Brain Struct Funct DOI 10.1007/s00429-014-0908-4

ORIGINAL ARTICLE

Cannabinoid CB1 receptor signaling dichotomously modulates inhibitory and excitatory synaptic transmission in rat inner retina Xiao-Han Wang • Yi Wu • Xiao-Fang Yang • Yanying Miao • Chuan-Qiang Zhang • Ling-Dan Dong • Xiong-Li Yang • Zhongfeng Wang

Received: 26 June 2014 / Accepted: 26 September 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract In the inner retina, ganglion cells (RGCs) integrate and process excitatory signal from bipolar cells (BCs) and inhibitory signal from amacrine cells (ACs). Using multiple labeling immunohistochemistry, we first revealed the expression of the cannabinoid CB1 receptor (CB1R) at the terminals of ACs and BCs in rat retina. By patch-clamp techniques, we then showed how the activation of this receptor dichotomously regulated miniature inhibitory postsynaptic currents (mIPSCs), mediated by GABAA receptors and glycine receptors, and miniature excitatory postsynaptic currents (mEPSCs), mediated by AMPA receptors, of RGCs in rat retinal slices. WIN552122 (WIN), a CB1R agonist, reduced the mIPSC frequency due to an inhibition of L-type Ca2? channels no matter whether AMPA receptors were blocked. In contrast, WIN reduced the mEPSC frequency by suppressing T-type Ca2? channels only when inhibitory inputs to RGCs were present, which could be in part due to less T-type Ca2? channels of cone BCs, presynaptic to RGCs, being in an inactivation state under such condition. This unique feature of CB1R-mediated retrograde regulation provides a novel mechanism for modulating excitatory synaptic transmission in the inner retina. Moreover, depolarization of RGCs suppressed mIPSCs of these cells, an effect that was

X.-H. Wang and Y. Wu contributed equally to this work. X.-H. Wang  Y. Wu  X.-F. Yang  Y. Miao  C.-Q. Zhang  L.-D. Dong  X.-L. Yang  Z. Wang (&) Institutes of Brain Science, Institute of Neurobiology, State Key Laboratory of Medical Neurobiology, Fudan University, 138 Yixueyuan Rd, Shanghai 200032, China e-mail: [email protected]

eliminated by the CB1R antagonist SR141716, suggesting that endocannabinoid is indeed released from RGCs. Keywords Cannabinoid CB1 receptor  Synaptic transmission  Inner retina  mIPSCs  mEPSCs  Calcium channels Abbreviations ACs AMPARs 2-AG Anandamide, AEA ACSF BCs CB1R CNS eCB eIPSCs ([Ca2?]o) FAAH GABAARs GlyRs INL IPL ([Ca2?]i) MIB mIPSCs mEPSCs NIM PPR PB PBS

Amacrine cells AMPA receptors 2-Arachidonoylglycerol N-Arachidonoylethanolamine Artificial cerebral spinal fluid Bipolar cells CB1 receptor Central nervous system Endocannabinoid Evoked inhibitory postsynaptic currents Extracellular calcium concentration Fatty acid amide hydrolase GABAA receptors Glycine receptors Inner nuclear layer Inner plexiform layer Intracellular Ca2? concentration Mibefradil Miniature inhibitory postsynaptic currents Miniature excitatory postsynaptic currents Nimodipine Paired-pulse ratio Phosphate buffer Phosphate buffer saline

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PFA RGCs SR WIN

Paraformaldehyde Retinal ganglion cells SR141716 WIN 55212-2

Introduction Endocannabinoid (eCB) signaling from postsynaptic neurons retrogradely regulates neurotransmitter release from presynaptic neurons by activating the CB1 receptor (CB1R), thus modulating synaptic strength in many regions of the central nervous system (CNS) (Kreitzer and Regehr 2001; Ohno-Shosaku et al. 2001; Wilson and Nicoll 2001; Kushmerick et al. 2004; Chevaleyre et al. 2006; Wang et al. 2006; Galve-Poperh et al. 2007; Hashimotodani et al. 2007; Kano et al. 2009). eCB signaling is also extensively distributed in the vertebrate retina, as evidenced by the presence of eCBs, along with CB1Rs and the eCB degradative enzyme fatty acid amide hydrolase (FAAH) in various retinal neurons (Straiker et al. 1999; Yazulla et al. 1999, 2000; Porcella et al. 2000; Lalonde et al. 2006; Yazulla 2008; Zabouri et al. 2011). Indeed, in the outer retina, WIN 55212-2 (WIN), a CB1R agonist, modulates various K? and Ca2? currents in lots of species (Straiker et al. 1999; Yazulla et al. 2000; Fang and Yazulla 2003; Straiker and Sullivan 2003; Yazulla 2008). Specifically, retrograde suppression of voltage-gated K? currents (IK(v)) by 2-arachidonoylglycerol (2-AG), an eCB released from bipolar cells (BCs) in a Ca2?-dependent manner, was found in goldfish cones, implying a downegulation of tonic glutamate release from these cells (Fan and Yazulla 2007). In the inner retina, BCs, amacrine cells (ACs), and ganglion cells (RGCs) form a neuronal circuit that integrates and processes visual signals. RGCs, output neurons of the retina, receive inhibitory inputs from ACs, mediated by GABAA and glycine receptors (GABAARs and GlyRs) and excitatory inputs directly from cone BCs, mediated by AMPA receptors (AMPARs) (Wa¨ssle and Boycott 1991; Protti et al. 1997; Marc and Liu 2000; Wa¨ssle 2004). Moreover, ACs have been demonstrated to modulate BCs through reciprocal synapses between these two cell types (Vigh and von Gersdorff 2005; Grimes et al. 2009; Vigh et al. 2011). While CB1Rs are localized to BCs and ACs (Yazulla 2008), few data are available concerning whether and how this signaling regulates synaptic transmission in the inner retina. Warrier and Wilson (2007) reported that WIN increased spontaneous minis mediated by GABAARs in cultured chick embryonic ACs. Most recently, Middleton and Protti (2011) demonstrated WIN-induced inhibition of the release of GABA from ACs and glutamate from BCs

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in mouse retinal slices by examining miniature inhibitory and excitatory postsynaptic currents (mIPSCs and mEPSCs) of RGCs. In this work, we first show the expression of CB1Rs at the terminals of rat BCs and ACs by multiple labeling immunohistochemistry. We further demonstrate, using patch-clamp techniques, how activation of the presynaptic CB1Rs at ACs and BCs differentially modulates mIPSCs and mEPSCs of RGCs by regulating L- and T-type Ca2? channels, respectively, in rat retinal slices. While activation of CB1Rs reduced the mIPSC frequency consistently, CB1R-mediated regulation of mEPSCs could be seen only when inhibitory inputs from ACs were present. Moreover, we also provide evidence, suggesting that eCBs may be indeed synthesized and released from RGCs in a Ca2?-dependent manner.

