Complementary expression of HSPG 6-O-endosulfatases and 6-O-sulfotransferase in the hindbrain of Xenopus laevis.

Heparan sulfate proteoglycans (HSPGs) are abundant cell surface molecules, consisting of glycosaminoglycan (GAG) chains bound to a protein core. There is high diversity in the sulfation pattern within each GAG chain, creating specific binding sites for many proteins including cell signalling factors, whose activities and distribution are modified by their association with HSPGs (Danesin et al., 2006; Freeman et al., 2008). Here, we describe the distinct expression of three enzymes which modify the 6-O-sulfation state of HSPGs: two 6-O-endosulfatases (Sulf1 and Sulf2), and a 6-O-sulfotransferase (6OST-1). We use in situ hybridisation to determine the spatial distribution of transcripts during tailbud stages of Xenopus laevis development, with a particular focus on neural regions where the 6-O-sulfatases are expressed ventrally and the 6-O-sulfotransferase is restricted dorsally. The complementary expression of these enzymes in the hindbrain and neural tube suggest a role for specific HSPG structure in dorsoventral patterning, possibly through modifying the activity or distribution of signalling molecules such as BMP, Sonic hedgehog, Wnt and/or FGF.

Characterization of primary neurospheres generated from mouse ventral rostral hindbrain.

Serotonergic (5-HT) neurons of the reticular formation play a key role in the modulation of behavior, and their dysfunction is associated with severe neurological and psychiatric disorders, such as depression and schizophrenia. However, the molecular mechanisms underlying the differentiation of the progenitor cells and the specification of the 5-HT phenotype are not fully understood. A primary neurosphere cell-culture system from mouse ventral rostral hindbrain at embryonic day 12 was therefore established. The generated primary neurospheres comprised progenitor cells and fully differentiated neurons. Bromodeoxyuridine incorporation experiments in combination with immunocytochemistry for neural markers revealed the proliferation capacity of the neural multipotent hindbrain progenitors within neurospheres and their ability to differentiate toward the neuronal lineage and serotonergic phenotype. Gene expression analysis by reverse transcription with the polymerase chain reaction showed that the neurospheres were regionally specified, as reflected by the expression of the transcription factors Gata2 and Pet1. Treatment of dissociated primary neurospheres with exogenous Shh significantly increased the number of 5-HT-immunopositive cells compared with controls, whereas neutralization of endogenous Shh significantly decreased the number of 5-HT neurons. Thus, the primary neurosphere culture system presented here allows the expansion of hindbrain progenitor cells and the experimental control of their differentiation toward the serotonergic phenotype. This culture system is therefore a useful model for in vitro studies dealing with the development of 5-HT neurons.

Zebrafish colgate/hdac1 functions in the non-canonical Wnt pathway during axial extension and in Wnt-independent branchiomotor neuron migration.

Vertebrate gastrulation involves the coordinated movements of populations of cells. These movements include cellular rearrangements in which cells polarize along their medio-lateral axes leading to cell intercalations that result in elongation of the body axis. Molecular analysis of this process has implicated the non-canonical Wnt/Frizzled signaling pathway that is similar to the planar cell polarity pathway (PCP) in Drosophila. Here we describe a zebrafish mutant, colgate (col), which displays defects in the extension of the body axis and the migration of branchiomotor neurons. Activation of the non-canonical Wnt/PCP pathway in these mutant embryos by overexpressing DeltaNdishevelled, rho kinase2 and van gogh-like protein 2 (vangl2) rescues the extension defects suggesting that col acts as a positive regulator of the non-canonical Wnt/PCP pathway. Further, we show that col normally regulates the caudal migration of nVII facial hindbrain branchiomotor neurons and that the mutant phenotype can be rescued by misexpression of vangl2 independent of the Wnt/PCP pathway. We cloned the col locus and found that it encodes histone deacetylase1 (hdac1). Our previous results and studies by others have implicated hdac1 in repressing the canonical Wnt pathway. Here, we demonstrate novel roles for zebrafish hdac1 in activating non-canonical Wnt/PCP signaling underlying axial extension and in promoting Wnt-independent caudal migration of a subset of hindbrain branchiomotor neurons.

Ablation of MEKK4 kinase activity causes neurulation and skeletal patterning defects in the mouse embryo.

