Neuronal calcium sensor-1 gene ncs-1a is essential for semicircular canal formation in zebrafish inner ear

We have analyzed the functional role of neuronal calcium sensor-1 (Ncs-1) in zebrafish development. We identified two orthologs of the mammalian NCS-1 gene. Full-length cDNAs encoding zebrafish Ncs-1a and Ncs-1b polypeptides were cloned and characterized. Whole-mount in situ hybridization revealed that ncs-1a mRNA was expressed beginning at early somitogenesis. As development progressed, ncs-1a mRNA was present throughout the embryo with expression detected in ventral hematopoietic mesoderm, pronephric tubules, CNS nuclei, and otic vesicle. By 4.5 days post fertilization (dpf), ncs-1a expression was detected primarily in the brain. Expression of ncs-1b mRNA was first detected at 36 hours post fertilization (hpf) and was restricted to the olfactory bulb. By 4.5 dpf, ncs-1b was expressed at low levels throughout the brain. Knockdown of ncs-1a mRNA translation with antisense morpholinos blocked formation of semicircular canals. These studies identify a novel function for ncs-1a in inner ear development and suggest that this calcium sensor plays an important role in vestibular function.     

Development of the Senegal sole, Solea senegalensis forebrain

The present paper deals with the ontogeny of the forebrain of the flatfish Senegal sole, Solea senegalensis, through different developmental stages before and after to metamorphosis. A first approach was made by conventional histological techniques, which allowed the determination of the main ontogenetic events. A second approach was to analyze the proliferation zones (PZ) during development and their locations, as well as the relation between them and the telencephalic asymmetry of the Senegal sole. The results show that before metamorphosis the Senegal sole goes through a fast development. The pituitary is visible 1 day after hatching (DAH), the inferior lobes of the hypothalamus appear 3 DAH, and the olfactory bulb and the differentiation between telencephalon and diencephalon are present around 4 DAH. In addition, by applying proliferating cell nuclear antigen (PCNA) immunohistochemistry by means of a monoclonal antibody against the PCNA and ABC complex, we were able to determine the PZs in the forebrain of pre- and post- metamorphic specimens. Although in both cases the PZs were similar, in premetamorphic animals they were thicker. However, PZs were observed in the pallium and subpallium, preoptic region, pretectum, epithalamus, dorsal and ventral thalamus, posterior tuberculum and hypothalamus. In all cases the PZs, mainly focusing on the telencephalon, were symmetrical in both hemispheres.     

Defects in thalamocortical axon pathfinding correlate with altered cell domains in Mash-1-deficient mice

We have analyzed the pathfinding of thalamocortical axons (TCAs) from dorsal thalamus to neocortex in relation to specific cell domains in the forebrain of wild-type and Mash-1-deficient mice. In wild-type mice, we identified four cell domains that constitute the proximal part of the TCA pathway. These domains are distinguished by patterns of gene expression and by the presence of neurons retrogradely labeled from dorsal thalamus. Since the cells that form these domains are generated in forebrain proliferative zones that express high levels of Mash-1, we studied Mash-1 mutant mice to assess the potential roles of these domains in TCA pathfinding. In null mutants, each of the domains is altered: the two Pax-6 domains, one in ventral thalamus and one in hypothalamus, are expanded in size; a complementary RPTP(delta) domain in ventral thalamus is correspondingly reduced and the normally graded expression of RPTP(delta) in that domain is no longer apparent. In ventral telencephalon, a domain characterized in the wild type by Netrin-1 and Nkx-2.1 expression and by retrogradely labeled neurons is absent in the mutant. Defects in TCA pathfinding are localized to the borders of each of these altered domains. Many TCAs fail to enter the expanded, ventral thalamic Pax-6 domain that constitutes the most proximal part of the TCA pathway, and form a dense whorl at the border between dorsal and ventral thalamus. A proportion of TCAs do extend further distally into ventral thalamus, but many of these stall at an aberrant, abrupt border of high RPTP(delta) expression. A small proportion of TCAs extend around the RPTP(delta) domain and reach the ventral thalamic-hypothalamic border, but few of these axons turn at that border to extend into the ventral telencephalon. These findings demonstrate that Mash-1 is required for the normal development of cell domains that in turn are required for normal TCA pathfinding. In addition, these findings support the hypothesis that ventral telencephalic neurons and their axons guide TCAs through ventral thalamus and into ventral telencephalon.     

