Serotonin neuron development: shaping molecular and structural identities.

The continuing fascination with serotonin (5-hydroxytryptamine, 5-HT) as a nervous system chemical messenger began with its discovery in the brains of mammals in 1953. Among the many reasons for this decades-long interest is that the small numbers of neurons that make 5-HT influence the excitability of neural circuits in nearly every region of the brain and spinal cord. A further reason is that 5-HT dysfunction has been linked to a range of psychiatric and neurological disorders many of which have a neurodevelopmental component. This has led to intense interest in understanding 5-HT neuron development with the aim of determining whether early alterations in their generation lead to brain disease susceptibility. Here, we present an overview of the neuroanatomical organization of vertebrate 5-HT neurons, their neurogenesis, and prodigious axonal architectures, which enables the expansive reach of 5-HT neuromodulation in the central nervous system. We review recent findings that have revealed the molecular basis for the tremendous diversity of 5-HT neuron subtypes, the impact of environmental factors on 5-HT neuron development, and how 5-HT axons are topographically organized through disparate signaling pathways. We summarize studies of the gene regulatory networks that control the differentiation, maturation, and maintenance of 5-HT neurons. These studies show that the regulatory factors controlling acquisition of 5-HT-type transmitter identity continue to play critical roles in the functional maturation and the maintenance of 5-HT neurons. New insights are presented into how continuously expressed 5-HT regulatory factors control 5-HT neurons at different stages of life and how the regulatory networks themselves are maintained. WIREs Dev Biol 2018, 7:e301. doi: 10.1002/wdev.301 This article is categorized under: Nervous System Development > Vertebrates: General Principles Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics Gene Expression and Transcriptional Hierarchies > Cellular Differentiation Nervous System Development > Secondary: Vertebrates: Regional Development.

Multi-Scale Molecular Deconstruction of the Serotonin Neuron System.

Serotonergic (5HT) neurons modulate diverse behaviors and physiology and are implicated in distinct clinical disorders. Corresponding diversity in 5HT neuronal phenotypes is becoming apparent and is likely rooted in molecular differences, yet a comprehensive approach characterizing molecular variation across the 5HT system is lacking, as is concomitant linkage to cellular phenotypes. Here we combine intersectional fate mapping, neuron sorting, and genome-wide RNA-seq to deconstruct the mouse 5HT system at multiple levels of granularity-from anatomy, to genetic sublineages, to single neurons. Our unbiased analyses reveal principles underlying system organization, 5HT neuron subtypes, constellations of differentially expressed genes distinguishing subtypes, and predictions of subtype-specific functions. Using electrophysiology, subtype-specific neuron silencing, and conditional gene knockout, we show that these molecularly defined 5HT neuron subtypes are functionally distinct. Collectively, this resource classifies molecular diversity across the 5HT system and discovers sertonergic subtypes, markers, organizing principles, and subtype-specific functions with potential disease relevance.

Functional and developmental identification of a molecular subtype of brain serotonergic neuron specialized to regulate breathing dynamics.

Serotonergic neurons modulate behavioral and physiological responses from aggression and anxiety to breathing and thermoregulation. Disorders involving serotonin (5HT) dysregulation are commensurately heterogeneous and numerous. We hypothesized that this breadth in functionality derives in part from a developmentally determined substructure of distinct subtypes of 5HT neurons each specialized to modulate specific behaviors. By manipulating developmentally defined subgroups one by one chemogenetically, we find that the Egr2-Pet1 subgroup is specialized to drive increased ventilation in response to carbon dioxide elevation and acidosis. Furthermore, this subtype exhibits intrinsic chemosensitivity and modality-specific projections-increasing firing during hypercapnic acidosis and selectively projecting to respiratory chemosensory but not motor centers, respectively. These findings show that serotonergic regulation of the respiratory chemoreflex is mediated by a specialized molecular subtype of 5HT neuron harboring unique physiological, biophysical, and hodological properties specified developmentally and demonstrate that the serotonergic system contains specialized modules contributing to its collective functional breadth.