Skeletal disorders and neural tube closure defects represent clinically significant human malformations. The signaling networks regulating normal skeletal patterning and neurulation are largely unknown. Targeted mutation of the active site lysine of MEK kinase 4 (MEKK4) produces a kinase-inactive MEKK4 protein (MEKK4(K1361R)). Embryos homozygous for this mutation die at birth as a result of skeletal malformations and neural tube defects. Hindbrains of exencephalic MEKK4(K1361R) embryos show a striking increase in neuroepithelial cell apoptosis and a dramatic loss of phosphorylation of MKK3 and -6, mitogen-activated protein kinase kinases (MKKs) regulated by MEKK4 in the p38 pathway. Phosphorylation of MAPK-activated protein kinase 2, a p38 substrate, is also inhibited, demonstrating a loss of p38 activity in MEKK4(K1361R) embryos. In contrast, the MEK1/2-extracellular signal-regulated kinase 1 (ERK1)/ERK2 and MKK4-Jun N-terminal protein kinase pathways were unaffected. The p38 pathway has been shown to regulate the phosphorylation and expression of the small heat shock protein HSP27. Compared to the wild type, MEKK4(K1361R) fibroblasts showed significantly reduced phosphorylation of p38 and HSP27, with a corresponding heat shock-induced instability of the actin cytoskeleton. Together, these data demonstrate MEKK4 regulation of p38 and that substrates downstream of p38 control cellular homeostasis. The findings are the first demonstration that MEKK4-regulated p38 activity is critical for neurulation.

Histone deacetylase 1 is essential for oligodendrocyte specification in the zebrafish CNS.

Histone deacetylases are critical components of transcriptional silencing mechanisms that regulate embryonic development. Recent work has shown that histone deacetylase 1 (hdac1) is required for neuronal specification during zebrafish CNS development. We show here that specification of oligodendrocytes, the myelinating cells of the CNS, also fails to occur in the hdac1 mutant hindbrain, but persistence of neural progenitors in the hindbrain ventricular zone, which express pax6a and sox2, is independent of hdac1 activity. Commitment of ventral neural progenitors to the oligodendrocyte fate is thought to require co-ordinate, hedgehog-dependent expression of olig2 and nkx2.2a in these cells, leading to expression of sox10 and subsequent differentiation of oligodendrocytes. Remarkably, transcription of olig2 is extinguished in ventral neural progenitors of the hdac1 mutant hindbrain, whereas expression of nkx2.2a is up-regulated in these cells, and sox10 expression is suppressed. Our results identify hdac1 as a novel, essential component of the mechanism that allocates neural progenitors to the oligodendrocyte fate, by attenuating expression of a subset of neural progenitor genes and rendering olig2 expression responsive to Hedgehog signalling.

HSD2 neurons in the hindbrain drive sodium appetite.

Sodium-depleted animals develop an appetite for aversive concentrations of sodium. Here we show that chemogenetic activation of aldosterone-sensitive neurons that express 11ß-hydroxysteroid dehydrogenase type 2 (HSD2) in the nucleus of the solitary tract is sufficient to drive consumption of sodium-containing solutions in mice, independently of thirst or hunger. These HSD2-positive neurons are necessary for full expression of sodium appetite and have distinct downstream targets that are activated during sodium depletion.

The connections between C75 and obesity drug-target pathways.

Obesity and its attendant disorders, such as Type II diabetes, have reached epidemic proportions in the USA, and their prevalence is increasing globally. C75 is a small-molecule inhibitor of fatty acid synthase (FAS) and a stimulator of carnitine palmitoyl 1 activity, which causes profound weight loss in mice. Although C75 is not a compound that is destined for human drug development, it has provided two potential pathways to target in obesity therapy: fatty acid synthesis and fatty acid oxidation. In this article, we discuss the latest data challenging the relationship between fatty acid synthase inhibition and C75-induced anorexia.

Calretinin immunoreactivity in the brain of the zebrafish, Danio rerio: distribution and comparison with some neuropeptides and neurotransmitter-synthesizing enzymes. II. Midbrain, hindbrain, and rostral spinal cord.

