Pretectal Region Research Articles

Application of diffusion tensor imaging and functional alterations in evaluating brain alterations related to heatstroke in a rat model

ObjectivesTo assess the alterations in resting-state functions and neural structures in the brain of a heatstroke rat model and explore the underlying relationship. MethodsIn total, 17 male Sprague Dawley rats were randomly divided into a control group (CTRL, n = 7) and a heatstroke group (HS, n = 10). All rats underwent 7.0 T magnetic resonance imaging (MRI). T2-weighted imaging, resting-state functional MRI (rs-fMRI), and diffusion tensor imaging (DTI) were obtained. On day 25, the surviving HS group rats (the follow-up group, FU, n = 7) were scanned again. ResultsHeatstroke resulted in functional alterations and structural damage in the cerebellar molecular layer (CML), right perirhinal area (PA), pretectal region (PR), right dentate gyrus, and external cortex of the inferior colliculus (ECIC). Further functional changes occur in the right temporal associative cortex (TAC), left retrosplenial cortex (RC), and CML during convalescence. The fractional anisotropy values were significantly positively correlated with the amplitude of low-frequency fluctuation (ALFF) (HS–CML: r = 0.746, p = 0.034; right PR: r = 0.648, p = 0.049; FU–right PA: r = 0.817, p = 0.025)/regional homogeneity (ReHo) ratio (HS–CML: r = 0.833, p = 0.008; ECIC: r = 0.678, p = 0.045) and negatively correlated with the ALFF (FU–left RC: r = −0.818, p = 0.024; right TAC: r = −0.813, p = 0.049). ConclusionDTI and rs-fMRI allow meticulous monitoring of the progression of neurological and functional alterations in the brain after heatstroke.

Noradrenergic substrates sensing light within brainstem reticular formation as targets for light-induced behavioral and cardiovascular plasticity.

The occurrence of pure light exerts a variety of effects in the human body, which span from behavioral alterations, such as light-driven automatic motor activity, cognition and mood to more archaic vegetative functions, which encompass most organs of the body with remarkable effects on the cardiovascular system. Although empirical evidence clearly indicates occurrence of these widespread effects, the anatomical correlates and long-lasting changes within putatively specific neuronal circuitries remain largely unexplored. A specific role is supposed to take place for catecholamine containing neurons in the core of the brainstem reticular formation, which produces a widespread release of noradrenaline in the forebrain while controlling the vegetative nervous system. An indirect as well as a direct (mono-synaptic) retino-brainstem pathway is hypothesized to rise from a subtype of intrinsically photosensitive retinal ganglion cells (iPRGCs), subtype M1, which do stain for Brn3b, and project to the pre-tectal region (including the olivary pre-tectal nucleus). This pathway provides profuse axon collaterals, which spread to the periacqueductal gray and dorsal raphe nuclei. According to this evidence, a retino-reticular monosynaptic system occurs, which powerfully modulate the noradrenergic hub of reticular nuclei in the lateral column of the brainstem reticular formation. These nuclei, which are evidenced in the present study, provide the anatomical basis to induce behavioral and cardiovascular modulation. The occurrence of a highly interconnected network within these nuclei is responsible for light driven plastic effects, which may alter persistently behavior and vegetative functions as the consequence of long-lasting alterations in the environmental light stimulation of the retina. These changes, which occur within the core of an archaic circuitry such as the noradrenaline-containing neurons of the reticular formation, recapitulate, within the CNS, ancestral effects of light-driven changes, which can be detected already within the retina itself at the level of multipotent photic cells.

Organization of the Catecholaminergic System in the Short-Lived Fish Nothobranchius furzeri.

