✕
Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jun;43(7):1623-1632.
doi: 10.1038/s41386-018-0013-0. Epub 2018 Feb 5.

Positive regulation of raphe serotonin neurons by serotonin 2B receptors

Arnauld Belmer  1   2   3   4 , Emily Quentin  1   2   3 , Silvina L Diaz  1   2   3   5 , Bruno P Guiard  6   7   8 , Sebastian P Fernandez  1   2   3   9 , Stéphane Doly  1   2   3   10 , Sophie M Banas  1   2   3 , Pothitos M Pitychoutis  1   2   3   11 , Imane Moutkine  1   2   3 , Aude Muzerelle  1   2   3 , Anna Tchenio  1   2   3   12 , Anne Roumier  1   2   3 , Manuel Mameli  1   2   3   12 , Luc Maroteaux  13   14   15
Affiliations

Positive regulation of raphe serotonin neurons by serotonin 2B receptors

Arnauld Belmer et al. Neuropsychopharmacology. 2018 Jun.

Abstract

Serotonin is a neurotransmitter involved in many psychiatric diseases. In humans, a lack of 5-HT2B receptors is associated with serotonin-dependent phenotypes, including impulsivity and suicidality. A lack of 5-HT2B receptors in mice eliminates the effects of molecules that directly target serotonergic neurons including amphetamine derivative serotonin releasers, and selective serotonin reuptake inhibitor antidepressants. In this work, we tested the hypothesis that 5-HT2B receptors directly and positively regulate raphe serotonin neuron activity. By ex vivo electrophysiological recordings, we report that stimulation by the 5-HT2B receptor agonist, BW723C86, increased the firing frequency of serotonin Pet1-positive neurons. Viral overexpression of 5-HT2B receptors in these neurons increased their excitability. Furthermore, in vivo 5-HT2B-receptor stimulation by BW723C86 counteracted 5-HT1A autoreceptor-dependent reduction in firing rate and hypothermic response in wild-type mice. By a conditional genetic ablation that eliminates 5-HT2B receptor expression specifically and exclusively from Pet1-positive serotonin neurons (Htr2b 5-HTKO mice), we demonstrated that behavioral and sensitizing effects of MDMA (3,4-methylenedioxy-methamphetamine), as well as acute behavioral and chronic neurogenic effects of the antidepressant fluoxetine, require 5-HT2B receptor expression in serotonergic neurons. In Htr2b 5-HTKO mice, dorsal raphe serotonin neurons displayed a lower firing frequency compared to control Htr2b lox/lox mice as assessed by in vivo extracellular recordings and a stronger hypothermic effect of 5-HT1A-autoreceptor stimulation was observed. The increase in head-twitch response to DOI (2,5-dimethoxy-4-iodoamphetamine) further confirmed the lower serotonergic tone resulting from the absence of 5-HT2B receptors in serotonin neurons. Together, these observations indicate that the 5-HT2B receptor acts as a direct positive modulator of serotonin Pet1-positive neurons in an opposite way as the known 5-HT1A-negative autoreceptor.

