GABA Immunostaining of the Olfactory Pathway in the Heliothine Moth Brain

γ-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in all known nervous systems, and it is widely distributed in the olfactory circuit as well as other parts of the invertebrate brain. In order to further understand the GABAergic olfactory circuitry, the distribution of GABA in the brain of a nuctoid moth, Heliothis virescens, was investigated. Immunohistochemical experiments with an antibody against GABA were performed in order to visualize GABAergic structures and neurons. The stainings revealed strong immunoreactivity in the antennal lobes, the lateral protocerebrum, and the mushroom bodies, all of which are areas involved in olfactory information processing. The dense immunostaining in the lateral protocerebrum included a brightly immunoreactive cell cluster linked to processes innervating the mushroom bodies; this likely corresponds to the protocerebral calycal tract described in other species. Of the main antenno-protocerebral tracts, fibers in the medio-lateral showed strong immunoreactivity. However, double labeling of two intracellularly pre-stained projection neurons passing in this tract showed that not all fibers therein are GABAergic. Looking at the projection pattern of these neurons, it seems likely that they belong to a different morphological class of projection neurons than the GABAergic ones.Some methodologically important issues were also discovered in the study, the most significant being that the identity of not only the primary but also of the secondary antibody is of great importance to the quality of the results