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The structure and distribution of PC5‐A, a prohormone convertase that is thought to be involved in post‐translational processing of peptide hormone and neuropeptide precursors, have not been investigated in submammalian vertebrates. In the present study, we characterized the cDNA encoding PC5‐A in the European green frog Rana esculenta. The frog PC5‐A cDNA encodes a 913‐amino acid protein that encompasses a 28‐amino acid signal peptide, the Asp/His/Ser catalytic triad found in all serine proteinases of the subtilisin family, and two potential N‐linked glycosylation sites located in a C‐terminal cysteine‐rich domain. Reverse transcriptase polymerase chain reaction amplification showed that PC5‐A mRNA is expressed in various organs including the brain, spinal cord, pituitary, lung, liver, intestine, and testis, but not in the stomach and pancreas. The distribution of PC5‐A mRNA in the frog brain was studied by in situ hybridization histochemistry. Intense expression was observed in the mitral cellular layer of the olfactory bulb, the nucleus of the diagonal band of Broca, the anterior preoptic area, and the suprachiasmatic and ventral hypothalamic nuclei. The expression pattern of PC5‐A mRNA in the central nervous system of anuran amphibians was consistent with the implication of this prohormone convertase in the processing of various neuropeptide precursors. J. Comp. Neurol. 456:60–72, 2003. © 2002 Wiley‐Liss, Inc.

The sequence of somatostatin‐14 (SS1) has been strongly preserved throughout the evolution of vertebrates from agnathans to mammals. In Acipenseridae (sturgeons), two isoforms of somatostatin have been characterized to date: somatostatin‐14 has been identified from the gastrointestinal tract of the pallid sturgeon Scaphirhynchus albusand [Pro2]somatostatin‐14 has been identified from the pituitary of the Russian sturgeon Acipenser gueldenstaedti. In the present study, we report the cloning of two distinct somatostatin cDNAs from the brain of the sturgeon Acipenser transmontanus. One of the cDNAs encodes a 116‐amino acid protein (PSS1) that contains the SS1 sequence at its C‐terminal extremity and, thus, is clearly orthologous to other vertebrate PSS1. The other cDNA encodes a 111‐amino acid protein that contains the somatostatin variant [Pro2]somatostatin‐14 at its C‐terminal extremity. This second precursor exhibits more than 67% identity with the recently characterized lungfish PSS2 and goldfish PSS2. Reverse transcriptase‐polymerase chain reaction analysis revealed that PSS1 is expressed in the central nervous system, the pancreas and the gut, whereas PSS2 is found in the central nervous system but not in the digestive system. In situ hybridization histochemistry showed that the PSS1 and PSS2 genes are differently expressed in numerous regions of the sturgeon brain. Interestingly, PSS1 and PSS2 mRNAs are present in the hypothalamus suggesting that, in sturgeon, both SS1 and SS2 may play hypophysiotropic functions. The PSS2 mRNA but not the PSS1 mRNA was found in the intermediate lobe of the pituitary. The present data demonstrate that two somatostatin genes are expressed in the sturgeon brain: one precursor generates somatostatin‐14 and the other one gives rise to a [Pro2]somatostatin‐14 variant, which is orthologous to goldfish, lungfish, and frog SS2. J. Comp. Neurol. 443:332–345, 2002. © 2002 Wiley‐Liss, Inc.

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 3β‐hydroxysteroid dehydrogenase (3β‐HSD) and 5α‐reductase (5α‐R) in the brain and pituitary of the African lungfish Protopterus annectensby using antibodies raised against type I human 3β‐HSD and type I human 5α‐R. The 3β‐HSD and 5α‐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, 3β‐HSD immunolabeling is expressed exclusively by neurons, whereas the 5α‐R‐immunoreactive material is contained in both neurons and glial cells. In the pituitary gland, 3β‐HSD‐ and 5α‐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. J. Comp. Neurol. 438:123–135, 2001. © 2001 Wiley‐Liss, Inc.

