Single-cell transcriptomes and whole-brain projections of serotonin neurons in the mouse dorsal and median raphe nuclei

Ren, Jing; Isakova, Alina; Friedmann, Drew; Zeng, Jiawei; Grutzner, Sophie M.; Pun, Albert; Zhao, Grace; Kolluru, Sai Saroja; Wang, Ruiyu; Lin, Rui; Li, Pengcheng; Li, Anan; Raymond, Jennifer L.; Luo, Qi; Luo, Minmin; Quake, Stephen R.; Luo, Liqun

doi:10.7554/elife.49424

Cited by 239 publications

(236 citation statements)

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“…We observed that stimulation of NPVF neurons results in increased GCaMP6s fluorescence in most serotonergic IRa neurons, although a significant minority of IRa neurons show reduced GCaMP6s fluorescence. These observations suggest that there are at least two functionally distinct neuronal populations among tph2 -expressing IRa neurons, consistent with studies of the mammalian RN that identified molecularly distinct sub-populations with distinct efferent projection patterns and functions (Huang et al, 2019; Ren et al, 2018; Ren et al, 2019). The NPVF neuron-induced inhibition of a sub-population of IRa neurons that we observed is consistent with a previous study which reported that optogenetic stimulation of NPVF neurons resulted in decreased GCaMP6s fluorescence in IRa neurons (Madelaine et al, 2017), although the effect was small and slow compared to our data.…”

Section: Discussionsupporting

confidence: 87%

Neuropeptide VF neurons promote sleep via the serotonergic raphe

Lee

Oikonomou

Cammidge

et al. 2019

Preprint

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15Although several sleep-regulating neurons have been identified, little is known about how they 16 interact with each other for sleep/wake control. We previously identified neuropeptide VF (NPVF) 17 and the hypothalamic neurons that produce it as a sleep-promoting system (Lee et al., 2017). 18Here we use zebrafish to describe a neural circuit in which neuropeptide VF (npvf)-expressing 19 neurons control sleep via the serotonergic raphe nuclei (RN), a hindbrain structure that promotes 20 sleep in both diurnal zebrafish and nocturnal mice. Using genetic labeling and calcium imaging, 21we show that npvf-expressing neurons innervate and activate serotonergic RN neurons. We 22 additionally demonstrate that optogenetic stimulation of npvf-expressing neurons induces sleep 23 in a manner that requires NPVF and is abolished when the RN are ablated or lack serotonin. 24Finally, genetic epistasis demonstrates that NPVF acts upstream of serotonin in the RN to 25 maintain normal sleep levels. These findings reveal a novel hypothalamic-hindbrain circuit for 26 sleep/wake control. 28While several sleep-and wake-promoting neuronal populations have been identified (reviewed in 30 (Bringmann, 2018; Liu and Dan, 2019; Saper and Fuller, 2017; Scammell et al., 2017)), 31 characterizing and understanding the functional and hierarchical relationships between these 32 populations is essential for understanding how the brain regulates sleep and wake states 33 (Oikonomou and Prober, 2017). Recent evidence from zebrafish and mice demonstrate that the 34 serotonergic raphe nuclei (RN) are critical for the initiation and maintenance of sleep (Oikonomou 35 et al., 2019), in contrast with previous models suggesting a wake-promoting role for the RN that 36 were largely based on their wake-active nature (Saper and Fuller, 2017; Scammell et al., 2017; 37Weber and Dan, 2016). In zebrafish, mutation of tryptophan hydroxylase 2 (tph2), which is 38 required for serotonin (5-HT) synthesis in the RN, results in reduced sleep, sleep depth, and 39 homeostatic response to sleep deprivation (Oikonomou et al., 2019). Pharmacological inhibition 40 of 5-HT synthesis or ablation of the RN also results in reduced sleep. Consistent with a sleep-41 promoting role for the raphe, optogenetic stimulation of raphe neurons results in increased sleep.42 Similarly, in mice, ablation of the RN results in increased wakefulness and an impaired 43 homeostatic response to sleep deprivation, whereas tonic optogenetic stimulation of the RN at a 44 rate similar to their baseline pattern of activity induces NREM sleep. These complementary results 45 in zebrafish and mice (Oikonomou et al., 2019), along with classical ablation and pharmacological 46 studies (Ursin, 2008), indicate an evolutionarily conserved role for the serotonergic system in 47 promoting vertebrate sleep. However, it is unclear how the RN are themselves regulated to 48 promote sleep. 50Trans-synaptic retrograde viral tracing studies have identified substantial inputs to the RN from 51 hypothalamic ...