Zebrafish dax1 is required for development of the interrenal organ, the adrenal cortex equivalent

Mutations in the human nuclear receptor, DAX1, cause X-linked adrenal hypoplasia congenita (AHC). We report the isolation and characterization of a DAX1 homolog, dax1, in zebrafish. The dax1 cDNA encodes a protein of 264 amino acids, including the conserved carboxy-terminal ligand binding-like motif; but the amino-terminal region lacks the unusual repeats of the DNA binding-like domain in mammals. Genomic sequence analysis indicates that the dax1 gene structure is conserved also. Whole-mount in situ hybridization revealed the onset of dax1 expression in the developing hypothalamus at approximately 26 h post fertilization (hpf). Later, at about 28 hpf, a novel expression domain for dax1 appeared in the trunk. This bilateral dax1-expressing structure was located immediately above the yolk sac, between the otic vesicle and the pronephros. Interestingly, weak and transient expression of dax1 was observed in the interrenal glands (adrenal cortical equivalents) at approximately 31 hpf. This gene was also expressed in the liver after 3 dpf in the zebrafish larvae. Disruption of dax1 function by morpholino oligonucleotides (MO) down-regulated expression of steroidogenic genes, cyp11a and star, and led to severe phenotypes similar to ff1b (SF1) MO-injected embryos. Injection of dax1 MO did not affect ff1b expression, whereas ff1b MO abolished dax1 expression in the interrenal organ. Based on these results, we propose that dax1 is the mammalian DAX1 ortholog, functions downstream of ff1b in the regulatory cascades, and is required for normal development and function of the zebrafish interrenal organ.     

Evidences for tangential migrations in Xenopus telencephalon: developmental patterns and cell tracking experiments

Extensive tangential cell migrations have been described in the developing mammalian, avian, and reptilian forebrain, and they are viewed as a powerful developmental mechanism to increase neuronal complexity in a given brain structure. Here, we report for the first time anatomical and cell tracking evidence for the presence of important migratory processes in the developing forebrain of the anamniote Xenopus laevis. Combining developmental gene expression patterns (Pax6, Nkx2.1, Isl1, Lhx5, Lhx9, and Dll3), neurotransmitter identity (GABA, NOS, ChAT), and connectivity information, several types of putative migratory cell populations and migration routes originating in the ventral pallium and the subpallium are proposed. By means of in vivo cell tracking experiments, pallio-subpallial and subpallio-pallial migrating neurons are visualized. Among them, populations of Nkx2.1(+) striatal interneurons and pallial GABAergic interneurons, which also express the migratory marker doublecortin, are identified. Finally, we find that these tangentially migrating pallial interneurons travel through an "isl1-free channel" that may guide their course through the subpallium. Our findings strongly suggest that the developing Xenopus telencephalon shares many similarities with amniotes in terms of neuronal specification and migrations. However, some differences are discussed, particularly with regard to the evolution of the pallium.     

肌间刺缺失对斑马鱼骨骼发育的影响

利用斑马鱼(Danio rerio)野生型与肌间刺完全缺失突变型个体, 从骨骼染色和骨骼发育相关基因表达两方面, 初步评价了肌间刺缺失对斑马鱼骨骼发育的影响。通过骨骼染色对比观察了两种肌间刺表型个体受精后8dpf(days post fertilization, dpf)到56dpf的骨骼发育情况, 结果显示, 两种肌间刺表型除肌间刺外, 其他骨骼发育基本同步。此外, 通过qRT-PCR实验检测分析了6个骨骼发育相关基因(bmp2a、bmp4、smad1、smad4a、runx2a和sp7)在不同肌间刺表型5个胚胎发育时期(3hpf囊胚期、6hpf原肠胚期、12hpf体节期、24hpf咽囊期和72hpf孵化期)和5个胚后生长阶段(15、30、45、60和75dpf)的表达情况。结果显示:在胚胎发育时期, 野生型和突变型个体中bmp2a、bmp4、smad1、smad4a基因和突变型个体中sp7基因的表达均呈现先升后降的变化趋势, 且在体节期达到最高表达水平;野生型和突变型个体中runx2a基因和野生型个体中sp7基因则表现为逐渐上升的趋势。6个基因在囊胚期和原肠胚期表达量无显著差异, bmp2a的表达水平在体节期、咽囊期和孵化期无显著差异, 野生型个体bmp4、smad1、smad4a、runx2a基因在体节期、咽囊期和孵化期的表达水平明显高于突变型, 而sp7基因则表现为突变型明显高于野生型。胚后发育阶段 6个基因在5个生长阶段均呈现逐渐下降的趋势, 且在两种肌间刺表型间其表达仅在个别时期差异显著。综上所述, 肌间刺的缺失对斑马鱼骨骼发育表现型无显著影响, 只在胚胎发育时期影响骨骼相关基因表达水平的变化;结合骨骼染色结果, 推测肌间刺缺失对斑马鱼骨骼发育无显著影响。     