The catecholaminergic system has received much attention based on its regulatory role in a wide range of brain functions and its relevance in aging and neurodegenerative diseases. In the present study, we analyzed the neuroanatomical distribution of catecholaminergic neurons based on tyrosine hydroxylase (TH) immunoreactivity in the brain of adult Nothobranchius furzeri. In the telencephalon, numerous TH+ neurons were observed in the olfactory bulbs and the ventral telencephalic area, arranged as strips extending through the rostrocaudal axis. We found the largest TH+ groups in the diencephalon at the preoptic region level, the ventral thalamus, the pretectal region, the posterior tuberculum, and the caudal hypothalamus. In the dorsal mesencephalic tegmentum, we identified a particular catecholaminergic group. The rostral rhombencephalon housed TH+ cells in the locus coeruleus and the medulla oblongata, distributing in a region dorsal to the inferior reticular formation, the vagal lobe, and the area postrema. Finally, scattered TH+ neurons were present in the ventral spinal cord and the retina. From a comparative perspective, the overall organization of catecholaminergic neurons is consistent with the general pattern reported for other teleosts. However, N. furzeri shows some particular features, including the presence of catecholaminergic cells in the midbrain. This work provides a detailed neuroanatomical map of the catecholaminergic system of N. furzeri, a powerful aging model, also contributing to the phylogenetic understanding of one of the most ancient neurochemical systems.

Neuroanatomical Distribution of the Serotonergic System in the Brain and Retina of Holostean Fishes, The Sister Group to Teleosts

Among actinopterygian fishes, holosteans are the phylogenetically closest group to teleosts but they have been much less studied, particularly regarding the neurochemical features of their central nervous system. The serotonergic system is one of the most important and conserved systems of neurotransmission in all vertebrates. By means of immunohistochemistry against serotonin (5-hydroxytryptamine), we have conducted a comprehensive and complete description of this system in the brain and retina of representative species of the 3 genera of holostean fishes, belonging to the only 2 extant orders, Amiiformes and Lepisosteiformes. Serotonin-immunoreactive cell groups were detected in the preoptic area, the hypothalamic paraventricular organ, the epiphysis, the pretectal region, the long and continuous column of the raphe, the spinal cord, and the inner nuclear layer of the retina. Specifically, the serotonergic cell groups in the preoptic area, the epiphysis, the pretectum, and the retina had never been identified in previous studies in this group of fishes. Widespread serotonergic innervation was observed in all main brain regions, but more abundantly in the subpallium, the hypothalamus, the habenula, the optic tectum, the so-called cerebellar nucleus, and the area postrema. The comparative analysis of these results with those in other groups of vertebrates reveals some extremely conserved features, such as the presence of serotonergic cells in the retina, the pineal organ, and the raphe column, while other characteristics, like the serotonergic populations in the preoptic area, the paraventricular organ, the pretectum, and the spinal cord are generally present in all fish groups, but have been lost in most amniotes.

Pretectal neurons control hunting behaviour.

For many species, hunting is an innate behaviour that is crucial for survival, yet the circuits that control predatory action sequences are poorly understood. We used larval zebrafish to identify a population of pretectal neurons that control hunting. By combining calcium imaging with a virtual hunting assay, we identified a discrete pretectal region that is selectively active when animals initiate hunting. Targeted genetic labelling allowed us to examine the function and morphology of individual cells and identify two classes of pretectal neuron that project to ipsilateral optic tectum or the contralateral tegmentum. Optogenetic stimulation of single neurons of either class was able to induce sustained hunting sequences, in the absence of prey. Furthermore, laser ablation of these neurons impaired prey-catching and prevented induction of hunting by optogenetic stimulation of the anterior-ventral tectum. We propose that this specific population of pretectal neurons functions as a command system to induce predatory behaviour.

Gene expression analysis of developing cell groups in the pretectal region of Xenopus laevis

ABSTRACTOur previous analysis of progenitor domains in the pretectum of Xenopus revealed three molecularly distinct anteroposterior subdivisions, identified as precommissural (PcP), juxtacommissural (JcP), and commissural (CoP) histogenetic domains (Morona et al. [2011] J Comp Neurol 519:1024–1050). Here we analyzed at later developmental stages the nuclei derived from these areas, attending to their gene expression patterns and histogenesis. Transcription‐factor gene markers were used to selectively map derivatives of each domain: Pax7 and Pax6 (CoP); Foxp1 and Six3 (JcP); and Xiro1, VGlut2, Ebf1, and Ebf3 (PcP). Additional genoarchitectural information was provided by the expression of Gbx2, NPY, Lhx1, and Lhx9. This allowed both unambiguous characterization of the anuran pretectal nuclei with regard to their origin in the three early anteroposterior progenitor domains, and their comparison with counterparts in the chick and mouse pretectum. Our observations demonstrated a molecular conservation, during practically all the stages analyzed, for most of the main markers used to define genoarchitecturally the main derivatives of each pretectal domain. We found molecular evidence to propose homologous derivatives from the CoP (olivary pretectal, parvocellular, and magnocellular posterior commissure and lateral terminal nuclei), JcP (spiriformis lateral and lateral terminal nuclei), and PcP (anterior pretectal nucleus) to those described in avian studies. These results represent significant progress in the comprehension of the diencephalic region of Xenopus and show that the organization of the pretectum possesses many features shared with birds. J. Comp. Neurol. 525:715–752, 2017. © 2016 Wiley Periodicals, Inc.

