Neuropeptide Y and corticotropin-releasing factor bi-directionally modulate inhibitory synaptic transmission in the bed nucleus of the stria terminalis
Introduction
Anxiety disorders are serious medical conditions that have been estimated to impact approximately 20% of the population (Greenberg et al., 1999). While anxiety is a normal reaction to stress, repeated or severe stressors can produce pathological behaviors such as those seen in post-traumatic stress disorder (PTSD) and generalized anxiety disorder (Shekhar et al., 2005). An understanding of the neuronal and molecular targets associated with these conditions will likely promote more effective treatments. While classical neurotransmitters, such as gamma-aminobutyric acid (GABA) (Nemeroff, 2003) and glutamate (Javitt, 2004), have been implicated in these disorders, another class of signaling compounds, neuropeptides, are also thought to be involved in these conditions (Valdez and Koob, 2004). Neuropeptides are potent neuromodulators in the CNS whose actions are mediated via G-protein coupled receptors (Ludwig and Leng, 2006). In contrast to classical neurotransmitters, they are released in a frequency dependent fashion and often have a longer half-life of activity after release. These factors among others enable neuropeptides to produce long-lasting effects on cellular functions such as excitatory (Acuna-Goycolea et al., 2005) and inhibitory (Nie et al., 2004) synaptic transmission, neuronal excitability (Acuna-Goycolea et al., 2005) and gene transcription (Zhao et al., 2002). Thus, a long-lasting dysregulation of neuropeptides could have significant effects on the activity of neurons and consequentially, behavior.
A large body of literature implicates neuropeptide Y (NPY) as one of several neuropeptides involved in regulation of both anxiety and stress. Early pharmacological studies demonstrated that central infusion of NPY can reduce anxiety-like behaviors (Heilig et al., 1989). Conversely, mice with targeted deletions of NPY (Bannon et al., 2000) exhibit anxiety-like behaviors. Moreover, a recent study conducted in veterans exposed to combat suggested that NPY may play a role as a “stress protective” factor for individuals at risk for PTSD (Yehuda et al., 2006).
In contrast to NPY, the neuropeptide corticotrophin releasing factor (CRF) is thought to be primarily anxiogenic. Central administration of CRF causes an increase in anxiety-like behaviors, as well as activation of the stress response (Campbell et al., 2004). CRF knockout mice have significantly lower levels of corticosterone, both basally and following exposure to acute stress (Dunn and Swiergiel, 1999). Further, elevated CRF levels in the cerebrospinal fluid have been reported in persons diagnosed with PTSD (Bremner et al., 1997).
It has been proposed that dysregulation of NPY and CRF play opposing roles in regulation of anxiety disorders (Shekhar et al., 2005, Valdez and Koob, 2004). To date, however, a common site of opposing action has not been identified in situ. The bed nucleus of the stria terminalis (BNST), a brain region associated with anxiety, has enriched expression of both NPY (Walter et al., 1991) and YRs (Parker and Herzog, 1999), and CRF (Ju and Han, 1989) and CRFRs (Van Pett et al., 2000). The BNST receives a dense GABAergic and CRF input from the central nucleus of the amygdala (CeA) (Sakanaka et al., 1986), suggesting that CRF regulation of function in the BNST is critical for shaping BNST output. In keeping with this, pharmacological studies suggest that CRF signaling in the BNST is involved in anxiety (Lee and Davis, 1997, Sahuque et al., 2006) and stress-induced relapse to cocaine self-administration (Erb and Stewart, 1999). Moreover, a stimulus that promotes anxiogenic responses, the withdrawal of rodents from chronic ethanol exposure, produces rises in extracellular levels of CRF in the BNST (Olive et al., 2002), while acute heroin administration increases NPY immunoreactivity in the region (D'Este et al., 2006).
CRF (Nie et al., 2004) and NPY (Cowley et al., 1999, Pronchuk et al., 2002, Sun et al., 2001) have been shown to modulate GABAergic transmission, albeit in different brain regions. Interactions between these neuropeptides and GABAergic transmission have been proposed to underlie aspects of their regulation of animal behavior (Nie et al., 2004). Thus the BNST, in particular GABAergic transmission within the region, provides a potential anatomic and cellular substrate for interaction of CRF and NPY in regulating stress and anxiety. We have explored this possibility by investigating the actions of CRF and NPY on GABAergic transmission in the ventrolateral region of the BNST (vlBNST). This region projects to both the ventral tegmental area (VTA) (Dumont and Williams, 2004, Georges and Aston-Jones, 2002) and the paraventricular nucleus (PVN) of the hypothalamus (Cullinan et al., 1993), thus providing a point of access to both reward and stress pathways. Functionally the vlBNST is critical both for maintaining inhibitory tone on the PVN (Cullinan et al., 1993) and modulating stress induced relapse to drug seeking behavior (Erb et al., 2001). Given these findings, we reasoned that this would be a relevant region to investigate the actions of NPY and CRF, two neuropeptides known to regulate stress/anxiety responses.
Section snippets
Brain slice preparation
All procedures were performed according to Institutional Animal Care and Use Committee approved procedures. Male C57Bl/6J mice (6–8 weeks old, Jackson Laboratories) were decapitated under anesthesia (Isoflurane). The brains were quickly removed and placed in ice-cold sucrose artificial cerebrospinal fluid (ACSF): (in mM) 194 sucrose, 20 NaCl, 4.4 KCl, 2 CaCl2, 1 MgCl2, 1.2 NaH2PO4, 10.0 glucose, and 26.0 NaHCO3 saturated with 95% O2/5% CO2. Slices 300 μm in thickness were prepared using a Tissue
Results
We examined the effects of both NPY and CRF on inhibitory synaptic transmission in the vlBNST utilizing whole-cell voltage clamp of neurons from acutely prepared brain slices of adult mice. Local stimulation in the BNST produced an eIPSC that was driven by activation of GABAARs, as the selective GABAAR antagonist, SR95531 (GABAzine), completely blocked the response (Fig. 1A). We examined the current-voltage relationship for these eIPSCs (Fig. 1B) and found that the reversal potential was
Discussion
Abundant evidence suggests an antagonistic relationship of NPY and CRF signaling in the regulation of a variety of neuropsychiatric conditions. Here we provide a potential cellular substrate mediating this interaction by showing that NPY and CRF can bi-directionally modulate synaptic inhibition on a common pool of neurons in the BNST, a region critical for regulation of stress, anxiety and addiction. When presented in context of the connectivity of the BNST, these data thus provide at least one
Acknowledgements
This work was funded by INIA-STRESS (NIAAA) and NIDA. We thank Dr. Robert Kesterson for providing us with the CRFR2 knockout animals, and Dr. Robert Matthews for comments on a previous version of the manuscript.
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