3C). The protein expression assessed by western blotting of CRF1, pro-relaxin-3, GAD65 and TPH2 was similar among the naïve, saline injected, true sham BMS-907351 price (surgery but no infusion) and sham-lesioned (blank saporin infusion) groups (Fig. 4A and B). However, protein levels of CRF1 receptor was considerably reduced in the NI-lesioned rats. There was also a consistent decrease in the levels of pro-relaxin-3 in the NI-lesioned rats as compared to the sham-lesioned rats (Fig. 4A). The same set of samples

was also checked for GAD65 and TPH2 protein levels. A significant decrease in the expression of GAD65 was also observed in the NI-lesioned rats while TPH2 protein levels remained unchanged. Densitometry analysis of the western blot demonstrated a statistically significant reduction in CRF1 (approximately 54%), pro-relaxin-3 (approximately 53%) and GAD65 (approximately

64%) protein expression (Fig. 4B). Further confirmation of the lesion through immunofluorescence labelling verified the loss of CRF1 receptor positive cells in the NI of the NI-lesioned rats as compared to the sham-lesioned rats (Fig. 5A and B). Similarly, relaxin-3 expressing cells Alectinib order also decreased considerably in the NI-lesioned rats (Fig. 5D). Examination of the TPH2 expression revealed that TPH2 positive cells lined the midline of the NI and were unaffected by the lesioning procedure (Fig. 5E and F). Positive CRF1 staining of the nearby locus coeruleus (LC) in both sham- and NI-lesioned rats was also detected (Fig. 5G and H). Immunostaining for the glial cell marker, glial fibrillary acidic protein (GFAP), showed up-regulation of glial cells in the NI of both sham-lesioned and NI-lesioned rats at 14 days after surgery (Fig. 6). The levels of pro-relaxin-3 in the MS in naïve, saline, true sham, sham- and NI-lesioned rats were assessed by western blotting. A decrease in relaxin-3 levels was Gemcitabine observed in the MS of NI-lesioned rats (Fig. 7A). Densitometry analysis of the blots showed an approximately 90% decrease in

pro-relaxin-3 levels (Fig. 7B). The NI-lesioned and sham-lesioned rats were tested in a cued fear conditioning paradigm. The rats were first exposed to tone-shock pairing and subsequently freezing behaviour in response to the tone was assessed 24 h later. To determine if the NI-lesioned rats had any locomotor deficits, the total distance travelled by the sham- and NI-lesioned rats were measured during the 15 min habituation phase. There was a slight but insignificant decrease in the distance covered by the NI-lesioned rats (Fig. 8A) possibly due to the increased periods of freezing observed during this phase (Fig. 8B). No significant difference between the percentage of freezing between sham- and NI-lesioned rats was observed during the tone-shock training phase (Fig. 8C), for 2 min before (Fig. 8D) and during the 30 s tone at the 24 h test phase (Fig. 8D).