Daam1a mediates asymmetric habenular morphogenesis by regulating dendritic and axonal outgrowth.

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.

Ontogenesis of the asymmetric parapineal organ in the zebrafish epithalamus.

The parapineal organ is a midline-derived epithalamic structure that in zebrafish adopts a left-sided position at embryonic stages to promote the development of left-right asymmetries in the habenular nuclei. Despite extensive knowledge about its embryonic and larval development, it is still unknown whether the parapineal organ and its profuse larval connectivity with the left habenula are present in the adult brain or whether, as assumed from historical conceptions, this organ degenerates during ontogeny. This paper addresses this question by performing an ontogenetic analysis using an integrative morphological, ultrastructural and neurochemical approach. We find that the parapineal organ is lost as a morphological entity during ontogeny, while parapineal cells are incorporated into the posterior wall of the adult left dorsal habenular nucleus as small clusters or as single cells. Despite this integration, parapineal cells retain their structural, neurochemical and connective features, establishing a reciprocal synaptic connection with the more dorsal habenular neuropil. Furthermore, we describe the ultrastructure of parapineal cells using transmission electron microscopy and report immunoreactivity in parapineal cells with antibodies against substance P, tachykinin, serotonin and the photoreceptor markers arrestin3a and rod opsin. Our findings suggest that parapineal cells form an integral part of a neural circuit associated with the left habenula, possibly acting as local modulators of the circuit. We argue that the incorporation of parapineal cells into the habenula may be part of an evolutionarily relevant developmental mechanism underlying the presence/absence of the parapineal organ in teleosts, and perhaps in a broader sense in vertebrates.

Specific activation of histaminergic neurons during daily feeding anticipatory behavior in rats.

When food is available during a restricted and predictable time of the day, animals show increased locomotor and food searching behaviors before the anticipated daily meal. We had shown that histamine-containing neurons are the only aminergic neurons related to arousal that become active in anticipation of an upcoming meal. To further map, the brain regions involved in the expression of the feeding-anticipatory behavior, we quantified the expression of Fos in hypothalamic areas involved in arousal. We found that nearly 35% of the histamine neurons from the tuberomammillary nucleus were Fos-immunoreactive immediately before mealtime. One hour before this transient increase in Fos-immunoreactivity, we found a similarly brief increase of fos mRNA in the tuberomammillary nucleus. In contrast, the activation of two types of perifornical hypothalamic neurons followed meal onset by 1-2 h. One neuron type was orexin/hypocretin-immunoreactive, while the other type was neither orexin nor melanin concentrating hormone-immunoreactive. The present work indicates that the increased locomotor activity that anticipates mealtime coincides with the activation of the tuberomammillary nucleus, and that the behavioral activation during the consummatory phase of feeding coincides more closely with the delayed activation of the perifornical hypothalamic area.

Evolutionary plasticity of habenular asymmetry with a conserved efferent connectivity pattern.

The vertebrate habenulae (Hb) is an evolutionary conserved dorsal diencephalic nuclear complex that relays information from limbic and striatal forebrain regions to the ventral midbrain. One key feature of this bilateral nucleus is the presence of left-right differences in size, cytoarchitecture, connectivity, neurochemistry and/or gene expression. In teleosts, habenular asymmetry has been associated with preferential innervation of left-right habenular efferents into dorso-ventral domains of the midbrain interpeduncular nucleus (IPN). However, the degree of conservation of this trait and its relation to the structural asymmetries of the Hb are currently unknown. To address these questions, we performed the first systematic comparative analysis of structural and connectional asymmetries of the Hb in teleosts. We found striking inter-species variability in the overall shape and cytoarchitecture of the Hb, and in the frequency, strength and to a lesser degree, laterality of habenular volume at the population level. Directional asymmetry of the Hb was either to the left in D. rerio, E. bicolor, O. latipes, P. reticulata, B. splendens, or to the right in F. gardneri females. In contrast, asymmetry was absent in P. scalare and F. gardneri males at the population level, although in these species the Hb displayed volumetric asymmetries at the individual level. Inter-species variability was more pronounced across orders than within a single order, and coexisted with an overall conserved laterotopic representation of left-right habenular efferents into dorso-ventral domains of the IPN. These results suggest that the circuit design involving the Hb of teleosts promotes structural flexibility depending on developmental, cognitive and/or behavioural pressures, without affecting the main midbrain connectivity output, thus unveiling a key conserved role of this connectivity trait in the function of the circuit. We propose that ontogenic plasticity in habenular morphogenesis underlies the observed inter-species variations in habenular asymmetric morphology.

Cell-autonomous alterations in dendritic arbor morphology and connectivity induced by overexpression of MeCP2 in Xenopus central neurons in vivo.

