Nature Neuroscience: doi: /nn Supplementary Figure 1

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1 Supplementary Figure 1 Distribution of GlyT2::eGFP fibers in the mouse thalamus at three different coronal levels. Note the innervation centered in the rostral (CL, PC) and caudal (PF) nuclear groups of the intralaminar nuclei (IL) and midline nuclei (CM). Less dense innervation is present in the ventromedial nucleus (VM) as well. Scalebar: 1mm. Abbreviations: CL, centrolateral thalamic nucleus; CM, central medial thalamic nucleus; cp, cerebral peduncle; DLG, dorsal lateral geniculate nucleus; fr, fasciculus retroflexus; Hb, habenula ic, internal capsule; LDDM, laterodorsal thalamic nucleus, dorsomedial part; LDVL, laterodorsal thalamic nucleus, ventrolateral part; LPLR, lateral posterior thalamic nucleus, laterorostral part; LPMR, lateral posterior thalamic nucleus, mediorostral part; MD, mediodorsal thalamic nucleus; MGP, medial globus pallidus; OPC, oval paracentral thalamic nucleus; PC, paracentral thalamic nucleus; PF, parafascicular thalamic nucleus; Po, posterior thalamic nuclear group; PV, paraventricular thalamic nucleus; Re, reuniens thalamic nucleus; st, stria terminalis; Sub, submedius thalamic nucleus; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; VPM, ventral posteromedial thalamic nucleus; Zi, zona incerta.

Supplementary Figure 2 Distribution of retrogradely labeled neurons in the brainstem following injection of fluorogold (FG) into the IL. a-b)injection site at two coronal levels.

The boxed area in E is shown enlarged in h) Yellow dots, double labeled cells, red dots FG only.

2 Supplementary Figure 2 Distribution of retrogradely labeled neurons in the brainstem following injection of fluorogold (FG) into the IL. a-b)injection site at two coronal levels. c-g) Distribution of retrogradely labeled cells in one animal. Yellow numbers, number of FGeGFP double labeled cells; Red numbers, FG-labeled cells lacking egfp. Right ipsilateral, left contralateral. The boxed area in E is shown enlarged in h) Yellow dots, double labeled cells, red dots FG only. Abbreviations: CL, centrolateral thalamic nucleus; CM, central medial thalamic nucleus; DMTg, dorsomedial tegmental area; DpMe, deep mesencephalic nucleus; fr, fasciculus retroflexus; IRt, intermediate reticular nucleus; LC, locus coeruleus; MDL, mediodorsal thalamic nucleus, lateral part; ml, medial lemniscus; MnR, median raphe nucleus; P5, peritrigeminal zone; PC, paracentral thalamic nucleus; PF, parafascicular thalamic nucleus; PMnR, paramedian raphe nucleus; PnC, pontine reticular nucleus, caudal part; PnO, pontine reticular nucleus, oral part; PPTg, pedunculo pontine tegmental nucleus; PV, paraventricular thalamic nucleus; RtTg, reticulotegmental nucleus of the pons; ts, tectospinal tract; VLL, ventral nucleus of the lateral lemniscus.

Supplementary Figure 3 Distribution of retrogradely labeled cells in the PRF following an injection of FG into the parafascicular nucleus. a) Injection site.

3 Supplementary Figure 3 Distribution of retrogradely labeled cells in the PRF following an injection of FG into the parafascicular nucleus. a) Injection site. b-c) Colocalization of FG (DAB-Ni, black reaction product) and egfp. Black arrowhead, double labeled cells; white arrow, egfp-negative, retrogradely labeled cell. d-h) Distribution of retrogradely labeled cells at five coronal levels of the PnO-PnC complex. x, double labeled cells; o, FG-positive, egfp-negative cells. Abbreviations: ATg, anterior tegmental nucleus; B9, B9 serotonin cells; ml, medial lemniscus; mlf, medial longitudinal fasciculus; MnR, median raphe nucleus; PMnR, paramedian raphe nucleus; PnO, pontine reticular nucleus, oral part; Rbd, rhabdoid nucleus; rs, rubrospinal tract; RtTg, reticulotegmental nucleus of the pons; RtTgP, reticulotegmental nucleus of the pons, pericentral part; VLL, ventral nucleus of the lateral lemniscus.

