCCAGATGGCGCAGGTTAC-3′ (reverse)	200 nM
CRF Mouse	5′-GAGGCATCCTGAGAGAAGTCC-3′ (forward) 5′-GTTAGGGGCGCTCTCTTCTC-3′ (reverse)	50 nM
Cyclophilin-A Rat	5′-GTGTTCTTCGACATCACGGCT-3′ (forward) 5′-CTGTCTTTGGAACTTTGTCTGCA-3′ (reverse)	200 nM
PACAP Rat	5′-GCCTCTCTGGTTGTGATTCCA-3′ (forward) 5′-GGTCATTCGCGGCTAGGAA-3′ (reverse)	200 nM
Enkephalin Rat	5′-GGACTGCGCTAAATGCAGCTA-3′ (forward) 5′-GTGTGCATGCCAGGAAGTTG-3′ (reverse)	100 nM
CRF Rat	5′-GCTCAGCAAGCTCACAGCAA-3′ (forward) 5′-GGCCAAGCGCAACATTTC-3′ (reverse)	200 nM

Primers used for quantitative real-time polymerase chain reaction

2.3. Immunofluorescent histochemistry

To determine differences between males and females in PACAP peptide across the PVT, immunofluorescent histochemistry was used. During their dark cycle, male and female mice (n = 8 males, 7 females) were deeply anesthetized with Euthasol solultion C3N (390 mg/kg pentobarbital sodium/ 50 mg/kg phenytoin sodium; Virbac, Fort Worth, TX, USA) (i.p.) and perfused transcardially with 200 ml of ice-cold 0.9% sodium chloride followed by 200 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. During their dark cycle, male and female Sprague-Dawley rats (n = 5/group) were deeply anesthetized with 75 mg/kg ketamine (Fort Dodge Animal Health, Overland Park, KS, USA) and 10 mg/kg xylazine (LLOYD Incorporated, Shenandoah, IA, USA) (i.p.) and perfused transcardially with 60 ml of ice-cold 0.9% sodium chloride followed by 240 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were then removed, post-fixed in 4% paraformaldehyde for 24 hours at 4 ºC, cryoprotected in 30% sucrose for 2 − 4 days at 4 ºC, and then frozen and stored at −80 ºC. A cryostat was then used to slice the brains into 30 μm sections, which were stored at −20 °C in antifreeze solution (37.5% ethylene glycol, 20% sucrose in 0.03 M phosphate buffered saline (PBS)). Every sixth section through the PVT was taken for processing and analysis, resulting in approximately 8 sections per brain for mice and 18 sections per brain for rats.

To label PACAP, free-floating sections were rinsed for 10 min in 0.1% hydrogen peroxide to remove endogenous peroxidase activity. After rinsing in 0.1 M PBS, they were blocked for 90 min in 5% normal goat serum containing 0.5% Triton X-100 in PBS and incubated overnight at 4 ºC in polyclonal rabbit anti-PACAP-27/38 (1:250, Bioss Antibodies, Woburn, MA, USA; cat# BS0190R, lot# AI11065308) (for mouse) or polyclonal guinea pig anti-PACAP-27 (1:250, Peninsula Laboratories International, Inc, San Carlos, CA, USA; cat# T-5039.0050, lot# 040119–1) and polyclonal rabbit anti-PACAP-38 (1:250, Peninsula Laboratories International, Inc, San Carlos, CA, USA; cat# T-4473.0050, lot# A14722 and A16369) (for rat) primary antibodies. The specificity of these antibodies has previously been validated using immunohistochemistry and western blot [44–47]. According to the manufacturer, the isoform-specific antibodies were generated by immunization of guinea pigs or rabbits with PACAP-27 or PACAP-38, respectively, coupled to a carrier protein [48, 49]. Cross-reactivity for the PACAP-38 antibody was found to be 100% with PACAP-38, PACAP-38(16–38), and PACAP(31–38), but only 0.01% with PACAP-27 peptide, as assessed with radioimmunoassay. Cross-reactivity for the PACAP-27 antibody was found to be 100% with PACAP-27, but 11% with PACAP-38 and 0% with PACAP-38(16–38) and PACAP(31–38) peptides, as assessed with radioimmunoassay. After the antibody incubation, the slices were rinsed in PBS and incubated for 2 h in goat anti-rabbit (Alexa Fluor^® 647) (1:200, Abcam, Cambridge, MA, USA; cat# ab150079, lot# GR3444080–1) (for mouse) or goat anti-guinea pig (Alexa Fluor^® 555) (1:200, Abcam, Cambridge, MA, USA; cat# ab150186, lot# GR128511–1) and goat anti-rabbit (Alexa Fluor^® 488) (1:200, Abcam, Cambridge, MA, USA; cat# ab150077, lot# GR239034–1) (for rat) secondary antibodies in block solution. Pilot experiments were run to determine optimal staining conditions. Alternate sections did not show immunofluorescence when processed with the primary or secondary antibody omitted. Sections were mounted on slides and dried overnight in the dark, coverslipped with ProLong^® Diamond Antifade Mountant with diamidino-2-phenylindole (DAPI; Life Technologies, Carlsbad, CA, USA), and allowed to set for 24 hours before imaging. All sections for all male and female mice were processed at the same time under the same conditions and, separately, all sections for male and female rats were processed at the same time under the same conditions.

