Research reportColocalization of NADPH-diaphorase and GABA-immunoreactivity in the olfactory and visual system of the locust
Introduction
Nitric oxide (NO) is a short lived signalling molecule which readily diffuses across membranes. In the nervous system, nitric oxide is generated by Ca2+/calmodulin stimulated NO synthase (NOS). A major signal transduction pathway for NO in target cells is stimulation of the soluble form of guanylyl cyclase (sGC) leading to cyclic GMP (cGMP) formation, although other signal transduction mechanisms are possible (reviewed in 9, 18). NO mediated signalling in the nervous system is not only common to vertebrates but appears to be evolutionary conserved across many phyla. Meanwhile, the presence of NOS or NOS-related markers has been reported in the major phyla ranging from lower invertebrates such as coelenterates to arthopods, molluscs, and echinoderms 3, 11, 12, 13, 17, 19, 22, 25, 28, 30, 38.
The locust is a useful invertebrate preparation for the cellular analysis of information processing in the nervous system [10]. There is compelling evidence that the nervous system of the locust employs NO/cGMP signalling. The presence of a Ca2+/calmodulin activated NOS and NO activated guanylyl cyclase has been demonstrated in homogenates of the locust brain [13]. In addition, the biochemical properties of NOS have been characterized in various areas of the nervous system showing that NOS activity and NADPH-diaphorase (NADPHd) activity after fixation are caused by identical enzymes. Measurements of NOS activity in homogenates of the various neuropils of locusts correlated well with the histochemical staining pattern for NADPHd 5, 14, 31. A rather high portion of NADPHd positive neurons in olfactory pathways allowed the chemical detection of a Ca2+-activated NO release in dissociated cell cultures prepared from these brains [31]. As in mammals the NOS expressing cells of insect nervous systems can be identified reliably by NADPHd histochemistry. Potential NO synthesizing neurons of the brain and the ventral nerve cord have been described by NADPHd staining [31]. The histochemical survey of the CNS revealed less than 1% NADPHd positive cells in the ventral nerve cord. The most striking neuronal NADPHd activity is concentrated in a cluster of about 45–50 intensely stained local interneurons 14, 31of the antennal lobe, the principal olfactory neuropil of the insect brain. These interneurons appear to innervate all glomeruli. The conventional transmitters of these neurons remain, however, unknown.
An electron microscopical study of the synaptology of olfactory pathways has demonstrated GABA-immunoreactivity (GABA-IR) in local interneurons of the antennal lobe [23]. This finding provided a rationale to check for a colocalization of the NO and the GABA transmitter systems in the olfactory pathways of an insect brain. Moreover, since two recent investigations 7, 15have described NADPHd activity in identifyable neuronal populations of the optic lobes, we have included the visual system in the search for neurons coexpressing NADPHd and GABA-IR. Thus, in this paper we compare the anatomical relations between putative NO and GABA releasing neurons in neuropils processing the two most important sensory modalities of the insect brain.
Section snippets
Animals
The experiments were performed on adult Locusta migratoria reared in a crowded colony by H.-J. Pflüger's research group at our institute. Cytochemical stainings performed in parallel on Schistocerca gregaria revealed no staining differences between the species.
Double labelling exeriments
After decapitation the brains were dissected out of the head capsule and fixed for 1.5 h in 4% formaldehyde and 0.1% glutaraldehyde in phosphate-buffered saline (PBS, pH 7.4) at 4°C. After washing several times in PBS the brains were
Olfactory system
The antennal lobes are the primary integration centers of the olfactory pathway. Sites of synaptic contacts between receptor terminals, local interneurons, and projection neurons are arranged as approximately 1000 spherical glomeruli [23]. The projection neurons send their axons via the tractus olfactorio-globularis [42], also termed antennoglomerular tract [23]to the calyx of the mushroom body. All glomeruli of the antennal lobe neuropil express GABA-IR (Fig. 1a). Since the antennoglomerular
Olfactory system
This paper demonstrates that the NADPHd positive cells of the antennal lobe form a subset of the GABA-IR local interneurons (Fig. 1). An electronmicroscopic analysis of the antennal lobe resolved GABA-IR in presynaptic terminals contacting immunonegative but also immunopositive fiber profiles [23]. This finding indicates that GABAergic local interneurons of the antennal lobe are not only linked to projection neurons and sensory afferents but also form connections to each other. In addition to
Acknowledgements
We wish to thank Florine Knolle for photographic assistance. This work was supported by grants of the Deutsche Forschungsgesellschaft to G.B. and the Graduiertenkolleg: Signalketten in lebenden Systemen.
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