Study of serotonin raphe neurons projections during development with an IDISCO method 

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Dorsal component of the dorsal raphe, DRD (B7d)

Ascending projections from the DRD were detected as ventrally and bilaterally towards the mlf, then serotonin fibers are spreading laterally to innervate the substantia nigra pars compacta (SNC) and ventral tegmental area (VTA). The most abundant terminal innervation was localized in the hypothalamus, followed by the lateral geniculate nuclei of the thalamus, SNC and parts of the basal ganglia, namely the nucleus accumbens, and caudal parts of the caudate-putamen and pallidum. These projections were mainly but not exclusively ipsilateral.
In the brainstem, DRD axons terminal innervation was dense in the superior colliculus and the periaqueductal gray (PAG). More ventrally it was essentially concentrated in the superior Oliver complex, 7N, and dorsal and ventral cochlear nuclei (DC, VC)

Ventral component of dorsal raphe, DRV (B7v)

Amygdala was strongly innervated by DRV serotonin fibers, where a strong accumulation of axon varicosities was observed with a particularly dense distribution in the central and basolateral components of the amygdala. A somewhat similar dense innervation was visible within the extended amygdala, such as the bed nucleus of the stria terminalis (BST). In contrast, despite the presence of ascending fiber tracts in the medial septum, no terminal innervation was visible in the lateral septal (LS) nuclei.
A dense innervation also reached the cerebral cortex, with a particular concentration in its rostral and ventrolateral parts including the orbital cortex and olfactory-related brain areas, in particular the piriform cortex, the anterior olfactory nuclei (AON) and the mitral and granular layers of the olfactory bulb. In the medial prefrontal cortex, varicose fibers were visible mainly in the infralimbic cortex, whereas the dorsal part and the cingulate cortex were more sparingly innervated.
In the thalamus, innervation was essentially concentrated in the midline thalamic nuclei. The habenula was only moderately innervated, with terminal fibers mainly in the lateral habenula (LHb). Strikingly, and contrasting with the heavy ascending fiber tract in the mfb, the hypothalamic nuclei received hardly no innervation. Similarly the hippocampus contained only few fibers that were essentially localized in the stratum lacunosum-moleculare of both the dorsal and ventral hippocampus. However, hardly any innervation was noted in the dentate gyrus. In the mesencephalon, innervation was particularly conspicuous in the substantia nigra pars reticulata with moderate innervation of the ventral periaqueductal gray (PAG) and the superior colliculus.

The development of serotonin neurons in raphe nuclei

During development, the serotonin neurons are divided into two groups based on their spatial difference as inferior group and superior group. The two groups of serotonin neurons have different migration patterns (Lidov and Molliver, 1982b; Wallace and Lauder, 1983), and within the superior group, there is evidence the 5-HT neurons form different subsets of cells. (Azmitia and Gannon, 1986).
In many studies, the neuroanatomical development and neuronal organization of 5-HT neurons has been analyzed in many different species, such as rats, primates and zebrafish (Steinbusch and Nieuwenhuys, 1981; Lidov and Molliver, 1982b; Wallace and Lauder, 1983; Azmitia and Gannon, 1986; Lauder, 1990; Lillesaar et al., 2007), and more recently in mice with genetic-based fate mapping (Jensen et al., 2008). Moreover, during embryogenesis, the hindbrain is transiently subdivided into several components called rhombomeres, which correspond to different subpopulations of serotonin neurons (Figure 7). Jensen et al. demonstrated that the raphe rostral group derive entirely from r1-r3, whereas the caudal from r5-r8. Among the rostral group, the DR nucleus (B7, B6) origioned from r1 whereas the median raphe nucleus (B8, B5) from entire r1-r3. (Jensen et al., 2008).
Mouse serotonin neurons are specified and appear initially in the ventral rhombencephalon during a brief period of neurogenesis, embryonic days 9.5–12. The initial contributing of the raphe nuclei is based on the ultimately cluster of newborn serotonin neurons in different disparate regions of the midbrain, pons and medulla. The 5-HT neuron generation consists in several developmentally recognizable stages.
During the first stage, serotonergic progenitors in the ventral hindbrain are specified in a spatial organization along the dorsoventral and anterior-posterior axes, which are modulated by signaling molecules sonic hedgehog and fibroblast growth factors 4 and 8 respectively (Goridis and Rohrer, 2002; Vitalis et al., 2003; Cordes, 2005). The initial serotonin neurons are generated by progenitors from about E9.5 to E10.5 in rhombomere 1 (r1) co(Pattyn et al., 2003). Then a second wave of serotonin neurons are born about a day later in r2 and r3, as progenitors at these longitudinal levels initially generate visceromotor neurons (VMN) before becoming competent to generate serotonin neurons (Pattyn et al., 2003). Progenitor fate of caudal serotonin groups occurs with similar temporal characteristics in r5–r8, such that caudal serotonin neurons are born virtually simultaneously with those in the rostral domain. Interestingly, the synthesis of 5-HT in these newborn caudal serotonin neurons is delayed for 1–2 days (Lidov and Molliver, 1982a). Moreover, the serotonergic neurogenesis is never launched under normal circumstances in r4 (Pattyn et al., 2003).
During the second stage, progenitors do not exhibit serotonergic characteristic as yet. Serotonergic identity is then acquired through coordinated expression of serotonergic-type gene battery. The newborn serotonin neurons are phenotypically immature and have not been integrated into neural circuitry. Thus, beginning immediately after the birth of the 5-HT neurons and extending to at least the end of the third postnatal week, mouse 5-HT neurons undergo a series of complex maturation events (Lidov and Molliver, 1982b, a; Pattyn et al., 2003). These events include cell migration, dendric growth, expression of serotonin autoregulatory pathways, formation of highly collateralized axonal pathways and synaptogenesis.

