The present study provides a detailed description of the distribution of neuropeptide FF (NPFF)-like immunoreactivity in the brain of the lizard Gekko gecko. NPFF is found to be involved in nociception, cardiovascular regulation, and endocrine function. Because of its known relationship with catecholamines in mammals, double staining with tyrosine hydroxylase (TH) antibodies was used to corroborate this for reptiles. The present study revealed that NPFF-like-immunoreactive (NPFF-ir) cells and fibers were widely distributed throughout the brain. Major NPFF-ir cell groups were observed in the diagonal band nucleus of Broca, hypothalamus, and dorsal horn of the spinal cord. Additional cells were found in the anterior olfactory nucleus, lateral and dorsal cortices, dorsolateral septum, and diencephalic intergeniculate leaflet formation. Dense plexuses of NPFF-ir fibers were identified in the diagonal band nucleus of Broca, septum, preoptic and hypothalamic areas, isthmic region, ventrolateral tegmentum, solitary tract nucleus, and dorsolateral funiculus of the spinal cord. Extensive fiber staining also occurred in the nucleus accumbens and the midbrain tectum. Although an intimate relationship between NPFF-ir and TH-ir structures was obvious at many places in the brain, colocalization of these two substances was not observed. In conclusion, the distribution of NPFF in the brain of Gekko shares more features with anamniotes in terms of number of cell groups, more elaborate networks of fibers, and lack of colocalization with catecholamines than with mammals, suggesting a decrease in the distribution of this peptide in the latter vertebrate group.

The relationship between dopaminergic neuronal structures and dopaminoceptive structures in the amphibian brain and spinal cord are assessed by means of single and double immunohistochemical techniques with antibodies directed against DARPP-32 (a phosphoprotein related to the dopamine D(1)-receptor) and tyrosine hydroxylase (TH) applied to the brain of the anurans Rana perezi and Xenopus laevis. The DARPP-32 antibody yielded a well-differentiated pattern of staining in the brain of these anurans. In general, areas that are densely innervated by TH-immunoreactive fibers such as the nucleus accumbens, striatum, amygdaloid complex, thalamus, optic tectum, torus semicircularis and spinal cord display a remarkable immunoreactivity for DARPP-32 in cell bodies and neuropil. Distinct cellular DARPP-32 immunoreactivity was also found in the septum, preoptic area, suprachiasmatic nucleus, tuberal hypothalamic region, habenula, retina, midbrain tegmentum, rhombencephalic reticular formation and solitary tract nucleus. Hodological data supported that striatal projection neurons were DARPP-32 immunoreactive. Double immunohistofluorescence staining revealed that catecholaminergic cells generally do not stain for DARPP-32, except for some cells in the ventral mesencephalic tegmentum of Xenopus and cells in the nucleus of the solitary tract of Rana. Several interspecies differences were noted for the DARPP-32 distribution in the brain of the two anurans, namely in the habenula, the thalamus and prethalamus, the cerebellum and octavolateral area and the structures with DARPP-32/TH colocalization. However, in general, the distribution of DARPP-32 in the brain of the anuran amphibians resembles in many aspects the pattern observed in amniotes, especially in reptiles.

The Australian lungfish Neoceratodus forsteri is the only extant species of the order Ceratodontiformes, which retained most of the primitive features of ancient lobe finned-fishes. Lungfishes are the closest living relatives of land vertebrates and their study is important for deducing the neural traits that were conserved, modified, or lost with the transition from fishes to land vertebrates. We have investigated the nitrergic system with neural nitric oxide synthase (NOS) immunohistochemistry and NADPH-diaphorase (NADPH-d) histochemistry, which yielded almost identical results except for the primary olfactory projections and the terminal and preoptic nerve fibers labeled only for NADPH-d. Combined immunohistochemistry was used for simultaneous detection of NOS with catecholaminergic, cholinergic, and serotonergic structures, aiming to establish accurately the localization of the nitrergic elements and to assess possible interactions between these neurotransmitter systems. The results demonstrated abundant nitrergic cells in the basal ganglia, amygdaloid complex, preoptic area, basal hypothalamus, mesencephalic tectum and tegmentum, laterodorsal tegmental nucleus, reticular formation, spinal cord, and retina. In addition, low numbers of nitrergic cells were observed in the olfactory bulb, all pallial divisions, lateral septum, suprachiasmatic nucleus, prethalamic and thalamic areas, posterior tubercle, pretectum, torus semicircularis, cerebellar nucleus, interpeduncular nucleus, the medial octavolateral nucleus, nucleus of the solitary tract, and the dorsal column nucleus. Colocalization of NOS and tyrosine hydroxylase was observed in numerous cells of the ventral tegmental area/substantia nigra complex. Comparison with other vertebrates, using a neuromeric analysis, reveals that the nitrergic system of Neoceratodus shares many neuroanatomical features with tetrapods and particularly with amphibians.

The organization of the serotonergic system, one of the most important neurotransmitter systems in the brain, has been carefully analyzed in most vertebrate groups, and major shared characteristics have been described, although traits particular to each vertebrate class have also been found. The present study is the first that provides a comprehensive and detailed map of the serotonergic structures in the brain of two representative species of lungfishes, the African lungfish (Protopterus dolloi) and the Australian lungfish (Neoceratodus forsteri), as revealed by immunohistochemistry against serotonin (5-HT). Lungfishes are currently considered the closest living relatives of tetrapods and represent an interesting group for the study of evolutionary traits in the transition from fishes to tetrapods. Distinct groups of serotonin immunoreactive cells were observed in the preoptic area, nucleus of the periventricular organ, pretectum, optic tectum and the long column of the raphe. Fiber labeling was widely distributed in all main brain subdivisions but was more abundant in regions such as the striatum, septum, amygdaloid complex, preoptic area, suprachiasmatic nucleus, lateral hypothalamic area, prethalamus, thalamus, mesencephalic tegmentum and rhombencephalic reticular formation. Comparison of these results with those from other classes of vertebrates highlights numerous common traits shared by most groups of fishes but also reveals that the serotonergic system in lungfishes largely resembles those of amphibians and other tetrapods.