Imaging was conducted with a Leica DM5500 automated microscope (Buffalo Grove, IL, USA), and the images were captured with an Olympus DP71 high resolution digital color camera (Waltham, MA, USA) with Slidebook V6 image acquisition and analysis software (3i, Denver, CO, USA). Exposure conditions were the same for each slice processed with each primary antibody. Analyses were confirmed with a Leica SP8 VIS/405 HyVolution confocal microscope (Buffalo Grove, IL, USA). For analysis, mouse sections were defined as anterior PVT (−0.22 to −0.82 mm) or middle/posterior PVT (−0.83 to −2.30 mm) relative to bregma [41], with the middle and posterior PVT being combined due to the small size of the mouse posterior PVT (−2.07 to −2.30 mm); rat sections were defined as anterior PVT (−0.84 to −2.04 mm), middle PVT (−2.05 mm to −2.99 mm), or posterior PVT (−3.00 to −4.08 mm) relative to bregma [42]. Quantification was conducted by an evaluator blind to the sex of the subject. Counting of PACAP⁺, PACAP-27⁺, and PACAP-38⁺ cells was performed manually, while counting of DAPI⁺ cells and evaluation of PVT area and integrated density (area * mean gray value) was performed with NIH software, ImageJ version 1.48v for Windows [50]. For DAPI⁺ cell counts and integrated density, each image in the appropriate channel was converted to grayscale, the PVT was isolated, and the threshold was adjusted, with the same limits used for each slice. Integrated density for PACAP, PACAP-27, and PACAP-38 evaluated all labeling, including both cell bodies and fibers. Software counts for DAPI were initially matched to manual counts, to ensure accuracy. The average number of labeled cells and the PVT area and integrated density per slice was determined for each subregion. Because the images were not spatially calibrated at the start of the analysis, area measurements are reported in square pixels rather than square units (eg. μm²).

2.4. Data analysis

Analyses of mRNA levels were run using ΔΔC_T values. When possible, cohorts were combined for analysis, to increase statistical power. To analyze differences between PACAP mRNA levels in the PVT of male and female mice, a one-way ANOVA was used, with sex and cohort as between-subject variables. To analyze differences between other neuropeptide mRNA levels in the PVT of male and female mice, an independent-samples t-test with two tails was used. To examine the difference in measures between subregions of the PVT in males and females, mixed ANOVAs were used, with subregion as the repeated measures and sex and, when appropriate, cohort as the between-subjects measures. Significant effects were followed up with a Sidak pairwise comparison test. As an a priori assumption was made that sex-related differences could be apparent in one but not the other PVT subregions, Sidak pairwise comparison tests were used to examine sex effects in specific subregions and subregion effects within each sex, even when interaction effects between sex and subregion did not attain significance. Sphericity was determined using Mauchly’s test, and a Greenhouse-Geisser correction was used when sphericity was violated. Significance was determined at p < 0.05. Data are reported as mean ± standard error of the mean (S.E.M.).

