Transcranial direct current stimulation of the mouse prefrontal cortex modulates serotonergic neural activity of the dorsal raphe nucleus

Transcranial direct current stimulation (tDCS) is being increasingly considered as a non-pharmacological alternative for various psychiatric and neurological disorders. In major depressive disorder (MDD), anodal tDCS applied over the left dorsolateral prefrontal cortex (DL-PFC) improves symptoms in drug-resistant patients [[1]Lefaucheur J.P. Antal A. Ayache S.S. Benninger D.H. Brunelin J. Cogiamanian F. et al.Evidence-based guidelines on the therapeutic use of transcranial direct current stimulation (tDCS).Clin Neurophysiol. 2017 Jan; 128: 56-92Crossref PubMed Scopus (868) Google Scholar]. However, the molecular mechanisms underlying the efficacy of tDCS in MDD are still unknown. One of the main neurotransmitters involved in the pathophysiology and psychopharmacology of MDD is serotonin (5-HT), released by neurons of the dorsal raphe nucleus (DRN), which have widespread projections to cortical, subcortical and brainstem regions. In rodents, the prefrontal cortex directly innervates the DRN [[2]Pollak Dorocic I. Fürth D. Xuan Y. Johansson Y. Pozzi L. Silberberg G. et al.A whole-brain atlas of inputs to serotonergic neurons of the dorsal and median raphe nuclei.Neuron. 2014 Aug 6; 83: 663-678Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar], and the activation of this pathway by deep brain stimulation (DBS) was shown to have antidepressant activity [[3]Srejic L.R. Hamani C. Hutchison W.D. High-frequency stimulation of the medial prefrontal cortex decreases cellular firing in the dorsal raphe.Eur J Neurosci. 2015 May; 41: 1219-1226Crossref PubMed Scopus (26) Google Scholar]. We investigated whether tDCS over the PFC in mice affects the activity of DRN 5-HT neurons using single-unit in-vivo electrophysiology [[4]Comai S. Ochoa-Sanchez R. Dominguez-Lopez S. Bambico F.R. Gobbi G. Melancholic-Like behaviors and circadian neurobiological abnormalities in melatonin MT1 receptor knockout mice.Int J Neuropsychopharmacol. 2015 Jan 31; 18Crossref PubMed Scopus (51) Google Scholar] and analysis of cFos levels (as a measure of cell activation) in the prelimbic cortex (PrL) and the dorsal portion of the DRN (DRD) of adult (2–3 months) anesthetized male C57BL/6 mice. For in-vivo electrophysiology, once a spontaneous and stable DRN 5-HT neuron was identified and isolated [[4]Comai S. Ochoa-Sanchez R. Dominguez-Lopez S. Bambico F.R. Gobbi G. Melancholic-Like behaviors and circadian neurobiological abnormalities in melatonin MT1 receptor knockout mice.Int J Neuropsychopharmacol. 2015 Jan 31; 18Crossref PubMed Scopus (51) Google Scholar], we recorded the activity for 4-min at rest, for 10 min during 250 μA anodal or cathodal left PFC tDCS and for further 10min after stimulation (1 neuron/mouse; 8 neurons for both anodal and cathodal stimulations). tDCS was applied through a plastic tube filled with saline solution just prior to stimulation. The centre of the active electrode (0.045 cm2) was positioned over the left frontal areas (1.5 mm lateral and 1.8 mm anterior to bregma). The counter electrode was a saline soaked sponge (5.2 cm2) adhering to the ventral thorax [[5]Cambiaghi M. Velikova S. Gonzalez-Rosa J.J. Cursi M. Comi G. Leocani L. Brain transcranial direct current stimulation modulates motor excitability in mice.Eur J Neurosci. 2010 Feb; 31: 704-709Crossref PubMed Scopus (76) Google Scholar]. Mean firing activity during and after stimulation was measured and calculated as percentage of baseline. For cell activation analysis [[6]Crupi R. Cambiaghi M. Spatz L. Hen R. Thorn M. Friedman E. et al.Reduced adult neurogenesis and altered emotional behaviors in autoimmune-prone B-cell activating factor transgenic mice.Biol Psychiatry. 