Materials and methods Animals Male Sprague–Dawley rats, aged 14–20 days and obtained from SLAC Laboratory Animal Co., Ltd (Shanghai, China), were housed under conditions of a 12-h light/dark cycle. All experimental procedures described here were in accordance with the National Institutes of Health (NIH) guidelines for the Care and Use of Laboratory Animals and the guidelines of Fudan University on the ethical use of animals. During this study, all efforts were made to minimize the number of animals used and their suffering. Immunohistochemistry Immunofluorescent staining was performed following the procedure described in detail previously (Chen et al. 2011; Ji et al. 2011; Yang et al. 2011; Ji et al. 2012; Miao et al. 2012) with some modifications. Animals were deeply anesthetized with 20 % urethane (10 ml/kg). The posterior eyecups were immersion fixed in fresh 4 % paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB, pH 7.4) for 1 h, and chilled in 0.1 M PB with 10, 20, and 30 % sucrose. The fixed eyecups were then embedded in OCT (Miles Inc., EIkhart, IN, USA) and vertically sectioned at 14 lm thickness on a freezing microtome (Leica, Nussloch, Germany). The sections were collected and mounted on chrome-alum-gelatin-coated slides (Fisher Scientific, Pittsburgh, PA, USA). After rinsing with 0.01 M phosphate buffer saline (PBS) (pH 7.4), they were blocked for 1 h in 5 % normal donkey serum (v/v) (Sigma, St. Louis, MO, USA) in PBS plus 0.3 % Triton X-100 at room temperature. For immunofluorescence triple labeling, the CB1R staining was first carried out by incubating the retinal sections with the primary antibody of goat polyclonal to

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CB1R (sc-10066, 1:500, Santa Cruz, Biotechnology, CA, USA) overnight at 4 °C. Binding sites of the primary antibody were revealed by incubating with the antibody of anti-goat conjugated biotin for 1 h, followed by the antibody of CyTM5-conjugated streptavidin (1:400, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature. Then, a cocktail of primary antibodies to label the cell terminals and different cell types were incubated with different CB1R-labeled sections overnight at 4 °C, respectively, followed by incubating with secondary antibodies for 1 h at room temperature. Primary antibody against synaptophysin (ab8049, 1:1,000, Abcam, Cambridge, MA, USA) was used to label cell terminals. Primary antibodies used for labeling different cell types were as follows: rabbit monoclonal to PKC alpha (ab32376, 1:1,000, Abcam, Cambridge, MA, USA) and rabbit polyclonal to recoverin (AB5585, 1:1,000, Chemicon, Millipore, MA, USA) for rod-driven and cone-driven BCs, respectively; rabbit polyclonal to GABA (A2052, 1:1,000, Sigma, St. Louis, MO, USA) and rabbit polyclonal to parvalbumin (PV) (ab11427, 1:1,000, Abcam, Cambridge, MA, USA) for GABAergic and glycinergic AII ACs, respectively. Synaptophysin was visualized with 555-conjugated donkey anti-mouse IgG antibody (1:400, Jackson ImmunoResearch Laboratories, West Grove, PA, USA), whereas the different cell markers were visualized with 488-conjugated donkey anti-rabbit IgG (1:400, Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The sections were mounted with anti-fade mounting medium (Vector Laboratories, Burlingame, CA, USA) and scanned with Leica SP2 confocal laser scanning microscope at a 609 oil immersion objective lens. To avoid any possible reconstruction stacking artifact, triple labeling was precisely evaluated by sequential scanning on single-layer optical sections at intervals of 1.0 lm. Final images were assembled using Adobe Photoshop version 7.0 (Adobe Systems, San Jose, CA, USA). Preparation of retinal slices After deeply anesthetized with 20 % urethane (10 ml/kg) and decapitated, the eyes of rats were quickly enucleated and immersed in ice-cold, oxygenated sucrose cutting solution (0–4 °C) containing (in mM): sucrose 124, KCl 3, NaHCO3 26, NaH2PO4 1.25, sodium pyruvate 3, glucose 10, CaCl2 0.2, MgCl2 3.8 (pH 7.4). Retinas were isolated carefully and embedded in low-melting agarose [4 % in artificial cerebral spinal fluid (ACSF)]. Slices (200 lm) were made using vibratome (Leica, VT1000s) and transferred to a holding chamber where they were completely submerged in ACSF containing (in mM): NaCl 125, KCl 3, NaHCO3 26, NaH2PO4 1.25, CaCl2 2, MgCl2 1, glucose 15 (pH 7.4), bubbled continuously with 95 % O2 and 5 %