3. Results

3.1. Gene expression of PACAP in the PVT of the mouse

To examine PACAP mRNA in the PVT of male and female C57BL/6J mice, gene expression in the whole PVT was analyzed using qRT-PCR. Comparing gene expression between the sexes in two combined cohorts of mice (cohort 1: n = 5/group, cohort 2: n = 6/group), females were found to have significantly higher levels of PACAP mRNA than males [F(1, 18) = 4.675, p = 0.044], by 105% (Figure 1A). While there was also a significant main effect of cohort [F(1, 18) = 6.868, p = 0.017], there was no significant interaction effect between sex and cohort [F(1, 18) = 2.525, not significant, ns]. Indeed, females were found to display significantly higher levels of PACAP mRNA than males in both cohort 1 [t(8) = −2.510, p = 0.036] and cohort 2 [t(10) = −2.653, p = 0.024]. In contrast, with enkephalin and CRF examined only in cohort 2, females showed no significant difference from males in levels of enkephalin mRNA [t(10) = 0.193, ns] or CRF mRNA [t(10) = −0.697, ns] (Figure 1B and Figure 1C). Thus, the level of gene expression of PACAP in the PVT of female mice, but not enkephalin or CRF, is greater than that of males.

Figure 1.

Neuropeptide gene expression in the whole paraventricular nucleus of the thalamus of male and female C57BL/6J mice, as assessed using quantitative real-time PCR (cohort 1: n = 5/group, cohort 2: n = 6/group). A. Gene expression of pituitary adenylate cyclase-activating polypeptide (PACAP). B. Gene expression of enkephalin. C. Gene expression of corticotropin releasing factor (CRF). Values are mean ± SEM; black circles indicate cohort 1, black squares indicate cohort 2; *p < 0.05 vs. comparison group.

3.2. Gene expression of PACAP in the anterior and posterior halves of the PVT of the rat

To examine neuropeptide mRNA in the PVT of male and female rats, gene expression in the anterior/middle PVT and middle/posterior PVT was analyzed using qRT-PCR. In combined cohorts of Sprague-Dawley rats (n = 5 males, 6 females) and Long-Evans rats (n = 9/group), comparing both sexes and subregions, analysis of PACAP mRNA revealed a trend for a main effect of sex [F(1, 25) = 4.028, p = 0.056] and a significant main effect of subregion [F(1, 25) = 35.013, p < 0.001], with females overall showing higher levels of PACAP mRNA than males, and the middle/posterior PVT showing higher levels of PACAP mRNA than the anterior/middle PVT (Figure 2A). There was no significant interaction between sex and subregion [F(1, 25) = 1.075, ns], and pairwise comparisons similarly showed no significant differences between the sexes in the individual subregions (p = ns), but the middle/posterior PVT showed higher levels of PACAP mRNA than the anterior/middle PVT in both males (p = 0.003) and females (p < 0.001). There was a significant main effect of cohort [F(1, 25) = 19.361, p < 0.001]; however, there was no significant interaction effect between sex and cohort [F(1, 25) = 0.076, ns] or between sex and subregion and cohort [F(1, 25) = 0.435, ns].

Figure 2.

Neuropeptide gene expression in the anterior and posterior halves of the paraventricular nucleus of the thalamus (PVT) of male and female Sprague-Dawley (cohort 1) and Long-Evans (cohort 2) rats, as assessed using quantitative real-time PCR (cohort 1: n = 5 males, 6 females; cohort 2: n = 9/group). A. Gene expression of pituitary adenylate cyclase-activating polypeptide (PACAP). B. Gene expression of enkephalin. C. Gene expression of corticotropin releasing factor (CRF). Values are mean ± SEM; black circles indicate cohort 1, black squares indicate cohort 2; *p < 0.05 vs. comparison group.

Analysis of enkephalin mRNA revealed no main effect of sex [F(1, 25) = 0.476, ns], but there was a main effect of subregion [F(1, 25) = 10.556, p = 0.003], with the middle/posterior PVT showing significantly higher levels of enkephalin mRNA than the anterior/middle PVT (Figure 2B). There was no significant interaction between sex and subregion [F(1, 25) = 2.851, ns], and pairwise comparisons similarly showed no significant differences between the sexes in the individual subregions (p = ns). Pairwise comparisons revealed that the main effect of subregion was driven by females, as the middle/posterior PVT showed higher levels of enkephalin mRNA than the anterior/middle PVT only in females (p = 0.001). There was again a significant main effect of cohort [F(1, 25) = 12.855, p < 0.001]; however, there was again no significant interaction effect between sex and cohort [F(1, 25) = 0.241, ns] or between sex and subregion and cohort [F(1, 25) = 4.010, ns].