2010 Mar 15; 67: 558-566Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar], cFos expression was quantified in 15 mice (5/group: sham, anodal, cathodal). Briefly, 90 minutes after tDCS anesthetized mice were transcardially perfused with PBS followed by PBS-4% paraformaldehyde. Frozen brains were cut into 35-μm sections and incubated in blocking solution for 1h at RT, followed by overnight incubation in the blocking solution containing anti c-Fos rabbit antibodies (Santa Cruz Biotechnology, USA; 1:500) at 4 °C. After washing, the sections were incubated with Alexa Fluor 555-conjugated anti-rabbit IgG antibodies (Thermo Fisher Scientific, USA; 1:1000) at RT for 2 hours. Images were acquired through a 40x objective (5 sections/animal) and c-Fos levels were quantified as described [[6]Crupi R. Cambiaghi M. Spatz L. Hen R. Thorn M. Friedman E. et al.Reduced adult neurogenesis and altered emotional behaviors in autoimmune-prone B-cell activating factor transgenic mice.Biol Psychiatry. 2010 Mar 15; 67: 558-566Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar] within the sections of interest (PFC: +1.54 to +1.98; DRD: 4.36 to −4.84 from bregma). Briefly, c-Fos+ cells were counted based on shape and fluorescence intensity (threshold settled with ImageJ software), blinded to experimental conditions All procedures were conducted in accordance with the Italian guidelines of the Ministry of Health. We found that both anodal and cathodal tDCS acutely decreased DRN 5-HT activity (stimulus: F(2,28) = 12.99, P = 0.0004; current: F(1,28) = 8.74, P = 0.01; interaction: F(2,28) = 0.97, P = 0.50) compared with baseline. DRN 5-HT firing rate remained low even after the stimulus ceased (Fig. 1A–C). Interestingly, Bonferroni post-hoc analysis showed that the acute inhibitory effect of cathodal tDCS was greater than that of the anodic stimulation during both the 10-min stimulus (P = 0.010) and the 10-min post-stimulus (P = 0.023). Although it was not possible to perform unbiased stereology, quantitative analysis of the immediate-early gene product cFos (Fig. 1D–E) suggested an increased number of cFos+ cells in PrL after tDCS in both polarities with respect to the sham group (Fig. 1D; F(2,12) = 31.25, P < 0.001) and in DRD (Fig. 1E; F(2,12) = 12.77, P = 0.001). Notably, in the PrL cortex, cathodal tDCS resulted in higher cFos+ cell activation compared with anodal tDCS (P = 0.023). This study is based on the hypothesis that the modulation of the top–down projections from the PFC to the DRN could be a key mechanism in the antidepressant effects of non-pharmacological treatments. In keeping, we found that acute left PFC-tDCS inhibited DRN 5-HT activity. A similar effect was observed with acute application of current antidepressants, including selective serotonin reuptake inhibitors (SSRIs) [[7]Gobbi G. Blier P. Effect of neurokinin-1 receptor antagonists on serotoninergic, noradrenergic and hippocampal neurons: comparison with antidepressant drugs.Peptides. 21 Apr 2005; 26: 1383-1393Crossref PubMed Scopus (67) Google Scholar]. However, this similarity does not imply that the effects of tDCS are mediated by inhibition of serotonin reuptake (see, 8). Indeed, Srejic et al. [[3]Srejic L.R. Hamani C. Hutchison W.D. High-frequency stimulation of the medial prefrontal cortex decreases cellular firing in the dorsal raphe.Eur J Neurosci. 