Neurobiology: Imaging Prey Capture Circuits in Zebrafish

Two recent studies used a virtual hunting assay and functional imaging to identify prey-capture circuits in zebrafish. Together they show that the optic tectum and a pretectal region are two retinorecipient areas important for the recognition and capture of prey.

A dedicated visual pathway for prey detection in larval zebrafish.

Zebrafish larvae show characteristic prey capture behavior in response to small moving objects. The neural mechanism used to recognize objects as prey remains largely unknown. We devised a machine learning behavior classification system to quantify hunting kinematics in semi-restrained animals exposed to a range of virtual stimuli. Two-photon calcium imaging revealed a small visual area, AF7, that was activated specifically by the optimal prey stimulus. This pretectal region is innervated by two types of retinal ganglion cells, which also send collaterals to the optic tectum. Laser ablation of AF7 markedly reduced prey capture behavior. We identified neurons with arbors in AF7 and found that they projected to multiple sensory and premotor areas: the optic tectum, the nucleus of the medial longitudinal fasciculus (nMLF) and the hindbrain. These findings indicate that computations in the retina give rise to a visual stream which transforms sensory information into a directed prey capture response.

Advances in understanding mechanisms of thalamic relays in cognition and behavior.

The main impetus for a mini-symposium on corticothalamic interrelationships was the recent number of studies highlighting the role of the thalamus in aspects of cognition beyond sensory processing. The thalamus contributes to a range of basic cognitive behaviors that include learning and memory, inhibitory control, decision-making, and the control of visual orienting responses. Its functions are deeply intertwined with those of the better studied cortex, although the principles governing its coordination with the cortex remain opaque, particularly in higher-level aspects of cognition. How should the thalamus be viewed in the context of the rest of the brain? Although its role extends well beyond relaying of sensory information from the periphery, the main function of many of its subdivisions does appear to be that of a relay station, transmitting neural signals primarily to the cerebral cortex from a number of brain areas. In cognition, its main contribution may thus be to coordinate signals between diverse regions of the telencephalon, including the neocortex, hippocampus, amygdala, and striatum. This central coordination is further subject to considerable extrinsic control, for example, inhibition from the basal ganglia, zona incerta, and pretectal regions, and chemical modulation from ascending neurotransmitter systems. What follows is a brief review on the role of the thalamus in aspects of cognition and behavior, focusing on a summary of the topics covered in a mini-symposium held at the Society for Neuroscience meeting, 2014.

Immunohistochemical analysis of Pax6 and Pax7 expression in the CNS of adult Xenopus laevis

Pax6 and Pax7 are transcription factors essential for the development of the CNS. In addition, increasing data, mainly obtained in amniotes, support that they are expressed in subsets of neurons in the adult, likely playing a role in maintaining neuron type identity. In the present study we analyzed the detailed distribution of Pax6 and Pax7 cells in the adult CNS of Xenopus laevis. Immunohistochemistry with antibodies that are required for high-resolution analysis of Pax-expressing cells was conducted. A wide distribution of Pax6 and Pax7 cells throughout the CNS was detected, with distinct patterns that showed only slight overlapping. Only Pax6 was expressed in the telencephalon, including the olfactory bulbs, septum, striatum and amygdaloid complex. In the diencephalon, Pax6 and Pax7 were distinct in the alar and basal parts, respectively, of prosomere 3. Large numbers of Pax6 and Pax7 cells were distributed in the pretectal region (alar plate of prosomere 1) but only Pax6 cells extended into basal plate. Pax7 specifically labeled cells in the optic tectum, including the ventricular zone, and Pax6 cells were the only cells found in the tegmentum. Pax6 was found in most granule cells of the cerebellum and Pax7 expression was found only in the ventricular zone. In the rostral rhombomere 1, Pax7 labeling was detected in cells of the ventricular zone of the alar plate, but numerous migrated cells were located in the basal plate, including the griseum centrale and the interpeduncular nucleus. 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 separated from the ventricle, which did not show immunoreactivity. Our results show that the expression of Pax6 and Pax7 is widely maintained in the adult brain of Xenopus, like in urodele amphibians and in contrast to the situation described in amniotes. Therefore, in amphibians these transcription factors seem to be needed to maintain specific entities of subpopulations of neurons in the adult CNS.

