Abstract The inhibition of sensory responsivity is considered a core serotonin function, yet this hypothesis lacks direct support due to methodological obstacles. We adapted an optogenetic approach to induce acute, robust and specific firing of dorsal raphe serotonergic neurons. In vitro, the responsiveness of individual dorsal raphe serotonergic neurons to trains of light pulses varied with frequency and intensity as well as between cells, and the photostimulation protocol was therefore adjusted to maximize their overall output rate. In vivo, the photoactivation of dorsal raphe serotonergic neurons gave rise to a prominent light-evoked field response that displayed some sensitivity to a 5-HT1A agonist, consistent with autoreceptor inhibition of raphe neurons. In behaving mice, the photostimulation of dorsal raphe serotonergic neurons produced a rapid and reversible decrease in the animals' responses to plantar stimulation, providing a new level of evidence that serotonin gates sensory-driven responses.

Citation: Dugué GP, Lörincz ML, Lottem E, Audero E, Matias S, Correia PA, et al. (2014) Optogenetic Recruitment of Dorsal Raphe Serotonergic Neurons Acutely Decreases Mechanosensory Responsivity in Behaving Mice. PLoS ONE 9(8): e105941. https://doi.org/10.1371/journal.pone.0105941 Editor: Maurice J. Chacron, McGill University, Canada Received: March 17, 2014; Accepted: May 29, 2014; Published: August 22, 2014 Copyright: © 2014 Dugué et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The data are available at: http://doi.org/10.5061/dryad.nh437. Funding: This work was supported by an European Research Council grant to ZM (N ° 250334), an Intra-European Marie Curie postdoctoral fellowship to GD (N ° 220098), a Human Frontier Science Programme postdoctoral fellowship to MLL (N ° LT001009/2010L), a Human Frontier Science Programme postdoctoral fellowship to EL (N ° LT000881/2011L) and an Agence Nationale pour la Recherche grant to CL (Sensocode 11-BSV4-028). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction The influences of central serotonin (5-hydroxytryptamine or 5-HT) impact a wide range of brain functions, from the control of autonomic responses [1], [2] to the regulation of complex emotional behaviors [3], [4]. These diverse influences may be systematized by considering possible core neurophysiological functions. Among these, serotonergic neuromodulation has long been implicated in the inhibition of sensory responsivity [5], [6], an idea chiefly supported by gain-of-function experiments. Pharmacological enhancement of 5-HT function inhibits primary afferent neurotransmission in vitro [7], [8], dampens sensory and nociceptively-evoked firing in vivo [9]-[11], [12] and decreases acoustic startle responses and their pre-pulse inhibition [6], [13]. Similarly, electrical microstimulation of the dorsal raphe nucleus (DRN), one of the largest sources of ascending 5-HT projections [14], reduces forebrain sensory and nociceptively-evoked activity [9], [11], [12], [15]–[17] and elevates vocalization thresholds to noxious stimuli [18]. Despite these observations, technical limitations have impeded a deeper understanding of the underlying mechanisms. Pharmacological upregulation of 5-HT pathways neither mimics phasic 5-HT release nor takes into account the effect of co-released substances [19], [20], and may exhibit paradoxical effects due to autoreceptor-mediated negative feedback [21] and drug-induced plastic changes [22]. Electrical stimulation, while spatially and temporally precise, can produce non-specific effects by activating non-5-HT neurons and fibers-of-passage [18]. To overcome these technical limitations, we optimized and validated a direct and specific optogenetic stimulation of DRN serotonergic neurons in mice. We then employed this strategy to test whether transient and specific activation of DRN 5-HT neurons in behaving mice can indeed interfere with sensory responsivity in a simple test of mechanosensitivity.