CO2, and maintained at room temperature (21–23 °C) for 40 min before recording. Preparation of isolated rat retinal BCs Retinal neurons were acutely dissociated by enzymatic and mechanical methods as previously described (Zhao et al. 2010; Ji et al. 2011) with minor modifications. After deeply anesthetized with 20 % urethane (10 ml/kg) and decapitated, the retinas were removed quickly and incubated in oxygenated Hank’s solution containing (in mM): NaCl 137, NaHCO3 0.5, NaH2PO4 1, KCl 3, CaCl2 2, MgSO4 1, sodium pyruvate 1, HEPES 20, glucose 16 adjusted to pH 7.4 with NaOH. The retinas were digested in 5 mg/ml papain (Calbiochemical, San Diego, CA, USA) containing Hank’s solution, supplemented with 0.75 mg/ml L-cysteine for 33 min at 33.5–34.5 °C, and retinal neurons were mechanically dissociated with fire-polished Pasteur pipettes. Dissociated retinal BCs were characterized by short bush-like dendrites emerging from one end of the soma and a long axon at the other end (Yu et al. 2006; Yang et al. 2011). Electrophysiology and data analysis Whole-cell voltage- and current-clamp recordings were performed using standard techniques (Zhao et al. 2010; Yang et al. 2011). Individual slices were transferred to a perfusing chamber and continuously superfused with oxygenated ACSF at a rate of 1–2 ml/min under room temperature. RGCs and BCs in retinal slices were identified by their locations and morphology with the help of an infrared-differential interference contrast (IR-DIC) video microscopy (Olympus, Japan), and further identified by intracellular injection of Lucifer Yellow. Patch pipettes were made by pulling BF150-86-10 glass (Sutter Instrument Co., Novato, CA, USA) on a P-97 Flaming/Brown micropipette puller (Sutter Instrument) and fire polished (Model MF-830, Narishige, Japan) before recording. The pipette resistance was typically 4–8 MX after filled with the internal solution. For mEPSCs and evoked EPSCs (eEPSCs) recordings, the internal solution consisted of (in mM) CsMeSO3 120, NaCl 5, EGTA 2, HEPES 10, ATPMg 2, GTP-Na 0.2, TEA-Cl 10, QX-314 3, pH 7.2 adjusted with CsOH, 280–290 mOsm/l. For mIPSCs and evoked IPSCs (eIPSCs) recordings, the internal solution consisted of (in mM) CsCl 150, CaCl2 0.1, MgCl2 1, HEPES 10, EGTA 1, GTP-Na 0.4, ATP-Mg 4, pH 7.2 adjusted with CsOH, 280–290 mOsm/l. For whole-cell current-clamp recordings in cone BCs, the internal solution consisted of (in mM): potassium D-gluconate 120, CaCl2 0.1, MgCl2 1, EGTA 1, HEPES 10, ATP-Mg 4, GTP-Na 0.3, phosphocreatine 10, Lucifer yellow 5, pH 7.2 adjusted with KOH

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and to 280–290 mOsm/l. For eEPSCs or eIPSCs recordings, the test stimuli were delivered at a frequency of 0.05 Hz through a patch pipette filled with ACSF and placed on the inner nuclear layer (INL) or the inner plexiform layer (IPL). Whole-cell membrane currents were recorded from RGCs or BCs, voltage-clamped at -65 mV (adjusted liquid junction potential), by a patch amplifier (Axopatch 700B; Molecular Devices, Foster City, CA, USA) with Digidata 1440A data acquisition board and pClamp 10.2 software. Analog signals were sampled at 10 kHz, filtered at 1 kHz, and stored for further analysis. After stable currents (mIPSCs and mEPSCs) had been recorded (control), drugs were delivered by a gravity-driven superfusion system for at least 5 min before testing effects of the drugs on the currents. For whole-cell Ca2? current recording in dissociated BCs, the cells were bathed in Ringer’s solution containing (mM) NaCl 115, KCl 2.5, BaCl2 10, CsCl 5, HEPES 15, TEA-Cl 15, glucose 10, and TTX 0.5 lM, pH adjusted to 7.4 with NaOH and to 310 mOsm/l. The pipette solution consisted of (in mM): CsCl 128, CaCl2 1, MgCl2 2, EGTA 10, HEPES 10, ATP-Mg 2, GTP-Na 0.4, and phosphocreatine 10, adjusted to pH 7.2 with CsOH and to 290–300 mOsm/l. Whole-cell membrane currents in BCs were picked up by a patch amplifier (Axonpatch 700B) at a sample rate of 5 kHz, filtered at 1 kHz. Fast capacitance was fully canceled and cell capacitance was partially canceled by the circuits of the amplifier as much as possible. Seventy percent of the series resistance of the recording electrode was compensated (Ji et al. 2012). For selective analysis of mEPSCs in mixed excitatory and inhibitory minis, we made a distinction between mEPSC and mIPSCs according to their distinct characteristic kinetics. Usually, mEPSCs have a rapid kinetics with a rising time ranging from 0.8 to 3 ms and a decay time ranging from 3.5 to 7.0 ms (Best et al. 2008; Alberto and Hirasawa 2010). In contrast, mIPSCs have a relatively slower kinetics with a decay time ranging from 7.4 to 79.6 ms (Ropert et al. 1990; Gleason et al. 1993; Protti et al. 1997; Takahashi et al. 2006; Warrier and Wilson 2007; Ke et al. 2010). In the present work, the average rising time of mEPSCs was 2.18 ± 0.26 ms and the decay time was 4.88 ± 1.03 ms. The steady-state inactivation curve was fitted by the Boltzmann equation of the form I/Imax = 1/ {1 ? exp[(Vm - VH)/k]}, where I is the peak current measured from each prepulse, Imax is the maximum peak current, VH is the prepulse voltage at which the current amplitude is half maximum, Vm is the prepulse, and k is the slope factor at VH. The data analysis was performed using Clampfit 10.2 (Molecular Devices, Foster City, CA, USA), MiniAnalysis,

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SigmaPlot 10.0 and Igor 4.0 (WaveMetrics, Lake Oswego, OR, USA). Data were all presented as mean ± SEM. Paired t test or one-way ANOVA was used for statistical analysis.

