Divergent in vivo activity of non‐serotonergic and serotonergic VGluT3–neurones in the median raphe region

Abstract

Key points The median raphe is a key subcortical modulatory centre involved in several brain functions, such as regulation of the sleep–wake cycle, emotions and memory storage.A large proportion of median raphe neurones are glutamatergic and implement a radically different mode of communication compared to serotonergic cells, although their in vivo activity is unknown.We provide the first description of the in vivo, brain state‐dependent firing properties of median raphe glutamatergic neurones identified by immunopositivity for the vesicular glutamate transporter type 3 (VGluT3) and serotonin (5‐HT). Glutamatergic populations (VGluT3+/5‐HT– and VGluT3+/5‐HT+) were compared with the purely serotonergic (VGluT3–/5‐HT+ and VGluT3–/5‐HT–) neurones.VGluT3+/5‐HT+ neurones fired similar to VGluT3–/5‐HT+ cells, whereas they significantly diverged from the VGluT3+/5‐HT– population. Activity of the latter subgroup resembled the spiking of VGluT3–/5‐HT– cells, except for their diverging response to sensory stimulation.The VGluT3+ population of the median raphe may broadcast rapidly varying signals on top of a state‐dependent, tonic modulation. AbstractSubcortical modulation is crucial for information processing in the cerebral cortex. Besides the canonical neuromodulators, glutamate has recently been identified as a key cotransmitter of numerous monoaminergic projections. In the median raphe, a pure glutamatergic neurone population projecting to limbic areas was also discovered with a possibly novel, yet undetermined function. In the present study, we report the first functional description of the vesicular glutamate transporter type 3 (VGluT3)‐expressing median raphe neurones. Because there is no appropriate genetic marker for the separation of serotonergic (5‐HT+) and non‐serotonergic (5‐HT–) VGluT3+ neurones, we utilized immunohistochemistry after recording and juxtacellular labelling in anaesthetized rats. VGluT3+/5‐HT– neurones fired faster, more variably and were permanently activated during sensory stimulation, as opposed to the transient response of the slow firing VGluT3–/5‐HT+ subgroup. VGluT3+/5‐HT– cells were also more active during hippocampal theta. In addition, the VGluT3–/5‐HT– population, comprising putative GABAergic cells, resembled the firing of VGluT3+/5‐HT– neurones but without any significant reaction to the sensory stimulus. Interestingly, the VGluT3+/5‐HT+ group, spiking slower than the VGluT3+/5‐HT– population, exhibited a mixed response (i.e. the initial transient activation was followed by a sustained elevation of firing). Phase coupling to hippocampal and prefrontal slow oscillations was found in VGluT3+/5‐HT– neurones, also differentiating them from the VGluT3+/5‐HT+ subpopulation. Taken together, glutamatergic neurones in the median raphe may implement multiple, highly divergent forms of modulation in parallel: a slow, tonic mode interrupted by sensory‐evoked rapid transients, as well as a fast one capable of conveying complex patterns influenced by sensory inputs.

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

“…The molecular features of specific serotonin cell types suggest their functional properties. For example, several studies have reported that subgroups of serotonin neurons in the MR and DR express Vglut3 and indeed, subsequent slice recording confirmed that serotonin terminals can co-release glutamate and serotonin (Domonkos et al, 2016; Liu et al, 2014; Ren et al, 2018; Sengupta et al, 2017; Varga et al, 2009; Wang et al, 2019). In addition to neurochemical properties, each serotonin neuron population expresses a specific combination of distinct genes responsible for electrophysiological (ion channels), connectivity (wiring molecules), and functional (neurotransmitter/peptide receptors) properties (Figure 4; Figure 4—figure supplement 1A).…”

“…The majority of the clusters express Slc17a8 , which encodes vesicular glutamate transporter-3 (Vglut3). These include almost all serotonin neurons from the cDR, the majority from MR clusters (Domonkos et al, 2016), and DR-3–6 clusters (Figure 1D). These observations suggest that glutamate is the most prevalent co-transmitter for serotonin neurons.…”

Reciprocal lateral hypothalamic and raphé GABAergic projections promote wakefulness

“…Our results demonstrate that LH GABA neurons directly inhibit DR GABA neurons and this probably results in a disinhibition of various DR neurons. Revealing the exact targets of this disinhibition awaits further investigation and are hard to predict given the extreme neurochemical and physiological diversity of the raphe nuclei (Domonkos et al, 2016; Szonyi et al, 2016; Sos et al, 2017). A phasic increase in 5-HT neuron activity can rapidly and prominently affect cortical activity (Lottem et al, 2016) and thus may lead to brain state changes.…”

Serotonin neuron development: shaping molecular and structural identities

“…VGLUT3 + /5-HT + neurons may be a specialized 5-HT subtype as these cells were found to have specific electrophysiological signatures in patch clamp analyses, with lower activation thresholds, suggesting that they may be recruited only by strong stimuli to boost serotonergic/glutamatergic signals (59). Further, in vivo recordings indicated that VGLUT3 + /5-HT − , VGLUT3 − /5-HT + , and VGLUT3 + /5-HT + neurons in the MRN possess significantly different firing rates, responses to sensory stimulation, and phase coupling to forebrain oscillations (82).…”