Self-Modulation of Neocortical Pyramidal Neurons by Endocannabinoids

Self-Modulation of Neocortical Pyramidal Neurons by Endocannabinoids Silvia Marinelli 1, Simone Pacioni 1, Astrid Cannich 2, Giovanni Marsicano 2 and Alberto Bacci 1* 1 European Brain Research Institute, via del Fosso di Fiorano 64, 00143 Rome, Italy; 2 NeuroCentre Magendie U862 INSERM Université Bordeaux 2, 146 rue Lèo Saignat, 33077 Bordeaux, France SUPPLEMENTARY MATERIAL 1

Supplementary Figure 1: Proportion of cortical glutamatergic neurons expressing CB1Rs By highly sensitive fluorescent double in situ hybridization, we labeled cortical neurons expressing CB1 mrna (red staining) and neurons containing the mrna of the marker of glutamatergic neurons VGluT1 (green staining) in WT (top panels) and CB1-KO mice (lower panels). In WT, the great majority of glutamatergic neurons do contain low levels of CB1 mrna (intermingled red/green staining or overlapping yellow staining in the merged image of WT mice). However, neurons containing only one of the two mrnas are also present. Indeed, cells expressing high levels of CB1 are known to be GABAergic interneurons in cortical areas 1 and, consequently, they do not contain VGluT1 mrna (arrows). Conversely, cells containing only VGluT1 mrna are also observed (arrowheads), indicating that not all glutamatergic neurons do contain CB1 receptors. Semi quantitative cell counting show that 77.1±1.8% of cortical glutamatergic neurons contain CB1 mrna, whereas 81.4±1.8% of CB1-positive neurons are glutamatergic in nature. The virtual absence of CB1 staining in CB1- KO mice shows the specificity of the CB1 riboprobe and excludes the possibility of confounding background problems with the low expression of CB1 mrna in glutamatergic neurons. Blue: DAPI staining. 2

Supplementary Figure 2: Weak somatic CB1 expression in some cortical glutamatergic neurons a-b: Double immunofluorescence confocal micrographs of a layer 2/3 neuron from a P19 rat, reacted with antibodies against CB1R (a) and SMI-32 (b), which preferentially stains glutamatergic neurons. c: Merged image of a-b showing coexpression of the two proteins by the same neuron. Immunostaining was performed using pepsin, which was shown to reveal hidden proteins (see Methods) 1, 2. In addition to the usual strong axonal and presumably presynaptic CB1 immunoreactivity, some SMI-32-positive cortical neurons showed a dim cytoplasmic staining, suggesting somatic CB1expression in glutamatergic neurons. d-f: Same as in a-c, but in a different rat neocortical pyramidal neuron immuno-negative for CB1R in its somatodendritic compartment, indicating that the weak immunoreactivity in a-c was not due to background CB1 staining. g-i: Same as in a-f, in a WT P30 mouse showing a similar staining pattern of a-c. j-l: Both the strong axonal and weak somatic CB1 staining were completely absent in age-matched CB1-KO mice (CBN), indicating selective CB1 immunoreactivity. Scale bar: 5 µm. 3

Supplementary Figure 3: SSI is due to stimulation of G protein-activated inward-rectifying K + (GIRK) channels. a: Summary plot of current-to-voltage relationships (I-Vs) performed in voltageclamp (in the continuous presence of 0.5 µm TTX, 50 µm APV, 20 µm DNQX and 10 µm gabazine) from layer 2/3 PNs, which did (left) and did not (right) respond to WIN 55,212-2 (WIN, 1 µm) applications with an outward shift of the holding current. In WIN-sensitive neurons, application of the CB1 agonist (opened circles) resulted I-Vs with a different slope than in pre-drug conditions (filled circles). The two I-V slopes intersected at -90 mv, near the calculated reversal potential for K +, suggesting activation of K + channels. WIN-insensitive PNs had similar I-Vs before and after WIN application. Black and gray lines are polynomial fits of I-Vs in control and after WIN applications respectively. *: p<0.05; **: p<0.001, paired t-test, n = 5 (left) and 9 (right). b: Current-clamp recording of a PN before, during and after local application of the GIRK channel blocker BaCl 2 (100 µm; gray bar). Spike trains (truncated) failed to induce SSI in the presence of BaCl 2, but membrane potential hyperpolarized when the GIRK channel 4

