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The amygdala of anurans is currently considered as a complex of nuclei that share many features with their counterparts in amniotes. In the present study, the subdivisions of the amygdala that are directly related to olfactory and vomeronasal information, were investigated in the anurans Rana perezi and Xenopus laevis. In particular, the connectivity of the main and accessory olfactory bulbs and their related amygdaloid nuclei was studied by means of in vivo and in vitro tract-tracing with dextran amines. The projections observed from the main olfactory bulb clearly innervate the newly redefined lateral amygdala within the ventral pallium and, to a lesser extent, the rostral portion of the medial amygdala. Injections into the accessory olfactory bulb exclusively revealed projections to the medial amygdala. Tracer applications into the lateral and medial nuclei revealed abundant intra-amygdaloid connections. The dual flow of olfactory and vomeronasal projections throughout the telencephalon was not strictly segregated since the lateral pallium and the lateral amygdala, both receiving olfactory information, were found to project to the medial amygdala (the only target of vomeronasal information), which in turn projects to the lateral amygdala. Additionally, both the lateral and the medial amygdala strongly project to the hypothalamus through the anuran equivalent of the stria terminalis. The main hodological features found in the present study suggest that forerunners of the olfactory and vomeronasal amygdaloid nuclei can be distinguished in anurans. This supports the notion that all tetrapods share a common pattern of organization of the amygdaloid complex, which links environmental (olfactory/vomeronasal) information and the behavioural response of the animal.

By means of double immunohistofluorescence techniques, we have investigated the colocalization of nitric oxide synthase and tyrosine hydroxylase (TH) or serotonin (5-HT) in the central nervous system of the anurans Rana perezi and Xenopus laevis and the urodele Pleurodeles waltl. A wide codistribution of neuronal populations, expressing these markers, was found throughout the brain and spinal cord. In contrast, colocalization of these markers was rather restricted. Only in the caudal portion of the brainstem raphe column in anurans, approximately 80% of the 5-HT-positive cells were also NOS-immunoreactive, whereas in the urodele brain, about 40% of the serotonergic cells at the level of the glossopharyngeal motor nucleus were simultaneously NOS-positive. In various brain regions, a wide codistribution of NOS- and TH-containing neurons was observed, but real colocalization of nitrergic and catecholaminergic cells was only found in a small neuron population in the posterior tubercle of anuran amphibians. Therefore, in amphibians, only a distinct and small cell population within the serotonergic raphe column (anurans and urodele) and in the catecholaminergic posterior tubercle (anurans) seem to produce simultaneously nitric oxide.

Immunohistochemistry for calbindin-D28k (CB) revealed that the spinal cord of Xenopus laevis possess a large number of CB-containing neurons widely distributed in both the dorsal and ventral horns, including areas which possess long ascending projections to supraspinal structures. In addition, the presence of CB-immunoreactive axons in the spinal funiculi suggested that descending projections containing this calcium binding protein may originate in different brainstem nuclei. Apart from mapping CB-containing elements in the spinal cord, a double labeling approach was used that combined the retrograde transport of dextran amines with CB immunohistochemistry. Thus, dextran amine injections into the lateral reticular region of the rhombencephalon, the parabrachial region, the mesencephalon and the dorsal thalamus revealed many retrogradely labeled cells in the spinal cord, a few number of which were double labeled for CB and found in the superficial dorsal horn and in the ventral medial region of the ventral horn. Their axons passed mainly via the lateral funiculus. Tracer application into the cervical spinal cord, combined with CB immunohistochemistry, resulted in retrogradely labeled cells throughout the brain, five groups of which showed CB immunoreactivity: (1) the mesencephalic trigeminal nucleus, (2) the laterodorsal tegmental nucleus, (3) the raphe nucleus, (4) the middle reticular nucleus and (5) the inferior reticular nucleus. The presence of CB in spinal pathways suggests that CB may play a role in controlling spinal cells, mainly subserving visceroceptive and nociceptive information to supraspinal levels, and might also modulate reticulospinal pathways.