Methyl CpG binding protein-2 (MeCP2) is an essential epigenetic regulator in human brain development. Mutations in the MeCP2 gene have been linked to Rett syndrome, a severe X-linked progressive neurodevelopmental disorder, and one of the most common causes of mental retardation in females. MeCP2 duplication and triplication have also been found to affect brain development, indicating that both loss of function and gain in MeCP2 dosage lead to similar neurological phenotypes. Here, we used the Xenopus laevis visual system as an in vivo model to examine the consequence of increased MeCP2 expression during the morphological maturation of individual central neurons in an otherwise intact brain. Single-cell overexpression of wild-type human MeCP2 was combined with time-lapse confocal microscopy imaging to study dynamic mechanisms by which MeCP2 influences tectal neuron dendritic arborization. Analysis of neurons co-expressing DsRed2 demonstrates that MeCP2 overexpression specifically interfered with dendritic elaboration, decreasing the rates of branch addition and elimination over a 48 hour observation period. Moreover, dynamic analysis of neurons co-expressing wt-hMeCP2 and PSD95-GFP revealed that even though neurons expressing wt-hMeCP2 possessed significantly fewer dendrites and simpler morphologies than control neurons at the same developmental stage, postsynaptic site density in wt-hMeCP2-expressing neurons was similar to controls and increased at a rate higher than controls. Together, our in vivo studies support an early, cell-autonomous role for MeCP2 during the morphological differentiation of neurons and indicate that perturbations in MeCP2 gene dosage result in deficits in dendritic arborization that can be compensated, at least in part, by synaptic connectivity changes.

Synaptic maturation of the Xenopus retinotectal system: effects of brain-derived neurotrophic factor on synapse ultrastructure.

Synaptogenesis is a dynamic process that involves structural changes in developing axons and dendrites as synapses form and mature. The visual system of Xenopus laevis has been used as a model to study dynamic changes in axons and dendrites as synapses form in the living brain and the molecular mechanisms that control these processes. Brain-derived neurotrophic factor (BDNF) contributes to the establishment and refinement of visual connectivity by modulating retinal ganglion cell (RGC) axon arborization and presynaptic differentiation. Here, we have analyzed the ultrastructural organization of the Xenopus retinotectal system to understand better the maturation of this synaptic circuit and the relation between synapse ultrastructure and the structural changes in connectivity that take place in response to BDNF. Expression of yellow fluorescent protein (YFP) followed by preembedding immunoelectron microscopy was used to identify RGC axons specifically in living tadpoles. Injection of recombinant BDNF was used to alter endogenous BDNF levels acutely in the optic tectum. Our studies reveal a rapid transition from a relatively immature synaptic circuit in which retinotectal synapses are formed on developing filopodial-like processes to a circuit in which RGC axon terminals establish synapses with dendritic shafts and spines. Moreover, our studies reveal that BDNF treatment increases the number of spine synapses and docked vesicle number at YFP-identified synaptic sites within 24 hours of treatment. These fine structural changes at retinotectal synapses are consistent with the role that BDNF plays in the functional maturation of synaptic circuits and with dynamic, rapid changes in synaptic connectivity during development.

BDNF increases synapse density in dendrites of developing tectal neurons in vivo.

Neuronal connections are established through a series of developmental events that involve close communication between pre- and postsynaptic neurons. In the visual system, BDNF modulates the development of neuronal connectivity by influencing presynaptic retinal ganglion cell (RGC) axons. Increasing BDNF levels in the optic tectum of Xenopus tadpoles significantly increases both axon arborization and synapse density per axon terminal within a few hours of treatment. Here, we have further explored the mechanisms by which BDNF shapes synaptic connectivity by imaging tectal neurons, the postsynaptic partners of RGCs. Individual neurons were co-labeled with DsRed2 and a GFP-tagged postsynaptic density protein (PSD95-GFP) to visualize dendritic morphology and postsynaptic specializations simultaneously in vivo. Immunoelectron microscopy confirmed that PSD95-GFP predominantly localized to ultrastructurally identified synapses. Time-lapse confocal microscopy of individual, double-labeled neurons revealed a coincident, activity-dependent mechanism of synaptogenesis and axon and dendritic arbor growth, which is differentially modulated by BDNF. Microinjection of BDNF into the optic tectum significantly increased synapse number in tectal neuron dendritic arbors within 24 hours, without significantly influencing arbor morphology. BDNF function-blocking antibodies had opposite effects. The BDNF-elicited increase in synapse number complements the previously observed increase in presynaptic sites on RGC axons. These results, together with the timescale of the response by tectal neurons, suggest that the effects of BDNF on dendritic synaptic connectivity are secondary to its effects on presynaptic RGCs. Thus, BDNF influences synaptic connectivity in multiple ways: it enhances axon arbor complexity expanding the synaptic territory of the axon, while simultaneously coordinating synapse formation and stabilization with individual postsynaptic cells.