Analysis of CRF mRNA similarly revealed no main effect of sex [F(1, 25) = 0.056, ns] but again a main effect of subregion [F(1, 25) = 108.428, p < 0.001], with the middle/posterior PVT again showing significantly higher levels of CRF mRNA than the anterior/middle PVT (Figure 2C). There was again no significant interaction between sex and subregion [F(1, 25) = 0.477, ns], and pairwise comparisons similarly showed no significant differences between the sexes in the individual subregions (p = ns), but the middle/posterior PVT showed higher levels of CRF mRNA than the anterior/middle PVT in both males (p < 0.001) and females (p < 0.001). While there was again a significant main effect of cohort [F(1, 25) = 129.860, p < 0.001], there was no significant interaction effect between sex and cohort [F(1, 25) = 0.048, ns] or between sex and subregion and cohort [F(1, 25) = 0.821, ns]. Thus, while female rats have somewhat greater gene expression of PACAP than males, this is not the case for enkephalin or CRF, and all three neuropeptides overall show greater gene expression in the middle/posterior PVT than the anterior/middle PVT.

3.3. Peptide expression of PACAP in the anterior and middle/posterior PVT of the mouse

To examine PACAP peptide across the PVT of male and female mice, the anterior and middle/posterior PVT was analyzed in C57BL/6J mice (n = 8 males, 7 females) using immunofluorescent histochemistry with a PACAP antibody that targeted both PACAP-27 and PACAP-38. In examining the area of each PVT subregion, the analysis revealed a trend for a main effect of subregion [F(1, 10) = 4.712, p = 0.055], with the anterior PVT tending to have a smaller area than the middle/posterior PVT, but no significant main effect of sex [F(1, 10) = 1.894, ns] and no significant interaction between sex and subregion [F(1, 10) = 1.429, ns]. In examining general cellular nuclei across the PVT subregions, the analysis revealed no significant main effect of sex [F(1, 10) = 1.005, ns] or subregion [F(1, 10) = 1.311, ns] on the average number of DAPI⁺ cells per 30 μm slice (1069 for males vs. 1135 for females).

Examining PACAP, analysis of the percentage of DAPI⁺ cells that co-labeled with PACAP in the two sexes and across the PVT subregions identified a trend for a main effect of sex [F(1, 10) = 4.405, p = 0.062], but no significant main effect of subregion [F(1, 10) = 0.344, ns] or interaction effect [F(1, 10) = 0.901, ns] (Figure 3A and Figure 4). Females demonstrated overall greater PACAP co-labeling than males (37% vs. 29%), and pairwise comparisons revealed that the trend for greater PACAP co-labeling in females was specifically driven by a significant difference in the middle/posterior PVT (p = 0.006). Analysis of the number of PACAP⁺ cells, as normalized to PVT area, identified a significant main effect of sex [F(1, 10) = 8.375, p = 0.016], but again no significant main effect of subregion [F(1, 10) = 4.300, ns] or interaction effect [F(1, 10) = 1.341, ns] (Figure 3B). Females demonstrated an overall greater number of PACAP⁺ cells, as normalized to area, than males, and pairwise comparisons revealed that this occurred in both the anterior PVT (p = 0.024) and the middle/posterior PVT (p = 0.022), and also that females but not males had a lower number of PACAP⁺ cells in the middle/posterior PVT compared to the anterior PVT (p = 0.045). On the other hand, analysis of overall labeling for PACAP, as a function of area (equivalent to mean gray value), failed to reveal a significant main effect of sex [F(1, 10) = 2.330, ns] and also failed to reveal a significant main effect of subregion [F(1, 10) = 0.004, ns] (Figure 3C). Thus, while mice overall show few differences in the distribution of PACAP⁺ cells across the PVT, females show greater PACAP labeling than males.

Figure 3.

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP) peptide and 4’,6-diamidino-2-phenylindole (DAPI) in the paraventricular nucleus of the thalamus (PVT) of male and female C57BL/6J mice (n = 8 males, 7 females), as assessed using immunofluorescent histochemistry. A. Percentage of cells expressing DAPI that co-express PACAP. B. Number of PACAP-expressing cells, as normalized to PVT area. C. Overall labeling for PACAP, as a function of area (equivalent to mean gray value). Values are mean ± SEM; *p < 0.05 vs. comparison group.

Figure 4.

Confocal photomicrographs showing pituitary adenylate cyclase-activating polypeptide (PACAP) peptide and 4’,6-diamidino-2-phenylindole (DAPI) in the paraventricular nucleus of the thalamus (PVT) of male and female C57BL/6J mice. A. Composite images at the level of the middle/posterior PVT. Scale bar = 150 μm. B. Single channel images of the female middle/posterior PVT composite image. C. High magnification composite and single channel images from a middle/posterior PVT in a female mouse. Scale bar = 200 μm. Red = PACAP, blue = DAPI.