The distribution of atrial natriuretic factor (ANF)‐like immunoreactivity in the central nervous system of the cave salamander Hydromantes genei(Amphibia, Plethodontidae) was investigated by using antisera raised against rat and human ANF(1–28). Concurrently, the location of ANF‐binding sites was determined by autoradiography, using radioiodinated human ANF(1–28) as a tracer. In several regions of the brain, including the olfactory bulb, the preoptic area, the ventral thalamus, the tectum of the mesencephalon, and the choroid plexuses inside the ventricles, a good correlation was observed between the distribution of ANF‐immunoreactive elements and the location of ANF‐binding sites. Mismatching was found in the habenular nucleus, the commissura habenularis, the fasciculum retroflexus, and the interpeduncular nucleus, which contained high levels of binding sites but were devoid of ANF‐immunoreactive structures. In contrast, a few other regions, such as the pineal gland and the subcommissural organ, showed a high concentration of ANF‐like immunoreactivity but did not contain ANF‐binding sites. This study provides the first localization of ANF‐like immunoreactivity and ANF‐binding sites in the brain of an urodele amphibian. The results show that the ANF peptidergic system in the cave salamander has an organization more simple than the organizations described for the brain of frog or other vertebrates. This feature is probably related to the expression of highly pedomorphic characters in plethodontids. The anatomical distribution of ANF‐immunoreactive elements and ANF‐binding sites suggests that ANF‐related peptides may act as hypophysiotropic hormones as well as neurotransmitters and/or neuromodulators in the salamander brain. J. Comp. Neurol. 437:240–258, 2001. © 2001 Wiley‐Liss, Inc.

The distribution of GABAergic neurons was investigated in the diencephalon of the African lungfish, Protopterus annectens, by using specific antibodies directed against glutamic acid decarboxylase (GAD). A dense population of immunoreactive perikarya was observed in the periventricular preoptic nucleus, whereas the caudal hypothalamus and the dorsal thalamus contained only scattered positive cell bodies. Clusters of GAD‐positive cells were found in the intermediate lobe of the pituitary. The diencephalon was richly innervated by GAD‐immunoreactive fibers that were particularly abundant in the hypothalamus. In the periventricular nucleus, GAD‐positive fibers exhibited a radial orientation, and a few neurons extended processes toward the third ventricle. More caudally, a dense bundle of GAD‐immunoreactive fibers coursing along the ventral wall of the hypothalamus terminated into the median eminence and the neural lobe of the pituitary. Double‐labeling immunocytochemistry revealed that GAD and neuropeptide tyrosine (NPY)‐like immunoreactivity was colocalized in a subpopulation of perikarya in the periventricular preoptic nucleus. The proportion of neurons that coexpressed GAD and NPY was higher in the caudal region of the preoptic nucleus. The distribution of GAD‐immunoreactive elements in the diencephalon and pituitary of the African lungfish indicates that GABA may act as a hypophysiotropic neurohormone in Dipnoans. The coexistence of GAD and NPY in a subset of neurons of the periventricular preoptic nucleus suggests that GABA and NPY may interact at the synaptic level. J. Comp. Neurol. 419:223–232, 2000. © 2000 Wiley‐Liss, Inc.

The distribution of GABAergic neurons was investigated in the diencephalon of the African lungfish, Protopterus annectens, by using specific antibodies directed against glutamic acid decarboxylase (GAD). A dense population of immunoreactive perikarya was observed in the periventricular preoptic nucleus, whereas the caudal hypothalamus and the dorsal thalamus contained only scattered positive cell bodies. Clusters of GAD-positive cells were found in the intermediate lobe of the pituitary. The diencephalon was richly innervated by GAD-immunoreactive fibers that were particularly abundant in the hypothalamus. In the periventricular nucleus, GAD-positive fibers exhibited a radial orientation, and a few neurons extended processes toward the third ventricle. More caudally, a dense bundle of GAD-immunoreactive fibers coursing along the ventral wall of the hypothalamus terminated into the median eminence and the neural lobe of the pituitary. Double-labeling immunocytochemistry revealed that GAD and neuropeptide tyrosine (NPY)-like immunoreactivity was colocalized in a subpopulation of perikarya in the periventricular preoptic nucleus. The proportion of neurons that coexpressed GAD and NPY was higher in the caudal region of the preoptic nucleus. The distribution of GAD-immunoreactive elements in the diencephalon and pituitary of the African lungfish indicates that GABA may act as a hypophysiotropic neurohormone in Dipnoans. The coexistence of GAD and NPY in a subset of neurons of the periventricular preoptic nucleus suggests that GABA and NPY may interact at the synaptic level. J. Comp. Neurol. 419:223–232, 2000. © 2000 Wiley-Liss, Inc.

