Previous research suggested that α2A and α2C adrenergic receptor (AR) subtypes

Previous research suggested that α2A and α2C adrenergic receptor (AR) subtypes have overlapping but unique physiological roles in neuronal signaling; however the basis for these dissimilarities is not completely known. α2CARs had relocated to somatodendritic and axonal sites and unlike α2AARs co-localized with SV2 at synaptic contact sites. Consistent with a functional role for α2ARs we also observed that dexmedetomidine stimulation of cultured SGN more efficiently inhibited depolarization-induced calcium entry into older compared to younger cultures. These results provide direct evidence of distinct developmental patterns of endogenous α2A CZC24832 and α2CAR targeting and function in a native cell system and that maturation of SGN in culture leads to alterations in neuronal properties required for proper targeting. More importantly the co-localization at Day 16 of α2CARs at sites of synaptic contact may partially explain the differential modulation of neurotransmitter release and responsiveness to action potential frequency observed between RFC37 α2A and α2CARs in SGN. ≤ 0.05. CZC24832 3 Results 3.1 Characterization of superior cervical sympathetic ganglion cultures Sympathetic ganglion neuron cultures were prepared from 1-3-day-old mice. Identification of neurons in these different cultures was determined using a neuronal-specific antibody recognizing MAP2 which specifically recognizes neuronal cell bodies and dendrites (e.g. somatodendritic) (Fig. 1A). The presence of noradrenergic neurons was verified by indirect immunocytochemistry using dopamine b-hydroxylase (DBH) a catecholamine biosynthesis enzyme (Fig. 1B). MAP2 staining was restricted to the neuronal somatodendritic region whereas DBH staining appeared more diffusely throughout dendrites and axons with some localized accumulation of enzyme within punctate vesicles. Fig. 1 Characterization of superior cervical sympathetic ganglion cultures by indirect immunofluorescence. SGN were obtained from wild-type mice and cultured on glass coverslips for 7 days and analyzed by indirect immunofluorescence microscopy as outlined in … Affinity purified rabbit polyclonal antisera recognizing the intracellular carboxyl termini of either α2A (C10 antibody) or α2CARs (C4 antibody) (Daunt et al. 1997 were used to examine endogenous expression and distribution of these receptor subtypes in cultured SGN. To verify antibody sensitivity and specificity 4 SGN cultures obtained from WT α2AARKO and α2CARKO mice were examined (Fig. 2). Neurons were co-labeled with either α2A- or α2CAR and MAP2 antisera. Fig. 2 Antibody specificity for endogenously expressed α2A and α2CARs in SGN. SGN were obtained from WT (A C E G) α2AARKO (B F) and α2CARKO (D H) mice. SGN were cultured for 4 days and analyzed by indirect immunofluorescence … MAP2 antibody labeled comparable intracellular somatodendritic regions of SGN cultured from all three mouse lines and can be used to distinguish dendrites from axons (Fig. 2A-D). α2AAR antibody labeling of WT SGN revealed predominantly membrane staining of somatodendritic regions and axons (Fig. 2E). In contrast it did not display specific staining of α2AARKO mouse SGN (Fig. 2F) consistent with the loss of CZC24832 α2AAR expression in these knockout mice. Immunocytochemical staining with α2CAR specific antisera revealed a different membrane localization pattern. α2CAR antisera labeling of WT SGN revealed restricted intracellular vesicular staining that was confined to the somatodendritic region of the neuron (Fig. 2G). As expected cultured SGN obtained from α2CARKO mice did not display specific staining with α2CAR antisera (Fig. 2H). No expression of α2A or α2CARs was detected in fibroblasts or glial cells (data not shown). These data demonstrate that both C10 and C4 antisera can specifically identify endogenously expressed α2A and α2CARs CZC24832 respectively. 3.2 Differential localization of α2A and α2CARs during maturation of cultured SGN Since preliminary immunohistochemical staining of endogenous α2A and α2CARs in cultured SGN suggested differential localization and targeting we next initiated a more detailed examination during CZC24832 maturation of SGN cultures. Thus membrane targeting and distribution of α2A and α2CARs in SGN isolated from WT mice were examined at 1 4 8 and 16 days in culture using confocal microscopy (Figs. 3 and ?and4).4). To allow for direct comparisons immunoctyochemical staining with α2A and α2CAR antisera and MAP2 antibody was done in parallel SGN cultures to eliminate possible culture artifacts. Fig. 3 Time course of differential localization of α2AARs in cultured SGN. SGN were obtained from WT.