The increase in discharge activity of locus coeruleus (LC) neurons following precipitated opiate withdrawal has been reported to be caused, in part, by excitatory amino acid release most likely originating from the nucleus paragigantocellularis lateralis (PGCl) in the rostral ventral medulla. Activation of glutamate-containing neurons in the PGCl may depend on changes in the occupancy of opioid receptive sites located on LC-projecting neurons which subsequently effect excitatory amino acid release in the LC during opiate withdrawal. To determine whether the mu-opioid receptor (MOR) is localized to plasmalemmal sites of LC-projecting neurons in the PGCl, we combined retrograde transport of the protein-gold tracer, wheat germ agglutinin-conjugated to inactive horseradish peroxidase (WGA-AU-apoHRP), from the LC with immunocytochemical detection of MOR in the same section of tissue throughout the rostral medulla. Light microscopic analysis indicated that neurons containing either the retrograde tracer or immunoperoxidase labeling for the MOR were numerous throughout the ventral medulla and that individual PGCl neurons contained both WGA-Au-apoHRP as well as MOR. By electron microscopy, WGA-Au-apoHRP was commonly identified in lysosomes within somata and large proximal dendrites. The somata contained either spherical or invaginated nuclei and were often surrounded by numerous myelinated axons. Gold deposits could also be identified in the cytoplasm of smaller dendritic processes in the PGCl, although these were not necessarily associated with lysosomes. The smaller dendritic processes were often the target of afferent input by axon terminals containing heterogeneous types of synaptic vesicles. Of 150 cellular profiles exhibiting WGA-Au-apoHRP retrograde labeling, 31% contained immunoperoxidase labeling for MOR. These results indicate that the MOR is distributed along plasmalemmal sites of morphologically diverse neurons in the PGCl which project to the LC.
GABA is a major inhibitory neurotransmitter in molluscs and other animals, but has not been well studied in bivalves. In several bivalves, including Crassostrea virginica, beating of gill lateral cilia is controlled by a reciprocal serotonergic‐dopaminergic innervation from their ganglia. Serotonin (HT) increases beating rates, dopamine slows it down. GABA has no direct effects on cilia beating in C. virginica, however, GABA blocked the cilio‐excitation of HT when both are applied to the cerebral ganglia. We hypothesize cerebral and visceral ganglia contain GABA neurons and the cilio‐excitatory HT neurons in the ganglia contain inhibitory GABA receptors. We used immunofluorescence histochemistry to visualize GABA receptors and HT neurons in cerebral and visceral ganglia. Tissues were dissected, snap frozen, sectioned on a cryostat, fixed with paraformaldehyde, treated with blockers, incubated with 1̊ and 2̊ antibodies and viewed with a fluorescence microscope with FITC and Texas Red filters. We found cerebral and visceral ganglia contained GABA neurons and some HT neurons had GABA receptors on their soma. This study confirms our earlier pharmacological studies indicating a role for GABA in this bivalve, working centrally as an inhibitory ganglionic neurotransmitter to inhibit cilio‐excitatory HT neurons that innervate the gill and helps to explain the ganglionic circuitry by which this could be done.
Grant Funding Source: NIH‐2R25GM0600309, NYSED‐0516041071, NSF‐0622197
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