PubMed Disclaimer

Conflict of interest statement

AR has been supported by grants from the Université Pierre et Marie Curie (Emergence-UPMC program) and the Bio-Psy Labex. SD has been supported by a fellowship of the Lefoulon-DeLalande foundation, SLD from the Region Ile-de France DIM STEM and from the ANPCyT (PICT 2013-3225), CONICET (PIP-11220130100157CO), and University of Moron (PID 2015), and EQ by a PhD fellowship from the Region Ile-de France DIM Cerveau et Pensée. The other authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Ex vivo electrophysiological recordings of wild-type Pet1-GFP mice. a Recordings of 5-HT neuron in ex vivo wild-type slices. Cell-attached recordings made in identified Pet1-GFP neuron did not detect firing in the presence of 100 nM phenylephrine (Phe) or of 1 µM of the 5-HT2B-receptor agonist BW723C86 (top line—5 cells). In the presence of higher concentration of phenylephrine (300 nM), Pet1-positive neuron firing was observed, and BW723C86 (1 µM) was able to increase this firing rate (8 cells). Representative traces (top) and quantification (bottom left) reveal a significant BW723C86-induced increase in firing frequency (one-way repeated measures (RM) ANOVA; bar graph and scatter plots and mean ± SEM; Bonferroni post test, *P < 0.05). b Current-clamp recordings of ex vivo raphe slices from AAVs injected Pet1-GFP mice, overexpressing 5-HT2B-HA receptor in Pet1-positive 5-HT neuron. (Left) Sample traces of action potential for a +200 pA current step in two experimental groups. (Right) Quantification of the number of action potentials obtained in function of injected currents showed a significant increase in action potential number in Pet1-positive 5-HT neurons of mice overexpressing 5-HT2B-HA receptor, compared to controls (n = 20 cells from three control mice and 21 cells from four 5-HT2B-HA mice; two-way ANOVA RM, Bonferroni post test, *P < 0.05; data are mean ± SEM). c Input resistance values. Bar graph and scatter plot for input resistance values showed that input resistance was also increased in mice overexpressing HA-5-HT2B receptor in Pet1-positive 5-HT neuron (unpaired ttest, *P < 0.05; data are mean ± SEM)
Fig. 2
Fig. 2
In vivo response of wild-type mice to combined 5-HT2B and 5-HT1A receptor agonists. a In vivo extracellular electrophysiological recordings of putative raphe neurons in anesthetized wild-type mice. (Left) BW723C86 (5 mg/kg subcutaneously (s.c.)) injected 20 min before test counteracted the 5-HT1A agonist 8-OHDPAT (0.05 mg/kg s.c.) inhibitory cumulative effects to putative 5-HT neuron firing (n = 6–5 mice per group; two-way ANOVA RM, followed by Bonferroni’s multiple comparisons test, *P < 0.05; data are mean ± SEM). (Right) Examples of typical recordings of putative DRN 5-HT neurons obtained in each experimental group. Each arrow represents an injection of 8-OHDPAT (0.05 mg/kg s.c.). The injection of the 5-HT1A receptor antagonist WAY100635 (0.3 mg/kg s.c.) completely reversed the inhibitory effect of 8-OHDPAT. b In vivo hypothermic effects. The 5-HT2B-receptor agonist BW723C86 (5 mg/kg s.c.) injected 20 min before the test was able to counteract 5-HT1A agonist 8-OHDPAT (0.3 mg/kg s.c.) hypothermic effects on wild-type mice (scattered plot, n = 4–4 mice two-way ANOVA RM, followed by Bonferroni’s multiple comparisons test, *P < 0.05)
Fig. 3
Fig. 3
Behavioral response to the 5-HT releaser MDMA in Htr2b5-HTKO mice. a MDMA-induced locomotion. Mice were injected with MDMA (20 mg/kg i.p.) (arrow) after 30 min habituation. A lack of MDMA-induced locomotion was observed in Htr2b5-HTKO mice, while control Htr2blox/lox mice showed a clear increase in locomotion. Data between −30 to +60 min were analyzed using two-way ANOVA RM (means ± SEM, n = 16 mice per group) and a Bonferroni post test was applied on each graph, *P < 0.05. b Cumulative MDMA-induced locomotion. Cumulative locomotion during the first hour following MDMA injection showed a significant difference between the two genotypes. Data were analyzed using two-way ANOVA (n = 16–16 mice, scattered plot, mean ± SEM). A Bonferroni post test was also applied on each graph (*P < 0.05 Htr2b5-HTKO vs. Htr2blox/lox; #P < 0.05 MDMA vs. Veh). c Locomotor sensitization by two MDMA injection protocol. The stimulant effect of a challenge dose of MDMA (20 mg/kg i.p.) 7 days after the first (2nd) was significantly enhanced compared to the first injection in control Htr2blox/lox mice, while it had no effect in Htr2b5-HTKO mice. Data were analyzed using two-way ANOVA RM (n = 8–8 mice, scattered plot, mean ± SEM). Bonferroni post test was also applied on each graph (*P < 0.05 Htr2b5-HTKO vs. Htr2blox/lox; #P < 0.05 1st vs. 2nd injection)
Fig. 4
Fig. 4
Antidepressant action in Htr2b5-HTKO mice. a Forced swimming test (FST). The time spent immobile in the FST was significantly reduced in control Htr2blox/lox mine but not in Htr2b5-HTKO mice 30 min after SSRI antidepressant fluoxetine (Flx 3 mg/kg i.p.) injection (two-way ANOVA, Bonferroni post tests, *P < 0.05; n = 7–7 mice, scattered plots with mean ± SEM). b Neurogenesis in subgranular zone (SGZ) of the hippocampus. Fluoxetine (3 mg/kg/day i.p.), daily injected for 4 weeks, induced a significant increase in BrdU incorporation in neuron of the SGZ of control Htr2blox/lox mice, but had no effect in Htr2b5-HTKO mice (two-way ANOVA, Bonferroni post test; *P < 0.05; n = 7–8 mice, scattered plots with mean ± SEM). c SERT expression and function. (Left) SERT expression in conditional Htr2b5-HTKO and Htr2blox/lox control mice was evaluated using heterologous competition binding assays of [3H]citalopram on synaptosome membranes prepared from whole brain. No differences in the affinity (Ki) or expression (Bmax) between Htr2b5-HTKO and Htr2blox/lox genotypes were observed. (Right) Saturation isotherms for [3H]5-HT uptake of this synaptosomal preparation were similar in conditional Htr2b5-HTKO and Htr2blox/lox control mice. Nonlinear regression analysis did not reveal differences in the Km or Vmax. Shown are representative curves of at least two independent experiments performed in duplicates. Individual values are presented
Fig. 5
Fig. 5
Hyposerotonergic phenotype of Htr2b5−HTKO mice. a In vivo extracellular recordings of putative DRN 5-HT neurons in anesthetized mice. (Left) The firing frequency of individual putative DRN 5-HT neuron was shifted from high to low firing rates in Htr2b5-HTKO mice. (Right) A shift in cumulative distribution was observed with significantly lower firing rate in Htr2b5−HTKO mice than in control Htr2blox/lox mice (n = 108 and 118 neurons, respectively; Kolmogorov–Smirnov test; *P < 0.05). b Head-twitch dose–response to DOI. Control Htr2blox/lox and Htr2b5−HTKO mice were i.p. injected with ±DOI (1, 3, and 5 mg/kg i.p.). Ten minutes later, the head-twitch response was scored for 10 min. DOI-induced head-twitch response was significantly increased in conditional Htr2b5-HTKO (*P < 0.05; multiple t test) compared to littermate control mice at 5 mg/kg of DOI (data are presented as scattered plot, n = 3–5 mice per group, and means ± SEM). c In vivo hypothermic effects of 8-OHDPAT. The hypothermic response to 8-OHDPAT (0.1 mg/kg s.c.) was significantly stronger in Htr2b5-HTKO mice compared to control Htr2blox/lox mice (n = 3–8 mice, scattered plot, two-way ANOVA RM, followed by Bonferroni’s multiple comparisons test, *P < 0.05)
See this image and copyright information in PMC