Cloning of two tryptophan hydroxylase genes expressed in the diencephalon of the developing zebrafish brain

The monoamine serotonin (5-HT) exerts key neuromodulatory activities in all animal phyla, but the development and function of the serotonergic system is still incompletely understood. The zebrafish Danio rerio is an excellent model to approach this question since it is amenable to a combination of genetic, molecular and embryological studies. In order to characterize the organization of serotonergic neurons in the zebrafish we cloned two cDNAs encoding distinct forms of tryptophan hydroxylase (Tph), the rate-limiting enzyme in serotonin synthesis. We report here the pattern of expression of these two genes in relation with immunoreactive TH and 5-HT nuclei in the developing zebrafish embryo and early larva. tphD1 expression starts at 22 h post-fertilization (hpf) in the epiphysis and in basal spinal cells. Expression persists in the epiphysis until at least 4 days (dpf). Between 48 hpf and 3 dpf, tphD1 expression is initiated in retinal amacrine cells and in restricted preoptic and posterior tubercular nuclei within the basal diencephalon. At 3 and 4 dpf, tphD1 expression is newly initiated in the caudal hypothalamus and in branchial arches-associated neurons. tphD2 mRNA is detected transiently (between 30 somites and 32 hpf) in a restricted preoptic nucleus. All sites of tphD1 or D2 expression within the anterior central nervous system are also immunoreactive for 5-HT, but are not positive for TH. However, neither tphD gene is expressed in raphe nuclei, suggesting that additional tph gene(s) exist in zebrafish to account for 5-HT synthesis in that location. The co-expression of tphD1, tphD2 and 5-HT in the zebrafish diencephalon appears in striking contrast to the situation in mammals, where diencephalic serotonin results from re-uptake rather than from local production.     

Cloning of two tryptophan hydroxylase genes expressed in the diencephalon of the developing zebrafish brain

The monoamine serotonin (5-HT) exerts key neuromodulatory activities in all animal phyla, but the development and function of the serotonergic system is still incompletely understood. The zebrafish Danio rerio is an excellent model to approach this question since it is amenable to a combination of genetic, molecular and embryological studies. In order to characterize the organization of serotonergic neurons in the zebrafish we cloned two cDNAs encoding distinct forms of tryptophan hydroxylase (Tph), the rate-limiting enzyme in serotonin synthesis. We report here the pattern of expression of these two genes in relation with immunoreactive TH and 5-HT nuclei in the developing zebrafish embryo and early larva. tphD1 expression starts at 22 h post-fertilization (hpf) in the epiphysis and in basal spinal cells. Expression persists in the epiphysis until at least 4 days (dpf). Between 48 hpf and 3 dpf, tphD1 expression is initiated in retinal amacrine cells and in restricted preoptic and posterior tubercular nuclei within the basal diencephalon. At 3 and 4 dpf, tphD1 expression is newly initiated in the caudal hypothalamus and in branchial arches-associated neurons. tphD2 mRNA is detected transiently (between 30 somites and 32 hpf) in a restricted preoptic nucleus. All sites of tphD1 or D2 expression within the anterior central nervous system are also immunoreactive for 5-HT, but are not positive for TH. However, neither tphD gene is expressed in raphe nuclei, suggesting that additional tph gene(s) exist in zebrafish to account for 5-HT synthesis in that location. The co-expression of tphD1, tphD2 and 5-HT in the zebrafish diencephalon appears in striking contrast to the situation in mammals, where diencephalic serotonin results from re-uptake rather than from local production.     

Neuronal calcium sensor‐1 gene ncs‐1a is essential for semicircular canal formation in zebrafish inner ear

We have analyzed the functional role of neuronal calcium sensor‐1 (Ncs‐1) in zebrafish development. We identified two orthologs of the mammalian NCS‐1 gene. Full‐length cDNAs encoding zebrafish Ncs‐1a and Ncs‐1b polypeptides were cloned and characterized. Whole‐mount in situ hybridization revealed that ncs‐1a mRNA was expressed beginning at early somitogenesis. As development progressed, ncs‐1a mRNA was present throughout the embryo with expression detected in ventral hematopoietic mesoderm, pronephric tubules, CNS nuclei, and otic vesicle. By 4.5 days post fertilization (dpf), ncs‐1a expression was detected primarily in the brain. Expression of ncs‐1b mRNA was first detected at 36 hours post fertilization (hpf) and was restricted to the olfactory bulb. By 4.5 dpf, ncs‐1b was expressed at low levels throughout the brain. Knockdown of ncs‐1a mRNA translation with antisense morpholinos blocked formation of semicircular canals. These studies identify a novel function for ncs‐1a in inner ear development and suggest that this calcium sensor plays an important role in vestibular function. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Morphofunctional Characteristics of Gonadotropin-Releasing Hormone Immunoreactive Structures in the Brain of the Sturgeon Acipenser güldenstädti before Spawning

Light-microscopic immunohistochemical study of the brain with use of the unlabeled antibody to gonadotropin-releasing hormone (GnRH) was carried out on sexually mature individuals of the sturgeon of stage IV of gonadal maturity (before spawning). The brain was examined as a whole; the GnRH-immunoreactive (GnRH-IR) structures were revealed in the olfactory bulb, forebrain, hypothalamus, and neuropituitary. In the fish studied at prespawning period, the highest density of GnRH-IR structure was noticed in the ventral region of forebrain, preoptic region, and anterior neuropituitary. The GnRH-IR cells of the forebrain ventral region, preoptic, and tuberal nuclei send their axons to the region of anterior neuropituitary, in which they contact vessels of the primary portal pituitary system. Thereby, the gonadotropin secretion regulation is performed by corresponding cells of adenopituitary. Rare GnRH-IR fibers in the posterior pituitary lobe contact the general circulation vessels. The dendrites of the GnRH-IR cells that we have revealed in the preoptic region and in the region of tuberal nucleus are located very close to the preoptic bay cavity and to the cerebral III ventricle, respectively. This indicates a possibility of secretion of the neurohormone into the cerebrospinal fluid.     

Expression of Kisspeptins and Kiss Receptors Suggests a Large Range of Functions for Kisspeptin Systems in the Brain of the European Sea Bass

This study, conducted in the brain of a perciform fish, the European sea bass, aimed at raising antibodies against the precursor of the kisspeptins in order to map the kiss systems and to correlate the expression of kisspeptins, kiss1 and kiss2, with that of kisspeptin receptors (kiss-R1 and kiss-R2). Specific antibodies could be raised against the preprokiss2, but not the preoprokiss1. The data indicate that kiss2 neurons are mainly located in the hypothalamus and project widely to the subpallium and pallium, the preoptic region, the thalamus, the pretectal area, the optic tectum, the torus semicircularis, the mediobasal medial and caudal hypothalamus, and the neurohypophysis. These results were compared to the expression of kiss-R1 and kiss-R2 messengers, indicating a very good correlation between the wide distribution of Kiss2-positive fibers and that of kiss-R2 expressing cells. The expression of kiss-R1 messengers was more limited to the habenula, the ventral telencephalon and the proximal pars distalis of the pituitary. Attempts to characterize the phenotype of the numerous cells expressing kiss-R2 showed that neurons expressing tyrosine hydroxylase, neuropeptide Y and neuronal nitric oxide synthase are targets for kisspeptins, while GnRH1 neurons did not appear to express kiss-R1 or kiss-R2 messengers. In addition, a striking result was that all somatostatin-positive neurons expressed-kissR2. These data show that kisspeptins are likely to regulate a wide range of neuronal systems in the brain of teleosts.     

Emx2 directs the development of diencephalon in cooperation with Otx2

The vertebrate brain is among the most complex biological structures of which the organization remains unclear. Increasing numbers of studies have accumulated on the molecular basis of midbrain/hindbrain development, yet relatively little is known about forebrain organization. Nested expression among Otx and Emx genes has implicated their roles in rostral brain regionalization, but single mutant phenotypes of these genes have not provided sufficient information. In order to genetically determine the interaction between Emx and Otx genes in forebrain development, we have examined Emx2(-/-)Otx2(+/-) double mutants and Emx2 knock-in mutants into the Otx2 locus (Otx2(+/Emx2)). Emx2(-/-)Otx2(+/-) double mutants did not develop diencephalic structures such as ventral thalamus, dorsal thalamus/epithalamus and anterior pretectum. The defects were attributed to the loss of the Emx2-positive region at the three- to four-somite stage, when its expression occurs in the laterocaudal forebrain primordia. Ventral structures such as the hypothalamus, mammillary region and tegmentum developed normally. Moreover, dorsally the posterior pretectum and posterior commissure were also present in the double mutants. In contrast, Otx2(+/Emx2) knock-in mutants displayed the majority of these diencephalic structures; however, the posterior pretectum and posterior commissure were specifically absent. Consequently, development of the dorsal and ventral thalamus and anterior pretectum requires cooperation between Emx2 and Otx2, whereas Emx2 expression is incompatible with development of the commissural region of the pretectum.     