Organization of the Serotonergic System in the Central Nervous System of Two Basal Actinopterygian Fishes: the Cladistians Polypterus senegalus and Erpetoichthys calabaricus

Cladistians (Polypteriformes) are currently considered basal to other living ray-finned fishes (actinopterygians), and their brain organization is therefore critical to providing information about the primitive neural characters that existed in the earliest ray-finned fishes. The organization of the serotonergic system in the brain has been carefully analyzed in most vertebrate groups, and in the present study we provide the first detailed information on the distribution of serotonergic cell bodies and fibers in the central nervous system of representative species of the two extant genera of cladistians, i.e. Polypterus senegalus and Erpetoichthys calabaricus, by means of immunohistochemistry against serotonin (5-HT). Distinct groups of immunoreactive cells were detected in the preoptic area, the hypothalamic paraventricular organ, the pineal organ, the pretectal region, the long column of the raphe in the rhombencephalic midline, the spinal cord, and amacrine cells in the inner nuclear layer of the retina. Fiber labeling was widely distributed in all main brain subdivisions but was more abundant in distinct pallial and subpallial areas, the preoptic area, the thalamus, the optic tectum, the tori semicircularis and lateralis, the rhombencephalic reticular formation, the nucleus of the solitary tract, and the dorsal aspect of the spinal cord. Our analysis makes it possible to establish which serotonergic structures characterized the earliest ray-finned fishes, and a comparison of these results with those from other classes of vertebrates, including a segmental analysis to correlate cell populations, reveals that most characteristics, such as the presence of serotonergic cells in the preoptic area and the basal hypothalamus, are preserved in all anamniotes. However, this system seems to be reduced in amniotes, mainly mammals, although important features are shared, such as the presence of serotonergic cells in the pineal organ, the retina, and the raphe nuclei.

Olfacto-retinalis pathway in Austrolebias charrua fishes: A neuronal tracer study

The olfacto-retinal centrifugal system, a constant component of the central nervous system that appears to exist in all vertebrate groups, is part of the terminal nerve (TN) complex. TN allows the integration of different sensory modalities, and its anatomic variability may have functional and evolutionary significance. We propose that the olfacto-retinal branch of TN is an important anatomical link that allows the functional interaction between olfactory and visual systems in Austrolebias. By injecting three different neuronal tracers (biocytin, horseradish peroxidase, and 1,1′-dioctadecyl-3,3,3′,3′tetramethyl-indocarbocyanine perchlorate (DiI)) in the left eye of Austrolebias charrua fishes, we identified the olfacto-retinal branch of TN and related neuronal somas that were differentiable by location, shape, and size. The olfacto-retinal TN branch is composed of numerous thin axons that run ventrally along the olfactory bulb (OB) and telencephalic lobes, and appears to originate from a group of many small monopolar neurons located in the rostral portion of both the ipsi- and contralateral OB (referred to as region 1). Labeled cells were found in two other regions: bipolar and multipolar neurons in the transition between the OB and telencephalic lobes (region 2) and two other groups in the preoptic/pretectal area (region 3). In this last region, the most rostral group is constituted by monopolar pear-shaped neurons and may belong to the septo-preoptic TN complex. The second group, putatively located in the pretectal region, is formed by pseudounipolar neurons and coincides with a conserved vertebrate nucleus of the centrifugal retinal system not involved in the TN complex. The found that connections between the olfactory and visual systems via the olfacto-retinal TN branch suggest an early interaction between these sensory modalities, and contribute to the identification of their currently unknown circuital organization.