References

    1. Commons KG. Ascending serotonin neuron diversity under two umbrellas. Brain Struct Funct. 2016;221:3347–60. doi: 10.1007/s00429-015-1176-7. - DOI - PMC - PubMed
    1. Okaty BW, Freret ME, Rood BD, Brust RD, Hennessy ML, Debairos D, et al. Multi-scale molecular deconstruction of the serotonin neuron system. Neuron. 2015;88:774–91. doi: 10.1016/j.neuron.2015.10.007. - DOI - PMC - PubMed
    1. Riad M, Garcia S, Watkins KC, Jodoin N, Doucet E, Langlois X, et al. Somatodendritic localization of 5-HT1A and preterminal axonal localization of 5-HT1B serotonin receptors in adult rat brain. J Comp Neurol. 2000;417:181–94. doi: 10.1002/(SICI)1096-9861(20000207)417:2<181::AID-CNE4>3.0.CO;2-A. - DOI - PubMed
    1. Aghajanian GK, Lakoski JM. Hyperpolarization of serotonergic neurons by serotonin and LSD: studies in brain slices showing increased K+ conductance. Brain Res. 1984;305:181–5. doi: 10.1016/0006-8993(84)91137-5. - DOI - PubMed
    1. Andrade R, Huereca D, Lyons JG, Andrade EM, Mcgregor KM. 5-HT1A receptor-mediated autoinhibition and the control of serotonergic cell firing. ACS Chem Neurosci. 2015;6:1110–5. doi: 10.1021/acschemneuro.5b00034. - DOI - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources