Parabrachial nucleus (PBn) pituitary adenylate cyclase activating polypeptide (PACAP) signaling in the amygdala: implication for the sensory and behavioral effects of pain

Abstract

The intricate relationships that associate pain, stress responses and emotional behavior have been well established. Acute stressful situations can decrease nociceptive sensations and conversely, chronic pain can enhance other pain experiences and heighten the emotional and behavioral consequences of stress. Accordingly, chronic pain is comorbid with a number of behavioral disorders including depression, anxiety abnormalities and associated stress-related disorders including post traumatic stress disorder (PTSD). The central nucleus of the amygdala (CeA) represents a convergence of pathways for pain, stress and emotion, and we have identified pituitary adenylate cyclase activating polypeptide (PACAP) immunoreactivity in fiber elements in the lateral capsular division of the CeA (CeLC). The PACAP staining patterns colocalized in part with those for calcitonin gene related peptide (CGRP); anterograde fiber tracing and excitotoxic lesion studies demonstrated that the CeLC PACAP/CGRP immunoreactivities represented sensory fiber projections from the lateral parabrachial nucleus (LPBn) along the spino-parabrachioamygdaloid tract. The same PBn PACAP/CGRP fiber system also projected to the BNST. As in the BNST, CeA PACAP signaling increased anxiety-like behaviors accompanied by weight loss and decreased feeding. But in addition to heightened anxiety-like responses, CeA PACAP signaling also altered nociception as reflected by decreased latency and threshold responses in thermal and mechanical sensitivity tests, respectively. From PACAP expression in major pain pathways, the current observations are novel and suggest that CeA PACAP nociceptive signaling and resulting neuroplasticity via the spino-parabrachioamygdaloid tract may represent mechanisms that associate chronic pain with sensory hypersensitivity, fear memory consolidation and severe behavioral disorders.

Keywords: CGRP; Central amygdala; Hypersensitivity; Lateral capsular division; PACAP; Parabrachial nucleus; Parabrachialamygdaloid tract.

Figure 1

PACAP and CRH immunoreactivities are differentially distributed and regulated in the CeA. Tissue sections from control (A) and chronically stressed (B) rats were examined for CeA PACAP (Cy2, green) and CRH (Cy3, red) staining patterns. In both groups, CeA fiber PACAP immunoreactivity was predominantly in the lateral capsular region (CeLC) with diffuse staining extending into the lateral division (CeL); CRH immunoreactivity was localized predominantly to the CeL. From quantitative image analyses, only CRH immunoreactivity was augmented by chronic variate stress (C, n = 3). These results complemented quantitative PCR measurements which also demonstrated increased CRH transcript expression after stress (D, n = 6). Data represent mean ∀ SEM. Asterisk, significantly different from control at p < 0.05. Scale bar, 250 :m

Figure 2

PACAP and CGRP immunoreactivities can be colocalized in the CeLC and BNST. Tissue sections for the amygdala (A - C) and BNST (D - F) were processed for dual PACAP (Cy3, red) and CGRP (Alexa488, green) immunocytochemical localization. The merged micrographs demonstrate that in both regions, PACAP and CGRP immunoreactivities were largely colocalized (yellow) in the same fiber structures. Amygdala, representative micrograph from 4 independent experiments; BNST, representative micrograph from 3 experiments. LV, lateral ventricle; CPu, caudate-putamen. Correlation coefficients described in text. Scale bar, 200 :m for corresponding tissues.

Figure 3

PBn projection fibers to the CeLC demonstrate PACAP immunoreactivity. Biotinylated dextran amine (BDA, 10 kD; 10%) was injected iontophoretically into the LPBn for anterograde transport into the CeLC over 14 days. BDA at the LPBn injection site (A) and in the projection fibers to the CeLC (B) were detected using streptavidin-conjugated Cy2 (green). Processing of the same CeLC sections for PACAP immunoreactivity (Cy3, red) demonstrated that the LPBn projection fibers can contain PACAP (B, merge in yellow). Representative data from 3 separate preparations. scp, superior cerebellar peduncle. Scale bar, 200 :m for corresponding tissues.