Lungfishes are a group of sarcopterygian fishes currently considered the closest living relatives of tetrapods, and represent an interesting group for the study of evolutionary traits in the transition from fishes to tetrapods. Catecholaminergic systems in the brain are among the most carefully analyzed neurotransmitter systems in the brain of most vertebrate groups. Their organization shows major shared characteristics, although traits particular to each vertebrate class have also been found, primarily between anamniotes and amniotes. Given the relevance of lungfishes in evolutionary terms, the present study provides the first comprehensive and detailed map of the catecholaminergic structures in the brain of two representative species of lungfishes, an African lungfish (Protopterus dolloi) and the Australian lungfish (Neoceratodus forsteri), as revealed by immunohistochemistry. Distinct groups of catecholaminergic cells were observed in the olfactory bulb, pallium, and preoptic area of the telencephalon, and the subpallium is devoid of these cells. Hypothalamic and diencephalic groups were detected and, in particular, the dopaminergic nucleus of the periventricular organ was evidenced with dopamine antibodies but not with anti-tyrosine hydroxylase. A well developed mesostriatal system was revealed formed by conspicuous groups of dopamine cells in the midbrain tegmentum and profuse innervation of the subpallium. Comparison of these results with those from other classes of vertebrates shows numerous common traits shared by most groups and also highlights particular features in lungfishes different from actinopterygian fishes that resemble those of amphibians and amniotes.

Satb1 and Satb2 belong to a family of homeodomain proteins with highly conserved functional and regulatory mechanisms and posttranslational modifications in evolution. However, although their distribution in the mouse brain has been analyzed, few data exist in other non-mammalian vertebrates. In the present study, we have analyzed in detail the sequence of SATB1 and SATB2 proteins and the immunolocalization of both, in combination with additional neuronal markers of highly conserved populations, in the brain of adult specimens of different bony fish models at key evolutionary points of vertebrate diversification, in particular including representative species of sarcopterygian and actinopterygian fishes. We observed a striking absence of both proteins in the pallial region of actinopterygians, only detected in lungfish, the only sarcopterygian fish. In the subpallium, including the amygdaloid complex, or comparable structures, we identified that the detected expressions of SATB1 and SATB2 have similar topologies in the studied models. In the caudal telencephalon, all models showed significant expression of SATB1 and SATB2 in the preoptic area, including the acroterminal domain of this region, where the cells were also dopaminergic. In the alar hypothalamus, all models showed SATB2 but not SATB1 in the subparaventricular area, whereas in the basal hypothalamus the cladistian species and the lungfish presented a SATB1 immunoreactive population in the tuberal hypothalamus, also labeled with SATB2 in the latter and colocalizing with the gen Orthopedia. In the diencephalon, all models, except the teleost fish, showed SATB1 in the prethalamus, thalamus and pretectum, whereas only lungfish showed also SATB2 in prethalamus and thalamus. At the midbrain level of actinopterygian fish, the optic tectum, the torus semicircularis and the tegmentum harbored populations of SATB1 cells, whereas lungfish housed SATB2 only in the torus and tegmentum. Similarly, the SATB1 expression in the rhombencephalic central gray and reticular formation was a common feature. The presence of SATB1 in the solitary tract nucleus is a peculiar feature only observed in non-teleost actinopterygian fishes. At these levels, none of the detected populations were catecholaminergic or serotonergic. In conclusion, the protein sequence analysis revealed a high degree of conservation of both proteins, especially in the functional domains, whereas the neuroanatomical pattern of SATB1 and SATB2 revealed significant differences between sarcopterygians and actinopterygians, and these divergences may be related to the different functional involvement of both in the acquisition of various neural phenotypes.

To gain insight into the evolution of the thyrotropin-releasing hormone (TRH) system in the brain of vertebrates we have conducted a comparative analysis of the distribution of TRH immunoreactive cell bodies and fibers in two reptiles, the turtle Pseudemys scripta elegans and the lizard Gekko gecko. Double labeling for TRH and tyrosine hydroxylase, the main catecholamine marker, was made to help the correct localization of the labeled structures and to evaluate the possible interaction of these two systems. Cells containing TRH were found in the hypothalamic paraventricular and periventricular nuclei and their projections to the median eminence were corroborated. In addition, with some species variation, extrahypothalamic cells were present in the medial amygdala, mesencephalic tegmentum, descending vestibular nucleus and in the retina. Fiber labeling was observed in all main brain subdivisions but was more abundant in regions such as the striatum, septum, amygdaloid complex, dorsal thalamus and tegmento-reticular zones. Actual colocalization of TRH and catecholamines in the same neurons was not observed but the codistribution of cells and fibers labeled for TRH and tyrosine hydroxylase strongly suggests that they can interact in diverse regions, not only in the hypothalamo-hypophysial system. The comparison of the distribution of TRH immunoreactive neurons and fibers found in reptiles with those reported for other vertebrates reveals a strong resemblance but also notable variations, not only across vertebrate classes but also within the same class.