The unusual response of serotonergic neurons after CNS Injury: lack of axonal dieback and enhanced sprouting within the inhibitory environment of the glial scar.

Serotonergic neurons possess an enhanced ability to regenerate or sprout after many types of injury. To understand the mechanisms that underlie their unusual properties, we used a combinatorial approach comparing the behavior of serotonergic and cortical axon tips over time in the same injury environment in vivo and to growth-promoting or growth-inhibitory substrates in vitro. After a thermocoagulatory lesion in the rat frontoparietal cortex, callosal axons become dystrophic and die back. Serotonergic axons, however, persist within the lesion edge. At the third week post-injury, 5-HT+ axons sprout robustly. The lesion environment contains both growth-inhibitory chondroitin sulfate proteoglycans (CSPGs) and growth-promoting laminin. Transgenic mouse serotonergic neurons specifically labeled by enhanced yellow fluorescent protein under control of the Pet-1 promoter/enhancer or cortical neurons were cultured on low amounts of laminin with or without relatively high concentrations of the CSPG aggrecan. Serotonergic neurons extended considerably longer neurites than did cortical neurons on low laminin and exhibited a remarkably more active growth cone on low laminin plus aggrecan during time-lapse imaging than did cortical neurons. Chondroitinase ABC treatment of laminin/CSPG substrates resulted in significantly longer serotonergic but not cortical neurite lengths. This increased ability of serotonergic neurons to robustly grow on high amounts of CSPG may be partially due to significantly higher amounts of growth-associated protein-43 and/or ß1 integrin than cortical neurons. Blocking ß1 integrin decreased serotonergic and cortical outgrowth on laminin. Determining the mechanism by which serotonergic fibers persist and sprout after lesion could lead to therapeutic strategies for both stroke and spinal cord injury.

Serotonergic neurons migrate radially through the neuroepithelium by dynamin-mediated somal translocation.

Embryonic CNS neurons can migrate from the ventricular zone to their final destination by radial glial-guided locomotion. Another less appreciated mechanism is somal translocation, where the young neuron maintains its primitive ventricular and pial processes, through which the cell body moves. A major problem in studying translocation has been the identification of neuronal-specific markers that appear in primitive, radially shaped cells. We used enhanced yellow fluorescent protein under control of the Pet-1 enhancer/promoter region (ePet-EYFP), a specific marker of early differentiated serotonergic neurons, to study their migration via immunohistology and time-lapse imaging of living slice cultures. As early as E10.0, ePet-EYFP-expressing neurons were axonless, radially oriented, and spanned the entire neuroepithelium. The soma translocated within the pial process toward the pial surface and could also translocate through its neurites, which sprouted from the pial process. The dynamin inhibitor dynasore significantly reduced translocation velocity, while the nonmuscle myosin II inhibitor blebbistatin and the kinesin inhibitor AMP-PNP had no significant effect. Here we show for the first time that serotonergic neurons migrate by somal translocation mediated, in part, by dynamin.

Distinct transcriptomes define rostral and caudal serotonin neurons.

The molecular architecture of developing serotonin (5HT) neurons is poorly understood, yet its determination is likely to be essential for elucidating functional heterogeneity of these cells and the contribution of serotonergic dysfunction to disease pathogenesis. Here, we describe the purification of postmitotic embryonic 5HT neurons by flow cytometry for whole-genome microarray expression profiling of this unitary monoaminergic neuron type. Our studies identified significantly enriched expression of hundreds of unique genes in 5HT neurons, thus providing an abundance of new serotonergic markers. Furthermore, we identified several hundred transcripts encoding homeodomain, axon guidance, cell adhesion, intracellular signaling, ion transport, and imprinted genes associated with various neurodevelopmental disorders that were differentially enriched in developing rostral and caudal 5HT neurons. These findings suggested a homeodomain code that distinguishes rostral and caudal 5HT neurons. Indeed, verification studies demonstrated that Hmx homeodomain and Hox gene expression defined an Hmx(+) rostral subtype and Hox(+) caudal subtype. Expression of engrailed genes in a subset of 5HT neurons in the rostral domain further distinguished two subtypes defined as Hmx(+)En(+) and Hmx(+)En(-). The differential enrichment of gene sets for different canonical pathways and gene ontology categories provided additional evidence for heterogeneity between rostral and caudal 5HT neurons. These findings demonstrate a deep transcriptome and biological pathway duality for neurons that give rise to the ascending and descending serotonergic subsystems. Our databases provide a rich, clinically relevant resource for definition of 5HT neuron subtypes and elucidation of the genetic networks required for serotonergic function.

A genetic approach to access serotonin neurons for in vivo and in vitro studies.

Serotonin (5HT) is a critical modulator of neural circuits that support diverse behaviors and physiological processes, and multiple lines of evidence implicate abnormal serotonergic signaling in psychiatric pathogenesis. The significance of 5HT underscores the importance of elucidating the molecular pathways involved in serotonergic system development, function, and plasticity. However, these mechanisms remain poorly defined, owing largely to the difficulty of accessing 5HT neurons for experimental manipulation. To address this methodological deficiency, we present a transgenic route to selectively alter 5HT neuron gene expression. This approach is based on the ability of a Pet-1 enhancer region to direct reliable 5HT neuron-specific transgene expression in the CNS. Its versatility is illustrated with several transgenic mouse lines, each of which provides a tool for 5HT neuron studies. Two lines allow Cre-mediated recombination at different stages of 5HT neuron development. A third line in which 5HT neurons are marked with yellow fluorescent protein will have numerous applications, including their electrophysiological characterization. To demonstrate this application, we have characterized active and passive membrane properties of midbrain reticular 5HT neurons, which heretofore have not been reported to our knowledge. A fourth line in which Pet-1 loss of function is rescued by expression of a Pet-1 transgene demonstrates biologically relevant levels of transgene expression and offers a route for investigating serotonergic protein structure and function in a behaving animal. These findings establish a straightforward and reliable approach for developing an array of tools for in vivo and in vitro studies of 5HT neurons.