Figure 4

Excitotoxic LPBn lesions diminish PACAP and CGRP fiber immunoreactivities in the CeLC and BNST. The LPBn was unilaterally lesioned with NMDA as described in Methods; the contralateral LPBn received vehicle. After 7 days, the PBn sections were processed for neuron-specific nuclear NeuN immunoreactivity (Cy3, red) to assess the specificity and extent of the lesion. Whereas vehicle injections had no apparent effects (A), NMDA injections produced substantial LPBn neuronal loss (B, dashed circled area). Representative vehicle treated and contralateral NMDA excitotoxic lesioned PBn in the same animal are shown; the lesioned image was flipped to facilitate comparison. CeA and BNST tissue sections from the NMDA excitotoxic lesioned animals were processed for dual PACAP and CGRP immunocytochemical localizations. Similar to Figure 2, tissue sections ipsilateral to LPBn - vehicle injections (left panels) demonstrated substantial PACAP (Cy3, red) and CGRP (AlexaFluor 488, green) colocalization in the CeLC (C and E) and BNST (G and I); colocalization in merged micrographs illustrated in yellow. By contrast, the same CeLC and BNST regions in the contralateral half that received LPBn NMDA excitotoxic lesion (PBn - lesion) demonstrated marked decreases in both PACAP and CGRP immunoreactivities. Again, micrographs from the stained CeLC and BNST regions from the PBn - lesioned side were flipped for comparisons with the control vehicle -injected side from the same animals to facilitate comparisons. These data were consistent with the colocalization of PACAP and CGRP in Figure 2. scp, superior cerebellar peduncle; LV, lateral ventricles; CPu, caudate putamen. Representative figures from 3 separate animals. Scale bar, 200 :m in corresponding tissues.

Figure 5

CeA and BNST peptide immunoreactivities are diminished after PBn lesions. PACAP and CGRP immunoreactivities in the CeA (A) and BNST (B) from studies described in Figure 4 were subjected to image analyses as described in Methods. The PBn lesions decreased PACAP and CGRP immunoreactivities in the limbic regions to a comparable extent compared to levels on the contralateral hemisphere with PBn - vehicle injections. n = 3, data represent mean ∀ SEM. *, different from vehicle control at p < 0.05.

Figure 6

PACAP infusions into the CeA decrease open arm entries on the elevated plus maze. Adult rats were cannulated as described in Methods for CeA PACAP infusions. Thirty minutes after PACAP injection, the animals were placed in the center square of the elevated plus maze, facing a closed arm, for behavior testing during a 5 min period. All movements were tracked digitally for data analyses. Total open arm entries (A) and open arm preference (B, open arm entries/total arm entries) were calculated. CeA PACAP signaling significantly increased anxiety-like behavior reflected by decreased number of open arm entries and open arm preference. There were no changes in the number of closed arm entries and there were no indications of freezing behaviors. n = 10 per group, data represent mean ∀ SEM, *, different from vehicle control p < 0.05.

Figure 7

CeA PACAP/PAC1 receptor signaling increases thermal sensitivity. A, Rats were habituated in Hargreave's thermal sensitivity apparatus with 2 days of baseline assessments (24 and 48 h). PACAP was subsequently infused into the CeA (single injection) for thermal testing at the indicated time (shaded area). Whereas vehicle injection produced no apparent responses changes compared to baseline (white bars), CeA PACAP infusions consistently decreased thermal latency responses (black bars) up to 4 h post treatment. The responses dissipated by 24 h; the small but significant decrease in thermal latency at 72 h may reflect latent plasticity events. n = 6 - 8 per group, data represent mean response ∀ SEM, *, different from corresponding vehicle control, p < 0.025. B, the PACAP-induced decrease in thermal latency was mirrored in CeA infusions with the PAC1 receptor specific agonist maxadilan. The maxadilan responses observed at 1 h was again dissipated by 24 h. n = 7 - 8 per group, data represent mean response ∀ SEM, *, different from corresponding vehicle control, p = 0.002.