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Islet-1 (Isl1) is one of the most conserved transcription factors in the evolution of vertebrates, due to its continuing involvement in such important functions as the differentiation of motoneurons, among other essential roles in cell fate in the forebrain. Although its functions are thought to be similar in all vertebrates, the knowledge about the conservation of its expression pattern in the central nervous system goes as far as teleosts, leaving the basal groups of actinopterygian fishes overlooked, despite their important phylogenetic position. In order to assess the extent of its conservation among vertebrates, we studied its expression pattern in the central nervous system of selected nonteleost actinopterygian fishes. By means of immunohistochemical techniques, we analyzed the Isl1 expression in the brain, spinal cord, and sensory ganglia of the cranial nerves of young adult specimens of the cladistian species Polypterus senegalus and Erpetoichthys calabaricus, the chondrostean Acipenser ruthenus, and the holostean Lepisosteus oculatus. We also detected the presence of the transcription factor Orthopedia and the enzymes tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) to better locate all the immunoreactive structures in the different brain areas and to reveal the possible coexpression with Isl1. Numerous conserved features in the expression pattern of Isl1 were observed in these groups of fishes, such as populations of cells in the subpallial nuclei, preoptic area, subparaventricular and tuberal hypothalamic regions, prethalamus, epiphysis, cranial motor nuclei and sensory ganglia of the cranial nerves, and the ventral horn of the spinal cord. Double labeling of TH and Isl1 was observed in cells of the preoptic area, the subparaventricular and tuberal hypothalamic regions, and the prethalamus, while virtually all motoneurons in the hindbrain and the spinal cord coexpressed ChAT and Isl1. Altogether, these results show the high degree of conservation of the expression pattern of the transcription factor Isl1, not only among fish, but in the subsequent evolution of vertebrates., (© 2023 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

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., (© 2023. The Author(s).)

During development, gene expression is tightly controlled to facilitate the generation of the diverse cell types that form the central nervous system. Brahma-related gene 1 (Brg1, also known as Smarca4) is the catalytic subunit of the SWItch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex that regulates transcription. We investigated the role of Brg1 between embryonic day 6.5 (E6.5) and E14.5 in Sox2-positive neural stem cells (NSCs). Being without major consequences at E6.5 and E14.5, loss of Brg1 between E7.5 and E12.5 resulted in the formation of rosette-like structures in the subventricular zone, as well as morphological alterations and enlargement of neural retina (NR). Additionally, Brg1-deficient cells showed decreased survival in vitro and in vivo. Furthermore, we uncovered distinct changes in gene expression upon Brg1 loss, pointing towards impaired neuron functions, especially those involving synaptic communication and altered composition of the extracellular matrix. Comparison with mice deficient for integrase interactor 1 (Ini1, also known as Smarcb1) revealed that the enlarged NR was Brg1 specific and was not caused by a general dysfunction of the SWI/SNF complex. These results suggest a crucial role for Brg1 in NSCs during brain and eye development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

The Pax6 gene encodes a regulatory transcription factor that is key in brain development. The molecular structure of Pax6, the roles it plays and its patterns of expression in the brain have been highly conserved during vertebrate evolution. As neurodevelopment proceeds, the Pax6 expression changes from the mitotic germinal zone in the ventricular zone to become distributed in cell groups in the adult brain. Studies in various vertebrates, from fish to mammals, found that the Pax6 expression is maintained in adults in most regions that express it during development. Specifically, in amphibians, Pax6 is widely expressed in the adult brain and its distribution pattern serves to highlight regional organization of the brain. In the present study, we analyzed the detailed distribution of Pax6 cells in the adult central nervous system of lungfishes, the closest living relatives of all tetrapods. Immunohistochemistry performed using double labeling techniques with several neuronal markers of known distribution patterns served to evaluate the actual location of Pax6 cells. Our results show that the Pax6 expression is maintained in the adult brain of lungfishes, in distinct regions of the telencephalon (pallium and subpallium), diencephalon, mesencephalon, hindbrain, spinal cord, and retina. The pattern of Pax6 expression is largely shared with amphibians and helps to understand the primitive condition that would have characterized the common ancestors to all sarcopterygians (lobe-finned fishes and tetrapods), in which Pax6 would be needed to maintain specific entities of subpopulations of neurons., (© 2019 Wiley Periodicals, Inc.)