Medullary serotonin neurons are CO2 sensitive in situ.

Brainstem central chemoreceptors are critical to the hypercapnic ventilatory response, but their location and identity are poorly understood. When studied in vitro, serotonin-synthesizing (5-HT) neurons within the rat medullary raphé are intrinsically stimulated by CO2/acidosis. The contributions of these neurons to central chemosensitivity in vivo, however, are controversial. Lacking is documentation of CO2-sensitive 5-HT neurons in intact experimental preparations and understanding of their spatial and proportional distribution. Here we test the hypothesis that 5-HT neurons in the rat medullary raphé are sensitive to arterial hypercapnia. We use extracellular recording and hypercapnic challenge of spontaneously active medullary raphé neurons in the unanesthetized in situ perfused decerebrate brainstem preparation to assess chemosensitivity of individual cells. Juxtacellular labeling of a subset of recorded neurons and subsequent immunohistochemistry for the 5-HT-synthesizing enzyme tryptophan hydroxylase (TPH) identify or exclude this neurotransmitter phenotype in electrophysiologically characterized chemosensitive and insensitive cells. We show that the medullary raphé houses a heterogeneous population, including chemosensitive and insensitive 5-HT neurons. Of 124 recorded cells, 16 cells were juxtacellularly filled, visualized, and immunohistochemically identified as 5-HT synthesizing, based on TPH-immunoreactivity. Forty-four percent of 5-HT cells were CO2 stimulated (increased firing rate with hypercapnia), while 56% were unstimulated. Our results demonstrate that medullary raphé neurons are heterogeneous and clearly include a subset of 5-HT neurons that are excited by arterial hypercapnia. Together with data identifying intrinsically CO2-sensitive 5-HT neurons in vitro, these results support a role for such cells as central chemoreceptors in the intact system.

Serotonin neuron diversity in the dorsal raphe.

The dorsal raphe nucleus contains one of the largest groups of serotonergic neurons in the mammalian brain and is the main site of origin of the serotonergic projection to the cerebral cortex. Early electrophysiological studies suggested that serotonergic neurons in this cell group formed a homogeneous cell class. More recent studies however have reported heterogeneity among the core anatomical and electrophysiological properties of these neurons, thus raising the possibility that serotonergic neurons of this cell group may form two or more distinct cell classes. In this Viewpoint, we review these findings and suggest ways to look at cellular heterogeneity among serotonergic neurons.

Development of the serotonergic cells in murine raphe nuclei and their relations with rhombomeric domains.

The raphe nuclei represent the origin of central serotonergic projections. The literature distinguishes seven nuclei grouped into rostral and caudal clusters relative to the pons. The boundaries of these nuclei have not been defined precisely enough, particularly with regard to developmental units, notably hindbrain rhombomeres. We hold that a developmental point of view considering rhombomeres may explain observed differences in connectivity and function. There are twelve rhombomeres characterized by particular genetic profiles, and each develops between one and four distinct serotonergic populations. We have studied the distribution of the conventional seven raphe nuclei among these twelve units. To this aim, we correlated 5-HT-immunoreacted neurons with rhombomeric boundary landmarks in sagittal mouse brain sections at different developmental stages. Furthermore, we performed a partial genoarchitectonic analysis of the developing raphe nuclei, mapping all known serotonergic differentiation markers, and compared these results, jointly with others found in the literature, with our map of serotonin-containing populations, in order to examine regional variations in correspondence. Examples of regionally selective gene patterns were identified. As a result, we produced a rhombomeric classification of some 45 serotonergic populations, and suggested a corresponding modified terminology. Only a minor rostral part of the dorsal raphe nucleus lies in the midbrain. Some serotonergic neurons were found in rhombomere 4, contrary to the conventional assumption that it lacks such neurons. We expect that our reclassification of raphe nuclei may be useful for causal analysis of their differential molecular specification, as well as for studies of differential connectivity and function.