From growth cone to axon guidance

Growing axons have a specialized structural differentiation at their tip known as the growth cone, which plays a crucial role in integrating the many signals that direct axons to their proper targets. The first detailed described of a growth cone was done by Cajal on spinal commissural axons, on three day old chick embryos. Morphologically, the growth cone is a fan-shaped structure with many long, thin spikes that radiate outward much like fingers on a glove. The prediction of Cajal that this might be a structure permitting the growing neuron to receive and integrate the variety of physico-chemical signals present along its pathway has now been validated by numerous cell biology experiments in vitro, but also with the improved visualizing techniques, in vivo. The signals that are sensed by the growth cones are produced by intermediate targets and the surrounding tissue and involve both chemical and biophysical cues. Such cues guide axons to their final target, where they establish synapses with one or more neurons (Mueller, 1999; Song and Poo, 1999). As predicted by Cajal, the growth cones are also highly motile structures. Video microscopic studies have shown that growth cones undergo continuous expansion and retraction before they reach their final targets. This was initially demonstrated in vitro on cell cultures (Harrison, 1959) and more recently in vivo (Tashiro et al., 2003).
We can explain the growth cone navigation system as driving. This system is similar to an experienced driver who can grasp different environmental changes, such as the traffic lights, street signs or bad road conditions in order to guarantee the right destination. Definitely, as the multiple environmental variables, the navigation system needs a series of molecules to form a signaling pathway in order to introduce spatial bias for steering the growth cone to the right targets.
To study the complex effects on the growth cone cytoskeletal machinery, recently, as interdisciplinary development, biophysical techniques became more and more popular to be used for cell biology assay. By performing the microfluidic devices has allowed the production of stable, precisely controlled gradients in various combinations of both diffusible and substrate-bound factors (Lang et al., 2008; Wang et al., 2008). These types of combined cues can more faithfully recapitulate the complexity of the in vivo environment, and thus, future experiments utilizing this method, in combination with high-resolution cytoskeletal imaging, hold considerable potential for refining our understanding of the growth cone guidance mechanism.

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Molecular mechanism of serotonin raphe neuronal wiring.

As described in part 1, serotonin raphe neurons provide a massive and diffuse axonal projections to almost all the forebrain, brainstem and spinal cord. Moreover, as the studies of serotonin axonal projections become more complete and more detailed, the question that now arises is to identify the specific signals that allow this specific targeting.
Here I remind the three step of the development of serotonergic projections in rat, for helping to understand the relation of axon guidance in each period. First neurons migrate from their original site of production in the ventricles both ventrally and toward the midline. They initially form 2 bands on either side of the midline, and eventually fuse on the midline. The initial organization and orientation of serotonergic raphe neurons, can influence the spatial orientation of axon growth and the direction in which they grow; Secondly long-range guidance cue can guide serotonin axons grow along the main tracts toward their main targets; Finally . The short-rang effect, which is the terminal innervation of the specific targets of the axons. The molecular mechanisms of these steps during serotonin projection development were unknown until recent years- the genetic tools were developed, many genetic mouse lines were used to study these mechanisms.

Slit—long projection pathways modulator of serotonin axon in the forebrain

Slit proteins have been implicated in axon guidance in both vertebrates and invertebrates. In mammals, the three Slit genes are expressed in, floor plate and roof plate of the spinal cord, and the septum area, hypothalamus, and hippocampus (Metin and Godement, 1996; Brose et al., 1999; Yuan et al., 1999). The expression patterns and in vitro observations suggest that Slit proteins are also likely to play important roles as repulsive guidance cues for multiple populations of migrating axons and cells, likely using Robo receptors for their functions as well (Brose et al., 1999; Hu, 1999; Nguyen Ba-Charvet et al., 1999; Piper et al., 2000; Ringstedt et al., 2000; Shu and Richards, 2001). A mammalian Slit protein was also independently identified as a positive regulator of branching and elongation of sensory axons.
Bagri et al. in 2002 studied on the long projection pathways of serotonin in forebrain in Slit1-/-, Slit2-/- and the double knockout mice. Their results indicated that in contrast to control mice serotonin fibers in the medial forebrain bundle of Slit2 mutants were displaced ventrally as they coursed through the diencephalon. In Slit1/Slit2 double mutants the medial forebrain bundle was commonly split in two components and numerous fibers descended ventrally into the hypothalamus, approaching the midline a significant percentage abnormally crossed the midline in the basal telencephalon.