The distribution of calretinin (CR) in the brainstem and rostral spinal cord of the adult zebrafish was studied by using immunocytochemical techniques. For analysis of some brainstem nuclei and regions, CR distribution was compared with that of complementary markers (choline acetyltransferase, glutamic acid decarboxylase, tyrosine hydroxylase, neuropeptide Y). The results reveal that CR is a marker of various neuronal populations distributed throughout the brainstem, including numerous cells in the optic tectum, torus semicircularis, secondary gustatory nucleus, reticular formation, somatomotor column, gustatory lobes, octavolateral area, and inferior olive, as well as of characteristic tracts of fibers and neuropil. These results indicate that CR may prove useful for characterizing a number of neuronal subpopulations in zebrafish. Comparison of the distribution of CR observed in the brainstem of zebrafish with that reported in an advanced teleost (the gray mullet) revealed a number of similarities, and also some interesting differences. Our results indicate that many brainstem neuronal populations have maintained the CR phenotype in widely divergent teleost lines, so CR studies may prove very useful for comparative analysis.

Role of estradiol in intrinsic hindbrain AMPK regulation of hypothalamic AMPK, metabolic neuropeptide, and norepinephrine activity and food intake in the female rat.

This study addressed the hypothesis that dorsomedial hindbrain adenosine 5'-monophosphate-activated protein kinase (AMPK) imposes inherent estradiol-dependent control of hypothalamic AMPK, neuropeptide, and norepinephrine (NE) activity and feeding in the female rat. Estradiol (E)- or oil (O)-implanted ovariectomized rats were injected with the AMPK inhibitor compound c (Cc) or vehicle into the caudal fourth ventricle (CV4) prior to micropunch-dissection of individual hypothalamic metabolic loci or assessment of food intake. Cc decreased hindbrain dorsal vagal complex phosphoAMPK (pAMPK) in both E and O; tissue ATP levels were reduced by this treatment in O only. In E/Cc, pAMPK expression was diminished in the lateral hypothalamic area (LHA) and ventromedial (VMH) and paraventricular (PVH) nuclei; only PVH pAMPK was suppressed by this treatment in O/Cc. Cc decreased PVH corticotropin-releasing hormone and arcuate (ARH) proopiomelanocortin (POMC) and neuropeptide Y in O, but suppressed only POMC in E. O/Cc exhibited both augmented (PVH, VMH) and decreased (LHA, ARH) hypothalamic NE content, whereas Cc treatment of E elevated preoptic and dorsomedial hypothalamic nucleus NE. Cc completely or incompletely repressed feeding in E versus O, respectively. Results implicate dorsomedial hindbrain AMPK in physiological stimulus-induced feeding in females. Excepting POMC, hypothalamic neuropeptide responses to this sensor may be contingent on estrogen. Estradiol likely designates hypothalamic targets of altered NE signaling due to hindbrain AMPK activation. Divergent changes in NE content of hypothalamic loci in O/Cc uniquely demonstrate sensor-induced bimodal catecholamine signaling to those sites.

An Eph-related receptor protein tyrosine kinase gene segmentally expressed in the developing mouse hindbrain.

In search of genes possibly involved in the regulation of hindbrain segmentation, we have isolated mouse cDNA clones corresponding to putative protein kinase genes by polymerase chain reaction amplification of cDNA from 9.5-day-old embryo hindbrains. In situ hybridization analysis revealed that one of these genes, Sek, was expressed in an alternating segment-restricted pattern in the developing hindbrain. Isolation and analysis of Sek cDNAs covering the entire coding sequence indicated that Sek encoded a putative receptor protein tyrosine kinase, belonging to the Eph family. These data are consistent with a role of the Sek gene product in a signal transduction process involved in pattern formation in the hindbrain.

Elk-L3, a novel transmembrane ligand for the Eph family of receptor tyrosine kinases, expressed in embryonic floor plate, roof plate and hindbrain segments.

The Eph family of receptor tyrosine kinases has 13 distinct members and seven ligands for these receptors have been described to date. These receptors and their ligands have been implicated in regulating neuronal axon guidance and in patterning of the developing nervous system and may also serve a patterning and compartmentalization role outside of the nervous system as well. The ligands are all membrane-attached, and this attachment appears to be crucial for their normal function; five of the known ligands are linked to the membrane via a glycosyl phosphotidylinositol (GPI) linkage, while two of the ligands are transmembrane proteins. Despite the large number of Eph family receptors and ligands, they can be divided into just two major subclasses based on their binding specificities. All the GPI-anchored ligands bind and activate one subclass of the Eph receptors (that represented by Eck) while the two transmembrane ligands bind and activate the other major subclass of receptors (represented by Elk). Here we report the identification and characterization of the third, and most divergent, member of the transmembrane group of Eph ligands, which we term Elk-L3 (Elk-related receptor ligand number 3). Elk-L3 is notable for its remarkably restricted and prominent expression in the floor plate and roof plate of the developing neural tube and its rhombomere-specific expression in the developing hindbrain. The Elk-L3 gene has been localized to mouse chromosome 11 and human chromosome 17.

Expression of cyp26b1 during zebrafish early development.

We have cloned the zebrafish ortholog of the mammalian cyp26b1 gene. The predicted zebrafish cyp26b1 protein shares greater than 73% identity with mammalian homologues. cDNA transfection assays showed that like human cyp26b1, zebrafish cyp26b1 is involved in limiting the activity of retinoic acid. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of embryonic RNAs suggested that no maternal cyp26b1 message is detectable. Zygotic cyp26b1 message could be detected at 75% epiboly by RT-PCR and localized to presumptive rhombomere 3 and rhombomere 4 at the early two-somite (2S) stage (10.5 hpf: hour post fertilization) by whole mount in situ hybridization. As development proceeds expression expands anteriorly to include rhombomere 2 at the 10S stage (14hpf). By 14S (16hpf) expression in the hindbrain has also expanded posteriorly and encompasses rhombomere 2 through rhombomere 6. At later stages, 24 through 48 hpf, additional expression was found in the eyes, diencephalon, midbrain-hindbrain boundary, cerebellum, pectoral fin and the pharyngeal arch primordia.

Cloning and developmental expression of zebrafish GTP cyclohydrolase I.

GTP cyclohydrolase I (GCH) catalyses the conversion of GTP to dihydroneopterin triphosphate, initiating the pteridine pathway. The final product tetrahydrobiopterin (H4biopterin) is the cofactor for neurotransmitter synthesis and for tyrosine supply during melanogenesis. Sepiapterin accumulates as a pigment. We cloned the zebrafish gch cDNA, which encodes a protein highly homologous to other vertebrate sequences and characterised the recombinant enzyme. By in situ hybridisation, we found that gch is expressed in both the melanophore and xanthophore lineages during early development. gch-expression almost disappears after 3 days post-fertilisation (dpf), despite further sepiapterin synthesis. gch-transcripts are also located in catecholaminergic neurons, within the central nervous system and in the arch-associated neurons.

NMDA receptors on zebrafish Mauthner cells require CaMKII-α for normal development.

Calcium/calmodulin dependent protein kinase 2 (CaMKII) is a multifunctional protein that is highly enriched in the synapse. It plays important roles in neuronal functions such as synaptic plasticity, synaptogenesis, and neural development. Gene duplication in zebrafish has resulted in the occurrence of seven CaMKII genes (camk2a, camk2b1, camk2b2, camk2g1, camk2g2, camk2d1, and camk2d2) that are developmentally expressed. In this study, we used single cell, real-time quantitative PCR to investigate the expression of CaMKII genes in individual Mauthner cells (M-cells) of 2 days post fertilization (dpf) zebrafish embryos. We found that out of seven different CaMKII genes, only the mRNA for CaMKII-α was expressed in the M-cell at detectable levels, while all other isoforms were undetectable. Morpholino knockdown of CaMKII-α had no significant effect on AMPA synaptic currents (mEPSCs) but decreased the amplitude of NMDA mEPSCs. NMDA events exhibited a biexponential decay with τfast ≈ 30 ms and τslow ≈ 300 ms. Knockdown of CaMKII-α specifically reduced the amplitude of the slow component of the NMDA-mediated currents (mEPSCs), without affecting the fast component, the frequency, or the kinetics of the mEPSCs. Immunolabelling of the M-cell showed increased dendritic arborizations in the morphants compared with controls, and knockdown of CaMKII-α altered locomotor behaviors of touch responses. These results suggest that CaMKII-α is present in embryonic M-cells and that it plays a role in the normal development of excitatory synapses. Our findings pave the way for determining the function of specific CaMKII isoforms during the early stages of M-cell development.

Localization of the calcium/calmodulin-dependent protein phosphatase, calcineurin, in the hindbrain and spinal cord of the rat.

The calcium/calmodulin-dependent protein phosphatase calcineurin was localized at the light microscopic level in the rat hindbrain and spinal cord by using an antibody against the alpha-isoform of the catalytic subunit. Calcineurin was highly concentrated in axons, dendrites, and cell bodies of a subpopulation of alpha-motoneurons in hindbrain motor nuclei and the lateral motor column along the length of the spinal cord. These calcineurin-positive alpha-motoneurons appeared to be randomly distributed and represented approximately 25% of the total alpha-motoneuron pool in the motor trigeminal nucleus and the spinal cord lateral motor column. Within the facial nucleus, calcineurin-containing motoneurons were present in the medial and dorsal subdivision but not in the lateral and intermediate subdivision. In addition to the enrichment in motoneurons, calcineurin was enriched in cells of the superficial laminae of the spinal cord dorsal horn and its extension into the medulla, the caudal spinal trigeminal nucleus. Axonal staining in the white matter of the spinal cord was generally weak, except in the dorsolateral funiculus, where strongly calcineurin-positive axons formed a putative ascending tract that appeared to terminate uncrossed in the caudal lateral reticular nucleus of the medulla. This tract may originate from calcineurin-positive cells in the dorsolateral funiculus. We also compared the distribution of calcineurin with calcium/calmodulin-dependent kinase II in the spinal cord and found that the kinase is more widely expressed. Thus, calcineurin is highly restricted to a few locations in the hindbrain and spinal cord. Selective staining in facial subnuclei that innervate phasically active muscles suggests that calcineurin-positive motoneurons represent a subset of alpha-motoneurons innervating a metabolic subtype of muscle fibers, possibly fast-twitch fibers.

Immunocytochemical localization of aromatase in sensory and integrating nuclei of the hindbrain in Japanese quail (Coturnix japonica).

The distribution of the estrogen synthesizing enzyme (aromatase) in the hindbrain (rhombencephalon and mesencephalon) of male adult quail was investigated by immunocytochemistry. Aromatase-immunoreactive neuronal structures (perikarya and fibers bearing punctate structures) were observed in sensory (trigeminal, solitary tract, vestibular, optic tectum) and integrating (parabrachial, periaqueductal, cerulean, raphe) nuclei. Besides the expression of aromatase in these well-delineated nuclei, dense to scattered networks of immunoreactive fibers were found dispersed throughout the hindbrain and, in particular, in its rostral and dorsal parts. To a lesser extent, they were also present throughout the premotor nuclei of the reticular formation and in various fiber tracts. In contrast, no immunoreactive signal was found in motor nuclei, and in most of the statoacoustic (cerebellum, cochlear, olive, pontine, part of vestibular) nuclei. The expression of aromatase in perikarya and fibers in areas of the adult hindbrain where estrogen receptors have been identified previously suggests a role for estrogens locally produced in the regulation of sensory and integrating functions, contrary to the widespread assumption that these functions are regulated exclusively by steroids produced in the gonads.

Distribution and characterization of cyclooxygenase immunoreactivity in the ovine brain.

Evidence from tissue culture studies suggests that glial cells are the principal source of prostaglandins in the brain. We have used immunohistochemistry, Western blot analysis, and enzyme activity assays to localize cyclooxygenase (COX), the enzyme responsible for the conversion of arachidonic acid to prostaglandins, in situ in the normal ovine brain. We observed very few immunoreactive glial cells. In contrast, an extensive distribution of COX-like immunoreactive (ir) neuronal cell bodies and dendrites and a corresponding pattern of COX enzyme activity were observed. COXir neurons were most abundant in forebrain sites involved in complex, integrative functions and autonomic regulation such as the cerebral cortex, hippocampus, amygdala, bed nucleus of the stria terminalis, substantia innominata, dorsomedial nucleus of the hypothalamus, and tuberomammillary nucleus. Moderate populations were observed in other regions of the central nervous system implicated in sensory afferent processing, including the dorsal column nuclei, spinal trigeminal nucleus, and superior colliculus, and in structures involved in autonomic regulation, such as the nucleus of the solitary tract, parabrachial nucleus, and the periaqueductal gray matter. We did not observe COXir axons or terminal fields, however. Our results suggest that neurons may use prostaglandins as intracellular or perhaps paracrine, but probably not synaptic, mediators in the normal brain.

Differential cellular and subcellular localization of protein phosphatase 1 isoforms in brain.