4 Supplementary Figure 4 Quantitative morphological features of GlyT2::eGFP terminals in the IL. a) 3D images of three GlyT2::eGFP terminals reconstructed from serial electron microscopic sections, displaying different size and synapse number. Green, synapses; magenta, puncta adhaerentia; dark blue, membrane of the terminal; light blue, glia. Scale bar, 0.5 μm. b) Correlation between the number of synapses an egfp bouton establishes in the IL and the volume of the bouton. c) Cumulative distribution of nearest neighbor synaptic distances in egfp IL terminals (right).

5 Supplementary Figure 5 Immunohistochemical localization of inhibitory postsynaptic receptor clusters. The apposition of GlyR and GABAR-γ2 subunit clusters with GFP-positive presynaptic varicosities was analyzed in the central lateral and parafascicular nuclei of GlyT2::eGFP transgenic mouse. a) Projection of a 1.5 µm z-stack through the central lateral nucleus showing the GFP-positive glycinergic axons and their large en-passant varicosities. Superimposed in red is the detected volume of thresholded putative varicosities. Scale bar 5 µm. b) Pan GlyR (blue) and GABAR-γ2 (red) immunoreactive clusters in the same stack. Varicosities are superimposed as yellow shadows. c) Density of detected varicosities, GlyR clusters and GABAR-γ2 clusters. Data were pooled from 4 stacks of the central lateral nucleus and 3 stacks of the parafascicular nucleus, representing a total volume of µm 3. d-e) Density of receptor clusters as a function of the distance from the edge of the GlyT2::eGFP varicosities. Both populations of receptor clusters showed an apposition peak which could be fitted by a mixture of a Gaussian (red and blue curves respectively; GABAR 150 ± 180 nm; GlyR 170 ± 135 nm) and a sigmoid curve (black, fitting the uniform distribution of receptor cluster density observed at larger distances). Error bars represent the s.e.m.

Supplementary Figure 6 Specificity of the GlyT2::Cre mouse line.

lines. We found that out of 198 Cre-positive neurons 196 (99%) were also egfp-positive (n=3 animals).

levels. Note intense bilateral labeling i) High power image of ChR2-eYFP cells in the PRF.

6 Supplementary Figure 6 Specificity of the GlyT2::Cre mouse line. a-c) Co-localization between Cre immunostaining and GlyT2::eGFP signal in the PnO following the crossing of GlyT2::Cre and GlyT2::eGFP mouse lines. We found that out of 198 Cre-positive neurons 196 (99%) were also egfp-positive (n=3 animals). d-h) Extent of viral labeling following the injection of floxed AAV-ChR2-eYFP construct into the PRF of aglyt2::cre mouse shown at five coronal levels. Note intense bilateral labeling i) High power image of ChR2-eYFP cells in the PRF. j-n) The resulting fiber labeling in the thalamus at five coronal levels. The position of the optic fiber is indicated in (j, arrows). o) High power image of the labeled ChR2- eyfppositive fibers in the IL. Boxed areas in e) and k) indicate the position of high power images. Scale bars: a-c, i, o, 10 μm; d-h, j-n, 500 μm.

7 Supplementary Figure 7 Dynamic behavior of optogenetically and electrically evoked inhibitory synaptic events in the IL a) In slices from virus-injected GlyT2::Cre mice, pairs of light stimulations were given at different ISIs, and the ratio between the amplitude of the second versus the first response (PP ratio) was calculated. The averaged traces (leipscs) for 3 distinct ISIs are also illustrated. The graph illustrates the dependence of the PP ratio on the ISI. b) In slices from GlyT2::eGFP mice, electrically induced glycinergic IPSCs (eipscs) were pharmacologically isolated with APV, NBQX and SR The glycinergic nature of the remaining eipscswas confirmed by their sensitivity to strychnine,(red trace in the upper panel) (n = 7; Wilcoxon signed rank test, p = 0.018; W(7) = 0). Averaged traces at 40 and 200Hz are shown. In the graph below, note the absence of significant short-term plasticity at all frequencies tested. Refer to Fig. 3 and to the main body of the manuscript for comparison with optogenetically-induced multiple stimulations. c) Comparison between the PP ratio of optogenetically and electrically evoked IPSCs at short ISIs. The paired pulse ratios (PPRs) of eipscs and of leipscs were similar, and close to 100%for frequencies up to 40 Hz (Mann-Whitney U-test; p = 0.10). However, the strong paired-pulse depression found for leipscs (51.49 ± 5.60%; n = 10) was absent in case of eipscs at 50 Hz ( ± %; n=14; Mann-Whitney U-test, p=0.005; U(14,10) = 135), which was the highest frequency tested with optogenetic stimulation.