Results Expression of CB1Rs at the terminals of rat retinal BCs and ACs The expression of CB1Rs in BCs and ACs was examined in rat retinal vertical sections, using immunofluorescence triple labeling techniques. As shown in Fig. 1, CB1Rpositive signals were co-localized with those for synaptophysin (red) at the terminals of PKC-labeled rod BCs (green) (Fig. 1a, a1–a3, arrows), recoverin-labeled conedriven OFF- (Fig. 1b, b1–b3, arrows) and ON-type BCs (green) (Fig. 1b, b4–b6, arrows), as well as at those of GABAergic ACs (green), labeled by GABA (Fig. 1c, c1– c3, arrows), and glycinergic AII ACs (green), labeled by parvalbumin (Fig. 1d, d1–d3, arrows). mIPSCs and mEPSCs in RGCs show distinct Ca2? dependence We first determined how mIPSCs of RGCs, mediated by GABAARs and GlyRs, depended on extracellular calcium concentration ([Ca2?]o), with non-NMDA receptors and NMDA receptors being blocked by CNQX (10 lM) and D-APV (50 lM), respectively. Perfusion of Ca2?-free ACSF almost eliminated all the events of GABAAR-mediated mIPSCs when GlyR-mediated events were blocked by strychnine (10 lM) (n = 6) (Fig. 2a). It was the same for GlyR-mediated mIPSCs when GABAAR-mediated events were blocked by bicuculline (10 lM) (n = 6) (Fig. 2b). In contrast, removal of extracellular Ca2? had no significant effect on both frequencies and amplitudes of mEPSCs (n = 5, P = 0.831 and 0.770) in the presence of bicuculline and strychnine (Fig. 2c, d). We also tested possible effects of changes in intracellular Ca2? concentration ([Ca2?]i) on mEPSCs by pretreating the slices with thapsigargin (10 lM), an inhibitor of sarco-endoplasmic reticulum Ca2?-ATPases, which increases calcium release from intracellular stores (Tsuzuki et al. 2004). As shown in Fig. 2d, the frequency, but not the amplitude of mEPSCs, was significantly increased (n = 4, P \ 0.001). These results suggest that Ca2? released from intracellular Ca2? stores plays an important role in triggering glutamate release from BCs when inhibitory inputs from ACs were blocked.

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Fig. 1 CB1 receptors (CB1Rs) are expressed at the terminals of rat retinal BCs and ACs. a Confocal microphotograph of a vertical section of the rat retina, triple stained with the antibodies against CB1R (gray), synaptophysin (red) and PKC (green), shows that CB1Rs are expressed at the terminals of PKC alpha-positive rod BCs. a1, a2, a3 Enlarged images, all taken from the square in a. a1 CB1Rlabeled image; a2 synaptophysin- and PKC-labeled image; a3 merged image of a1 and a2. Note that CB1R-positive signal is co-localized with signals for synaptophysin and PKC (arrows). b Triple immunofluorescence-labeled microphotograph shows that CB1Rs are expressed at the terminals of recoverin-positive cone-driven OFFand ON-type BCs. b1, b2, b4, b5 Enlarged unmerged images, while b3 and b6 are merged ones, taken from the white (OFF-type BC) and

blue (ON-type BC) squares in b, respectively. b1, b4 CB1R-labeled images; b2, b5 synaptophysin and recoverin-labeled images; b3, b6 merged images of b1 and b2, b4 and b5, respectively. Note that CB1R-positive signal is co-localized with signals for synaptophysin and recoverin (arrows). CB1Rs are expressed at the terminals of GABA-positive GABAergic (c) and PV-positive glycinergic AII ACs (d). c1, c2, d1, d2 Enlarged unmerged images, while c3 and d3 are merged ones, taken from the squares in c and d, respectively. CB1Rpositive signals are co-localized with signals for synaptophysin in GABA- and PV-positive ACs (arrows). All the scale bars represent 10 lm. GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, AC amacrine cell, BC bipolar cell

Activation of CB1Rs dichotomously regulates mIPSCs and mEPSCs of RGCs separated pharmacologically

antagonist, blocked the WIN-induced change in GABAARmediated mIPSC frequency (92.1 ± 9.2 % of control, n = 4, P = 0.803) (Fig. 3c). WIN application did not significantly change the amplitude (76.1 ± 9.6 % of control, n = 8, P = 0.064) (Fig. 3d). Moreover, WIN hardly changed the kinetics of mIPSCs [rising time 4.86 ± 0.43 ms (WIN) vs. 4.85 ± 0.32 ms (control), n = 8, P = 0.973; decay time 12.7 ± 1.4 ms (WIN) vs. 12.3 ± 0.6 ms (control), n = 8, P = 0.793]. Furthermore, WIN (5 lM)

Perfusion of WIN (5 lM) significantly suppressed the frequency of GABAAR-mediated mIPSCs of RGCs separated pharmacologically in a progressive manner (Fig. 3a, b), with the average being reduced to 44.8 ± 7.3 % of control after 5 min perfusion (n = 8, P = 0.013) (Fig. 3b, c). Pretreatment with SR141716 (SR, 300 nM), a specific CB1R

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Fig. 2 Different Ca2?-dependence of mIPSCs and mEPSCs in rat RGCs. Representative traces showing that removal of extracellular Ca2? (Ca2?-free extracellular solution) almost eliminated the GABAAR- (a) and GlyR- (b) mediated mIPSCs, recorded in the presence of CNQX (10 lM) and D-APV (50 lM). c Sample traces showing that perfusion of Ca2?-free extracellular solution did not change the mEPSCs, recorded in the presence of bicuculline (10 lM) and strychnine (10 lM). d Summary data showing the changes in frequency (left) and amplitude (right) of mEPSCs under different conditions. Note that thapsigargin (10 lM) significantly increased the frequency of mEPSCs. All data are normalized to control and presented as mean ± SEM. n = 4–6. ***P \ 0.001 vs. control

induced an elevation of the paired-pulse ratio (PPR) of GABAAR-mediated evoked IPSCs (eIPSCs) [2.66 ± 0.22 (WIN) vs. 1.88 ± 0.10 (control), n = 7, P = 0.038] (Fig. 3e, f), suggesting a reduction in presynaptic GABA release probability (Kreitzer and Regehr 2001). The effect of CB1R activation on GlyR-mediated mIPSCs of RGCs was basically similar. That is, WIN (5 lM) progressively suppressed the frequency of the currents (Fig. 3g, h), with the average being reduced to 37.5 ± 13.8 % of control (n = 9, P = 0.001) after 5 min perfusion (Fig. 3i). The WIN-induced change in GlyRmediated mIPSCs was rescued when SR (300 nM) was coapplied (94.0 ± 5.8 % of control, n = 4, P = 0.846) (Fig. 3i). Again, WIN did not change either the average amplitude (97.4 ± 9.3 % of control, n = 9, P = 0.751) (Fig. 3j) or the kinetics of the mIPSCs [rising time 6.08 ± 0.65 ms (WIN) vs. 6.29 ± 0.75 ms (control),