3.4. Peptide expression of PACAP-27 and PACAP-38 in the anterior, middle, and posterior PVT of the rat

To examine the PACAP isoforms across the PVT of male and female rats, PACAP-27 and PACAP-38 in the anterior, middle, and posterior PVT were analyzed in Sprague Dawley rats (n = 5/group) using immunofluorescent histochemistry. In examining the area of each PVT subregion, the analysis revealed a significant main effect of subregion [F(2, 16) = 5.285, p = 0.017], but no significant main effect of sex [F(1, 8) = 1.412, ns] and no significant interaction between sex and subregion [F(2, 16) = 0.759, ns]. Pairwise comparisons revealed that the middle PVT had a smaller average area than the posterior PVT (p = 0.041). In examining general cellular nuclei across the PVT subregions, the analysis revealed no significant main effect of sex [F(1, 8) = 1.130, ns] or subregion [F(2, 16) = 2.704, ns] on the average number of DAPI⁺ cells per 30 μm slice (428 for males vs. 422 for females).

Examining PACAP-27, analysis of the percentage of DAPI⁺ cells that co-labeled with PACAP-27 in the two sexes and across the PVT subregions identified a significant main effect of sex [F(1, 8) = 13.867, p = 0.006] and subregion [F(1.23, 9.84) = 7.758, p = 0.016], but no significant interaction effect [F(1.23, 9.84) = 0.044, ns] (Figure 5A and Figure 6). Females demonstrated overall greater PACAP-27 co-labeling than males (52% vs. 44%), and pairwise comparisons revealed that DAPI had significantly lower co-labeling with PACAP-27 in the anterior PVT compared to both the middle PVT (p = 0.003) and posterior PVT (p = 0.015). Pairwise comparisons further revealed that the greater PACAP-27 co-labeling in females was specifically driven by a significant difference in the anterior PVT (p = 0.007), and that while both females and males had lower co-labeling in the anterior PVT compared to the middle PVT (p = 0.013 and p = 0.031), there was a trend for a difference compared to the posterior PVT in males (p = 0.058) but no significant difference in females (p = ns). Analysis of the number of PACAP-27⁺ cells, as normalized to PVT area, similarly identified a significant main effect of sex [F(1, 8) = 7.094, p = 0.029] and subregion [F(2, 16) = 10.188, p = 0.001], but no significant interaction effect [F(2, 16) = 0.100, ns] (Figure 5B). Females demonstrated an overall greater number of PACAP-27⁺ cells, as normalized to area, than males, and pairwise comparisons revealed that the overall number of PACAP-27⁺ cells as normalized to area was significantly lower in the anterior PVT compared to both the middle PVT (p = 0.002) and posterior PVT (p = 0.016). Further pairwise comparisons revealed that the greater number of PACAP-27⁺ cells in females was specifically driven by a significant difference in the anterior PVT (p = 0.030), and that while both females and males had a lower number of PACAP-27⁺ cells in the anterior PVT compared to the middle PVT (p = 0.020 and p = 0.008), neither sex alone showed a significant difference compared to the posterior PVT (p = ns). On the other hand, analysis of overall labeling for PACAP-27, as a function of area (equivalent to mean gray value), failed to reveal a significant main effect of sex [F(1, 8) = 1.110, ns] and also failed to reveal a significant main effect of subregion [F(2, 16) = 1.332, ns] (Figure 5C). Thus, while rats overall show the lowest number of PACAP-27⁺ cells in the anterior subregion of the PVT, this is where females show greater PACAP-27 labeling than males.

Figure 5.

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP) peptide and 4’,6-diamidino-2-phenylindole (DAPI) in the paraventricular nucleus of the thalamus (PVT) of male and female Sprague-Dawley rats (n = 5/group), as assessed using immunofluorescent histochemistry. A. Percentage of cells expressing DAPI that co-express PACAP-27. B. Number of PACAP-27-expressing cells, as normalized to PVT area. C. Overall labeling for PACAP-27, as a function of area (equivalent to mean gray value). D. Percentage of cells expressing DAPI that co-express PACAP-38. E. Number of PACAP-38-expressing cells, as normalized to PVT area. F. Overall labeling for PACAP-38, as a function of area (equivalent to mean gray value). G. Percentage of cells expressing PACAP-27 that co-express PACAP-38. Values are mean ± SEM; *p < 0.05 vs. comparison group.

