Jesús M. López

Distribution of choline acetyltransferase immunoreactivity in the brain of an elasmobranch, the lesser spotted dogfish (Scyliorhinus canicula).

Although the distribution of cholinergic cells is remarkably similar across the vertebrate species, no data are available on more primitive species, such as cartilaginous fishes. To extend the evolutionary analysis of the cholinergic systems, we studied the distribution of cholinergic neurons in the brain and rostral spinal cord of Scyliorhinus canicula by immunocytochemistry using an antibody against the enzyme choline acetyltransferase (ChAT). Western blot analysis of brain extracts of dogfish, sturgeon, trout, and rat showed that this antibody recognized similar bands in the four species. Putative cholinergic neurons were observed in most brain regions, including the telencephalon, diencephalon, cerebellum, and brainstem. In the retrobulbar region and superficial dorsal pallium of the telencephalon, numerous small pallial cells were ChAT‐like immunoreactive. In addition, tufted cells of the olfactory bulb and some cells in the lateral pallium showed faint immunoreactivity. In the preoptic‐hypothalamic region, ChAT‐immunoreactive (ChAT‐ir) cells were found in the preoptic nucleus, the vascular organ of the terminal lamina, and a small population in the caudal tuber. In the epithalamus, the pineal photoreceptors were intensely positive. Many cells of the habenula were faintly ChAT‐ir, but the neuropil of the interpeduncular nucleus showed intense ChAT immunoreactivity. In the pretectal region, ChAT‐ir cells were observed only in the superficial pretectal nucleus. In the brainstem, the somatomotor and branchiomotor nuclei, the octavolateral efferent nucleus, and a cell group just rostral to the Edinger‐Westphal (EW) nucleus contained ChAT‐ir neurons. In addition, the trigeminal mesencephalic nucleus, the nucleus G of the isthmus, some locus coeruleus cells, and some cell populations of the vestibular nuclei and of the electroreceptive nucleus of the octavolateral region exhibited ChAT immunoreactivity. In the reticular areas of the brainstem, the nucleus of the medial longitudinal fascicle, many reticular neurons of the rhombencephalon, and cells of the nucleus of the lateral funiculus were immunoreactive to this antibody. In the cerebellum, Golgi cells of the granule cell layer and some cells of the cerebellar nucleus were also ChAT‐ir. In the rostral spinal cord, ChAT immunoreactivity was observed in cells of the motor column, the dorsal horn, the marginal nucleus (a putative stretch‐receptor organ), and in interstitial cells of the ventral funiculus. These results demonstrate for the first time that cholinergic neurons are distributed widely in the central nervous system of elasmobranchs and that their cholinergic systems have evolved several characteristics that are unique to this group. J. Comp. Neurol. 420:139–170, 2000.

Regional expression of the homeobox gene NKX2-1 defines pallidal and interneuronal populations in the basal ganglia of amphibians.

The distribution of gene expression domains during development constitutes a novel tool for the identification of distinct brain regions. This is particularly useful in the brain of amphibians where cell migration is very limited and most neurons organize in a periventricular layer. Here we report the expression pattern of NKX2-1 protein in the developing Xenopus telencephalon. In mammals, the Nkx2-1 gene is expressed in distinct subpallial regions such as the septum, the medial ganglionic eminence and preoptic region. The results of the present study demonstrate that the expression of NKX2-1 delineates the pallidal anlage and its derivatives in amphibians, as in mammals and birds. In addition, double-labeling immunohistochemistry and the combination of tracing experiments with NKX2-1 immunohistochemistry demonstrate that the amphibian striatum contains interneurons, which express NKX2-1 and produce, among other possible neurotransmitters, nitric oxide and acetylcholine. In sum, the results of the present study strengthen the notion that similar developmental programs exist during basal ganglia development in all tetrapods.

Ontogeny of NADPH diaphorase/nitric oxide synthase reactivity in the brain of Xenopus laevis

The development of nitric oxide synthase (NOS) expression in the brain of Xenopus laevis tadpoles was studied by means of immunohistochemistry using specific antibodies against NOS and enzyme histochemistry for nicotinamide adenine dinucleotide phosphate (NADPH)‐diaphorase. Both techniques yielded identical results and were equally suitable for demonstrating the nitrergic system in the brain. The only mismatches were observed in the olfactory nerve and glomeruli and in the terminal nerve; they were intensely labeled with the NADPH‐diaphorase technique but failed to stain with NOS immunohistochemistry. As early as stage 33, nitrergic cells were observed in the caudal rhombencephalon within the developing inferior reticular nucleus. At later embryonic stages, different sets of reticular and tegmental neurons were labeled in the middle reticular nucleus and, more conspicuously, in the laterodorsal and pedunculopontine tegmental nuclei. As development proceeded, new nitrergic cell groups gradually appeared in the mesencephalon, diencephalon, and telencephalon. A general caudorostral temporal sequence was observed, both in the whole brain and within each main brain subdivision. The premetamorphic period was mainly characterized by the maturation of the cell populations developed in the embryonic period. During prometamorphosis, the nitrergic system reached an enormous development, and many new cell groups were observed for the first time, in particular in the telencephalon. By the climax of metamorphosis, the pattern of organization of nitrergic cells and fibers observed in the brain was similar to that present in the adult brain. Transient expression of NOS was not detected in any brain region. Our data suggest that nitric oxide plays an important role during brain development of Xenopus. Comparison with the developmental pattern of nitrergic systems in other vertebrates shows that amphibians possess more common features with amniotes than with anamniotes. J. Comp. Neurol. 445:59–77, 2002.