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n = 9, P = 0.470; decay time 16.4 ± 2.3 ms (WIN) vs. 16.9 ± 2.6 ms (control), n = 9, P = 0.356]. Similarly, WIN also increased the PPR of GlyR-mediated eIPSCs [2.56 ± 0.37 (WIN) vs. 1.84 ± 0.25 (control), n = 5, P = 0.023] (Fig. 3k, l). The effect of WIN on the frequency of mIPSCs may involve L-type Ca2? channels. Figure 3m shows that nimodipine (NIM, 10 lM), an L-type Ca2? channel blocker, dramatically reduced the frequencies of mIPSCs mediated by GABAARs (upper panel) and GlyRs (lower panel), and addition of 5 lM WIN did not further alter these frequencies. On average, the frequency was reduced to 20.8 ± 3.6 % of control (n = 6, P = 0.010) for GABAARmediated mIPSCs and to 16.1 ± 5.4 % of control (n = 4, P = 0.010) for GlyR-mediated mIPSCs. These values were not significantly changed by adding 5 lM WIN (NIM ? WIN) [20.0 ± 5.6 % of control vs. 20.8 ± 3.6 % of control (NIM), n = 6, P = 0.310 for GABAAR-mediated mIPSCs; 13.1 ± 5.5 % of control vs. 16.1 ± 5.4 % of control (NIM), n = 4, P = 0.329 for GlyR-mediated ones] (Fig. 3n). NIM (10 lM) did not significantly change the amplitude of either GABAAR- or GlyR-mediated mIPSCs (P = 0.091 and 0.612, respectively) (Fig. 3o). Effects of WIN on mEPSCs, recorded when GABAARs and GlyRs were blocked, were rather different. As shown in Fig. 4a, perfusion of WIN (5 lM) did not influence the mEPSCs, with the average frequency and amplitude being 99.6 ± 12.9 % and 98.6 ± 14.6 % of control (n = 5, P = 0.958 and 0.897), respectively (Fig. 4c, d). The rising time and decay time of the mEPSCs were also unchanged (rising time 2.17 ± 0.12 ms vs. 2.16 ± 0.14 ms, n = 5, P = 0.854; decay time 4.88 ± 0.46 ms vs. 5.04 ± 0.47 ms, n = 5, P = 0.228). Moreover, WIN failed to change the elevated frequency of mEPSCs by thapsigargin (10 lM) (99.6 ± 2.6 % of control, n = 4, P = 0.900) (Fig. 4b, c). Moreover, unlike observed in mIPSCs, extracellular application of NIM (10 lM) had no significant effect on the mEPSCs under this condition (upper panel, Fig. 4e). The average frequency and amplitude were 94.8 ± 2.6 % (n = 6, P = 0.126) and 105 ± 7.3 % (n = 6, P = 0.681) of control, respectively (Fig. 4f, g). Perfusion of mibefradil (MIB, 10 lM), a T-type Ca2? channel blocker, also failed to influence the mEPSCs (frequency 98.1 ± 4.6 %; amplitude 91.7 ± 9.4 % of control, n = 5, P = 0.356 and 0.122, respectively) (Fig. 4e, lower panel, f, g). Activation of CB1Rs suppresses mEPSCs of RGCs in the presence of inhibitory inputs We further studied how WIN affects mEPSCs of RGCs when inhibitory inputs from ACs were present. We first identified mEPSC events in mixed minis, containing both

Brain Struct Funct

Fig. 3 Activation of CB1Rs suppresses RGC mIPSCs. a Representative traces show that 5 lM WIN suppressed GABAAR-mediated mIPSCs of an RGC, recorded in the presence of CNQX (10 lM), D-APV (50 lM), and strychnine (10 lM). b Bar chart showing the WIN-induced decrease of GABAAR-mediated mIPSC frequency as a function of time. Summary data showing that WIN reduced the frequency (c), but not the amplitude (d) of GABAAR-mediated mIPSCs (n = 8), and pretreatment with SR141716 (SR), a specific CB1R antagonist, abolished the WIN effect (n = 4). e Sample traces show that WIN decreased the amplitude of GABAAR-mediated eIPSCs and increased the PPR of eIPSCs (left panel). f Cumulative data showing that WIN increased the PPR of eIPSCs (n = 7). g Representative traces show that WIN (5 lM) suppressed GlyR-mediated mIPSCs, recorded in the presence of CNQX (10 lM), D-APV (50 lM) and bicuculline (10 lM). h Bar chart

showing the WIN-induced changes of GlyR-mediated mIPSC frequency as a function of time. Summary data showing that WIN reduced the frequency (i), but not the amplitude (j) of GlyR-mediated mIPSCs (n = 9), an effect that was blocked by SR (n = 4). k Sample traces show that WIN decreased the amplitude of GlyR-mediated eIPSCs and increased the PPR of eIPSCs (left panel). l Cumulative data showing that WIN increased the PPR of eIPSCs (n = 5). m Sample traces show the effect of 10 lM nimodipine (NIM), applied alone or along with 5 lM WIN, on GABAAR- (upper) and GlyR- (lower) mediated mIPSCs. Bar charts showing that NIM remarkably suppressed the frequency (n), but not the amplitude (o) of GABAAR- and GlyRmediated mIPSCs. n = 4–6. Note that WIN had no further effect on the mIPSCs in the presence of NIM. All data are normalized to control and presented as mean ± SEM. *P \ 0.05 and **P \ 0.01 vs. control