8 Supplementary Figure 8 Activation of GlyT2 fibers with increasing laser power in the IL evokes graded motor inhibition. The experimental configuration depicted in a) is the same as in Fig. 4 of the main body of the manuscript. The mice were periodically stimulated using varying laser powers while freely moving in the open field arena. The effect on the distance traveled was quantified. b) The box and whisker plots represent the distribution of distances traveled (sampling rate: 33Hz) during control (10 seconds preceding light onset; yellow boxes) and test (between seconds 5 and 10 following light onset; blue boxes) periods in a representative animal. The distances are normalized to the average distance traveled in control conditions over all trials. At approximately 3 mw, 6 mw and 19 mw laser powers, the distance traveled decreased to 67.3 ± 3.3%, 26.7 ± 1.8% and 12.3 ± 1.0% of control values, respectively. Asterisks represent the level of statistical significance (p ) calculated with the Mann-Whitney test. Error bars represent the s.e.m.

Supplementary Figure 9 Distribution of cortico-prf neurons. The distribution of cells was used to establish the localization of cortical LFP electrode (Br +1.7 mm, Lat 0.8mm).

9 Supplementary Figure 9 Distribution of cortico-prf neurons. The distribution of cells was used to establish the localization of cortical LFP electrode (Br +1.7 mm, Lat 0.8mm). a) FG injection in the PnO. b) Schematic representation of the retrogradely labeled (FG positive) layer 5 neurons (green shading) in the frontal cortex at three anteroposterior levels. c) FG positive L5 pyramidal neurons in the cortex (red box in b). The experiments were performed in 4 animals. Scalebar: c, 20µm Abbreviations: aca, anterior commissure; Cg1-2, cingulate cortex area 1-2; CPu, caudate putamen; DpMe, deep mesencephalic nucleus; DR dorsal raphe nucleus; fmi, forceps minor of the corpus callosum; IL, infralimbic cortex; M1, 2, primary and secondary motor cortex; MR, median raphe nucleus; PAG, periaqueductal gray; PnO, pontine reticular nucleus, oral part; PrL, prelimbic cortex; SC superior colliculus

c-d) The autocorrelogram and spike triggered LFP averages (STA) of the neuron shown in Fig. 6 b-d. The cell displays rhythmic phase modulation.

10 Supplementary Figure 10 Activity of in vivo recorded PnO cells. a) Electrode arrangement. b) Antidromic response of a GlyT2::eGFP-positive, PnO cell stimulated from the IL thalamus (left, 20 stimulation overlaid, right response latencies of individual stimulations). c-d) The autocorrelogram and spike triggered LFP averages (STA) of the neuron shown in Fig. 6 b-d. The cell displays rhythmic phase modulation. e-i) Activity of a GlyT2::eGFP cell in vivo under ketamine-xylazine anesthesia (bottom trace) together with the cortical LFP (top trace) and filtered cortical multiunit activity (MUA, middle trace). g-h) Autocorrelogram and STA of the same unit. This GlyT2::eGFP neuron is active out of phase, i.e. during the DOWN state of the cortical slow oscillation Scalebar: e, 20µm.

Nature Neuroscience: doi: /nn Supplementary Figure 1

Nature Neuroscience: doi: /nn Supplementary Figure 1 Supplementary Figure 1 Atlas representations of the midcingulate (MCC) region targeted in this study compared against the anterior cingulate (ACC) region commonly reported. Coronal sections are shown on