AP193B Sigma-AldrichDonkey Anti-Guinea Pig IgG Antibody, biotin conjugate, Species Adsorbed

It has been shown that endomorphin-2-like immunoreactive (EM2-LI) neurons in dorsal root ganglion play important roles in regulating somatic information transmission. Although EM2-ergic neurons have been found in nodose ganglion (NG) which is mainly involved in transmitting visceral information into the nucleus tractus solitarii (NTS), the neurochemical phenotypes of EM2-ergic neurons have not yet been investigated. In the present study, immunofluorescent histochemical staining showed that 43.5% of the NG neurons contained EM2 and these neurons were small to medium in size. 15.2%, 27.8%, 74.4% and 25.2% of the EM2-LI NG neurons expressed substance P (SP), calcitonin gene-related peptide (CGRP), nitric oxide synthase (NOS) and vasoactive intestinal peptide (VIP), respectively. In addition, about 90.8% of EM2-LI NG neurons also contained mu-opioid receptor (MOR). EM2/MOR and EM2/SP double-labeled peripheral axons were observed in the vagal trunk. Anterograde tracing combined with immunofluorescent staining showed EM2/MOR and EM2/SP double-labeled vagal afferents in the NTS. EM2/MOR/SP and EM2/MOR/CGRP triple-labeled neurons and axons were observed in the NG. Importantly, at the ultrastructrual level, post-embedding electron microscopy revealed that EM2-LI and SP-LI gold particles coexisted in the same large dense-cored synaptic vesicles in the pre-synaptic button, while MOR-LI gold particles existed on both pre- and post-synaptic membranes in the NTS. These results suggest that EM2 in axon terminals of NG neurons might be involved in visceral information transmission and homeostatic control through modulating the release of other neurotransmitters (such as SP, CGRP, NO, VIP) via pre-synaptic MOR and through post-synaptic mechanisms in the NTS.

Neostriatal projection neurons are known to be largely divided into two groups, striatoentopeduncular/striatonigral and striatopallidal neurons, which mainly express D1 and D2 dopamine receptors, respectively. Recently, a small population of neostriatal neurons have been reported to produce neurokinin B (NKB), and send their axons mainly to the basal forebrain regions. To reveal which type of dopamine receptors were expressed by these NKB-producing neurons, we examined rat striatal neurons by combining immunofluorescence labeling for preprotachykinin B (PPTB), the precursor of NKB, and fluorescence in situ hybridization labeling for dopamine receptors. Fluorescent signals for D1 receptor mRNA were detected in 85-89% of PPTB-immunopositive neurons in the neostriatum, accumbens nucleus and lateral stripe of the striatum, whereas almost no signal for D2 receptor was observed in PPTB-positive striatal neurons. To further reveal intracellular signaling downstream of D1 receptor in PPTB-producing neurons, we used a double immunofluorescence labeling method to study the localization of some substrates for protein kinase A (PKA), which was known to be activated by D1 receptor. Although only 3-7% of PPTB-immunopositive striatal neurons displayed immunoreactivity for dopamine- and cAMP-regulated phosphoprotein of 32 kDa, a well-known PKA substrate expressed in the two major groups of neostriatal projection neurons, 60-64% of PPTB-positive striatal neurons exhibited immunoreactivity for striatal-enriched tyrosine phosphatase. These results suggest that NKB-producing neostriatal neurons are similar to striatoentopeduncular/striatonigral neurons in the usage of dopamine receptor subtypes, but different from the two major groups of neostriatal projection neurons in terms of the downstream signaling of dopamine receptors.