Comparative Analysis of the Organization of the Cholinergic System in the Brains of Two Holostean Fishes, the Florida Gar Lepisosteus platyrhincus and the Bowfin Amia calva

The cholinergic system in the brain has been widely studied in most vertebrate groups, but there is no information available about this neurotransmission system in the brains of holostean fishes, a primitive and poorly understood group of actinopterygian fishes. The present study provides the first detailed information on the distribution of cholinergic cell bodies and fibers in the central nervous system in two holostean species, the Florida gar, Lepisosteus platyrhincus, and the bowfin, Amia calva. Immmunohistochemistry against the enzyme choline acetyltransferase (ChAT) revealed distinct groups of ChAT-immunoreactive (ChAT-ir) cells in the habenula, isthmic nucleus, laterodorsal tegmental nucleus, octavolateral area, reticular formation, cranial nerve motor nuclei and the motor column of the spinal cord, all of which seem to be highly conserved among vertebrates. Some ChAT-ir cells were detected in the basal telencephalon that appear in actinopterygians for the first time in the evolution of this neurotransmission system, whereas the remarkable cholinergic population in the optic tectum is a peculiar characteristic, the presence of which varies throughout evolution, although it is present in all teleosts studied. Abundant cholinergic fibers were found in the pretectal region and optic tectum, where they probably modulate vision, and in the hypothalamus and the interpeduncular neuropil. Some interspecific differences were also observed, such as the presence of ChAT-ir cells in the supraoptoparaventricular band only in Lepisosteus and in in the nucleus subglomerulosus only in Amia. In addition, ChAT-ir fibers in the olfactory bulb were detected only in Amia. Comparison of these results with those from other classes of vertebrates, and a segmental analysis to correlate cell populations, reveal that the pattern of the cholinergic system in holosteans is very close to that in ancestral actinopterygian fishes, as recently described in the bichir (Cladistia), although an important evolutionary novelty in holosteans is the presence of cholinergic cells in the basal telencephalon.

Organization of the cholinergic systems in the brain of two lungfishes, Protopterus dolloi and Neoceratodus forsteri

Lungfishes (dipnoans) are currently considered the closest living relatives of tetrapods. The organization of the cholinergic systems in the brain has been carefully analyzed in most vertebrate groups, and major shared characteristics have been described, although traits particular to each vertebrate class have also been found. In the present study, we provide the first detailed information on the distribution of cholinergic cell bodies and fibers in the central nervous system in two representative species of lungfishes, the African lungfish (Protopterus dolloi) and the Australian lungfish (Neoceratodus forsteri), as revealed by immunohistochemistry against the enzyme choline acetyltransferase (ChAT). Distinct groups of ChAT immunoreactive (ChAT-ir) cells were observed in the basal telencephalon, habenula, isthmic nucleus, laterodorsal tegmental nucleus, cranial nerve motor nuclei, and the motor column of the spinal cord, and these groups seem to be highly conserved among vertebrates. In lungfishes, the presence of a cholinergic cell group in the thalamus and the absence of ChAT-ir cells in the tectum are variable traits, unique to this group and appearing several times during evolution. Other characters were observed exclusively in Neoceratodus, such as the presence of cholinergic cells in the suprachiasmatic nucleus, the pretectal region and the superior raphe nucleus. Cholinergic fibers were found in the medial pallium, basal telencephalon, thalamus and prethalamus, optic tectum and interpeduncular nucleus. Comparison of these results with those from other classes of vertebrates, including a segmental analysis to correlate cell populations, reveals that the cholinergic systems in lungfishes largely resemble those of amphibians and other tetrapods.

Glomerular nucleus of the weakly electric fish, Gymnotus sp.: Cytoarchitecture, histochemistry, and fiber connections—Insights from neuroanatomy to evolution and behavior