Materials and Methods All procedures were performed in accordance with the Champalimaud Foundation Ethics Committee guidelines, and approved by the Portuguese Veterinary General Board (Direcção Geral de Veterinária, approval ID 014315). Detailed methods are available in Supporting Information S1. Viral transduction of DRN neurons Adeno-associated viral vectors carrying a Cre-activated ChR2-YFP expression cassette (AV-1-20298P, University of Pennsylvania, 1013 GC/mL) were stereotaxically injected (volume: 1.0–1.2 µL) into the DRN of adult (8–16 weeks) transgenic SERT-Cre or wild-type mice. The SERT-Cre mouse line [23] was obtained from the Mutant Mouse Regional Resource Centers (stock number: 017260-UCD). For behavioral testing, an optical fiber (200 µm, 0.37 NA) housed inside a connectorized implant (M3, Doric Lenses) was positioned over the DRN after the injection. The detailed coordinates of the injection and fiber placement are provided in Supporting Information S1. Immunohistochemistry Animals were anesthetized with 5% chloral hydrate (500 mg/kg) and perfused with 4% PFA. After cryoprotection (in 30% sucrose), coronal sections (12 µm) were cut in a cryostat, mounted on glass slides and incubated in 10 mM citrate buffer (pH 6.0, 0.05% Tween) at 96°C for 10 min. After washing with TBS, sections were preincubated for 1 hour at room temperature in a blocking solution containing 5% fetal bovine serum, 2% bovine serum albumin and 0.25% Triton X-100 diluted in TBS. Section were incubated overnight at 4°C in a blocking solution containing a mouse monoclonal anti-TPH antibody (T0678, Sigma-Aldrich, 1∶400) and a rabbit polyclonal anti-GFP antibody (A6455, Life Technologies, 1∶400). Sections were then incubated for 1 h at room temperature in a blocking solution containing an Alexa Fluor 488 goat anti-rabbit antibody and an Alexa Fluor 594 goat anti-mouse antibody (Life Technologies, diluted 1∶1000). ChR2-YFP- and TPH-positive cell bodies were counted on projections of 20 confocal images taken every 0.5 µm. In vitro recordings and photostimulation of DRN neurons DRN slices were prepared from SERT-Cre mice aged 8–20 weeks (2–4 weeks post-infection). DRN cells were recorded in the loose cell-attached (n = 4 non-fluorescent and 28 fluorescent cells) and whole-cell configurations (n = 3 non-fluorescent and 6 fluorescent cells). Cells were illuminated using a 465 nm LED coupled to a 200 µm 0.37 NA optical fiber (Doric Lenses) held at 34° at a fixed distance above the slice. For each cell, the incident irradiance was calculated by dividing the power measured at the fiber tip by the area of the illuminated zone. Blue light propagation in the DRN The spread of blue light in the DRN was assessed in freshly dissected brains hemisected in the sagittal plane. The blocks of tissue were immersed in PBS and the plane of cut was imaged from the top. Blue light was delivered through an LED-coupled optical fiber positioned next to the dorsal aspect of the DRN. The detailed procedure is available in Supporting Information S1. Fluorescence mapping of ChR2-EYFP-expressing DRN neurons and recordings of local photoevoked activity in vivo Custom optrodes were assembled by gluing a 200 µm 0.37 NA multimode optical fiber (BFL37-200, Thorlabs) onto a platinum microelectrode (0.8–1.0 MΩ at 1 kHz, FHC). Light pulses were generated using a laser beam (473 nm, 100 mW DPSS laser, Laserglow) gated by a mechanical shutter (VS14S2ZM1, Uniblitz) and attenuated by a set of neutral density filters. The tissue fluorescence collected by the optical fiber was monitored through a dichroic mirror using a CCD camera. Von Frey test 17 SERT-Cre mice and 17 wild type littermates (control group) were infected with the Cre-dependent viral vector and implanted with connectorized optical fibers. After recovery (one week), animals were allowed to habituate to the testing box (a 9×7×14 cm Plexiglas box placed on an elevated metal mesh platform) 5 minutes per day for 5 days, during which the 4.0 g filament was applied 5 times per hind paw. In the testing phase, filaments ranging 0.4–8 g were applied consecutively in an ascending fashion (each filament was applied 5 times successively to the right and left hind paws). This procedure was repeated 3 times per session: before (“baseline”), in conjunction with (“stim”) and after (“recovery”) photostimulation. The “stim” and “recovery” blocks were separated by a 5 minutes delay. In the “stim” condition, filament application was restricted to the last three quarters of a 12 s photostimulation train (10 ms, 20 Hz, 318 mW.mm−2 at the fiber tip). The 3 s delay between photostimulation onset and first filament application are meant to allow the establishment of a potentially slow serotonergic neuromodulatory mechanism.