Figure 6.

Confocal photomicrographs showing pituitary adenylate cyclase-activating polypeptide (PACAP) peptide and 4’,6-diamidino-2-phenylindole (DAPI) in the paraventricular nucleus of the thalamus (PVT) of male and female Sprague-Dawley rats. A. Composite images at the level of the anterior, middle, and posterior PVT. Scale bar = 100 μm. B. Single channel images of the middle PVT composite images. C. High magnification composite and single channel images from a middle PVT in a female rat. Scale bar = 200 μm. Red = PACAP-27, green = PACAP-38, blue = DAPI.

Examining PACAP-38, visual inspection revealed that all cells containing PACAP-38 co-labeled with PACAP-27, and that a sizeable portion of PACAP-38 labeling occured fibers; the origin of these fibers was not directly measured. Analysis of the percentage of DAPI⁺ cells that co-labeled with PACAP-38 in the two sexes and across the PVT subregions identified a significant main effect of sex [F(1, 8) = 19.866, p = 0.002] but, unlike with PACAP-27, failed to reveal a significant main effect of subregion [F(2, 16) = 2.465, ns] or interaction effect [F(2, 16) = 0.078, ns] (Figure 5D and Figure 6). Females again demonstrated overall greater PACAP-38 co-labeling than males (17% vs. 11%). Pairwise comparisons revealed that the greater PACAP-38 co-labeling in females occurred in the anterior PVT (p = 0.003), middle PVT (p < 0.001), and posterior PVT (p = 0.031). Analysis of the number of PACAP-38⁺ cells, as normalized to area, similarly identified a significant main effect of sex [F(1, 8) = 37.340, p < 0.001], but this time also revealed a significant main effect of subregion [F(2, 16) = 7.219, p = 0.006], although there was still no significant interaction effect [F(2, 16) = 0.143, ns] (Figure 5E). Females demonstrated an overall greater number of PACAP-38⁺ cells, as normalized to area, than males, and pairwise comparisons revealed that the overall number of PACAP-38⁺ cells as normalized to area was significantly lower in the anterior PVT compared to the middle PVT (p = 0.040). Further pairwise comparisons revealed that the greater number of PACAP-38⁺ cells in females occurred in the anterior PVT (p = 0.005), middle PVT (p < 0.001), and posterior PVT (p = 0.005), but that neither females nor males on their own had a significantly different number of PACAP-38⁺ cells in any specific subregion compared to any other subregion (p = ns). Similar to the analysis with PACAP-27, analysis of overall labeling for PACAP-38, as a function of area (equivalent to mean gray value), failed to reveal a significant main effect of sex [F(1, 8) = 0.004, ns] and also failed to reveal a significant main effect of subregion [F(2, 16) = 0.129, ns] (Figure 5F). Thus, rats show more uniform numbers of PACAP-38⁺ cells across the PVT subregions, and females show greater PACAP-38 labeling than males across the whole PVT.

As all cells containing PACAP-38 were found to co-label with PACAP-27, the percentage of PACAP-27⁺ cells that co-labeled with PACAP-38 was analyzed in the two sexes and across the PVT subregions. This analysis revealed a significant main effect of sex [F(1, 8) = 5.845, p = 0.042] but no significant main effect of subregion [F(2, 16) = 2.350, ns], and no significant interaction effect [F(2, 16) = 0.139, ns]. Females showed overall greater co-labeling of PACAP-27⁺ cells than males (33% vs. 26%), but pairwise comparisons revealed no specific PVT subregion where this occurred (p = ns) (Figure 5G and Figure 6). Thus, with females having more PACAP-27 and PACAP-38 than males, more of their PACAP-27⁺ cells co-label with PACAP-38.

4. Discussion

Our results indicate that, in both mice and rats, PACAP expression in the PVT is higher in females compared to males, contrary to our original hypothesis. In mice, this sexual dimorphism was found for PACAP in the middle/posterior subregion of the PVT. In rats, PACAP-27 labeling was higher in females compared to males in the anterior the PVT, and PACAP-38 was higher across the whole PVT. While previous research has shown that the PVT is sexually dimorphic in its neuronal activity responses [4–6], our study is the first to our knowledge to document a sex-related difference in neuropeptide expression in the PVT or of PACAP peptide labeling in the brain. With both the PVT and PACAP being implicated in motivated and affective behavior [8, 9, 12, 24, 51], and these behaviors having been found to be sexually dimorphic in both clinical and pre-clinical studies [1–3], our findings have particular implications for understanding of the neurobiological bases of sex-related differences in behavior.