Ontogeny of gonadotropin-releasing hormone (GnRH) neurons in hybrid striped bass Morone sp.: whole-mount in situ hybridization analysis

The ontogeny of gonadotropin-releasing hormone (GnRH) mRNA-producing neurons was studied in developing hybrid striped bass (white bass Morone chrysops female × striped bass Morone saxatilis male), 1–55 days post-fertilization (dpf), by whole-mount in situ hybridization. Neurons that produce salmon (s) GnRH were first detected at 32 h post-fertilization in the olfactory placodes. These neurons migrated posteriorly during development and reached their final position at the olfactory bulbs-telencephalon boundary, possibly the terminal nerve ganglion (TNg) by 11 dpf. First signal of chicken (c) GnRH-II neurons appeared in the midbrain 2 dpf and remained there throughout development. A signal of seabream (sb) GnRH mRNA was first detected at 21 dpf in the preoptic area (POA) and as a bilateral continuum along the ventral telencephalon at 32–55 dpf. The expression of all three forms of GnRH increased throughout development. These results suggest that cGnRH-II neurons originate in the mid-brain, and that sGnRH neurons originate in the olfactory placodes and migrate caudally to the TNg. Neurons that express sbGnRH seem to originate at the preoptic area and then to migrate anteriorly along the ventral telencephalon. An olfactory placodal origin of these neurons, however, cannot be ruled out.     

Basic helix-loop-helix transcription factors regulate the neuroendocrine differentiation of fetal mouse pulmonary epithelium

To clarify the mechanisms that regulate neuroendocrine differentiation of fetal lung epithelia, we have studied the expression of the mammalian homologs of achaete-scute complex (Mash1) (Ascl1 - Mouse Genome Informatics); hairy and enhancer of split1 (Hes1); and the expression of Notch/Notch-ligand system in the fetal and adult mouse lungs, and in the lungs of Mash1- or Hes1-deficient mice. Immunohistochemical studies revealed that Mash1-positive cells seemed to belong to pulmonary neuroendocrine cells (PNEC) and their precursors. In mice deficient for Mash1, no PNEC were detected. Hes1-positive cells belong to non-neuroendocrine cells. In the mice deficient in Hes1, in which Mash1 mRNA was upregulated, PNEC appeared precociously, and the number of PNEC was markedly increased. NeuroD (Neurod1 - Mouse Genome Informatics) expression in the lung was detected in the adult, and was enhanced in the fetal lungs of Hes1-null mice. Expression of Notch1, Notch2, Notch3 and Notch4 mRNAs in the mouse lung increased with age, and Notch1 mRNA was expressed in a Hes1-dependent manner. Notch1, Notch2 and Notch3 were immunohistochemically detected in non-neuroendocrine cells. Moreover, analyses of the lungs from the gene-targeted mice suggested that expression of Delta-like 1 (Dll1 - Mouse Genome Informatics) mRNA depends on Mash1. Thus, the neuroendocrine differentiation depends on basic helix-loop-helix factors, and Notch/Notch-ligand pathways may be involved in determining the cell differentiation fate in fetal airway epithelium.     

Tuberoinfundibular Peptide of 39 Residues- Immunoreactive Fibers in the Zona Incerta and the Supraoptic Decussations Terminate in the Neuroendocrine Hypothalamus