The occurrence of two somatostatin precursors, PSS1 and PSS2, yielding S‐14 (SS1) and the variant [Pro2, Met13]S‐14 (SS2), has been recently reported in the frog Rana ridibunda. The evolutionary significance of frog PSS2 is unclear because its sequence exhibits very little similarity with other known vertebrate somatostatin precursors. In the present study, we report on the characterization of two somatostatin precursor cDNAs from the brain of the African lungfish Protopterus annectens. One of the cDNAs encodes a 115‐amino‐acid protein that contains the SS1 sequence at its C‐terminal extremity and thus is clearly homologous to PSS1. Comparison with other vertebrate PSS1 showed that lungfish PSS1 is more closely related to PSS1 from tetrapods than to PSS1 from fish. The other cDNA encodes a 109‐amino‐acid protein that contains a somatostatin variant [Pro2]S‐14 at its C‐terminal extremity. Sequence analysis of this second precursor indicated that it is the lungfish counterpart of frog PSS2. Northern blot analysis showed that lungfish PSS1 mRNA is widely distributed in the central nervous system and in peripheral organs, including the pancreas and gastrointestinal tract. In contrast, PSS2 mRNA was primarily found in the central nervous system but not in the pancreas or gut. In situ hybridization studies showed that the two genes are differentially expressed in various regions of the lungfish brain. The present data indicate that the PSS2 gene, initially discovered in frog, appeared early in vertebrate evolution, before the emergence of the tetrapod lineage. The recent isolation of a [Pro2]S‐14 variant in the sturgeon, whose sequence is identical to that of lungfish SS2, suggests that the PSS2 gene may actually be present in the genome of all Osteichthyii. J. Comp. Neurol. 410:643–652, 1999. © 1999 Wiley‐Liss, Inc.

The distribution of various opioid peptides derived from proenkephalin A and B was studied in the brain of the African lungfish Protopterus annectensby using a series of antibodies directed against mammalian opioid peptides. The results show that both Met‐enkephalin‐ and Leu‐enkephalin‐immunoreactive peptides are present in the lungfish brain. In contrast, enkephalin forms similar to Met‐enkephalin‐Arg‐Phe, or Met‐enkephalin‐Arg‐Gly‐Leu, as well as mammalian α‐neoendorphin, dynorphin A (1–8), dynorphin A (1–13), or dynorphin A (1–17) were not detected. In all major subdivisions of the brain, the overwhelming majority of Met‐enkephalin‐ and Leu‐enkephalin‐immunoreactive cells were distinct. In particular, cell bodies reacting only with Leu‐enkephalin antibodies were detected in the medial subpallium of the telencephalon, the griseum centrale, the reticular formation, the nucleus of the solitary tract, and the visceral sensory area of the rhombencephalon. Cell bodies reacting only with Met‐enkephalin antibodies were found in the lateral subpallium of the telencephalon, the caudal hypothalamus, and the tegmentum of the mesencephalon. The preoptic periventricular nucleus of the hypothalamus exhibited a high density of Met‐enkephalin‐immunoreactive neurons and only a few Leu‐enkephalin‐immunoreactive neurons. The distribution of Met‐enkephalin‐ and Leu‐enkephalin‐immunoreactive cell bodies and fibers in the lungfish brain showed similarities to the distribution of proenkephalin A‐derived peptides described previously in the brain of land vertebrates. The presence of Met‐enkephalin‐ and Leu‐enkephalin‐like peptides in distinct regions, together with the absence of dynorphin‐related peptides, suggests that, in the lungfish, Met‐enkephalin and Leu‐enkephalin may originate from distinct precursors. J. Comp. Neurol. 396:275–287, 1998. © 1998 Wiley‐Liss, Inc.

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. annectenssuggests 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.

The anatomical distribution of somatostatin‐immunoreactive structures and the autoradiographic localization of somatostatin binding sites were investigated in the brain of the African lungfish, Protopterus annectens. In general, there was a good correlation between the distribution of somatostatin‐immunoreactive elements and the location of somatostatin binding sites in several areas of the brain, particularly in the anterior olfactory nucleus, the rostral part of the dorsal pallium, the medial subpallium, the anterior preoptic area, the tectum, and the tegmentum of the mesencephalon. However, mismatching was found in the mid‐caudal dorsal pallium, the reticular formation, and the cerebellum, which contained moderate to high concentrations of binding sites and very low densities of immunoreactive fibers. In contrast, the caudal hypothalamus and the neural lobe of the pituitary exhibited low concentrations of binding sites and a high to moderate density of somatostatin‐immunoreactive fibers. The present results provide the first localization of somatostatin in the brain of a dipnoan and the first anatomical distribution of somatostatin binding sites in the brain of a fish. The location of somatostatin‐immunoreactive elements in the brain of P. annectensis consistent with that reported in anuran amphibians, suggesting that the general organization of the somatostatin peptidergic systems occurred in a common ancestor of dipnoans and tetrapods. The anatomical distribution of somatostatin‐immunoreactive elements and somatostatin binding sites suggests that somatostatin acts as a hypophysiotropic neurohormone as well as a neurotransmitter and/or neuromodulator in the lungfish brain. J. Comp. Neurol. 388:337–353, 1997. © 1997 Wiley‐Liss, Inc.