NADPH diaphorase histochemistry in the thoracic ganglia of locusts, crickets, and cockroaches: species differences and the impact of fixation

Abstract

The NADPH diaphorase (NADPHd) reaction is widely used as a histochemical marker for nitric oxide synthase (NOS). In this study on locusts, crickets, and cockroaches, we demonstrate 1) that related species can differ considerably in the fixation sensitivity of putatively NOS-related NADPHd; and 2) that prolonged fixation can induce NADPHd activity in cells that are diaphorase negative under mild fixation regimes. These two phenomena reconcile previous, contradictory reports on the distribution of NADPHd in locusts and crickets. In locusts, neuronal NADPHd is found exclusively in interneurones. The projection neuropiles of the exteroceptors contain a dense NADPHd-positive fibre meshwork, but sensory afferents do not stain. In crickets, staining has been reported in sensory afferents, in motor neurones and dorsal unpaired median (DUM) neurones, and in a non-fibrous distribution throughout the sensory neuropiles. We demonstrate that this widespread, non-selective staining is induced by strong formaldehyde fixation. Weak fixation resulted in a highly selective labelling of a few individual interneurones and of a fibre meshwork in the projection neuropiles of the exteroceptive afferents. Staining was absent in the afferents themselves, in motor neurones, and in efferent DUM neurones. Thus, after weak fixation, the staining pattern closely matched that in the locust. The similar distribution of putatively NOS-related NADPHd in the thoracic nervous systems of orthopteroid insects suggests a species-independent role for nitric oxide in the processing of mechanosensory information. Histopharmacological techniques such as permanganate oxidation, or incubation in the NOS inhibitors methylene blue or dichlorophenolindophenol, did not allow discrimination between the selective and the fixation-induced staining. The species-specific impact of different fixation regimes may necessitate reconsideration of results obtained in other cross-species comparisons.