Pdcha— the forbrain terminal innervation effector of serotonergic neurons

The largest family in the cadherin superfamily is the clustered protocadherins, which are mainly expressed in neurons and localized to axons and synapses (Obata et al., 1995; Kohmura et al., 1998; Phillips et al., 2003; Junghans et al., 2008). Katori et al (Deneris and Wyler, 2012). In 2009, they detected a high level expression of pdcha in mouse raphe nuclei, particularly in rostral raphe. The maximum expression is from embryonic stage to the second postnatal week. Moreover, they analyse the serotonergic innervation in their forebrain targets of the pdcha-/- mice. In pdcha-/- mice, the abnormal distributed serotonergic fibers were detected in the hippocampus, substantia nigra and olfactory bulb at the endpoint of the third postnatal week in contrast to the wild type mice. Moreover, the globus pallidus and substantia nigra, in the mutants, exhibited as dense at the periphery of each region, but sparse in the center. In the stratum lacunosum-moleculare of the hippocampus, the mutants showed denser serotonergic innervation than in wild-type, and in the dentate gyrus of the hippocampus and the caudateputamen, the innervation was sparser. Their findings in the pdcha-/-mice indicated the pdcha is required for the terminal organization of serotonergic fibers.
Overall these studies have provided new clues on molecules that modulate serotonin raphe neurons outgrowth, and are starting to explain the different routes followed by axons from the rostral/caudal serotonin raphe neurons. However, we are still far from understanding what molecules guide the different subpopulation of serotonin neurons from the DR or MnR to their specific targets. A first step towards identification of these molecular cues is provided by gene expression screen in the raphe. A first screen was obtained by the team of Deneris and colleagues. Using the Pet1 promoter that is exclusively expressed in serotonin raphe neurons, they generated a transgenic mouse line, which expresses a fluorescent reporter (enhanced green fluorescent protein, eGFP) selectively in the serotonin neurons of the hindbrain. Using this transgenic mouse line, they sorted GFP+/GFP-neurons from microdissected raphe tissue of rostral and caudal hindbrain at embryonic day 12. Whole-genome screen was then done with microarrays comparing serotonin/non serotonin neurons, and rostral versus the caudal raphe cell groups. This first set of data identified hundreds of genes that are expressed specifically in serotonin neurons. Not surprisingly, in this gene list, axon guidance related genes were detected such as ephrins and Ephs, semaphorins and plexins, cadherins and protocadherins. A further analysis of the difference between rostral raphe and caudal raphe showed differential expression of several classes of molecules, suggesting that these factors could be involved in the different targeting of the rostral and caudal serotonin cell groups. In a more recent study, the team of Susan Dymecki provided a detailed description of the differential genetic expression related to the subgroups difference. They used intersectional genetics combining a Pet1 driver to select for serotonin neurons and rhombomere specific drivers to identify the developmental origin of the serotonin neurons. With their animal models, serotonergic neurons arising from a given rhombomere (R)—R1, R2, R3, R5, or R6 and posterior could be distinguished in the fourth postnatal week, as a GFP-labeled 5HT neuron sublineage, and mRNA expression studies were performed both as population analysis and at the level of single cells. This provides a very valuable data set to compare gene expression patterns of serotonin neurons originating from different subgroup of serotonin neurons.

Table of contents :

Part 1: Serotonin raphe neurons and topographic mapping of serotonergic projections
1. History
2. Biochemistry of serotonin system
3. Serotonin neuroanatomy – Anatomical
organization of the raphe nuclei and serotonergic raphe neurons
4. The development of serotonin neurons in raphe nuclei
Part 2: Molecular pathway of serotonin system and Eph family
1. From growth cone to axon guidance
2. Molecular mechanism of serotonin raphe neuronal wiring.
3. Eph receptors and their ligands ephrins
4. Eph/ephrin signaling mechanisms
5. EphA5 and ephrinA5 from past to present
6. The mechanism of EphA5 modulated growth cone collapse
7. The role of EphA5-ephrinA5 interaction within the hypothalamus from glutamine system to aggressive behavior
Results
Part 1: EphAs-EphrinAs signaling is required for the distinctive targeting of raphe 5-HT neurons in the telencephalon
Part 2: Study of serotonin raphe neurons projections during development with an IDISCO method 
Discussion
Bibliography

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