antagonist was washed out, indicating that this particular cell was a SSI+ PN. Inset shows membrane potential responses of the same neuron to negative current injections (-30 pa) before BaCl 2 application and before spike trains (left, gray trace), in the presence of BaCl 2 but before spike trains (left, black trace), in the presence of BaCl 2 and after spike trains (right, black trace), and after BaCl 2 washout (right, gray trace). Traces are average of 5 responses each. Numbers refer to Rm test traces during the course of the experiment shown in b. c: Rm vs. ΔVm plot of all PNs tested in the presence of BaCl 2 after SSI-inducing stimuli. Note that the GIRK channel blocker prevented SSI in all tested cells. d: Same plot of as in c but with values taken during BaCl 2 washout, indicating that SSI could be recovered in some neurons (black circles). 5

Supplementary Figure 4: SSI is associated to CB1-dependent short- and long-term changes of inhibitory synaptic transmission. a: Representative voltage-clamp traces of monosynaptic inhibitory postsynaptic currents (IPSCs) extracellularly evoked in the continuous presence of ionotropic glutamate receptor blockers APV (50 µm) and DNQX (20 µm) from a layer 2/3 PN. Ten intracellular depolarizations to 0 mv (5 sec; delivered every 27 seconds) resulted in strong reduction of IPSC amplitudes. IPSC depression lasted for several minutes following the depolarization steps and was terminated by late application of CB1 antagonist AM-251. Traces are averages of 30 sweeps evoked every 3 seconds at the time points indicated. b: Average time-course of relative IPSC changes (upper graph) in 9 PNs that showed SSI, evidenced as a positive shift of the holding current (lower graph, see also Fig 1 j-m). Gray areas indicate the time of voltage step applications, inducing long-term depression of inhibitory responses (d-ltdi). c: Immediately after each voltage step to 0 mv, it was possible to measure another known cortical endocannabinoid-mediated signaling: depolarization-induced suppression of inhibition (DSI) 3, 4. DSI was present in a higher percentage of SSI+ than SSI- pyramidal neurons (89% and 35%, n = 9 and 31, respectively). Interestingly, d-ltdi was expressed by all SSI+ PNs (100%; n = 9), but by a smaller fraction of SSI- PNs (25%; n = 31). 6

Supplementary Table 1. Summary of SSI parameters in layer 2/3 PNs SSI+ SSI- Incidence (n = 163) 30% 69% Control ΔVm (mv) -5.6 + 0.4 (**) 0.75+0.28 (ns) %Rm 79.4+2.5(**) 105.6+0.7(ns) Incidence (n = 46) 9% 91% AM-251 ΔVm (mv) -3.93+0.6(*) -0.5+0.4(ns) %Rm 96.4+1.31(*) 102.9+0.8(*) Incidence (n = 43) 5% 95% BAPTA ΔVm (mv) -2.7+0.2-0.25+0.4(ns) %Rm 95.48+0.34 99.9 +1.66(ns) Incidence (n = 48) 2% 98% THL ΔVm (mv) -2.82 1.42+0.37 (ns) %Rm 89.5 111.28+2(*) Incidence (n = 31) 2% 98% Ba 2+ ΔVm (mv) -2.1+0.7 0.6+0.2(*) %Rm 94 109.2+2.4(*) Incidence (n = 20) 35% 65% WIN 55,212 ΔVm (mv) -6.3 + 0.9(**) 1.19+0.8(ns) %Rm 89.39+3.1(*) 99.97+2(ns) Incidence (n = 19) 31% 69% 2-AG ΔVm (mv) -4.1 +0.4(**) 0.99+0.8(ns) %Rm 92.5+1.24(*) 104.4+1.2(ns) Incidence (n = 87) 26% 78% WT mice ΔVm (mv) -4.75 + 0.4(**) 0.34+0.39(ns) %Rm 78+0.8(**) 124+1.1(**) a Incidence (n = 67) 8.9% 91.8% CB1 -/- mice ΔVm (mv) -4.03+0.9(*) 0.2+0.3(ns) %Rm 70+0.87(**) 119+1.3(**) a *: p<0.05 paired t-test; **: p<0.01, paired t-test; n.s.: non significantly different. a Both in WT and in CB1 -/- SSI- PNs, Rm significantly increased after spike trains. 7

Supplementary Table 2. Firing properties of SSI+ and SSI- PNs SSI+ SSI- Spike frequency adaptation a 0.75 + 0.03 (n = 12) 0.81 + 0.04 (n = 12) AHP b 20.4 + 2.4 mv (n = 20) 20.5 + 1.3 mv (n = 19) Action potential half-width 0.68 + 0.25 ms (n = 20) 0.8 + 0.19 ms (n = 19) a Spike frequency adaptation was calculated as the ratio of the instantaneous frequency at the 1st and the last spike intervals in an action potential train b AHP = after-hyperpolarization, calculated as the difference between the action potential threshold and the most negative level reached during the repolarizing phase All values are non significantly different (p>0.05; independent t-test). 8

SUPPLEMENTARY METHODS In vitro slice preparation and electrophysiology Sprague Dawley rats, wild type and CB1 -/- mice (C57BL6 strain) aged postnatal day (P) 13-21 (rats) and P18-30 (mice) were deeply anesthetized with isofluorane inhalation, decapitated, and brains removed and immersed in cold cutting solution (4 C) containing (in mm): 234 sucrose, 11 glucose, 24 NaHCO 3, 2.5 KCl, 1.25 NaH 2 PO 4, 10 MgSO 4 and 0.5 CaCl 2, gassed with 95% O 2 / 5% CO 2. Coronal slices (300 µm) were cut from somatosensory cortex (parietal area 1) with a vibratome (Leica) and then incubated in oxygenated artificial cerebrospinal fluid (ACSF) containing (in mm): 126 NaCl, 26 NaHCO 3, 2.5 KCl, 1.25 NaH 2 PO 4, 2 MgSO 4, 2 CaCl 2 and 10 glucose; ph 7.4, initially at 32 C for one hour, and subsequently at room temperature, before being transferred to the recording chamber and maintained at 34 C. Recordings were obtained from visually-identified pyramidal neurons in layer 2/3, easily distinguished by the presence of an emerging apical dendrite. Some pyramidal neurons were filled with biocytin (5 mg/ml) for subsequent anatomical reconstruction and analysis (see below). Experiments were performed in the whole-cell configuration of the patch-clamp technique. Electrodes (tip resistance = 2-3 MΩ) were filled with an intracellular solution containing (in mm): Kgluconate 70, KCl 70, NaCl 2, HEPES 10, EGTA 10, MgCl 2, Mg-ATP 4 and Na-GTP; ph adjusted to 7.3 with KOH; 290 mosm. AM-251 was added in the bath perfusion. CB1R agonists and BaCl 2 were delivered using a local perfusion system composed of multiple fine tubes ending in a common outlet tube, positioned in proximity (~250 µm) to the recorded neuron. In the majority of the experiments, the ionotropic glutamate receptor blockers 6,7-dinitroquinoxaline-2,3,dione (DNQX, 10 µm) and DL-2-amino-5- posphonovaleric acid (DL-APV, 100 µm), and the GABA A receptor blocker gabazine (10 µm) were included in the bath and local perfusate. For voltageclamp experiments, ionotropic glutamate and GABA receptor antagonists, as well the Na + channel blocker tetrodotoxin (TTX; 0.5 µm), were always present. 9

Tetrahydrolipstatin (THL) was from Sigma-Aldrich (Milano, Italy), AM-251, DHPG, TTX, DNQX, gabazine, and DL-AP5 were from Tocris Bioscience (Bristol, UK). Signals were amplified, using a Multiclamp 700B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA), sampled at 20 KHz and filtered at 10 KHz, unless otherwise noted. A Digidata 1320 digitizer and PClamp9 (Molecular Devices, Sunnyvale, CA) were used for data acquisition and analysis. Membrane resistance was measured from responses to small current injections (-15-30 pa, 250 ms, 0.2 Hz). SSI-inducing stimuli consisted of 10 trains of either 10 or 50 Hz APs evoked every 20 sec. Trains were evoked by brief (1 ms) suprathreshold current injections delivered at fixed intervals in order to produce spike trains at 10 or 50 Hz. Each train had 60 APs, regardless of whether it was induced at 10 or 50 Hz. Experiments on evoked GABAergic synaptic transmission were performed in voltage-clamp in the continuous presence of DL-APV (100 µm) and DNQX (10 µm). Intracellular chloride concentration was imposed by the pipette intracellular solution, which yielded a calculated reversal potential for chloride of ~-16 mv. Layer 2/3 PNs were held at -70 mv, thus GABA-mediated events could be detected as inward currents. Evoked inhibitory postsynaptic currents (IPSCs) were evoked by a monopolar patch pipette filled with ACSF and positioned at a distance of 100-200 µm from the soma of each recorded neuron. Threshold responses were identified as failing with a rate of ~50%. Stimulus intensity or duration was therefore increased in order to have relatively stable suprathreshold IPSC amplitudes. IPSCs were evoked every 3 seconds. Depolarization-induced suppression of inhibition (DSI) was induced with voltage steps to 0 mv (5 sec). SSI was induced with 10 of such stimuli with an inter-step interval of 27 seconds to allow partial DSI recovery. Series resistance was monitored throughout the experiment and recordings were discarded if Rs changed 25% of its initial value. Statistics Results are presented as means + SEM. Unless otherwise noted, paired Student s t-test was used to compare control data with those obtained in the 10

same neurons after drug applications or 5-8 minutes following SSI-inducing stimuli. To assess whether different experimental manipulations changed the occurrence of SSI+ PNs, we used the χ 2 test to compare data originating from different neuronal populations. Differences were considered significant if p<0.05. Histology Biocytin (5 mg/ml, Sigma) was included in the internal solution to fill neurons during electrophysiological recordings. Slices were subsequently fixed overnight in 4% paraformaldehyde in phosphate buffer (PB, ph 7.4) at 4ºC, and were not re-sectioned to prevent loss of sample and thus allow for a more complete reconstruction. Fixed slices were rinsed in phosphate buffer solution (PBS) for 10 min and quenched in 1% H2O2 (in 10% MetOH) for 5 min. After rinsing two times in PBS, slices were permeabilized in 2% Triton X-100 in PBS for 1 hr at room temperature, and incubated in ABC reagent (Avidin and Biotinylated horseradish peroxidase [HRP] Complex; Vectastain) for 2 hr at room temperature. Slices were rinsed four times in PBS (2x 10 min; 1x 15 min; 1x 1hr) and reacted with 3,3'-diaminobenzidine (DAB; Vectastain). After 2 rinses in PBS (10 min), slices were whole-mounted and coverslipped with 85% glycerol. Neurons were reconstructed using the Neurolucida software (MicroBrightField Inc., Colchester VT, USA) using a 100 X objective. During electrophysiological recordings, both axons and dendrites were successfully filled by biocytin. However, axonal arborization was often incomplete, as it was cut during slice preparation. For this reason, filled axons were not included in the morphological analysis. Dendritic length and Scholl analysis used NeuroExplorer software (MicroBrightField Inc., Colchester VT, USA). Scholl analysis was performed to quantify dendritic branching by counting the number of intersections of both apical and basal dendrites with virtual concentric circles of increasing (10 µm) radii, centered on the neuronal cell bodies. 11