One of the most intriguing questions in neuroscience is to understand the mechanism of information transfer between different brain areas. Recently, network theory has gained traction and is at the forefront of providing a possible explanation to not only the mechanism of information transfer but also in the identification of different neuropathologies. The perception of a phantom ringing in the ear called tinnitus, similar to other neuropathologies, has been shown to be accompanied by aberrant functional connectivity between different brain areas. Although, there have been independent studies showing that specific groups of areas encode individual symptoms of tinnitus, there has not been one study to show that tinnitus is the unified percept of distinguishable subnetworks encoding different behavioral aspects. This study combines resting-state functional connectivity obtained from the source-localized electroencephalography of 311 tinnitus patients and 264 controls, and a k-fold cross-validation machine learning algorithm to develop a predictive model that verifies the presence of behaviorally specific, spatiotemporally overlapping subnetworks in tinnitus. This reorganization is found to be exclusive to tinnitus, even when compared to physiologically similar disorders such as chronic pain, with each behavioral symptom having a unique oscillatory signature. This frequency-specific transmission of information, called multiplexing, enables different types of information to be carried between two brain regions through the same anatomical connection. In addition to understanding the efficient compensation mechanism of the brain in the presence of multisymptom disorders, the exclusivity of the prediction model presents an encouraging possibility for an objective neural marker for tinnitus.

The onset and developmental dynamics of Pax3, Pax6, and Pax7 expressions were analyzed by immunohistochemical techniques in the central nervous system (CNS) of embryos, larvae, and recently metamorphosed juveniles of the urodele amphibian Pleurodeles waltl. During the embryonic period, the Pax proteins start being detectable in neuroepithelial domains. Subsequently, they become restricted to subsets of cells in distinct brain regions, maintaining different degrees of expression in late larvae and juvenile brains. Specifically, Pax6 is broadly expressed all along the urodele CNS (olfactory bulbs, pallium, basal ganglia, diencephalon, mesencephalic tegmentum, rhombencephalon, and spinal cord) and the developing olfactory organ and retina. Pax3 and Pax7 are excluded from the rostral forebrain and were usually observed in overlapping regions during embryonic development, whereas Pax3 expression is highly downregulated as development proceeds. Thus, Pax3 is restricted to the roof plate of prosomere 2, pretectum, optic tectum, rhombencephalon, and spinal cord. Comparatively, Pax7 was more conspicuous in all these regions. Pax7 cells were also found in the paraphysis, intermediate lobe of the hypophysis, and basal plate of prosomere 3. Our data show that the expression patterns of the three Pax genes studied are overall evolutionarily conserved, and therefore could unequivocally be used to identify subdivisions in the urodele brain similar to other vertebrates, which are not clearly discernable with classical techniques. In addition, the spatiotemporal sequences of expression provide indirect evidence of putative migratory routes across neuromeric limits and the alar-basal boundary., (Copyright © 2013 Wiley Periodicals, Inc.)

The sequence of appearance of calretinin and calbindin-D28k immunoreactive (CRir and CBir, respectively) cells and fibers has been studied in the brain of the urodele amphibian Pleurodeles waltl. Embryonic, larval and juvenile stages were studied. The early expression and the dynamics of the distribution of CBir and CRir structures have been used as markers for developmental aspects of distinct neuronal populations, highlighting the accurate extent of many regions in the developing brain, not observed on the basis of cytoarchitecture alone. CR and, to a lesser extent, CB are expressed early in the central nervous system and show a progressively increasing expression from the embryonic stages throughout the larval life and, in general, the labeled structures in the developing brain retain their ability to express these proteins in the adult brain. The onset of CRir cells primarily served to follow the development of the olfactory bulbs, subpallium, thalamus, alar hypothalamus, mesencephalic tegmentum, and distinct cell populations in the rhombencephalic reticular formation. CBir cells highlighted the development of, among others, the pallidum, hypothalamus, dorsal habenula, midbrain tegmentum, cerebellum, and central gray of the rostral rhombencephalon. However, it was the relative and mostly segregated distribution of both proteins in distinct cell populations which evidenced the developing regionalization of the brain. The results have shown the usefulness in neuroanatomy of the analysis during development of the onset of CBir and CRir structures, but the comparison with previous data has shown extensive variability across vertebrate classes. Therefore, one should be cautious when comparing possible homologue structures across species only on the basis of the expression of these proteins, due to the variation of the content of calcium-binding proteins observed in well-established homologous regions in the brain of different vertebrates.

Expression patterns of Pax6, Pax7, and, to a lesser extent, Pax3 genes were analyzed by a combination of immunohistochemical techniques in the central nervous system of adult specimens of the urodele amphibian Pleurodeles waltl. Only Pax6 was found in the telencephalon, specifically the olfactory bulbs, striatum, septum, and lateral and central parts of the amygdala. In the diencephalon, Pax6 and Pax7 were distinct in the alar and basal parts, respectively, of prosomere 3. The distribution of Pax6, Pax7, and Pax3 cells correlated with the three pretectal domains. Pax7 specifically labeled cells in the dorsal mesencephalon, mainly in the optic tectum, and Pax6 cells were the only cells found in the tegmentum. Large populations of Pax7 cells occupied the rostral rhombencephalon, along with lower numbers of Pax6 and Pax3 cells. Pax6 was found in most granule cells of the cerebellum. Pax6 cells also formed a column of scattered neurons in the reticular formation and were found in the octavolateral area. The rhombencephalic ventricular zone of the alar plate expressed Pax7. Dorsal Pax7 cells and ventral Pax6 cells were found along the spinal cord. Our results show that the expression of Pax6 and Pax7 is widely maintained in the brains of adult urodeles, in contrast to the situation in other tetrapods. This discrepancy could be due to the generally pedomorphic features of urodele brains. Although the precise role of these transcription factors in adult brains remains to be determined, our findings support the idea that they may also function in adult urodeles., (Copyright © 2012 Wiley Periodicals, Inc.)

The orexins (hypocretins) are peptides found primarily in neurons of the hypothalamus of all vertebrates. Many differences were reported about the precise location of orexin containing cells and their projections throughout the brain in different species. However, there are few direct cross-species comparisons. Previous studies in anuran amphibians have also reported notable species differences. We examined and directly compared the distribution of orexinergic neurons and fibers within the brains of representatives of the three amphibian orders, anurans, urodeles and gymnophionans. Simultaneous detection of orexins and tyrosine hydroxylase was used to assess the precise location of the orexins in the brain and to evaluate the possible influence of the orexin system on the catecholaminergic cell groups. Although some differences were noted, a common pattern for the distribution of orexins in amphibians was observed. In all species, most immunoreactive neurons were observed in the suprachiasmatic nucleus, whereas the cells in the preoptic area and the tuberal region were more variable. Orexin immunoreactive fibers in the brain of all species included abundant fibers throughout the preoptic area and hypothalamus, whereas moderate amounts of fibers were present in the pallium, striatum, septum, thalamus, optic tectum, torus semicircularis, rhombencephalon and spinal cord. The use of double immunohistochemistry in amphibians revealed orexinergic innervation in dopaminergic and noradrenergic cell groups, such as the midbrain tegmentum, locus coeruleus and nucleus of the solitary tract, as was previously reported in mammals.

A previous study in the lizard Gekko gecko has revealed that neuropeptide FF (NPFF, a neuropeptide involved in nociception, cardiovascular regulation, and endocrine function) is widely distributed throughout the brain and spinal cord. Although the distribution of NPFF immunoreactivity shares many features with that found in other vertebrates, it was noted that Gekko shared 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. To assess the primitive or derived character of these features, NPFF and tyrosine hydroxylase (TH) antibodies have been applied to the brain and spinal cord of the turtle, Pseudemys scripta elegans, which belongs to a different radiation of reptiles. As in Gekko, major NPFF-ir cell groups were found in the diagonal band nucleus of Broca and in the hypothalamus, whereas additional cells were identified in the anterior olfactory nucleus, lateral and dorsal cortices, dorsal ventricular ridge, and the intergeniculate leaflet formation. Notable differences are the presence of NPFF-ir cells in the medial cortex and striatum of Pseudemys, which are lacking in Gekko. On the other hand, no NPFF-ir cells could be detected in the septal region and dorsal horn of the spinal cord in Pseudemys. Double staining with NPFF and TH antibodies revealed an intimate relationship between NPFF-ir and TH-ir structures but colocalization could not be established. In conclusion, the distribution of NPFF in the brain of Pseudemys has corroborated previous results in Gekko, but also revealed some notable species differences.

The organization of the somatostatin-like-immunoreactive (SOM-ir) structures in the brain of anuran and urodele amphibians has been well documented, and significant differences were noted between the two amphibian orders. However, comparable data are not available for the third order of amphibians, the gymnophionans (caecilians). In the present study, we analyzed the anatomical distribution of SOM-ir cells and fibers in the brain of the gymnophionan Dermophis mexicanus. In addition, because of its known relationship with catecholamines in other vertebrates, double immunostaining for SOM and tyrosine hydroxylase was used to investigate this situation in the gymnophionan. Abundant SOM-ir cell bodies and fibers were widely distributed throughout the brain. In the telencephalon, pallial and subpallial cells were labeled, being most numerous in the medial pallium and amygdaloid region. Most of the SOM-ir neurons were found in the preoptic area and hypothalamus and showed a clear projection to the median eminence. Less conspicuously, SOM-ir structures were found in the thalamus, tectum, tegmentum, and reticular formation. Both SOM-ir cells and fibers were demonstrated in the spinal cord. The double-immunohistofluorescence technique revealed that catecholaminergic neurons and SOM-ir cells are largely intermingled in many brain regions but form totally separated populations. Many differences were found between the distribution of SOM-ir structures in Dermophis and that in anurans or urodeles. Some features were shared only with anurans, such as the abundant pallial SOM-ir cells, whereas others were common only to urodeles, such as the organization of the hypothalamohypophysial SOM-ir system. In addition, some characteristics were found only in Dermophis, such as the localization of the SOM-ir spinal cells and the lack of colocalization of catecholamines and SOM throughout the brain. Therefore, any conclusions concerning the SOM system in amphibians are incomplete without considering evidence for gymnophionans., (2007 Wiley-Liss, Inc.)

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.

The concentrations of neurokinin A (NKA) and substance P (SP), members of tachykinins family, have been studied in all seasons of the year in frontal cortex, striatum and testes of male offspring 21-, 31-, or 60 days old of mother Wistar rats: control, pinealectomized (PIN-X) and pinealectomized + melatonin during pregnancy (PIN- X + MEL) kept under 12h:12h L:D. Control-offspring: in spite of having been kept under constant environmental conditions throughout the year, had marked differences in tachykinin concentrations. The highest tachykinin concentrations in the frontal cortex were found in summer and fall and the lowest in winter and spring. Maternal PIN-X resulted in alterations of this developmental pattern, mainly in PIN-X- and PIN- X + MEL-offspring in which the highest tachykinin concentrations at 21 and 31 days of age were only observed during summer. The alterations were observed up to 60 days of age for both tachykinins, when at this age control-offspring showed similar NKA concentrations. Seasonal variations were still observed in PIN-X- and PIN- X + MEL-offspring. In striatum and testes no mayor modifications throughout the four seasons of the year were found, with very few exceptions. PIN-X did not alter tachykinin concentrations, neither treatment with melatonin did it. In conclusion, our data clearly indicate for the first time that NKA and SP do indeed have seasonal rhythms in frontal cortex and that the maternal pineal gland plays a role in their entrainment already during fetal life.