One of the major findings of this study was that, compared to males, female mice and rats display higher expression of PACAP in the PVT. Prior work on PACAP, which examined the whole hypothalamus of rats, reported no sex-related differences in the gene expression of this neuropeptide [38, 39]. Similarly, female compared to male rats were found to show only a non-significant elevation in peptide levels of PACAP-38 in the whole hypothalamus, whole brainstem, and sections of the temporal area and telencephalon [40], and female compared to male human subjects showed no difference in levels of PACAP-38 in the circulation [52]. Thus, our work is the first to demonstrate significant sex-related differences in levels of PACAP in the brain. On the other hand, published studies have shown that levels of PACAP can differentially change between the sexes under specific challenges or conditions. For example, levels of PACAP-38 did not rise as significantly in the hypothalamus of female compared to male rats following food deprivation [40], and they were reduced in the circulation of female but not male human subjects with generalized anxiety disorder [52]. Thus, our previous finding that levels of PACAP-27 in cells of the PVT of male rats are significantly increased by intermittent-access 20% ethanol drinking [11] may not generalize to female rats, despite females drinking more than males under this paradigm [8, 53, 54]. Whether or not females show a different PACAP response to the intake of drugs of abuse remains to be tested.

Similar to our published results in males [11], we found that co-labeling of PACAP-27 with DAPI was higher than that of PACAP-38 in the PVT of female rats (52% vs 17%). While it is possible that this difference was due to differences in the efficacy of the antibodies used, there is a known difference in the levels in the PVT of the prohormone processing enzymes that lead to the formation of PACAP-27 and PACAP-38. Although PACAP-27 contains the 27 residues corresponding to the N-terminal 27 amino acids of PACAP-38 [28], PACAP-38 and PACAP-27 appear to be independently cleaved from pro-PACAP [28, 30]. In fact, while processing of the PACAP precursor by the prohormone processing enzyme, SPC3 (also called PC1, PC3, and BDP) is required for mature, bioactive PACAP-38, it is a different prohormone processing enzyme, SPC2 (also called PC2 and RPC2), that is required for PACAP-27 [29], and it is SPC2 and not SPC3 that is enriched in the PVT [55]. Thus, while PACAP-38 is expressed at higher levels than PACAP-27 in much of the brain [28, 36, 56], it is not surprising that PACAP-27 was observed to be more highly expressed than PACAP-38 in the PVT of both females and males. It should be noted that PACAP-38 did appear to be enriched in fibers in the PVT. While we did not attempt to confirm the origin of these fibers, it is known that soma in several areas of the hypothalamus have moderate-to-high levels of PACAP-38 and also send projections throughout the PVT [16, 56–58]. We speculate that the PACAP-38 that we observed in the PVT represented axons from cells that originated in the hypothalamus.

Another notable finding in this study was that there was a difference in the distribution of neuropeptides across the subregions of the PVT. With male rats previously found to have lower co-labeling of DAPI with PACAP-27 in the anterior compared to posterior portions of the PVT [11], we found here that female rats similarly demonstrated this distribution across the antero-posterior axis of this nucleus. Moreover, as we previously found that male mice have relatively more even distribution of PACAP-27 across the PVT [11], it was notable that we found here that female mice demonstrated higher expression of PACAP in the anterior compared to posterior portions of the PVT. Interestingly, this is in line with the gene expression of PACAP depicted in the Allen Brain Atlas with in situ hybridization, which showed male mice to have slightly greater expression in the anterior compared to posterior PVT [10]. Similar to the greater expression of PACAP in the posterior aspects of the rat PVT, our current results show gene expression of enkephalin and CRF to generally be greater in the posterior compared to anterior half of the rat PVT, which also contrasts somewhat with prior studies. Consistent with our current findings, gene expression of CRF in male mice and peptide levels of CRF in male and female mice were found to show somewhat higher density in the posterior portion of the PVT [10, 59]. Conversely, gene expression of CRF in male and female rats was found using in situ hybridization to have similar levels along the antero-posterior axis [19]. Also, gene expression of enkephalin in male mice, as depicted in the Allen Brain Atlas with in situ hybridization, and peptide levels of enkephalin in male rats, using immunohistochemistry, were shown to be slightly elevated in the anterior compared to posterior half of the PVT [10, 60], in contrast to our current findings using qRT-PCR. It is likely that differences in the sex (female vs. male) and species (rat vs. mouse) of the subjects, as well as the form of the neuropeptide (mRNA vs. peptide) and the method with which it is assessed (in situ hybridization vs. qRT-PCR), account for these discrepancies in the findings. More specifically, we have observed even in our own results here and previously [11] that males and females can show different levels of neuropeptide expression, that neuropeptide distribution across the PVT can be different between mice and rats, and that gene expression does not completely predict peptide measurements, particularly when the assessment is of peptide levels. We believe that the greater differences in PACAP levels with qRT-PCR compared to immunohistochemistry are likely not due to any ability of qRT-PCR to detect PACAP mRNA in processes [61]; rather, they may be due to differences in the regulation of translation [62], stability of the peptide [63], and/or the efficacy of the primers compared to the antibodies. While our current results suggest that peptide labeling in rats of PACAP, enkephalin, and CRF could be greater in cells of the posterior aspects of the PVT, possibly within the same cells, this remains to be determined.