In situ hybridization Probes and tissues were prepared as described using DIG-labeled riboprobes against mouse CB1 receptor 5, 6 and FITC-labeled riboprobes against mouse VGluT1 7. For signal amplification we used the TSATM Plus System Cyanine 3/Fluorescein (Perkin Elmer). Blocking buffer TNB and wash buffer TNT were prepared according to manufacturer protocol. Slides were analyzed by epifluorescence microscopy at 40X (Leica). Quantitative co-expression data were obtained using the program ImageJ (http://rsbweb.nih.gov/ij/), by separately counting VGluT1 (green), CB1 (red) and co-expressing neurons. A total of 3433 neurons were counted in 12 images acquired at a 20X magnification in the layer 2-3 of the motor cortex of two mice. Immunohistochemistry Sprague Dawley rats, wild type and CB1 -/- mice (C57BL6 strain) aged postnatal day (P) 19-20 (rats) and P29-30 (mice) were deeply anesthetized with chloral hydrate and transcardially perfused with a saline solution (0.9% NaCl), followed by 4% paraformaldehyde in PB. Brains were removed, immerse in 4% paraformaldehyde in PB overnight and cryoprotected in 30% sucrose. Coronal brain slices (60 µm thick), including the sensorimotor area of the neocortex, were cut using a cryostat (Leica). Slices were rinsed three times at room temperature (10 min each), and subsequently warmed at 37 C for 5-10 min in PB. Slices were then incubated for 10 minutes at 37 C in pepsin (DAKO, Milano, Italy; 1 mg/ml in 0.2 N HCl). Slices were rinsed three times at room temperature (10 min each) in PB and incubated for 36 hrs at 4 C in PB with 0.3% Triton X-1000, 0.1% normal donkey serum (NDS) and with primary guinea pig anti-cb1 (1:300, a kind gift of Dr M. Watanabe, Hokkaido University School of Medicine, Sapporo, Japan) and mouse anti-smi-32 (Sternberger Monoclonals, 1:1000) antibodies. Slices were then rinsed three times in PB (10 min each) at room temperature and incubated with Cy-3-conjugated donkey anti-guinea pig (1:100; Jackson IR) and Cy-2- conjugated donkey anti-mouse secondary antibodies (1:100; Jackson IR) for 3.5 hrs at room temperature. Slices were then rinsed three times in PB (10 min each) 12

at room temperature and coverslipped in mounting medium. Immunofluorescence was then observed with a laser confocal microscope (Leica SP5) and images were acquired. References 1. Lorincz,A. & Nusser,Z. Specificity of immunoreactions: the importance of testing specificity in each method. J. Neurosci. 28, 9083-9086 (2008). 2. Watanabe,M. et al. Selective scarcity of NMDA receptor channel subunits in the stratum lucidum (mossy fibre-recipient layer) of the mouse hippocampal CA3 subfield. Eur. J. Neurosci. 10, 478-487 (1998). 3. Kano,M., Ohno-Shosaku,T., Hashimotodani,Y., Uchigashima,M., & Watanabe,M. Endocannabinoid-mediated control of synaptic transmission. Physiol Rev. 89, 309-380 (2009). 4. Freund,T.F., Katona,I., & Piomelli,D. Role of endogenous cannabinoids in synaptic signaling. Physiol Rev. 83, 1017-1066 (2003). 5. Marsicano,G. & Lutz,B. Expression of the cannabinoid receptor CB1 in distinct neuronal subpopulations in the adult mouse forebrain. Eur. J. Neurosci. 11, 4213-4225 (1999). 6. Marsicano,G. et al. CB1 cannabinoid receptors and on-demand defense against excitotoxicity. Science 302, 84-88 (2003). 7. Monory,K. et al. The endocannabinoid system controls key epileptogenic circuits in the hippocampus. Neuron 51, 455-466 (2006). 13