2015 May; 41: 1219-1226Crossref PubMed Scopus (26) Google Scholar] showed in rats that DBS in the PFC affected the activity of DRN 5-HT neurons likely through the modulation of the local GABAergic tone, that could be the same underlying mechanism we observed with tDCS. Notably, in that work the significant decrease in the firing rate of DRN 5-HT neurons was present only during the stimulation, while we found that acute tDCS inhibitory effects outlasted the stimulation period. The intensity used here corresponds to a charge density of 33.3 kC/m2, which is higher with respect to typical human studies (33.3 vs. 0.3 kC/m2). It has been chosen to be consistent with our previous findings in mice [[5]Cambiaghi M. Velikova S. Gonzalez-Rosa J.J. Cursi M. Comi G. Leocani L. Brain transcranial direct current stimulation modulates motor excitability in mice.Eur J Neurosci. 2010 Feb; 31: 704-709Crossref PubMed Scopus (76) Google Scholar] and other studies performed in rats [[9]Chhatbar P.Y. George M.S. Kautz S.A. Feng W. Charge density, not current density, is a more comprehensive safety measure of transcranial direct current stimulation.Brain Behav Immun. 2017 Nov; 66: 414-415Crossref PubMed Scopus (8) Google Scholar]. Moreover, these parameters fall within the safety range suggested by similar stimulation condition in animal models [[9]Chhatbar P.Y. George M.S. Kautz S.A. Feng W. Charge density, not current density, is a more comprehensive safety measure of transcranial direct current stimulation.Brain Behav Immun. 2017 Nov; 66: 414-415Crossref PubMed Scopus (8) Google Scholar]. Of note, in addition to the different current density, it has to be taken into account a different electric field direction and magnitude between mice and humans, due to their vast anatomical differences. In summary, these novel findings show that 10-min anodal or cathodal tDCS over left PFC produces a significant acute inhibition of DRN 5-HT neurons, thus resembling the effects exerted by SSRIs such as fluoxetine or by PFC DBS [[3]Srejic L.R. Hamani C. Hutchison W.D. High-frequency stimulation of the medial prefrontal cortex decreases cellular firing in the dorsal raphe.Eur J Neurosci. 2015 May; 41: 1219-1226Crossref PubMed Scopus (26) Google Scholar,[7]Gobbi G. Blier P. Effect of neurokinin-1 receptor antagonists on serotoninergic, noradrenergic and hippocampal neurons: comparison with antidepressant drugs.Peptides. 21 Apr 2005; 26: 1383-1393Crossref PubMed Scopus (67) Google Scholar,[8]Bregman T. Nona C. Volle J. Diwan M. Raymond R. Fletcher P.J. et al.Deep brain stimulation induces antidepressant-like effects in serotonin transporter knockout mice.Brain Stimul. 2018 Mar - Apr; 11: 423-425Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar]. Therefore, it is conceivable that the antidepressant effects deriving from DL-PFC tDCS may occur through the direct or indirect modulation of DRN 5-HT neurons, as also suggested by human studies [[10]Brunoni A.R. Kemp A.H. Shiozawa P. Cordeiro Q. Valiengo L.C. Goulart A.C. et al.Impact of 5-HTTLPR and BDNF polymorphisms on response to sertraline versus transcranial direct current stimulation: implications for the serotonergic system.Eur Neuropsychopharmacol. 2013 Nov; 23: 1530-1540Crossref PubMed Scopus (58) Google Scholar]. Interestingly, although for the treatment of depression in humans anodic tDCS is mostly used, we have found that acute cathodal tDCS induces the activation of PrL cortex neurons and the inhibition of DRN 5-HT neurons at higher magnitude than acute anodal tDCS of the same intensity. As a final remark, it has to be considered that in the clinical setting tDCS has the great advantage of being a non-invasive and side-effect free technique compared with DBS or pharmacological therapies. MC is in part supported by a 2019 grant from the Brain Research Foundation Verona (BRFV); SC is supported in part by a 2017 NARSAD Young Investigator Grant from the Brain & Behavior Research Foundation.

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