Discussion The initial optogenetic approaches used to study DRN functions employed non-specific promoters [28], [29] or targeted local or distal neurons presynaptic to 5-HT neurons [28], [30]. Recently, specific optogenetic stimulation of DRN 5-HT neurons has been achieved using transgenic mouse lines [31], [32] or viral injections [33] but these studies have not provided a detailed account of optimal photostimulation parameters. Here we devoted substantial efforts to optimizing the yield of direct and specific photostimulation of DRN 5-HT neurons. ChR2-EYFP-expressing cells were sensitive to low irradiance in vitro (<1 mW·mm−2, Fig. 1H–I). However high irradiances (>100 mW·mm−2 at fiber tip) were necessary to saturate the photoevoked local field potential in vivo, a measure that might prove useful for optimally positioning optical fibers and assessing levels of ChR2-YFP expression in target structures. Using irradiance of >250 mW·mm−2 at the fiber tip, we estimated that the entire DRN received irradiances over 5–6 mW·mm−2, a value at which the output of ChR2-EYFP neurons could be maximized (up to 16 Hz) in vitro by using 20 Hz stimulations, despite their strong frequency-dependent adaptation. These parameters are appropriate to attempt to mimic episodes of increased DRN activity, which occur in association with a variety of behavioral conditions such as oro-buccal movements [24] and defensive encounters [26], and in relation to reward outcome [34] and waiting for delayed rewards [25]. Such episodes typically last several seconds, during which the activity of DRN neurons can peak up to 10–20 Hz. Our protocol for DRN 5-HT neuron photostimulation in behaving mice (20 Hz for 12 seconds) evoked robust decreases in behavioral responses to hind paw stimulation. Previous observations have shown that chronic elevation of 5-HT levels can increase response thresholds in rodent models of mechanical allodynia [35]–[37]. Our result extends this observation by showing that a similar effect can be reversibly induced on a faster timescale in non-pathological conditions by recruiting DRN 5-HT neurons. Whether the threshold calculated using von Frey filaments in naïve animals is a measure of sensory or nociceptive sensitivity is still a debated question [27]. Therefore the question whether the stimulation of DRN 5-HT neurons in our conditions primarily acts upon sensory or nociceptive pathways remains open. Nevertheless, our result helps to resolve the ambiguity of previous gain-of-function experiments testing the influence of DRN output by directly showing that DRN 5-HT neurons can indeed tone down the influence of sensory and/or nociceptive inputs, as opposed to what has been recently observed in zebrafish [38]. Given the projection pattern of DRN 5-HT cells [14], this effect is likely to be mediated by the modulation of anterior structures, as suggested by evidence highlighting a role for 5-HT in the modulation of thalamic [11], [12], [16], [39] and cortical [5], [10] sensory and nociceptive responses. It is not unlikely that other co-released substances may play a role in the observed effect. In particular, a recent study has shown that the glutamatergic phenotype of certain 5-HT neurons seems to be partly responsible for the effects produced by the photostimulation of DRN 5-HT neurons on reward-related behaviors [33]. More refined targeting strategies, e.g. retrograde infection [40] or intersectional genetics [41], will allow the assessment of contributions of specific sub-populations of DRN 5-HT neurons [14]. Overall these results provide a new level of evidence for the involvement of DRN 5-HT neurons in gating the access of sensory inputs to behavioral output, a key physiological role which will help constrain larger-scale theories of 5-HT function.

Acknowledgments We thank Michel Barrot and Ipek Yalcin for their help in designing the behavioral experiment and Matthieu Pasquet for his technical assistance.

Author Contributions Conceived and designed the experiments: GD CL ZM. Performed the experiments: GD ML EL EA SM PC. Analyzed the data: GD CL ZM. Contributed to the writing of the manuscript: GD CL ZM.