The present study provides a detailed description of morphological and hodological aspects of the glomerular nucleus in the weakly electric fish Gymnotus sp., and explores the evolutionary and functional implications flowing from this analysis. The glomerular nucleus of Gymnotus shows numerous morphological similarities with the glomerular nucleus of percomorph fish, although cytoarchitectonically simpler. In addition, congruence of the histochemical acetylcholinesterase (AChE) distribution with cytoarchitectonic data suggests that the glomerular nucleus, together with the ventromedial cell group of the medial subdivision of the preglomerular complex (PGm-vmc) rostrally, and the subglomerular nucleus (as identified by Maler et al. [1991] J Chem Neuroanat 4:1–38) caudally, may form a distinct longitudinally organized glomerular complex. Our results show that an important source of sensory afferents to the glomerular nucleus originates in the pretectal and electrosensorius nuclei. The glomerular nucleus in turn projects to the hypothalamus (inferior lobe and anterior hypothalamus), to the anterior tuberal nucleus, and to the medial region of the preglomerular nucleus (PGm). These data suggest that visual and electrosensory information reach the glomerular nucleus and are relayed to the hypothalamus and, via PGm, to the pallium. Such connections are similar to those of the glomerular nucleus in percomorphs and the posterior pretectal nucleus in osteoglossomorph, esocids, and salmonids, where they comprise one component of a visual processing pathway. In Gymnotiform fish, however, the pretectal region that projects to the glomerular nucleus is dominated by electrosensory input (visual input is minor), which is consistent with the dominant role of electroreception in these fish.

Embryonic genoarchitecture of the pretectum in Xenopus laevis: A conserved pattern in tetrapods

Networked gene activities control the evolutionarily conserved histogenetic organization of the central nervous system of vertebrates. Genoarchitectonic studies contribute to the analysis of each morphogenetic field by identifying distinct progenitor domains and corresponding derivatives whose pattern of gene expression shows a unique combinatory code. Previous studies in the pretectal region (caudal diencephalon) have defined three anteroposterior genoarchitectonic domains that are conserved in birds and mammals. Here, we have studied the embryonic pretectal genoarchitecture in the amphibian Xenopus laevis, in order to determine whether it is possible to define a comparable anteroposterior tripartition of the amphibian pretectal area. The expression patterns of 14 genes mapped from early embryonic stages to metamorphic climax allowed us to define the boundaries of the pretectum, the expected precommissural, juxtacommissural, and commissural anteroposterior domains, and some dorsoventral subdivisions. Taken together, our data provide evidence for a conserved pattern of pretectal domains and subdomains, shared by amniotes and amphibian anamniotes (tetrapods), understandable as part of a general Bauplan in vertebrates.

Comparison of Pretectal Genoarchitectonic Pattern between Quail and Chicken Embryos.

Regionalization of the central nervous system is controlled by local networks of transcription factors that establish and maintain the identities of neuroepithelial progenitor areas and their neuronal derivatives. The conserved cerebral Bauplan of vertebrates must result essentially from conserved patterns of developmentally expressed transcription factors. We have previously produced detailed molecular maps for the alar plate of prosomere 1 (the pretectal region) in chicken (Ferran et al., 2007, 2008, 2009). Here we compare the early molecular signature of the pretectum of two closely related avian species of the family Phasianidae, Coturnix japonica (Japanese quail) and Gallus gallus (chicken), aiming to test conservation of the described pattern at a microevolutionary level. We studied the developmental pretectal expression of Bhlhb4, Dbx1, Ebf1, Gata3, Gbx2, Lim1, Meis1, Meis2, Pax3, Pax6, Six3, Tal2, and Tcf7l2 (Tcf4) mRNA, using in situ hybridization, and PAX7 immunohistochemistry. The genoarchitectonic profile of individual pretectal domains and strata was produced, using comparable section planes. Remarkable conservation of the combinatorial genoarchitectonic code was observed, fundamented in a tripartite anteroposterior subdivision. However, we found that at corresponding developmental stages the pretectal region of G. gallus was approximately 30% larger than that of C. japonica, but seemed relatively less mature. Altogether, our results on a conserved genoarchitectonic pattern highlight the importance of early developmental gene networks that causally underlie the production of homologous derivatives in these two evolutionarily closely related species. The shared patterns probably apply to sauropsids in general, as well as to more distantly related vertebrate species.