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Section: Discussionsupporting

confidence: 87%

Neuropeptide VF neurons promote sleep via the serotonergic raphe

Lee

Oikonomou

Cammidge

et al. 2019

Preprint

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“…Interestingly, simultaneous recording 5-HT1.0 in OFC and BNST revealed tight correlation in fluorescence during NREM sleep (Fig. 3h,i), suggesting global synchrony of the 5-HT signaling despite of the region-specific innervation by different subpopulations of the serotonergic neurons in DRN 23, 24 . Lastly, consistent with our previous findings, we found that treating mice with the 5-HT receptor antagonist Met largely blocked the fluorescence change of the 5-HT1.0 sensor (Fig.…”

Section: Mainmentioning

confidence: 90%

A genetically encoded GRAB sensor for measuring serotonin dynamicsin vivo

Wan

Peng

et al. 2020

Preprint

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28Serotonin (5-HT) is a phylogenetically conserved monoamine neurotransmitter modulating 29 a variety of processes in the brain. To directly visualize the dynamics of 5-HT, we developed 30 a genetically encoded GPCR-Activation-Based 5-HT (GRAB5-HT) sensor with high 31 sensitivity, selectivity, and spatiotemporal resolution. GRAB5-HT, detected 5-HT release in 32 multiple physiological and pathological conditions in both flies and mice, and thus provides 33 new insights into the dynamics and mechanisms of 5-HT signaling. 34 Main 35Serotonergic signaling in the brain plays a critical role in a wide range of physiological 36 processes, including mood control, reward processing, and sleep-wake homeostatic 37 regulation 1-3 . Given its functional importance, drugs targeting central serotonergic activity 38have been used to treat virtually every psychiatric disorder, with the best example being 39 the use of selective serotonin reuptake inhibitors (SSRIs) for depression 4 . Despite the 40 importance of 5-HT, understanding of cell-specific 5-HT signaling during behaviors is 41 greatly hampered by the lack of ability to measure 5-HT in vivo with high sensitivity and 42 precise spatiotemporal resolution 5,6,7 . Using molecular engineering, we developed a 43 genetically encoded fluorescent sensor for directly measuring extracellular 5-HT. 44Previously, we and others independently developed GPCR activation based sensors for 45 detecting different neurotransmitters by converting the conformational change in the 46 respective GPCR to a sensitive fluorescence change in circular permutated GFP (cpGFP) 8, 47 9,10,11 . Using similar strategy, we initiated the engineering of 5-HT-specific GRAB sensor 48 by inserting a cpGFP into the third intracellular loop (ICL3) of various 5-HT receptors. 49Based on the performance of membrane trafficking and affinity of receptor-cpGFP 50 chimeras, we selected and focused on the 5-HT2C receptor-based chimera for further 51 optimization (Extended Data Fig. 1a,b). Mutagenesis and screening in its linker regions 52 and cpGFP moiety, resulted in a sensor with a 250% ∆F/F0 in response to 5-HT, which we 53 named GRAB5-HT1.0 (referred to hereafter as simply 5-HT1.0; Fig 1a and Extended Data 54 Fig. 2). In addition, we generated a 5-HT-insensitive version of this sensor by introducing 55 the D134 3.32 Q mutation in the receptor 12 , resulting in GRAB5-HTmut (referred to hereafter as 56 5-HTmut). This mutant sensor has the similar membrane trafficking as 5-HT1.0, but <2% 57 ∆F/F0 even in response to 100 µM 5-HT ( Fig. 1a and Extended Data Fig. 3a-d). In cultured 58 neurons, the 5-HT1.0 sensor produced a robust fluorescence increase (280% ∆F/F0) in 59both the soma and neurites in response to 5-HT application, whereas 5-HTmut sensor had 60 no measurable change in fluorescence ( Fig. 1b and Extended Data Fig. 3l). 61Next, we characterized the properties of the 5-HT1.0 sensor, including the brightness and 62 photostability, dose-response relationship between 5-HT concentration and fluorescence 63 change, res...