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mEPSCs and mIPSCs, of RGCs recorded in normal ACSF, based on their characteristic kinetics (inset of Fig. 5a) (see ‘‘Materials and methods’’ for details), and analyzed effects of WIN on these events. Under this condition, WIN (5 lM) remarkably suppressed the frequency of these events (Fig. 5a), with the average frequency being reduced to 55.4 ± 3.9 % of control (n = 5, P \ 0.001) (Fig. 5b). The WIN effect was abolished by co-application of SR (300 nM) (99.9 ± 11.7 % of control, n = 4, P = 0.958) (Fig. 5a, b). No change in the average amplitude was found (96.8 ± 10.5 % of control, n = 5, P = 0.850) (Fig. 5c). WIN-induced suppression of mEPSC frequency could be seen as long as either one of glycinergic and GABAergic inputs was present (Fig. 5d, e). The average frequency was reduced to 77.7 ± 1.8 % of control (n = 5, P = 0.001) with glycinergic input being blocked by 10 lM strychnine and to 65.3 ± 4.4 % of control (n = 5, P = 0.001) with GABAergic input being blocked by 10 lM bicuculline, respectively (Fig. 5f). To test whether T- and/or L-type dominant Ca2? channels, expressed in rat cone BCs (Pan 2000; Hu et al. 2009), may be involved in glutamate release from these cells, we examined effects of MIB and NIM on mEPSCs of RGCs. NIM (10 lM) hardly changed either frequency or amplitude of mEPSCs (n = 5). In contrast, perfusion of MIB (10 lM) markedly reduced the average frequency of mEPSCs to 35.1 ± 5.2 % of control (n = 5, P \ 0.001) (Fig. 5h, i), suggesting the involvement of T-type Ca2? channels, even though MIB perfusion did not change the amplitude (99.3 ± 5.5 % of control, n = 5, P = 0.850) (Fig. 5j). This notion was further strengthened by recording Ca2? currents from dissociated cone BCs. T-type Ca2? currents could be induced when the cells were depolarized from a holding potential of -80 to -40 mV (Fig. 6a, left panel), and these currents were completely suppressed by MIB (10 lM) (n = 6) (Fig. 6a, right panel). L-type Ca2? currents failed to be detected in all cells tested (n = 24) when these cells, clamped at -40 mV, were stepped to 0 mV. These results suggest that it was T-type Ca2? channels that may mediate Ca2?-dependent glutamate release. Indeed, these channels may be a target mediating the action of presynaptic CB1Rs on the cone BCs. As shown in Fig. 6b, T-type Ca2? currents of dissociated cone BCs, induced by depolarizing the cells to 40 mV from a holding potential of -80 mV, were reduced to 58.7 ± 5.6 % of control (n = 6, P \ 0.001) by WIN (5 lM). While T-type Ca2? channels mediated the WIN-induced suppression of mEPSCs, we further characterized changes in inactivation curve of these channels in dissociated cone BCs. Figure 6c shows the steady-state inactivation curve of T-type Ca2? channels, determined when the cells were first hyperpolarized to different potentials and then depolarized to -40 mV (right panel, Fig. 6c), which exhibited a VH

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Fig. 4 Effect of activation of CB1Rs on mEPSCs of RGCs in the presence of bicuculline (10 lM) and strychnine (10 lM). a Representative traces show that 5 lM WIN had no effect on AMPARmediated mEPSCs of an RGC. b Representative traces show that 5 lM WIN did not suppress AMPAR-mediated mEPSCs recorded from an RGC predialyzed with thapsigargin (10 lM). Summary data showing that WIN did not affect the frequency (c) and the amplitude (d) of AMPAR-mediated mEPSCs in normal ACSF and with predialysis of thapsigargin (n = 4–6). e Representative traces show the effects of NIM (10 lM) and mibefradil (MIB, 10 lM) on mEPSCs of RGCs. Summary data showing that NIM and MIB did not influence the frequency (f) and amplitude (g) of mEPSCs (n = 6). All data are normalized to control and presented as mean ± SEM

value of -65.8 ± 0.8 mV (n = 6). It means that most of T-type Ca2? channels in cone BCs may be in an inactivated state around the resting membrane potential level. Furthermore, we examined how the inhibitory inputs may change the membrane potentials of cone BCs (Fig. 7a). Figure 7b

Brain Struct Funct Fig. 5 Activation of CB1Rs suppresses mEPSCs when GABAergic and glycinergic inhibitory inputs are present. a Sample traces show that 5 lM WIN suppressed mixed minis of an RGC when mIPSCs were not blocked by bicuculline and strychnine (upper traces). Lower traces are those from the rectangles shown in a faster time scale, demonstrating that WIN suppressed mEPSCs (red), and SR blocked the WINinduced suppression of mEPSCs (red). The inset shows different kinetics of mIPSCs and mEPSCs of an RGC. Summary data showing the WIN-induced changes in the frequency (b) and amplitude (c) of mEPSCs (n = 5) under different conditions. Representative traces show that WIN reduced the frequency of mEPSCs (red) in the presence of strychnine (STRY, 10 lM) (d) and of bicuculline (BIC, 10 lM) (e) obtained in two different RGCs. Summary data showing the WIN-induced changes in the frequency (f) and amplitude (g) of mEPSCs (n = 5) under different conditions. h Sample traces show that 10 lM MIB suppressed mixed minis of an RGC. Lower traces are those from the rectangles shown in a faster time scale, demonstrating that MIB selectively reduced the frequency of mEPSCs (red). Summary data show that MIB suppressed the frequency (i), but not the amplitude (j) of mEPSCs (n = 5). All data are normalized to control and presented as mean ± SEM. **P \ 0.01 and ***P \ 0.001 vs. control

shows that the membrane potential of a cone BC, recorded by current clamping in a retinal slice, was depolarized by coapplication of bicuculline (10 lM) and strychnine (10 lM).

