Chronic stress selectively augmented PACAP expression 10-fold in the dorsal aspect of the anterolateral BNST when compared with non-stressed rats. PACAP transcript was also reliably elevated in the PVN, but did not differ from non stressed rats in other stress-associated brain regions including the basolateral amygdala, central amygdala, locus coeruleus, hippocampus, or ventral aspect of the BNST. Chronic stress also produced an anxiogenic profile on baseline responding in the light-enhanced startle test (see description below), and intra-BNST infusion of PACAP was anxiogenic in a dose-dependent manner that persisted for at least 7 days following PACAP infusion. These data suggest that chronic stress may selectively alter BNST function via a PACAP-dependent mechanism to produce enhanced anxiety; hence, the BNST PACAP system may represent an important therapeutic target for the treatment of anxiety disorders.
As mentioned above, the behavioral and catabolic responses to stressful stimuli are all coordinated by CNS nuclei such as the BNST, and the sustained activation of these regions has been argued to lead to maladaptive changes underlying pathological states (Schulkin, et al. 1998
; Vyas, et al. 2003
; Pego, et al. 2008
). Hence, chronic stress may facilitate fear and anxiety disorders by promoting neuronal plasticity and/or remodeling within the BNST. Chronic stress has been shown to increase dendritic arborization in the BNST (Vyas, et al. 2003
; Pego, et al. 2008
); however, the mechanisms underlying this plasticity are still not well understood, but may include PACAP signaling. PACAP has well established trophic fiber outgrowth functions during development and in culture paradigms (Wolf and Krieglstein 1995
; Gonzalez, et al. 1997
; Lu and DiCicco-Bloom 1997
; Lioudyno, et al. 1998
; Nielsen, et al. 2004
; Falluel-Morel, et al. 2005
; Braas, et al. 2007
), and PAC1
receptor-mediated activation of MAPK signaling pathways in the BNST may mediate the observed dendritic remodeling. Alternatively, PACAP has been shown to stimulate both BDNF and TrkB expression in central and peripheral neurons (Lu and DiCicco-Bloom 1997
; Yaka, et al. 2003
; Braas, et al. 2007
); hence the changes in BNST neuronal cytoarchitecture (Vyas, et al. 2003
; Pego, et al. 2008
) and functional plasticity may be dependent on PACAP-stimulated BDNF and TrkB expression and neurotrophic signaling. Indeed, in addition to increasing PACAP transcript, chronic stress was associated with increased BDNF and TrKB expression in the BNST. Many studies have suggested that altered BDNF and TrkB expression may be related to behavioral disorders (Martinowich, et al. 2007
); hence, the integrated effects of PACAP, BDNF and TrkB expression and function, through coordinated or sequential gene regulatory events, or via G-protein receptor crosstalk with Trk signaling, may enhance BNST plasticity and function to affect behavior. Regardless of mechanism, these studies show that altered PACAP expression and signaling in the BNST may mediate anxiety-related disorders after chronic stress.
Hypothalamic and extrahypothalamic CRH have been shown to mediate the endocrine, sympathetic, and behavioral stress response. Importantly, chronic stress has been shown to increase BNST CRH, and this increase has been argued to mediate an increase in anxiety-like behavior (Stout, et al. 2000
). PACAP-immunoreactive fibers have been suggested to heavily innervate hypothalamic CRH neurons in the PVN and extrahypothalamic CRH neurons in the BNST (Kozicz, et al. 1997
; Legradi, et al. 1998
). PACAP expression in these two brain regions was elevated after chronic stress in the present study, and BNST PACAP injection enhanced baseline startle. CRH injection also has been shown to enhance baseline startle (Lee and Davis 1997
); hence, PACAP may mediate increased plasticity and function specifically within BNST CRH neurons. The endogenous sources of the PACAP-containing axons in the BNST appear to originate in part from PACAP neurons in the PVN (Kozicz, et al. 1998
). As specific regions of the BNST also project to the PVN, these results also support the suggestion that the hypothalamus and BNST may have reciprocal regulatory functions (Herman, et al. 2005
). The observation that both PACAP and PAC1
receptor transcripts are increased in the same dBNST tissues suggests that local PACAP circuits within the BNST may be activated by chronic stressor exposure. However, as PVN PACAP mRNA was also increased by the same treatment, and the PVN projects to the BNST, hypothalamic PACAP afferents may have also contributed to the stress-induced increase in the mRNA for these peptides.
Studies examining the role of PACAP mediating the effects of stress have been limited. Early studies showed that some stressors increased PACAP mRNA expression in the hypothalamus while other stressors did not (Hannibal, et al. 1995
). Intraventricular or PVN infusion of PACAP increased plasma corticosterone levels, stimulated hypothalamic c-fos and phosphorylated CREB immunoreactivity, increased PVN CRH mRNA levels, and altered grooming behavior and motor activity (Agarwal, et al. 2005
; Norrholm, et al. 2005
). Moreover, PAC1
receptor knockout mice demonstrated abnormal social and sexual behavior associated with pheromone processing (Nicot, et al. 2004
) and impaired contextual fear conditioning (Sauvage, et al. 2000
; Otto, et al. 2001a
). Consistent with an anxiogenic role, PACAP and PAC1
receptor null mice demonstrate reduced anxiety-like behavior across several tests; null mice spent more time in the middle of an open field, more time in the open arms of the elevated-plus maze, showed a shorter latency to emerge from a covered cylinder, and increased exploration of novel objects (Hashimoto, et al. 2001
; Otto, et al. 2001b
; Girard, et al. 2006
). Despite the anxiolytic effects observed in these knockout mice, the neuroanatomical mechanisms of PACAP action on anxiety-like behavior have not been identified.
