Stria medullaris innervation follows the transcriptomic division of the habenula.

The habenula is a complex neuronal population integrated in a pivotal functional position into the vertebrate limbic system. Its main afference is the stria medullaris and its main efference the fasciculus retroflexus. This neuronal complex is composed by two main components, the medial and lateral habenula. Transcriptomic and single cell RNAseq studies have unveiled the morphological complexity of both components. The aim of our work was to analyze the relation between the origin of the axonal fibers and their final distribution in the habenula. We analyzed 754 tracing experiments from Mouse Brain Connectivity Atlas, Allen Brain Map databases, and selected 12 neuronal populations projecting into the habenular territory. Our analysis demonstrated that the projections into the medial habenula discriminate between the different subnuclei and are generally originated in the septal territory. The innervation of the lateral habenula displayed instead a less restricted distribution from preoptic, terminal hypothalamic and peduncular nuclei. Only the lateral oval subnucleus of the lateral habenula presented a specific innervation from the dorsal entopeduncular nucleus. Our results unveiled the necessity of novel sorts of behavioral experiments to dissect the different functions associated with the habenular complex and their correlation with the distinct neuronal populations that generate them.

Adhesion molecule Amigo2 is involved in the fasciculation process of the fasciculus retroflexus.

BACKGROUND: The fasciculus retroflexus is the prominent efferent pathway from the habenular complex. Medial habenular axons form a core packet whereas lateral habenular axons course in a surrounding shell. Both groups of fibers share the same initial pathway but differ in the final segment of the tract, supposedly regulated by surface molecules. The gene Amigo2 codes for a membrane adhesion molecule with an immunoglobulin-like domain 2 and is selectively expressed in the medial habenula. We present it as a candidate for controlling the fasciculation behavior of medial habenula axons. RESULTS: First, we studied the development of the habenular efferents in an Amigo2 lack of function mouse model. The fasciculus retroflexus showed a variable defasciculation phenotype. Gain of function experiments allowed us to generate a more condensed tract and rescued the Amigo2 knock-out phenotype. Changes in Amigo2 function did not alter the course of habenular fibers. CONCLUSION: We have demonstrated that Amigo2 plays a subtle role in the fasciculation of the fasciculus retroflexus.

Gli2-Mediated Shh Signaling Is Required for Thalamocortical Projection Guidance.

The thalamocortical projections are part of the most important higher level processing connections in the vertebrates and follow a highly ordered pathway from their origin in the thalamus to the cerebral cortex. Their functional complexities are not only due to an extremely elaborate axon guidance process but also due to activity-dependent mechanisms. Gli2 is an intermediary transcription factor in the Sonic hedgehog (Shh) pathway. During neural early development, Shh has an important role in dorsoventral patterning, diencephalic anteroposterior patterning, and many later developmental processes, such as axon guidance and cell migration. Using a Gli2 knockout mouse line, we have studied the role of Shh signaling mediated by Gli2 in the development of the thalamocortical projections during embryonic development. In wild-type brains, we have described the normal trajectory of the thalamocortical axons into the context of the prosomeric model. Then, we have compared it with the altered thalamocortical axons course in Gli2 homozygous embryos. The thalamocortical axons followed different trajectories and were misdirected to other territories probably due to alterations in the Robo/Slit signaling mechanism. In conclusion, the alteration of Gli2-mediated Shh signaling produces an erroneous specification of several territories related with the thalamocortical axons. This is translated into a huge modification in the pathfinding signaling mechanisms needed for the correct wiring of the thalamocortical axons.

Wnt1 Role in the Development of the Habenula and the Fasciculus Retroflexus.

Wnt1 is one of the morphogenes that controls the specification and differentiation of neuronal populations in the developing central nervous system. The habenula is a diencephalic neuronal complex located in the most dorsal aspect of the thalamic prosomere. This diencephalic neuronal population is involved in the limbic system and its malfunction is related with several psychiatric disorders. Our aim is to elucidate the Wnt1 role in the habenula and its main efferent tract, the fasciculus retroflexus, development. In order to achieve these objectives, we analyzed these structures development in a Wnt1 lack of function mouse model. The habenula was generated in our model, but it presented an enlarged volume. This alteration was due to an increment in habenular neuroblasts proliferation rate. The fasciculus retroflexus also presented a wider and disorganized distribution and a disturbed final trajectory toward its target. The mid-hindbrain territories that the tract must cross were miss-differentiated in our model. The specification of the habenula is Wnt1 independent. Nevertheless, it controls its precursors proliferation rate. Wnt1 expressed in the isthmic organizer is vital to induce the midbrain and rostral hindbrain territories. The alteration of these areas is responsible for the fasciculus retroflexus axons misroute.

Netrin 1-Mediated Role of the Substantia Nigra Pars Compacta and Ventral Tegmental Area in the Guidance of the Medial Habenular Axons.

The fasciculus retroflexus is an important fascicle that mediates reward-related behaviors and is associated with different psychiatric diseases. It is the main habenular efference and constitutes a link between forebrain regions, the midbrain, and the rostral hindbrain. The proper functional organization of habenular circuitry requires complex molecular programs to control the wiring of the habenula during development. However, the mechanisms guiding the habenular axons toward their targets remain mostly unknown. Here, we demonstrate the role of the mesodiencephalic dopaminergic neurons (substantia nigra pars compacta and ventral tegmental area) as an intermediate target for the correct medial habenular axons navigation along the anteroposterior axis. These neuronal populations are distributed along the anteroposterior trajectory of these axons in the mesodiencephalic basal plate. Using in vitro and in vivo experiments, we determined that this navigation is the result of netrin 1 attraction generated by the mesodiencephalic dopaminergic neurons. This attraction is mediated by the receptor deleted in colorectal cancer (DCC), which is strongly expressed in the medial habenular axons. The increment in our knowledge on the fasciculus retroflexus trajectory guidance mechanisms opens the possibility of analyzing if its alteration in mental health patients could account for some of their symptoms.

Neuronal tangential migration from Nkx2.1-positive hypothalamus.

During the development of the central nervous system, the immature neurons suffer different migration processes. It is well known that Nkx2.1-positive ventricular layer give rise to critical tangential migrations into different regions of the developing forebrain. Our aim was to study this phenomenon in the hypothalamic region. With this purpose, we used a transgenic mouse line that expresses the tdTomato reporter driven by the promotor of Nkx2.1. Analysing the Nkx2.1-positive derivatives at E18.5, we found neural contributions to the prethalamic region, mainly in the zona incerta and in the mes-diencephalic tegmental region. We studied the developing hypothalamus along the embryonic period. From E10.5 we detected that the Nkx2.1 expression domain was narrower than the reporter distribution. Therefore, the Nkx2.1 expression fades in a great number of the early-born neurons from the Nkx2.1-positive territory. At the most caudal positive part, we detected a thin stream of positive neurons migrating caudally into the mes-diencephalic tegmental region using time-lapse experiments on open neural tube explants. Late in development, we found a second migratory stream into the prethalamic territory. All these tangentially migrated neurons developed a gabaergic phenotype. In summary, we have described the contribution of interneurons from the Nkx2.1-positive hypothalamic territory into two different rostrocaudal territories: the mes-diencephalic reticular formation through a caudal tangential migration and the prethalamic zona incerta complex through a dorsocaudal tangential migration.