Ultrastructural localization of NADPH diaphorase and nitric oxide synthase in the neuropils of the snail CNS

Abstract

This manuscript is contextually identical with the following published paper:For the first time, we have shown that NADPH-diaphorase reactivity and NOS immunoreactivity are bound to transmitter vesicle membranes in varicosities of ganglionic neuropil of an invertebrate. The presence of NOS in certain synaptic configurations of the snail CNS suggests a role of NO in fast and target specific interneuronal signaling processes. 3 ABSTRACTComparative studies on the nervous system revealed that nitric oxide (NO) retains its function through the evolution. In vertebrates NO can act in different ways: it is released solely or as a co-transmitter, released from presynaptic or postsynaptic site, spreads as a volumetric signal or targets synaptic proteins. In invertebrates, however, the possible sites of NO release have not yet been identified. Therefore, in the present study, the subcellular distribution of the NO synthase (NOS) was examined in the central nervous system (CNS) of two gastropod species, the terrestrial snail, Helix pomatia and the pond snail, Lymnaea stagnalis, which are model species in comparative neurobiology. For the visualization of NOS NADPH-diaphorase histochemistry and an immunohistochemical procedure using a universal anti-NOS antibody were applied. At light microscopic level both techniques labeled identical structures in sensory tracts ramifying in the neuropils of central ganglia and cell bodies of the Lymnaea and Helix CNS. At ultrastructural level NADPH-d reactive/NOS-immunoreactive materials were localized on the nuclear envelope and membrane segments of the rough and smooth endoplasmic reticulum, as well as the cell membrane and axolemma of positive perikarya. NADPH-d reactive and NOS-immunoreactive varicosities connected to neighboring neurons with both unspecialized and specialized synaptic contacts. In the varicosities, the majority of the NADPH-d reactive/NOS-immunoreactive membrane segments were detected in round and pleomorph agranular vesicles of small size (50-200 nm). However, only a small portion (16%) of the vesicles displayed the NADPH-d reactivity/NOSimmunoreactivity. No evidence for the postsynaptic location of NOS was found. Our results suggest that the localization of NADPH-diaphorase and NOS is identical in the snail nervous system. In contrast to vertebrates, however, NO seems to act exclusively in an anterograde way possibly released from membrane segments of the presynaptic transmitter vesicle surface. Based on the subcellular distribution of NOS, NO could be both a volume and a synaptic mediator, in addition NO may function as a co-transmitter.

Nitric oxide-coupled signaling in odor elicited molecular events in the olfactory center of the terrestrial snail, Helix pomatia

“…PC [41,72]. These observations, taken together with the findings of other authors [26,40,41,69] and our recent data [27,28] indicate that NO is a prominent neurochemical substance in the olfactory center of terrestrial snails. Applying NO-scavenger, fluorescence microscopy, and flow cytometry, we have provided further evidence that both the NB and B cell types of isolated PC neurons [1] are capable of synthesizing NO.…”

“…2C) was highly similar to that of NOS [27,28]. Interperikaryonal trajectories within the cell mass, the lateral regions of the terminal neuropil area, and the internal neuropil, were labeled by the anti-NMDARζ1 antibody (Fig.…”

“…However, the intensity of labeling was much lower in Helix pomatia than in Lymnaea stagnalis, suggesting difference in the epitope regions of the putative NMDA-receptor in the two species. Second, on PC tissue sections the NMDA-receptor-like protein was localized in the same neuronal PC elements (PC cell surface, trajectories projecting towards the terminal and internal neuropils), where the presence of NOS is also prominent [26,27,28,41,42]. Since NB cells, but not B cells, project to the internal neuropil it is assumed that NMDA-receptor-like immunoreactivity belongs to NB cells in the PC.…”

“…Various types of transmitter-containing vesicles and granules in the axon profiles [22] were also described. The presence of neurotransmitters (glutamate, Glu [and its vesicular transporter, vGluT, 23], GABA [24,25], 5-HT [22], nitric oxide [NO, 26,27,28], acetylcholine [29], histamine [30]), and different neuropeptides [31,32,33,34,35] were demonstrated immunohistochemically. This, together with the findings on their effect following exogenous application [23,29,30,35,36,37,38,39,40], underlined the complex intrinsic and extrinsic regulation of neuronal activity in the PC.…”

“…NOS was localized in the Helix spp. PC [27,28,64,65], with applications of NO inducing reliable increases in cGMP synthesis [66]. Moreover, NOS inhibition was found to impair food-finding behavior, suggesting a role for NO signaling in Helix pomatia olfaction [67].…”