The ontogeny of cholinergic neurons has been studied in the brain of the urodele amphibian Pleurodeles waltl by means of choline acetyltransferase (ChAT) immunohistochemistry. Embryonic and larval stages were studied. The earliest ChAT immunoreactive (ChATi) cells were the primary motoneurons in the upper spinal cord, at embryonic stage 29. Slightly later, before hatching, the cranial nerve motor nuclei were immunopositive as well as non-motor populations in the developing inferior reticular nucleus, the nucleus of the solitary tract, the dorsal column nucleus and the retina. At initial larval stages, ChATi cells were located for the first time in the suprachiasmatic nucleus and the isthmic tegmentum. In addition, moderate immunoreactivity appeared in the Mauthner cells. During the period of active larval life the cholinergic systems maturated progressively and new ChATi cell groups were found in the caudal telencephalon and the habenula. Also extensive fiber labeling occurred at active larval stages. No transient ChAT expression was observed and even the Mauthner cells maintained their immunoreactivity after metamorphosis. A general caudorostral spatio-temporal sequence of appearance of cholinergic structures was found in the brain. Comparison of the results observed in the urodele with previous data available only in amniotes shows numerous similarities and suggests a conservative developmental pattern of cholinergic systems in vertebrates.

The distribution of nitrergic cells was studied in the brain of the urodele amphibian Pleurodeles waltl during embryonic and larval stages by means of NADPH-diaphorase histochemistry. The first positive neurons were observed at embryonic stage 30 in the ventrolateral area of the caudal rhombencephalon. Subsequently (stage 33b), weakly reactive cells appeared in the isthmic tegmentum, the mesencephalic tegmentum, the hypothalamus, and the nucleus of the solitary tract. At initial larval stages (34-38), two new groups of NADPH-diaphorase-positive cells appeared in the caudal telencephalon (the amygdaloid region) and in the middle reticular nucleus. During the beginning of the active larval life (stages 39-42), reactive cells were found in the granule cell layer of the olfactory bulb and in the telencephalic pallium. As in the adult, the nitrergic cells in the central nervous system are widely distributed during early development, pointing to important roles of nitric oxide through ontogenesis. The sequence of appearance of nitrergic cells suggests an early involvement in reticulospinal control most likely related to locomotor behavior.

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.

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., (Copyright 2002 S. Karger AG, Basel)

The origin and development of the supraspinal catecholaminergic (CA) innervation of the spinal cord was studied in representative species of the three amphibian orders (Anura: Xenopus laevis and Rana perezi; Urodela: Pleurodeles waltl; Gymnophiona: Dermophis mexicanus). Using retrograde dextran amine tracing in combination with tyrosine hydroxylase (TH)-immunohistochemistry, we showed that only four brain centers contribute to the CA innervation of the adult spinal cord: (1) the ventrolateral component of the posterior tubercle, (2) the periventricular nucleus of the zona incerta, (3) the locus coeruleus, and (4) the nucleus of the solitary tract (except for gymnophionans). The pattern observed is largely similar in all amphibian species studied. The development of the CA innervation of the spinal cord was studied with in vitro double labeling methods in Xenopus laevis tadpoles. At stage 40/41, the first CA neurons projecting to the spinal cord were found to originate in the posterior tubercle. At stage 43, spinal projections were found from the periventricular nucleus of the zona incerta and the locus coeruleus, whereas spinal projections from the nucleus of the solitary tract were not observed before stage 53. These results demonstrate a temporal sequence in the appearance of the CA cell groups projecting to the anuran spinal cord, organized along a rostrocaudal gradient. [Copyright &y& Elsevier]