A single-cell transcriptomic and anatomic atlas of mouse dorsal raphe Pet1 neurons.

Among the brainstem raphe nuclei, the dorsal raphe nucleus (DR) contains the greatest number of Pet1-lineage neurons, a predominantly serotonergic group distributed throughout DR subdomains. These neurons collectively regulate diverse physiology and behavior and are often therapeutically targeted to treat affective disorders. Characterizing Pet1 neuron molecular heterogeneity and relating it to anatomy is vital for understanding DR functional organization, with potential to inform therapeutic separability. Here we use high-throughput and DR subdomain-targeted single-cell transcriptomics and intersectional genetic tools to map molecular and anatomical diversity of DR-Pet1 neurons. We describe up to fourteen neuron subtypes, many showing biased cell body distributions across the DR. We further show that P2ry1-Pet1 DR neurons - the most molecularly distinct subtype - possess unique efferent projections and electrophysiological properties. These data complement and extend previous DR characterizations, combining intersectional genetics with multiple transcriptomic modalities to achieve fine-scale molecular and anatomic identification of Pet1 neuron subtypes.

Molecular and anatomical organization of the dorsal raphe nucleus.

The dorsal raphe nucleus (DRN) is an important source of neuromodulators and has been implicated in a wide variety of behavioral and neurological disorders. The DRN is subdivided into distinct anatomical subregions comprised of multiple cell types, and its complex cellular organization has impeded efforts to investigate the distinct circuit and behavioral functions of its subdomains. Here we used single-cell RNA sequencing, in situ hybridization, anatomical tracing, and spatial correlation analysis to map the transcriptional and spatial profiles of cells from the mouse DRN. Our analysis of 39,411 single-cell transcriptomes revealed at least 18 distinct neuron subtypes and 5 serotonergic neuron subtypes with distinct molecular and anatomical properties, including a serotonergic neuron subtype that preferentially innervates the basal ganglia. Our study lays out the molecular organization of distinct serotonergic and non-serotonergic subsystems, and will facilitate the design of strategies for further dissection of the DRN and its diverse functions.

Are periaqueductal gray and dorsal raphe the foundation of appetitive and aversive control? A comprehensive review.

Many see the periaqueductal gray (PAG) as a region responsible for the downstream control of defensive reactions. Here we provide a detailed review of anatomical and functional data on the different parts of the PAG together with the dorsal raphe, which completes the circle of periaqueductal nuclei. Based on anatomical features, we propose a new subdivision of the periaqueductal gray that accounts for the distinct characteristics of the area. We provide a comprehensive functional view of the periaqueductal gray, going beyond simple panic and escape to integrate data on fear, anxiety, and depression. Importantly, we conclude that this periaqueductal cluster of nuclei is broadly involved in motivated behavior controlling not only aversive but also appetitive behavior and with some involvement in more complex motivational processes such as approach-avoidance conflict resolution. In sum, these highly conserved nuclei surrounding the aqueduct appear to be the simplest, foundational, elements of integrated motivated goal-directed control of all types.

Anatomically Defined and Functionally Distinct Dorsal Raphe Serotonin Sub-systems.

The dorsal raphe (DR) constitutes a major serotonergic input to the forebrain and modulates diverse functions and brain states, including mood, anxiety, and sensory and motor functions. Most functional studies to date have treated DR serotonin neurons as a single population. Using viral-genetic methods, we found that subcortical- and cortical-projecting serotonin neurons have distinct cell-body distributions within the DR and differentially co-express a vesicular glutamate transporter. Further, amygdala- and frontal-cortex-projecting DR serotonin neurons have largely complementary whole-brain collateralization patterns, receive biased inputs from presynaptic partners, and exhibit opposite responses to aversive stimuli. Gain- and loss-of-function experiments suggest that amygdala-projecting DR serotonin neurons promote anxiety-like behavior, whereas frontal-cortex-projecting neurons promote active coping in the face of challenge. These results provide compelling evidence that the DR serotonin system contains parallel sub-systems that differ in input and output connectivity, physiological response properties, and behavioral functions.