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“…Single serotonergic neurons can span sensory networks within the same modality and therefore have the potential to modulate different processing stages simultaneously. Recent work mapping the projection patterns of 50 serotonergic Raphe neurons demonstrated that similarly to the CSDn, a single neuron can target several olfactory areas including the main olfactory bulb, accessory olfactory bulb, and piriform cortex [19]. The degree to which the influence of a single serotonergic neuron is conserved across processing stages is unknown, however work in mice suggests that the functional roles of serotonergic neurons can differ between networks.…”

Section: Discussionmentioning

confidence: 99%

The wiring logic of an identified serotonergic neuron that spans sensory networks

Coates

Calle-Schuler

Helmick

et al. 2020

Preprint

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19Serotonergic neurons modulate diverse physiological and behavioral processes in a 20 context-dependent manner, based on their complex connectivity. However, their 21 connectivity has not been comprehensively explored at a single-cell resolution. Using a 22 whole-brain EM dataset we determined the wiring logic of a broadly projecting 23 serotonergic neuron (the "CSDn") in Drosophila. Within the antennal lobe (AL; first-order 24 olfactory region), the CSDn receives glomerulus-specific input and preferentially targets 25 distinct local interneuron subtypes. Furthermore, the wiring logic of the CSDn differs 26 between olfactory regions. The CSDn innervates the AL and lateral horn (LH), yet does 27 not maintain the same synaptic relationship with individual projection neurons that also 28 span both regions. Consistent with this, the CSDn has more distributed connectivity in 29 the LH relative to the AL, preferentially synapsing with principal neuron types based on 30 presumptive transmitter content. Lastly, we identify protocerebral neurons that provide 31 abundant synaptic input to the CSDn. Our study demonstrates how an individual 32 modulatory neuron can interact with local networks and integrate input from non-33 olfactory sources. 35Introduction 36 Every neural network receives modulatory input from a variety of sources [1, 2], 37 and in some cases from heterogeneous populations of neurons that release the same 38 modulatory transmitter [3][4][5]. In mammals, one ubiquitous neuromodulator, serotonin, is 39 released by tens of thousands to hundreds of thousands of neurons which originate in 40 the raphe nuclei and project throughout the brain [6, 7]. Serotonergic raphe neurons are 41 highly diverse in their projections, connectivity, and electrophysiological properties, and 42 2 are implicated in a wide breadth of behaviors and physiological processes [4,[8][9][10][11][12][13][14][15][16]. 43 Further, the raphe system receives monosynaptic input from up to 80 anatomical areas 44 [8, 9]. As a result, a significant amount of work has focused on disentangling the 45 functional and behavioral roles of serotonergic neurons. Several recent studies have 46 suggested that serotonergic raphe neurons may be organized into functional 47 subpopulations based on neuroanatomy, electrophysiology, and behavior [4, 12,[17][18][19][20][21]. 48For example, two parallel sub-systems of serotonergic raphe neurons collateralize 49 complimentarily and are both activated by reward, yet have opposing responses to 50 aversive stimuli and promote distinct behaviors [18]. Sparse neuron reconstructions in 51 mice show that a single serotonin neuron can interconnect the olfactory bulb, piriform 52 cortex, and anterior olfactory nucleus [19], demonstrating that a single serotonergic 53 neuron can arborize several processing stages within the same sensory modality. Thus, 54 determining the precise patterns of connectivity of single serotonergic neurons within 55 and across the brain regions will be critical for understanding t...

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Single-cell transcriptomes and whole-brain projections of serotonin neurons in the mouse dorsal and median raphe nuclei

Cited by 239 publications

References 92 publications

Neuropeptide VF neurons promote sleep via the serotonergic raphe

Neuropeptide VF neurons promote sleep via the serotonergic raphe

A genetically encoded GRAB sensor for measuring serotonin dynamicsin vivo

The wiring logic of an identified serotonergic neuron that spans sensory networks

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