Tuberoinfundibular peptide of 39 residues (TIP39) is expressed by neurons in the subparafascicular area, the posterior intralaminar complex of the thalamus and the pontine medial paralemniscal nucleus. TIP39-positive fibers from these areas do not form individual bundles or fascicles, they join other pathways to reach their innervated brain areas. Fibers arise from TIP39 perikarya located in the subparafascicular area and the posterior intralaminar complex of the thalamus could be followed to the hypothalamus. After uni- and bilateral posterolateral surgical deafferentations of the hypothalamus, accumulation of TIP39 immunoreactivity was observed in the fibers caudal to the knife cut, while it disappeared completely rostral to the transection. In serial sections of the forebrain, we could follow TIP39-ir fibers coursing within the zona incerta and the supraoptic decussations. TIP39-positive fibers that join the incerto-hypothalamic pathway reach the medio-dorsal part of the hypothalamus and form moderate to high density networks in the dorsomedial and paraventricular nuclei. The other set of TIP39-positive axons from the subthalamic area join the fibers of the supraoptic decussations and run in an antero-medial direction through the most ventral portion of the hypothalamus up to the retrochiasmatic area, where they crossover. A certain portion of these TIP39-positive fibers terminates in the territories of the arcuate and the medial preoptic nuclei, as well as in the retrochiasmatic area.     

Presenilin-1 Targeted Morpholino Induces Cognitive Deficits,Increased Brain Aβ1−42 and Decreased Synaptic Marker PSD-95 in Zebrafish Larvae

Presenilins are transmembrane proteases required for the proteolytic cleavage of Notch and also act as the catalytic core of the γ-secretase complex, which is responsible for the final cleavage of the amyloid precursor protein into Amyloid-β (Aβ) peptides of varying lengths. Presenilin-1 gene (psen1) mutations are the main cause of early-onset autosomal-dominant Familial Alzheimer Disease. Elucidating the roles of Presenilin-1 and other hallmark proteins involved in Alzheimer’s disease is crucial for understanding the disease etiology and underlying molecular mechanisms. In our study, we used a morpholino antisense nucleotide that targets exon 8 splicing site of psen1 resulting in a dominant negative protein previously validated to investigate behavioral and molecular effects in 5 days post fertilization (dpf) zebrafish larvae. Morphants showed specific cognitive deficits in two optomotor tasks and morphological phenotypes similar to those induced by suppression of Notch signaling pathway. They also had increased mRNA levels of neurog1 at 5 dpf, confirming the potential interaction of Presenilin-1 and Notch in our model. We also evaluated levels of apoptotic markers including p53, PAR-4, Caspase-8 and bax-alpha and found only bax-a decreased at 5dpf. Western Blot analysis showed an increase in Aβ_1?42 and a decrease in the selective post-synaptic marker PSD-95 at 5 dpf. Our data demonstrates that psen1 splicing interference induces phenotypes that resemble early-stage AD, including cognitive deficit, Aβ_1?42 accumulation and synaptic reduction, reinforcing the potential contribution of zebrafish larvae to studies of human brain diseases.

     

Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system

With the use of an antiserum generated in rabbits against synthetic human calcitonin gene-related peptide (CGRP) the distribution of CGRP-like immunoreactive cell bodies and nerve fibers was studied in the rat central nervous system. A detailed stereotaxic atlas of CGRP-like neurons was prepared. CGRP-like immunoreactivity was widely distributed in the rat central nervous system. CGRP positive cell bodies were observed in the preoptic area and hypothalamus (medial preoptic, periventricular, anterior hypothalamic nuclei, perifornical area, medial forebrain bundle), premamillary nucleus, amygdala medialis, hippocampus and dentate gyrus, central gray and the ventromedial nucleus of the thalamus. In the midbrain a large cluster of cells was contained in the peripeduncular area ventral to the medial geniculate body. In the hindbrain cholinergic motor nuclei (III, IV, V, VI, VII XII) contained CGRP-immunoreactivity. Cell bodies were also observed in the ventral tegmental nucleus, the parabrachial nuclei, superior olive and nucleus ambiguus. The ventral horn cells of the spinal cord, the trigeminal and dorsal root ganglia also contained CGRP-immunoreactivity. Dense accumulations of fibers were observed in the amydala centralis, caudal portion of the caudate putamen, sensory trigeminal area, substantia gelatinosa, dorsal horn of the spinal cord (laminae I and II). Other areas containing CGRP-immunoreactive fibers are the septal area, nucleus of the stria terminalis, preoptic and hypothalamic nuclei (e.g., medial preoptic, periventricular, dorsomedial, median eminence), medial forebrain bundle, central gray, medial geniculate body, peripeduncular area, interpeduncular nucleus, cochlear nucleus, parabrachial nuclei, superior olive, nucleus tractus solitarii, and in the confines of clusters of cell bodies. Some fibers were also noted in the anterior and posterior pituitary and the sensory ganglia. As with other newly described brain neuropeptides it can only be conjectured that CGRP has a neuroregulatory action on a variety of functions throughout the brain and spinal cord.