Multiscale single-cell analysis reveals unique phenotypes of raphe 5-HT neurons projecting to the forebrain
Serotonergic neurons of the raphe nuclei exhibit anatomical, neurochemical and elecrophysiological heterogeneity that likely underpins their specific role in multiple behaviors. However, the precise organization of serotonin (5-HT) neurons to orchestrate 5-HT release patterns throughout the brain is not well understood. We compared the electrophysiological and neurochemical properties of dorsal and median raphe 5-HT neurons projecting to the medial prefrontal cortex (mPFC), amygdala (BLA) and dorsal hippocampus (dHP), combining retrograde tract tracing with brain slice electrophysiology and single-cell RT-PCR in Pet1-EGFP mice. Our results show that 5-HT neurons projecting to the dHP and the mPFC and the BLA form largely non-overlapping populations and that BLA-projecting neurons have characteristic excitability and membrane properties. In addition, using an unbiased clustering method that correlates anatomical, molecular and electrophysiological phenotypes, we find that 5-HT neurons with projections to the mPFC and the dHP segregate from those projecting to the BLA. Single-cell gene profiling showed a restricted expression of the peptide galanin in the population of 5-HT neurons projecting to the mPFC. Finally, cluster analysis allowed identifying an atypical subtype of 5-HT neuron with low excitability, long firing delays and preferential expression of the vesicular glutamate transporter type 3. Overall, these findings allow to define correlated anatomical and physiological identities of serotonin raphe neurons that help understanding how discrete raphe cells subpopulations account for the heterogeneous activities of the midbrain serotonergic system.
KeywordsSerotonin Raphe Galanin Amygdala Hippocampus Prefrontal cortex Single-cell PCR Patch-clamp
This work was funded by INSERM, the University Pierre et Marie Curie, the European Commission (FP7-health-2007-A-201714), The Fondation pour la Recherche Medicale (equipe FRM to PG) and the Agence Nationale pour la recherche (ANR605-neur-046 and ANR 2011 MALZ 003 01). SPF received a Fellowship from the Fondation pour la Recherche Medicale. The teams of PG and JCP are part of the Ecole de Neuroscience de Paris (ENP) training network and Labex Bio-Psy (Investissement d’Avenir, ANR). We thank Evan Deneris and Gord Fishell for providing the founders of ePet1-Cre and RCE:LoxP mouse lines. Sophie Scotto-Lomassese is acknowledged for help with confocal microscopy and Emma Martinelli and Mariano Soiza-Reilly for their helpful comments on the manuscript. The authors declare no competing financial interests.
- Alonso A, Merchán P, Sandoval JE, Sánchez-Arrones L, Garcia-Cazorla A, Artuch R, Ferrán JL, Martínez-de-la-Torre M, Puelles L (2012) Development of the serotonergic cells in murine raphe nuclei and their relations with rhombomeric domains. Brain Struct Functi 18:1229–1277Google Scholar
- Amilhon B, Lepicard E, Renoir T, Mongeau R, Popa D, Poirel O, Miot S, Gras C, Gardier AM, Gallego J, Hamon M, Lanfumey L, Gasnier B, Giros B, El Mestikawy S (2010) VGLUT3 (vesicular glutamate transporter type 3) contribution to the regulation of serotonergic transmission and anxiety. J Neurosci 30:2198–2210CrossRefPubMedGoogle Scholar
- Gras C, Amilhon B, Lepicard EM, Poirel O, Vinatier J, Herbin M, Dumas S, Tzavara ET, Wade MR, Nomikos GG, Hanoun N, Saurini F, Kemel M-L, Gasnier B, Giros B, El Mestikawy S (2008) The vesicular glutamate transporter VGLUT3 synergizes striatal acetylcholine tone. Nat Neurosci 11:292–300CrossRefPubMedGoogle Scholar
- Köhler C, Steinbusch H (1982) Identification of serotonin and non-serotonin-containing neurons of the mid-brain raphe projecting to the entorhinal area and the hippocampal formation. A combined immunohistochemical and fluorescent retrograde tracing study in the rat brain. Neuroscience 7:951–975CrossRefPubMedGoogle Scholar
- Muzerelle A, Scotto-Lomassese S, Bernard JF, Soiza-Reilly M, Gaspar P (2014) Conditional anterograde tracing reveals distinct targeting of individual serotonin cell groups (B5-B9) to the forebrain and brainstem. Brain Struct Funct 1–27. doi: 10.1007/s00429-014-0924-4
- Trulson ME, Frederickson CJ (1987) A comparison of the electrophysiological and pharmacological properties of serotonin-containing neurons in the nucleus raphe dorsalis, raphe medianus and raphe pallidus recorded from mouse brain slices in vitro: role of autoreceptors. Brain Res Bull 18:179–190CrossRefPubMedGoogle Scholar