Lungfishes, Like Tetrapods, Possess a Vomeronasal System

The vomeronasal system (VNS) is an accessory olfactory system that in tetrapod vertebrates is composed of specific receptor neurons in the nasal organ and a set of centers in the forebrain that receive and relay the information consecutively towards the hypothalamus. Thus, only in tetrapods the VNS comprises a discrete vomeronasal (Jacobsons) organ, which contains receptor cells that are morphologically distinct from those of the olfactory epithelium and use different transduction mechanisms. The axons of the vomeronasal receptors in tetrapods project to the accessory olfactory bulb (AOB) in the rostral telencephalon. Secondary vomeronasal connections exist through the medial amygdala to the hypothalamus. Currently, the lungfishes are considered the closest living relatives of tetrapods. Here we show that the African lungfish, Protopterus dolloi, has epithelial crypts at the base of the lamellae of the olfactory epithelium that express markers of the vomeronasal receptors in tetrapods. The projections of these crypts allow us to identify an AOB on the lateral margin of the main olfactory bulb. The projections of this AOB reach a region that is topologically, hodologically, and immunohistochemically identical to the medial amygdala and could represent its homolog. Neurons of this putative medial amygdala were demonstrated to project to the lateral hypothalamus, as they do in tetrapods. All these features that lungfishes share with tetrapods indicate that lungfishes have the complete set of brain centers and connections involved in processing vomeronasal information and that these features were already present in the last common ancestor of lungfishes and tetrapods.

Characterization of the bed nucleus of the stria terminalis in the forebrain of anuran amphibians

Major common features have been reported for the organization of the basal telencephalon in amniotes, and most characteristics were thought to be acquired in the transition from anamniotes to amniotes. However, gene expression, neurochemical, and hodological data obtained for the basal ganglia and septal and amygdaloid complexes in amphibians (anamniotic tetrapods) have strengthened the idea of a conserved organization in tetrapods. A poorly characterized region in the forebrain of amniotes has been the bed nucleus of the stria terminalis (BST), but numerous recent investigations have characterized it as a member of the extended amygdala. Our study analyzes the main features of the BST in anuran amphibians to establish putative homologies with amniotes. Gene expression patterns during development identified the anuran BST as a subpallial, nonstriatal territory. The BST shows Nkx2.1 and Lhx7 expression and contains an Islet1‐positive cell subpopulation derived from the lateral ganglionic eminence. Immunohistochemistry for diverse peptides and neurotransmitters revealed that the distinct chemoarchitecture of the BST is strongly conserved among tetrapods. In vitro tracing techniques with dextran amines revealed important connections between the BST and the central and medial amygdala, septal territories, medial pallium, preoptic area, lateral hypothalamus, thalamus, and prethalamus. The BST receives dopaminergic projections from the ventral tegmental area and is connected with the laterodorsal tegmental nucleus and the rostral raphe in the brainstem. All these data suggest that the anuran BST shares many features with its counterpart in amniotes and belongs to a basal continuum, likely controlling similar reflexes, reponses, and behaviors in tetrapods. J. Comp. Neurol. 520:330–363, 2012.

A forerunner of septohippocampal cholinergic system is present in amphibians

The medial pallium of anuran amphibians (frogs and toads) has been considered homologue to the mammalian hippocampus. While physiological and behavioral studies have strengthened this homology, basic anatomical features of the hippocampus were not observed in amphibians. The septohippocampal cholinergic pathway is among the most prominent afferent connection of the hippocampus and is involved in learning and memory processes. In the present study, double labeling techniques revealed that up to 18% of the retrograde labeled cells in the medial septal nucleus and nucleus of the diagonal band of the frog were cholinergic. This pathway can be considered as a forerunner of the septohippocampal pathway of mammals and might represent the anatomical substrate for learning processes in which the medial pallium is involved in amphibians.

Expression pattern of the homeobox protein NKX2-1 in the developing Xenopus forebrain.

Although morphological data suggest that the amphibian forebrain contains similar subdivisions to those observed in birds and mammals, it is presently unclear whether the same patterning mechanisms are conserved among all three classes of tetrapods. Here we report that NKX2-1, a transcription factor that is essential for the ventral patterning of the forebrain in birds and mammals, is expressed in corresponding (homologous) domains in the developing Xenopus forebrain. NKX2-1 expression is restricted to two domains in the amphibian forebrain: (1) a ventral diencephalic domain, with expression limited to hypothalamic structures; and (2) a telencephalic domain, with expression in the medial ganglionic eminence, preoptic area and part of the septum. Thus, the detailed analysis of the distribution of NKX2-1 provides the first unequivocal evidence for distinct progenitor zones within the amphibian forebrain through embryonic and larval development.