We note here that the effects of PACAP in the PVT on behavior were not tested in this study; however, the PACAP expressed in cells of the PVT likely exerts effects through release into its projection regions, which are known to have major roles in motivated and affective behavior [12, 24, 51]. Exhibiting a high degree of collateralization, major projections from the PVT include the nucleus accumbens shell, dorsolateral BNST, and central nucleus of the amygdala [23, 64]. In the nucleus accumbens shell, injection of PACAP-27 has been found to reduce intermittent-access ethanol drinking in both male and female rats, with females exhibiting a more prolonged reduction than males and a greater sensitivity to a lower concentration of PACAP-27 [8]. While the effects of PACAP-27 have not been studied in the BNST or the central amygdala, PACAP-38 in these regions has been found to have clear interactions with drug self-administration and affective behavior. For example, in the BNST of male rats, levels of PACAP-38 in afferent fibers are increased by acute withdrawal from chronic intermittent ethanol vapor or footshock stress [65, 66], and injection of PACAP-38 promotes cocaine-seeking [67] and increases the acoustic startle response [66], while the PACAP receptor antagonist, PACAP(6–38), blocks dependence-induced excessive ethanol intake and withdrawal-induced anxiety-like behavior in a light-dark box [65]. In the central amygdala of male rats, while levels of PACAP-38 in afferent fibers are not affected by acute withdrawal from chronic intermittent ethanol vapor [65], they are nevertheless increased by footshock stress [66], and injection of PACAP-38 promotes the acoustic startle response [66], anxiety-like behavior in an elevated plus maze [68], and a shift from active to passive coping in a defensive burying test [69]. Moreover, with all PACAP-27⁺ cells in the PVT found to co-label with glutamate [11], it is notable that chemogenetic inhibition of the glutamatergic pathway from the PVT to the BNST has been found to promote ethanol drinking in female but not male mice [7]. Together, these published findings not only suggest that PACAP from the PVT may modulate addictive and affective behavior, with a possible switch from negative to positive feedback as animals develop drug dependence or experience chronic stress [70], but that they could do so to a greater extent in females compared to males. We postulate that, in light of the elevations in PACAP that occur with drug withdrawal and stress, the higher endogenous levels of PACAP in the PVT of females may contribute to their greater susceptibility to drug use and affective disorders [1–3].

5. Conclusions

Our results demonstrate that female mice and rats, compared to male mice and rats, have higher expression of PACAP mRNA and more PACAP peptide positive cells across the PVT. With both the PVT and PACAP implicated in aspects of motivated and affective behavior, it is possible that these observed sex-related differences in PVT PACAP could underly the clinically and preclinically established sex-related differences in motivated and affective behavior. Future studies should directly test the functional relevance of the sexual dimorphism of PACAP in the PVT and determine if, in fact, the greater expression of PACAP in females could explain differences observed in their behavior.

Abbreviations:

BNST

bed nucleus of the stria terminalis

CRF

corticotropin releasing factor

DAPI

4',6-diamidino-2-phenylindole

PAC1

PACAP type 1 receptor

PACAP

pituitary adenylate cyclase-activating polypeptide

PVT

paraventricular nucleus of the thalamus

qRT-PCR

quantitative real-time PCR

VPAC1

vasoactive intestinal polypeptide type 1 receptor

VPAC2

vasoactive intestinal polypeptide type 2 receptor.