Protein phosphatase 1 (PP1) is a gene family with a number of important functions in brain. Association with a wide variety of regulatory/targeting subunits is thought to be instrumental in directing the phosphatase to specific subcellular locations and substrates. By using antibodies directed against specific PP1 isoforms, we asked whether PP1 isoforms are differentially distributed in brain. Immunoblotting detects in brain the PP1gamma2 isoform, which had previously been thought to be testis specific, in addition to alpha, beta, and gamma1 isoforms. PP1 isoform expression varies modestly in extracts from different subdissected brain regions and is relatively constant during postnatal development, except for an about twofold increase in PP1gamma2. By immunohistochemical analyses of rat brain, PP1beta and PP1gamma1 cellular expression is widespread but quite distinct from one another. Subcellular fractionation studies demonstrate that PP1beta and PP1gamma1 are selectively associated with different cytoskeletal elements: PP1beta with microtubules, PP1gamma1 with the actin cytoskeleton. Double-immunofluorescence labeling of cultured cortical neurons further reveals a strikingly different and nonoverlapping localization of PP1beta and PP1gamma1: whereas PP1beta localizes to a discrete area of the soma, PP1gamma1 is highly enriched in dendritic spines and presynaptic terminals of cultured neurons. These results show that PP1 isoforms are targeted to different neuronal cytoskeletal compartments with a high degree of specificity, presumably by isoform-specific association with regulatory/targeting proteins. Furthermore, the synaptic localization of PP1gamma1 indicates that it is this isoform that is involved in the regulation of synaptic phosphoproteins such as neurotransmitter receptors and ion channels implicated in synaptic plasticity.

Distribution of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) immunoreactivity in the central nervous system of two chondrostean fishes (Acipenser baeri and Huso huso).

To obtain a better understanding of the evolution of the brain catecholaminergic systems of fishes, we have examined the distribution of catecholamine-synthesizing enzymes in two species of sturgeon (Acipenser baeri and Huso huso) using antibodies against tyrosine hydroxylase (TH) and dopamine-beta -hydroxylase (DBH; only analyzed in Acipenser). Both sturgeons showed TH-immunoreactive (THir) neurons widely distributed in most regions of the brain, the highest number of THir cells being located in the forebrain (olfactory bulb, preoptic area, and posterior tuberculum). THir cells were also seen in other forebrain areas (retrobulbar area, dorsal and ventral telencephalic areas, hypothalamus, ventral thalamus, pretectal area) and in the brainstem (locus coeruleus, viscerosensory area, caudal reticular formation, and area postrema). Immunoreactive fibers and varicosities showed a wide distribution, being particularly abundant in the diencephalon and mesencephalon. DBH-immunoreactive (DBHir) cells were observed in the anterior tuberal nucleus, where these cells were TH-negative, and in the locus coeruleus and the caudal rhombencephalon (vagal reticular formation), where the DBHir cells were also THir. DBHir fibers were scarce in the telencephalon and very abundant in the diencephalon, mesencephalon, and rhombencephalon. The comparative analysis of the catecholaminergic systems of chondrosteans and those observed in other groups of fishes and tetrapods indicate a similar organization of many nuclei, as well as characteristics that are probably primitive, such as the presence of a large number of forebrain catecholaminergic groups.

Development of tyrosine hydroxylase-immunoreactive systems in the brain of the larval lamprey Lampetra fluviatilis.

The development of the catecholaminergic system of the brain of the lamprey (Lampetra fluviatilis) was studied with immunocytochemistry in a series of larvae of different sizes by using two different antibodies directed against tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis. In group 1 larvae (length: 29-54 mm, ages: 8 months to 1.5 years), the only TH-immunoreactive somata observed were located in the caudal wall of the recessus praeopticus (RP) and in the nucleus tuberculi posterioris (NTP). In group 2 larvae (length: 55-80 mm, ages: 1.5-2.5 years), the somata of immunolabeled cells of the NTP give rise to fibers, most of which are ascending and terminate in the corpus striatum. Additional immunoreactive cells are observed in the nucleus praeopticus (NP), which has differentiated, and in the spinal cord. In group 3 larvae (length: 81-110 mm, ages: 2.5-4 years), the spatial distribution of TH-immunoreactive elements (somata, fibers, and terminals) bears many resemblances to that seen in the adult. Immunolabeled cells may be observed in the olfactory bulb, in the nucleus commissurae postopticae (NCP), and in the nucleus dorsalis hypothalami (NDH). Nevertheless, some groups of TH-immunoreactive cells found in the adult are not observed in group 3 larvae; these may appear during the metamorphic phase. By comparative analysis, we show that, in spite of several differences, the spatiotemporal sequence of appearance of TH-immunoreactive cell bodies and fibers in the lamprey presents many similarities to that described in gnathostomes.