The average membrane potential determined in the presence of bicuculline and strychnine was -34.1 ± 2.0 mV (n = 5), which was more depolarized than that obtained when the

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types, a possibility may be that the inhibitory inputs may have changed the properties of cone BCs. For testing this possibility, we examined effects of WIN on cone BCs, and did find that WIN (5 lM) significantly suppressed the mIPSC frequency of these cells (33.6 ± 5.6 % of control, n = 4, P = 0.004) (Fig. 8a, b), but not the amplitude (87.3 ± 7.4 % of control, n = 4, P = 0.461) (Fig. 8c). Depolarization of RGCs suppresses mIPSCs of these cells

Fig. 6 Inhibition of T-type Ca2? currents by activating CB1Rs and inactivation curves of the channels in dissociated cone BCs. a Lower voltage-activated T-type Ca2? currents of a cone BC, induced by various voltage steps, were completely inhibited by mibefradil (MIB) (10 lM) (left panel). I–V relationships obtained in normal Ringer’s solution (control) and in the presence of 10 lM MIB are shown in the right panel. b Sample traces in the left panel show that WIN (5 lM) suppressed T-type Ca2? current in a cone BC. The current was induced by a 200 ms depolarization pulse from a holding potential of -80 mV stepping to -40 mV. Summary data (n = 6) are shown in the right panel. c Representative Ca2? currents of a dissociated cone BC are shown in the left panel. The currents were evoked by conditioning prepulses from a holding potential of -80 mV to different membrane potentials prior to a 200 ms test pulse of -40 mV. Inactivation curve of steady-state Ca2? currents is shown in the right panel. Normalized points are fitted with a Boltzmann function. All data are normalized to control and presented as mean ± SEM. n = 6. ***P \ 0.001 vs. control

inhibitory inputs were present (-41.0 ± 1.0 mV, n = 5, P = 0.016) (Fig. 7c). Since ACs provide inhibitory inputs to cone BCs through the reciprocal synapses between these two cell

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To test if eCB is indeed released from RGCs that regulates minis of these cells, we determined changes in GlyRmediated mIPSCs of RGCs separated pharmacologically when a 5 s depolarization pulse (from -70 to 0 mV) was repeatedly applied to these cells for four times at intervals of 1 s (Fig. 9a). In cerebellar slices, depolarizationinduced suppression of inhibitory synaptic transmission was firstly observed in Purkinje cells (Llano et al. 1991). Figure 9b shows that the depolarization pulses induced a transient decrease of the mIPSC frequency and the average value was reduced to 73.3 ± 1.3 % of control (n = 8, P = 0.010) at 10 s after the depolarization (Fig. 9c), but then gradually recovered. This effect of the depolarization pulses was blocked by co-application of SR (300 nM) (99.8 ± 1.4 % of control, n = 4, P = 0.986) (Fig. 9c), implying that eCBs were indeed released from RGCs and acted presynaptically. When the retinal slices were preincubated with AM404 (20 lM), an eCB transporter inhibitor, the depolarization-induced suppression of GlyR-mediated mIPSCs was not observed (Fig. 9e), with the average frequency of mIPSCs being 97.3 ± 4.9 % of control (n = 4, P = 0.343) (Fig. 9f), further demonstrating that the depolarization-induced suppression of mIPSC frequency was mediated by eCB released from RGCs. Moreover, when the cells were dialyzed with BAPTA (10 mM) through recording pipettes, the depolarizationinduced effect on GlyR-mediated mIPSCs was hardly observed (Fig. 9e), and the average frequency of mIPSCs was almost unchanged (96.0 ± 12.7 % of control, n = 5, P = 0.611) at 10 s after the depolarization (Fig. 9f), indicating that eCBs are released from RGCs in a calcium-dependent manner. These results are similar to the findings observed in various brain areas (Llano et al. 1991; Ohno-Shosaku et al. 2001, 2002; Maejima et al. 2005). To determine which type(s) of eCBs may be involved in the depolarization-induced effects, we examined how the frequency of GlyR-mediated mIPSCs of RGCs could be changed in retinal slices pretreated with either URB597 (50 lM), a FAAH inhibitor, or URB602 (50 lM), an inhibitor of 2-AG hydrolyzing enzyme (monoacylglycerol lipase, MGL), for 8 min. In the URB597 pretreated slices,

Brain Struct Funct

Fig. 7 Blockade of inhibitory inputs depolarizes cone BCs. a Micrograph showing a typical cone BC for recording in a rat retinal slice. Scale bar 10 lm. b Sample trace, recorded under current clamping, showing that application of the inhibitory synaptic receptor blockers bicuculline (BIC, 10 lM) and strychnine (STRY, 10 lM) induced a

depolarization of the membrane potential of a cone BC. c Summary data show the changes in membrane potentials of cone BCs after coapplication of BIC and STRY. All data are normalized to control and presented as mean ± SEM. n = 5. *P \ 0.05 vs. control

Discussion mIPSCs and mEPSCs in RGCs are mediated by different mechanisms

Fig. 8 Activation of CB1Rs suppresses mIPSCs in rat cone BCs. a Sample traces showing that WIN inhibited mIPSCs of a cone BC recorded in a retinal slice. b, c Summary data showing that WIN inhibited the frequency, but not the amplitude of cone BC mIPSCs. All data are normalized to control and presented as mean ± SEM. n = 4. **P \ 0.01 vs. control

the frequency was reduced to 77.9 ± 3.4 % of control at 10 s after the depolarization (n = 5, P \ 0.001) (Fig. 9h), quite comparable to that obtained in slice preparations without URB597 pretreatment (73.3 ± 1.3 % of control, Fig. 9c). However, the depolarization-induced suppression of the mIPSC frequency was significantly increased in the URB602 pretreated slices, and the average frequency at 10 s after the depolarization was lower (60.6 ± 5.0 % of control, n = 5, P = 0.001), as compared to that obtained in preparations without URB602 pretreatment (P = 0.035) (Fig. 9h). These results suggest that it was 2-AG, but not N-arachidonoylethanolamine (anandamide, AEA), that may be involved in the depolarization-induced suppression of GlyR-mediated mIPSCs.

