Asymmetric innervation of the habenula in zebrafish
Developmental Neurobiology Group, Temasek Lifesciences Laboratory, 1 Research Link, Singapore 117604.The Journal of Comparative Neurology (Impact Factor: 3.23). 06/2007; 502(4):611-9. DOI: 10.1002/cne.21339
The habenular complex is a paired structure found in the diencephalon of all vertebrates, linking the forebrain and midbrain. Habenulae are asymmetrical and may contribute to lateralized behavior. Recent studies in zebrafish have characterized molecular pathways that give rise to the habenular asymmetry and the distinct projections of the left and right habenula to the midbrain. However, it is unclear whether there are asymmetries in habenula afferents from the forebrain. By lipophilic dye tracing, we find that axons innervating the habenula derive primarily from a region in the lateral diencephalon containing migrated neurons of the eminentia thalami (EmT). EmT neurons terminate in neuropils in both ipsilateral and contralateral habenula. These axons, together with axons from migrated neurons of the posterior tuberculum and pallial neurons, cross the midline via the habenular commissure. Subsets of pallial neurons terminate only in the medial right habenula, regardless of which side of the brain they originate from. These include an unusual type of forebrain projection: axons that cross the midline twice, at both the anterior and habenular commissures. Our data establish that there is asymmetric innervation of the habenula from the telencephalon, suggesting a mechanism by which habenula asymmetry might contribute to lateralized behavior.
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- "Besides the observed cellular and molecular asymmetries, the zebrafish d-Hb also develops structural asymmetries in the size and organisation of neuropil domains (Concha et al., 2000) within which dendritic extensions from habenular neurons mix and synapse with axonal afferents arriving from other regions of the forebrain through the stria medullaris (Hendricks and Jesuthasan, 2007a; Bianco et al., 2008; Miyasaka et al., 2009). Habenular neuropil formation begins at ∼48 hpf, subsequent to the specification of d-HbL and d-HbM cellular domains, and becomes progressively enlarged on the left side reaching a conspicuous asymmetric array by 4 days of development (Concha et al., 2000; Concha et al., 2003). "
ABSTRACT: Although progress has been made in resolving the genetic pathways that specify neuronal asymmetries in the brain, little is known about genes that mediate the development of structural asymmetries between neurons on left and right. In this study, we identify daam1a as an asymmetric component of the signalling pathways leading to asymmetric morphogenesis of the habenulae in zebrafish. Daam1a is a member of the Formin family of actin-binding proteins and the extent of Daam1a expression in habenular neuron dendrites mirrors the asymmetric growth of habenular neuropil between left and right. Local loss and gain of Daam1a function affects neither cell number nor subtype organisation but leads to a decrease or increase of neuropil, respectively. Daam1a therefore plays a key role in the asymmetric growth of habenular neuropil downstream of the pathways that specify asymmetric cellular domains in the habenulae. In addition, Daam1a mediates the development of habenular efferent connectivity as local loss and gain of Daam1a function impairs or enhances, respectively, the growth of habenular neuron terminals in the interpeduncular nucleus. Abrogation of Daam1a disrupts the growth of both dendritic and axonal processes and results in disorganised filamentous actin and α-tubulin. Our results indicate that Daam1a plays a key role in asymmetric habenular morphogenesis mediating the growth of dendritic and axonal processes in dorsal habenular neurons.
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- "Accumulations of neuropil differ between the left and right dorsal habenulae of larval zebrafish, as visualized by immunolabeling with antibodies against acetylated α-Tubulin (Concha et al., 2000; Taylor et al., 2011) or Synaptic Vesicle Protein 2 (SV2) (Hendricks and Jesuthasan, 2007; Miyasaka et al., 2009), and by labeling of membrane-tagged GFP in live Tg(gng8:nfsB- CAAX-GFP) c375 larvae. The left side has an expanded neuropil that extends the width of the dorsal habenula, while the right dorsal habenula has three more distinct, small clusters (Figure 1E). "
ABSTRACT: The dorsal habenular nuclei of the zebrafish epithalamus have become a valuable model for studying the development of left-right (L-R) asymmetry and its function in the vertebrate brain. The bilaterally paired dorsal habenulae exhibit striking differences in size, neuroanatomical organization, and molecular properties. They also display differences in their efferent connections with the interpeduncular nucleus (IPN) and in their afferent input, with a subset of mitral cells distributed on both sides of the olfactory bulb innervating only the right habenula. Previous studies have implicated the dorsal habenulae in modulating fear/anxiety responses in juvenile and adult zebrafish. It has been suggested that the asymmetric olfactory-habenula pathway (OB-Ha), revealed by selective labeling from an lhx2a:YFP transgene, mediates fear behaviors elicited by alarm pheromone. Here we show that expression of the fam84b gene demarcates a unique region of the right habenula that is the site of innervation by lhx2a:YFP-labeled olfactory axons. Upon ablation of the parapineal, which normally promotes left habenular identity; the fam84b domain is present in both dorsal habenulae and lhx2a:YFP-labeled olfactory bulb neurons form synapses on the left and the right side. To explore the relevance of the asymmetric olfactory projection and how it might influence habenular function, we tested activation of this pathway using odorants known to evoke behaviors. We find that alarm substance or other aversive odors, and attractive cues, activate fos expression in subsets of cells in the olfactory bulb but not in the lhx2a:YFP expressing population. Moreover, neither alarm pheromone nor chondroitin sulfate elicited fos activation in the dorsal habenulae. The results indicate that L-R asymmetry of the epithalamus sets the directionality of olfactory innervation, however, the lhx2a:YFP OB-Ha pathway does not appear to mediate fear responses to aversive odorants.
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- "In contrast to the optic tectum, Cx35 immunoreactivity in the habenulae and the cerebellum developed later, starting at 4 dpf. Although the habenulae and the habenular commissure can be distinguished by 2 dpf (Hendricks and Jesuthasan, 2007), we did not see Cx35 immunoreactivity in them until 4 dpf. We saw intense labeling in habenular neurons and in the commissure indicating gap junctional connectivity among habenular neurons from 4 dpf onwards. "
ABSTRACT: Gap junctions are membrane specializations that allow the passage of ions and small molecules from one cell to another. In vertebrates, connexins are the protein subunits that assemble to form gap junctional plaques. Connexin-35 (Cx35) is the fish ortholog of mammalian Cx36, which is enriched in the retina and the brain and has been shown to form neuronal gap junctions. As a first step toward understanding the role of neuronal gap junctions in central nervous system (CNS) development, we describe here the distribution of Cx35 in the CNS during zebrafish development. Cx35 expression is first seen at 1 day post fertilization (dpf) along cell boundaries throughout the nervous system. At 2 dpf, Cx35 immunoreactivity appears in commissures and fiber tracts throughout the CNS and along the edges of the tectal neuropil. In the rhombencephalon, the Mauthner neurons and fiber tracts show strong Cx35 immunoreactivity. As the larva develops, the commissures and fiber tracts continue to be immunoreactive for Cx35. In addition, the area of the tectal neuropil stained increases vastly and tectal commissures are visible. Furthermore, at 4-5 dpf, Cx35 is seen in the habenulae, cerebellum and in radial glia lining the rhombencephalic ventricle. This pattern of Cx35 immunoreactivity is stable at least until 15 dpf. To test whether the Cx35 immunoreactivity seen corresponds to functional gap junctional coupling, we documented the number of dye-coupled neurons in the hindbrain. We found several dye-coupled neurons within the reticulospinal network indicating functional gap junctional connectivity in the developing zebrafish brain.