The potential at a carbon-fiber electrode was held at ?0
The potential at a carbon-fiber electrode was held at ?0.4 V versus Ag/AgCl, ramped to +1.3 V and back to ?0.4 V (400V/s) every 100 ms. this effect without recovery for at least 90 days. This loss of CRFs capacity to regulate dopamine release in the nucleus accumbens is accompanied by a switch in the reaction to CRF from appetitive to aversive, indicating a diametric change in the emotional response to acute stressors. Thus, the current findings offer a biological substrate for the switch in affect which is central to stress-induced depressive disorders. CRF initiates neuroendocrine signaling in the hypothalamic-pituitary-adrenal axis, and also regulates neurotransmission directly via two receptor subtypes, CRF R1 and CRF R2, which 3-Methylglutaric acid are distributed widely throughout the brain7,8. In the nucleus accumbens, CRF facilitates cue-elicited motivation9 and social bonding10, behaviors thought to be mediated by dopamine transmission11,12. Therefore, we sought evidence for CRF-dopamine interactions in the nucleus accumbens, first using fluorescent immunohistochemistry. Dense CRF immunoreactivity was present throughout the rostro-caudal axis of the nucleus accumbens core and lateral shell and in the most rostral portion of the medial shell in sparsely located large cell bodies (cholinergic interneurons, see Supplementary Fig. 1) and fiber terminals that were interdigitated with tyrosine-hydroxylase (TH) immunoreactive fibers that are indicative of dopamine-containing axons (Fig. 1a). Immunoreactivity for the CRF R1 receptor displayed punctate staining with co-localization of TH immunoreactivity on fiber segments in addition to localization on cell bodies within the nucleus accumbens (Fig. 1b and Supplementary Fig. 2). CRF R2 immunoreactivity had a more diffuse, but still, punctate pattern of staining, similar to that in other regions13, with some co-localization with Rabbit Polyclonal to GPRIN1 TH-immunoreactivity (Fig. 1c and Supplementary Fig. 3). Expression of CRF receptors on subcellular profiles in the nucleus accumbens, including TH-positive terminals, was confirmed at higher spatial resolution using transmission electron microscopy (Fig. 1d; quantified in Supplementary Table 1). Together, these data indicate that the localization of 3-Methylglutaric acid CRF and its receptors in the nucleus accumbens is well suited for modulation of dopamine release. Open in a separate window Figure 1 Cellular localization of CRF peptide, CRF R1 and CRF R2 in the nucleus accumbensa, Immunoreactivity for CRF peptide (top), CRF R1 (middle) or CRF R2 (bottom) is shown in red and for tyrosine hydroxylase (TH) is shown in green. The arrows highlight examples of co-localization (yellow in the merged images). Scale bar = 10 m. b, Transmission electron microscopy photomicrographs demonstrating CRF receptors (labeled with immunogold particles; arrows) present on both TH positive (immunoperoxidase labeled) and TH negative profiles. Top scale bar = 0.5 m; bottom scale bars = 1 m. To directly test the functional effects of CRF on dopamine release in the nucleus accumbens, we selectively monitored dopamine release evoked by a single biphasic electrical pulse (2 ms/phase, 100-500 A delivered once per minute) in acute coronal brain slices using fast-scan cyclic voltammetry at carbon-fiber microelectrodes (Fig. 2a and Supplementary Fig. 4). Vehicle or CRF (10, 100 or 1000 nM) was applied to the slice for 15 minutes following five minutes of 3-Methylglutaric acid stable baseline and the resultant effect was quantified by averaging the evoked dopamine current in the last ten minutes. Following application of vehicle, there was a modest decrease (~7 %) in dopamine release (Fig. 2b), whereas CRF increased dopamine 3-Methylglutaric acid release in a concentration-dependent manner eliciting effects significantly greater than vehicle at 100 and 1000 nM (27.8 6.7 and 30.0 8.4 % respectively, mean s.e.m.; F3, 49 = 5.026, p < 0.01, one-way ANOVA with Dunnetts post-hoc t-tests; Fig. 2b and Supplementary Fig. 5). Interestingly, this 3-Methylglutaric acid effect could be blocked by application of either the selective CRF R1 antagonist, antalarmin (1 M), or the selective CRF R2 antagonist, anti-sauvagine 30 (ASVG 30; 250 nM) to the slice beginning 20 minutes before CRF application (F2, 50 = 5.142, p < 0.01, one-way ANOVA with Dunnetts post-hoc t-tests; Fig. 2c) indicating that co-activation of both receptors is required. Consistently, CRF (10, 100, 1000 nM) failed to increase dopamine release in the nucleus accumbens of mice with deletion of either the CRF R114 or R215 gene (Fig. 2d). Application of the selective CRF R1 agonist Stressin 1 (100 or 300 nM) or the selective CRF R2 agonist Urocortin 3 (100 or 300 nM) failed to significantly increase dopamine release when applied individually (p > 0.05 compared to respective vehicles; Fig. 2e and f), but significantly increased dopamine release when co-applied (F3,36 = 3.528, p < 0.05 vs vehicle, one-way ANOVA with Dunnetts post-hoc t-tests). The.