Organization of the catecholaminergic systems in the brain of lungfishes,the closest living relatives of terrestrial vertebrates

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.     

Immunocytochemical localization of atrial natriuretic factor and autoradiographic distribution of atria natriuretic factor binding sites in the brain of the African lungfish,Protopterus annectens

The localization of atrial natriuretic factor (ANF)-immunoreactive elements was investigated in the brain of the African lungfish, Protopterus annectens, by using antisera raised against rat and human ANF(1–28). Concurrently, the distribution of ANF binding sites was studied by autoradiography using radioiodinated human ANF(1–28) as a tracer. In general, there was a good correlation between the distribution of ANF-immunoreactive structures and the location of ANF binding sites in several areas of the brain, particularly in the ventral part of the medial subpallium, the rostral preoptic region, the preoptic periventricular nucleus, the caudal hypothalamus, the neural lobe of the pituitary, and the mesencephalic tectum. In contrast, mismatching was observed in the pallium (which contained a high density of binding sites and a low concentration of ANF immunoreactive elements) as well as in the lateral subpallium and the medial region of the ventral thalamus, in which a low concentration of binding sites but a high density of ANF-immunoreactive fibers were detected. The present data provide the first localization of ANF-related peptides in the brain of dipnoans and the first anatomical distribution of ANF binding sites in the brain of fish. The results show that the ANF peptidergic systems of P. annectens exhibit similarities with those previously described in the frog Rana ridibunda, supporting the existence of relationships between dipnoans and amphibians. The location of ANF-like immunoreactivity and the distribution of ANF binding sites suggest that ANF-related peptides may act as hypothalamic neurohormones as well as neurotransmitters and/or neuromodulators in the lungfish brain. © 1996 Wiley-Liss, Inc.     

Pattern of nitrergic cells and fibers organization in the central nervous system of the Australian lungfish,Neoceratodus forsteri (Sarcopterygii: Dipnoi)

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.     

Melanin-concentrating hormone system in the brain of the lungfish Protopterus annectens

The neurochemical anatomy of the lungfish brain is of particular interest, because many features in these animals might be representative of the common ancestor of land vertebrates. In the present study, we have investigated the localization and biochemical characteristics of melanin-concentrating hormone (MCH)-immunoreactive material in the central nervous system of the African lungfish, Protopterus annectens. The most prominent group of MCH-immunoreactive cell bodies was found in the dorsal hypothalamus. Additional groups of MCH-immunoreactive perikarya were detected in the telencephalon within the medial and dorsal pallium, the medial subpallium, and the ventral part of the lateral subpallium. Brightly immunofluorescent nerve fibers were seen in the anterior olfactory nucleus, the ventral part of the medial pallium, the medial subpallium, and the anterior preoptic area. In the diencephalon, the hypothalamus and the medial region of the dorsal thalamus exhibited a dense accumulation of fibers. MCH-immunoreactive fibers were also found in the tectum and the tegmentum of the mesencephalon and within the reticular formation of the rhombencephalon. In the pituitary, several small groups of cells of the intermediate lobe showed a bright fluorescence. Reversed-phase high-performance liquid chromatography (HPLC) analysis of diencephalon and pituitary extracts resolved a major MCH-immunoreactive peak that coeluted with synthetic salmon MCH. The distribution of MCH in the brain of P. annectens suggests that, in lungfishes, this peptide may exert neuromodulator or neurotransmitter functions. The presence of MCH-like immunoreactivity in the intermediate lobe of the pituitary indicates that, in dipnoans, MCH may also act as a typical pituitary hormone. J. Comp. Neurol. 390:41–51, 1998. © 1998 Wiley-Liss, Inc.     

Regional chemoarchitecture of the brain of lungfishes based on calbindin D‐28K and calretinin immunohistochemistry

Lungfishes are the closest living relatives of land vertebrates, and their neuroanatomical organization is particularly relevant for deducing the neural traits that have been conserved, modified, or lost with the transition from fishes to land vertebrates. The immunohistochemical localization of calbindin (CB) and calretinin (CR) provides a powerful method for discerning segregated neuronal populations, fiber tracts, and neuropils and is here applied to the brains of Neoceratodus and Protopterus, representing the two extant orders of lungfishes. The results showed abundant cells containing these proteins in pallial and subpallial telencephalic regions, with particular distinct distribution in the basal ganglia, amygdaloid complex, and septum. Similarly, the distribution of CB and CR containing cells supports the division of the hypothalamus of lungfishes into neuromeric regions, as in tetrapods. The dense concentrations of CB and CR positive cells and fibers highlight the extent of the thalamus. As in other vertebrates, the optic tectum is characterized by numerous CB positive cells and fibers and smaller numbers of CR cells. The so‐called cerebellar nucleus contains abundant CB and CR cells with long ascending axons, which raises the possibility that it could be homologized to the secondary gustatory nucleus of other vertebrates. The corpus of the cerebellum is devoid of CB and CR and cells positive for both proteins are found in the cerebellar auricles and the octavolateralis nuclei. Comparison with other vertebrates reveals that lungfishes share most of their features of calcium binding protein distribution with amphibians, particularly with salamanders.     

Immunohistochemical localization of 3 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase in the brain of the African lungfish Protopterus annectens

The localization of the enzymes responsible for the biosynthesis of neurosteroids in the brain of dipnoans has not yet been determined. In the present study, we investigated the immunohistochemical distribution of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 5 alpha-reductase (5 alpha-R) in the brain and pituitary of the African lungfish Protopterus annectens by using antibodies raised against type I human 3 beta-HSD and type I human 5 alpha-R. The 3 beta-HSD and 5 alpha-R immunoreactivities were detected in cell bodies and fibers located in the same areas of the lungfish brain, namely, in the pallium, thalamus, hypothalamus, tectum, and periaqueductal gray. Identification of astrocytes, oligodendrocytes, and neurons with antisera against glial fibrillary acidic protein, galactocerebroside and neurofilaments revealed that, in the lungfish brain, 3 beta-HSD immunolabeling is expressed exclusively by neurons, whereas the 5 alpha-R-immunoreactive material is contained in both neurons and glial cells. In the pituitary gland, 3 beta-HSD- and 5 alpha-R-like immunoreactivity was localized in both the pars distalis and the pars intermedia. The present study provides the first immunocytochemical mapping of two key steroidogenic enzymes in the brain and pituitary of a lungfish. These data strongly suggest that neurosteroid biosynthesis occurs in the brain of fishes, as previously shown for amphibians, birds, and mammals.