Development of NADPH-diaphorase/nitric oxide synthase in the brain of the urodele amphibian Pleurodeles waltl

In the present study, the ontogenesis of nitrergic neurons has been studied in the urodele amphibian Pleurodeles waltl by means of NADPH-diaphorase (NADPHd) histochemistry and neuronal nitric oxide synthase (NOS) immunohistochemistry. Embryonic and larval stages were studied. Except for the olfactory fibers and glomeruli, both methods were equally suitable to reveal nitrergic structures in the brain. The earliest positive neurons were observed in the inferior reticular nucleus (Ri) in the caudal rhombencephalon at embryonic stage 30. At stage 33b, weakly reactive cells appeared in the tegmentum of the mesencephalon and isthmus, in the ventral hypothalamus (VH), and in the proximity of the solitary tract (sol). At initial larval stages (stages 34-38), two new groups appeared in the caudal telencephalon (future amygdaloid complex (Am)) and in the middle reticular nucleus (Rm) of the rhombencephalon. During the active larval life (stages 39-55c) the nitrergic system developed progressively both in number of cells and fiber tracts. At stages 39-42 reactive cells were found in the inner granular layer (igl) of the olfactory bulb, the telencephalic pallium, the pretectal region, the optic tectum (OT) and retina. New populations of nitrergic cells appear during the second half of the larval period (stages 52-55). Rostrally, reactive cells were found in the telencephalic diagonal band (DB) nucleus, medial septum and in the thalamic eminence (TE), whereas caudally cells appeared in the raphe (Ra) and the descending trigeminal nucleus (Vd). The last changes occurred during the juvenile period (metamorphic climax), when cells of the spinal cord (sc) and the preoptic area became positive. The sequence of appearance of nitrergic cells revealed a first involvement of this system in reticulospinal control, likely influencing locomotor behavior. As development proceeds, cells in different sensory systems expressed progressively nitric oxide synthase in a pattern that shows many similarities with amniotes.

Distribution of NADPH-Diaphorase/Nitric Oxide Synthase in the Brain of the Caecilian Dermophis mexicanus (Amphibia: Gymnophiona): Comparative Aspects in Amphibians

The organization of nitrergic systems in the brains of anuran and urodele amphibians was recently studied and significant differences were noted between both amphibian orders. However, comparable data are not available for the third order of amphibians, the gymnophionans (caecilians). In the present study we have investigated the distribution of neuronal elements that express nitric oxide synthase (NOS) in the brain of the gymnophionan amphibian Dermophis mexicanus by means of immunohistochemistry with specific antibodies against NOS and enzyme histochemistry for NADPH-diaphorase. Both techniques yielded identical results and were equally suitable to demonstrate the nitrergic system. In addition, they were useful tools in the identification of cell groups and brain structures, otherwise indistinct in the brains of caecilians. The distribution of nitrergic structures observed in Dermophis conforms to the overall amphibian pattern but numerous distinct peculiarities were also noted. These included a dense innervation of the olfactory bulbs but a lack of reactivity in olfactory and vomeronasal fibers and glomeruli. A large population of nitrergic cells in the striatum and the presence of thalamic neurons, as well as the specific distribution of nitrergic cells in the isthmic region, are some of the differential features in the gymnophionan brain. Given the variability among species in the same class of vertebrates any discussion including amphibians should also include evidence for gymnophionans.

Choline acetyltransferase immunoreactivity in the developing brain of Xenopus laevis.

The spatiotemporal sequence of the appearance of cholinergic structures in the brain of Xenopus laevis during development was studied by means of choline acetyltransferase (ChAT) immunohistochemistry. The first ChAT labeling in the central nervous system of Xenopus was obtained at late embryonic stages in the spinal motoneurons, the cranial nerve motor nuclei of the brainstem, and in amacrine cells of the retina. During premetamorphosis, these cholinergic structures maturated significantly and new ChAT‐immunoreactive cells were observed in several other nuclei such as the solitary tract nucleus, isthmic nucleus, laterodorsal and pedunculopontine tegmental nuclei, epiphysis, dorsal habenular nucleus, medial amygdala, bed nucleus of the stria terminalis, and dorsal pallidum. Further maturation continued through prometamorphosis and the climax of the metamorphosis together with the appearance of new cell groups in the efferent octaval nucleus, ventral hypothalamic nucleus, anterior preoptic area, suprachiasmatic nucleus, and medial septum. Transient expression of ChAT was only seen in the large Mauthner cells that showed moderate ChAT labeling during pre‐ and prometamorphosis but became immunonegative at the end of the metamorphosis. The gradual appearance, in general from caudal to rostral brain levels, of ChAT immunoreactivity in Xenopus, was correlated with other developmental events to get insight into the possible roles of acetylcholine during ontogeny. Comparison with the developmental pattern of cholinergic systems in other vertebrates shows that Xenopus possesses abundant features in common with amniotes, suggesting a conservative developmental plan for tetrapods. J. Comp. Neurol. 453:418–434, 2002.