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. Abstract: Subcortical 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.
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