Localization of tyrosine hydroxylase immunoreactive neurons in the forebrain of the guppy Poecilia reticulata

The current study reports for the first time the distribution of tyrosine hydroxylase immunoreactive (TH-ir) neurons in the forebrain of the guppy Poecilia reticulata. Numerous small TH-ir neurons were observed in the olfactory bulbs, located mainly in the periphery of the bulbs. The TH-ir telencephalic neurons are localized in the ventral telencephalic area where they are grouped in three distinct nuclei (Vv,Vd and Vp) composed of a small number of cells forming a continuous strip. The largest number of forebrain TH-ir neurons was observed in the diencephalon where both small and larger neurons are present. Diencephalic TH-ir neurons are subdivided in large nuclei located in the preoptic region (nSC, nPOp and nPOm), the thalamus (nDM), the pretectal region (nPPv and nAP), the hypothalamus (nPP and nRP) and the posterior tuberculum (nPT). Many diencephalic nuclei are distributed in periventricular regions and no TH-ir cells were observed in the paraventricular organ. A comparative analysis indicates that the present observations are consistent with the general pattern of TH-ir neurons distribution reported for the forebrain of other teleosts, but with some interspecies variability present, mainly in the diencephalon. This paper also provides valuable neuroanatomical information for P. reticulata, a teleost frequently used in toxicological tests, for future studies investigating the effects of environmental pollutants on the catecholaminergic system.

Cis-acting elements responsible for dopaminergic neuron-specific expression of zebrafish slc6a3 (dopamine transporter) in vivo are located remote from the transcriptional start site

The purpose of this study was to analyze the transcriptional regulation of the zebrafish solute carrier family 6 member 3 gene (slc6a3, dopamine transporter, dat), as a first step towards isolating regulatory sequences useful for driving transgene expression within dopaminergic neurons of the zebrafish CNS in vivo. We found that the 3.0 kb slc6a3 mRNA is expressed in each of the major groups of dopaminergic neurons previously identified in the zebrafish CNS. The slc6a3 gene spans >20 kb of genomic DNA and contains 15 exons. The genomic organization of slc6a3 is highly conserved with respect to its human orthologue, including the presence of an untranslated first exon. The promoter lacks a canonical TATA box and there are multiple transcriptional start sites. Functional analysis of cis-acting elements responsible for the expression pattern of slc6a3 was carried out by generating stable transgenic zebrafish lines expressing fluorescent reporters under transcriptional control of fragments of slc6a3 genomic sequence. The region between −2 kb and +5 kb with respect to the transcriptional start site contains the core slc6a3 promoter, in addition to neuronal enhancers and/or non-neuronal repressors that restrict expression to the CNS, but this region lacks cis-acting elements responsible for slc6a3 expression in dopaminergic neurons. The upstream sequence between −6 kb and −2 kb contains an enhancer element that drives slc6a3 expression in dopaminergic neurons of the pretectal region, and additional sequences that partially repress expression in non-dopaminergic neurons. However, expression of slc6a3 in dopaminergic neurons of the ventral diencephalon and telencephalon is dependent on elements that lie outside the region −6 kb to +5 kb. These data provide a detailed analysis of the slc6a3 gene and show that its expression in different populations of dopamine neurons is driven by discrete enhancers, rather than a single target sequence for a terminal factor involved in specifying neurochemical phenotype.

03-P108 Role of highly-conserved non-coding DNA regions as regulatory modules controlling the expression of Msx1 in the chicken pretectum

Gene regulatory networks are key elements for understanding how the different central nervous system regions differentiate. In recent years our group has done an extensive molecular characterization of the chicken pretectal region, obtaining a precise map of AP and DV molecular codes with respect to differential histogenetic pattern (Ferran et al., 2007; 2009, in press). As in other CNS regions, such as the spinal cord, Bmps signals from the roof plate and Shh signals from the floor and basal plates are clearcut candidates for governing the dorso-ventral patterning of the pretectum. The Msx1 transcription factor plays a role in diencephalic development, acting as intermediary between Bmp and Wnt (Bach et al., 2006), but it is still unclear how its expression is controlled by the gene regulatory network. Regulatory modules can be located in introns, exons or non-coding DNA regions, sometimes far away from the gene. In this work, we used comparative analysis of genomes to identify several highly-conserved non-coding DNA regions (HCNRs) located upstream and downstream of Msx1, and tested their ability for regulating Msx1 expression specifically in the pretectal region. This kind of approach is necessary for illuminating the specific regulatory network that patterns dorsoventral subdivisions in the pretectum, and possibly also in other CNS domains. Supported by NIH Grant 1-R01-MH070370-01A2, CIBERER (FISMinistry of Health), and MEC grant BFU2005-09378-C02-01 to L.P. (P.M. is a postdoctoral NIH fellow; J.L.F. is a postdoctoral CIBERER fellow).