Sub-regions of the dorsal raphé nucleus receive different inputs from the brainstem.

The dorsal raphe nucleus (DRN) through its extensive efferent projections has been implicated in a great variety of physiological and behavioral functions including the regulation of the sleep-wake cycle. This nucleus is composed of five sub-regions defined according to the distribution of its serotonergic (5-HT) neurons. In addition to its heterogeneity in neuronal populations, the DRN contains a great diversity of 5-HT neuronal subtypes identified based on their electrophysiological characteristics, morphology and sub-regional distribution. This suggests that the DRN sub-regions may play different functional roles. Recent studies reported long-range inputs specific to the 5-HT neurons of the DRN; but they did not differentiate whether some inputs were specific to a DRN sub-region, or another region. To fulfill this gap, we have previously described the forebrain afferents to the different sub-regions of the DRN using cholera toxin b subunit and Phaseolus vulgaris-leucoagglutinin, as retrograde and anterograde tracers respectively. In the present work, we provide a detailed map of the brainstem projections to these different sub-regions. We show that if some brainstem structures project homogeneously to all sub-regions, most of the brainstem long-range inputs project in a topographically organized manner onto the DRN and, moreover, that a rich interconnected network is present within the DRN.

The rostromedial tegmental nucleus is essential for non-rapid eye movement sleep.

The rostromedial tegmental nucleus (RMTg), also called the GABAergic tail of the ventral tegmental area, projects to the midbrain dopaminergic system, dorsal raphe nucleus, locus coeruleus, and other regions. Whether the RMTg is involved in sleep-wake regulation is unknown. In the present study, pharmacogenetic activation of rat RMTg neurons promoted non-rapid eye movement (NREM) sleep with increased slow-wave activity (SWA). Conversely, rats after neurotoxic lesions of 8 or 16 days showed decreased NREM sleep with reduced SWA at lights on. The reduced SWA persisted at least 25 days after lesions. Similarly, pharmacological and pharmacogenetic inactivation of rat RMTg neurons decreased NREM sleep. Electrophysiological experiments combined with optogenetics showed a direct inhibitory connection between the terminals of RMTg neurons and midbrain dopaminergic neurons. The bidirectional effects of the RMTg on the sleep-wake cycle were mimicked by the modulation of ventral tegmental area (VTA)/substantia nigra compacta (SNc) dopaminergic neuronal activity using a pharmacogenetic approach. Furthermore, during the 2-hour recovery period following 6-hour sleep deprivation, the amount of NREM sleep in both the lesion and control rats was significantly increased compared with baseline levels; however, only the control rats showed a significant increase in SWA compared with baseline levels. Collectively, our findings reveal an essential role of the RMTg in the promotion of NREM sleep and homeostatic regulation.

Unraveling the architecture of the dorsal raphe synaptic neuropil using high-resolution neuroanatomy.

The dorsal raphe nucleus (DRN), representing the main source of brain's serotonin, is implicated in the pathophysiology and therapeutics of several mental disorders that can be debilitating and life-long including depression, anxiety and autism. The activity of DRN neurons is precisely regulated, both phasically and tonically, by excitatory glutamate and inhibitory GABAergic axons arising from extra-raphe areas as well as from local sources within the nucleus. Changes in serotonin neurotransmission associated with pathophysiology may be encoded by alterations within this network of regulatory afferents. However, the complex organization of the DRN circuitry remains still poorly understood. Using a recently developed high-resolution immunofluorescence technique called array tomography (AT) we quantitatively analyzed the relative contribution of different populations of glutamate axons originating from different brain regions to the excitatory drive of the DRN. Additionally, we examined the presence of GABA axons within the DRN and their possible association with glutamate axons. In this review, we summarize our findings on the architecture of the rodent DRN synaptic neuropil using high-resolution neuroanatomy, and discuss possible functional implications for the nucleus. Understanding of the synaptic architecture of neural circuits at high resolution will pave the way to understand how neural structure and function may be perturbed in pathological states.