The data presented here suggest that a primary locus for PACAP signaling in anxiety-like behavioral responding to stressor exposure is the BNST. This complicated brain region has been divided into as many as 30 subdivisions based on cytoarchitecture, chemoarchitecture and connectivity (Dong, et al. 2001
). Interestingly, PACAP fibers target the dorsal aspect of the anterolateral BNST (Kozicz, et al. 1997
), and this subdivision has been associated with anxiety-like responding (Walker, et al. 2003
). While PACAP infusion into the anterolateral BNST was anxiogenic, this effect may have been mediated by spread into surrounding areas; however, chronic stress selectively increased PACAP transcript in this BNST subregion, suggesting that the dorsal anterolateral BNST mediates the anxiogenic effects of PACAP. Interestingly, chronic stress did not increase PACAP transcript in the anatomically-related central nucleus of the amygdala (CeA) or the basolateral amygdala (BLA). Connections between the BLA and CeA have been argued to mediate behavioral responding to threatening stimuli that are predictable and of short duration, whereas the BNST has been argued to mediate a more maladaptive response to unpredictable long-duration threat (Walker, et al. 2003
). Hence, the prevention of chronic stress-induced increases in PACAP could selectively prevent the formation of pathological anxiety-like state, while leaving adaptive fear-responding intact, unlike many present pharmaco-therapies that modulate activity in both pathways.
While intra-BNST PACAP injection increased baseline startle amplitude, an anxiogenic behavioral change after chronic stress was only observed using the light-enhanced startle paradigm. Importantly, in this paradigm, chronic stress exaggerated startle responding in response to both light exposure and the incidental handling that occurred between experimental phases. These observations may suggest some complementary but mechanistic differences between the acute and chronic anxiogenic effects of PACAP in the BNST. The anxiogenic effects of acute PACAP infusion at relatively high concentrations may be mediated by rapid PAC1
receptor-dependent activation of BNST neurons. In contrast, the chronic effects of stress are likely associated with enhanced BNST neuroplasticity, which may be related to long-term induction of BNST PACAP expression and signaling. In fact, BNST neuroplasticity may be the mechanism underlying the persistent anxiety-like behavior 7 days after PACAP infusion. In these latter cases, evoked BNST stimulation, through incidental handling or light exposure, may be a necessary requisite to observe increases in anxiety-like behavior. In fact, animals were handled extensively prior to testing in both experiments, when PACAP was infused into the BNST and also in the light-enhanced startle paradigm following chronic stress. Hence, the anxiogenic effects of handling-enhanced startle were potentiated by both treatments. As previously reported (Walker et al., 1997
), animal handling can enhance startle amplitude, and this anxiogenic effect is dependent on BNST activity. While it would be interesting to determine the effects of BNST PACAP injection in a light-enhanced startle paradigm, the large changes in baseline startle produced by BNST PACAP would make such an experiment difficult to interpret. Although other experiments may clarify these observations, the current studies nevertheless demonstrate that PACAP signaling and stress are behaviorally relevant and may share common pathways.
Interestingly, BDNF transcript was also reliably elevated in the dorsal raphe nucleus (DRN) following chronic stress. The activation of specific populations of DRN serotonergic neurons has also been argued to mediate anxiety-like responding (Lowry, et al. 2005
), which may be mediated via projections from the DRN to the BNST (Phelix, et al. 1992
; Commons, et al. 2003
). Indeed, the behavioral changes associated with learned helplessness, which has been argued to model human anxiety disorders such as post-traumatic stress disorder (PTSD), are blocked by the inactivation of either the DRN or BNST (Maier, et al. 1993
; Hammack, et al. 2004
). Moreover, PACAP-containing neurons within the DRN target the dBNST, and represent an important source of PACAP input (Kozicz, et al. 1998
). Hence, chronic stress-mediated changes in raphe BDNF expression may alter DRN morphology, function and synaptic connectivity within the DRN - dBNST pathway to mediate some anxiogenic effects of stressor exposure.
These data suggest that chronic stress dramatically increases BNST PACAP and is anxiogenic. Moreover, BNST PACAP produces a sustained increase in anxiety-like behavior. Hence, BNST PACAP may mediate the anxiogenic effects of chronic stress. We suggest that BNST PACAP may mediate stress-induced BNST neuroplasticity, and that this mechanism may underlie some human anxiety disorders. Because chronic stress seems to selectively increase PACAP within the BNST, BNST PACAP signaling may represent an important target for the treatment of anxiety disorders in humans.