Removal of extracellular Ca2? almost eliminated mIPSCs of RGCs (Fig. 2a, b) and application of NIM largely suppressed these events (Fig. 3m, n). These results strongly suggest that the release of GABA and glycine from ACs may be primarily dependent on Ca2? influx through L-type Ca2? channels in the plasma membrane, which is consistent with the observation made in rat ACs in culture (Ke et al. 2010). Ca2?-dependence of mEPSCs of rat RGCs, primarily generated by glutamate released from cone BCs (Wa¨ssle and Boycott 1991; Wa¨ssle 2004), however, may be rather complicated. Application of NIM/MIB or removal of extracellular Ca2? did not significantly affect the mEPSC frequency of RGCs when the inhibitory inputs to RGCs were blocked. We failed to record L-type Ca2? currents from cone BCs. While T-type Ca2? channels are expressed in cone BCs, most of these channels are inactivated at the resting membrane potential level, as indicated by the inactivation curve (Fig. 6c). On the other hand, thapsigargin greatly increased mEPSC frequency (Fig. 2d), suggesting that the mEPSCs may be largely mediated by Ca2? released from intracellular stores in cone BCs (Smith and Cunnane 1996). This may be in part resulted from the specialized ribbon synapses between BCs and RGCs (Morgans 2000; Singer et al. 2004; Heidelberger et al. 2005; tom Dieck and Brandstatter 2006). In contrast to action potential-initiated burst release at conventional synapses, glutamate is released tonically in ribbon synapses, and proteins involved in synaptic vesicle recruitment to the active zone and in synaptic vesicle fusion may be different. When the inhibitory inputs were present, glutamate release from cone BCs turned out to be dependent on

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Fig. 9 Depolarization of RGCs induces a decrease in GlyR-mediated mIPSC frequency. a Membrane depolarization of 5 s from -70 to 0 mV (arrows), which was applied four times at intervals of 1 s, suppressed GlyR-mediated mIPSCs recorded from an RGC in a retinal slice (upper panel). Recordings in the red rectangles are shown in a faster time scale in the lower panel. b Bar chart showing the changes of GlyR-mediated mIPSC frequency following a short depolarization as a function of time (n = 8). Summary data showing that membrane depolarization inhibited the frequency (c), but not the amplitude (d) of GlyR-mediated mIPSCs in RGCs. e Representative traces show that depolarization-induced suppression of GlyR-

mediated mIPSCs recorded from two RGCs was blocked either by AM404, an eCB transporter inhibitor (upper traces), or by intracellular dialysis of BAPTA (lower traces). Summary data showing the changes of frequency (f) and amplitude (g) of GlyR-mediated mIPSCs under the different conditions. h Summary data showing the changes in GlyR-mediated mIPSC frequency in slices pretreated with either URB597 (50 lM), a FAAH inhibitor (n = 5) or URB602 (50 lM), an inhibitor of the 2-AG hydrolyzing enzyme (n = 5). All data are normalized to control and presented as mean ± SEM. **P \ 0.01 and ***P \ 0.001 vs. control, and #P \ 0.05 vs. depolarization alone

Ca2? influx through the plasma membrane. But unlike observed for mIPSCs, it was T-type Ca2? channels, instead of L-type ones, that were involved (Fig. 6) (Pan 2000; Pan et al. 2001; Hu et al. 2009). Although L-type Ca2? channels are found in cone BCs (Pan 2000), they are unlikely to be involved in the generation of mEPSCs of rat RGCs, as these events were insensitive to NIM (Fig. 4e).

through modulating L-type Ca2? channels (Fig. 3n). Similar WIN-induced suppression of the mIPSC frequency was observed when mEPSCs were present (Fig. 5a). Consistently, WIN induced an increase of PPR in eIPSCs mediated by both receptor types. These results are reminiscent of what reported previously in mouse RGCs (Middleton and Protti 2011), pyramidal neurons of cerebral cortex, cerebellar Purkinje cells and neurons of hypothalamic arcuate nucleus (Takahashi and Linden 2000; Trettel and Levine 2003; Nguyen and Wagner 2006; Yamasaki et al. 2006), but different from the results obtained in hippocampal pyramidal neurons, in which WIN reduced the frequency of spontaneous, action potential-dependent

WIN differentially modulates inhibitory and excitatory signaling in the rat inner retina Application of WIN suppressed the frequency of mIPSCs mediated by GABAARs or GlyRs to similar extents,

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IPSCs (sIPSCs), without altering action potential-independent mIPSCs (Ha´jos et al. 2000; Hoffman and Lupica 2000). Miniature EPSCs of a neuron are studied commonly when inhibitory input is pharmacologically blocked (Trettel and Levine 2003; Yamasaki et al. 2006). With GABAAR- and GlyR-mediated inhibitory inputs being suppressed, mEPSCs of rat RGCs were insensitive to CB1R activation (Fig. 4a). Co-application of bicuculline and strychnine did not only block GABAergic and glycinergic signals from ACs to RGCs, but also those to BCs through the reciprocal synapses between ACs and these cells. Therefore, no changes in mEPSCs mean that WIN did not retrogradely modulate the release of glutamate from cone BCs through CB1Rs under such condition. When either GABAergic or glycinergic input existed, however, WIN significantly suppressed the frequency of mEPSCs. It was recently reported that WIN suppressed the frequency of mEPSCs of mouse RGCs when GABAARs were blocked (Middleton and Protti 2011). This was obviously due to the fact that the glycinergic input was still present in those experiments. As shown in Fig. 5f, WIN indeed suppressed mEPSCs of rat RGCs with the glycinergic input being not inhibited. This suppression should be resulted from the inhibition by WIN of T-type Ca2? channels that are responsible for glutamate release from cone BCs. CB1Rs are presynaptically expressed at the terminals of rat cone BCs (Fig. 1). Therefore, a possible explanation for the absence of WIN-induced regulation of mEPSCs when inhibitory inputs were blocked is that the T-type Ca2? channels might be in an inactivated state under such condition so that these channels failed to be regulated by CB1R activation. The inactivation curve, shown in Fig. 6c, indicates that most of the T-type Ca2? channels were inactivated when the membrane potential of cone BCs was more positive than -40 mV and more of the channels could be activated, in a voltage-dependent manner, with the membrane potential becoming more negative than 40 mV. We did find that the membrane potential of cone BCs determined in the presence of bicuculline and strychnine was more depolarized by 6.9 mV than that obtained when the inhibitory inputs were present (Fig. 7). This was obviously because tonic GABAergic and glycinergic inputs to cone BCs pushed the membrane potential of cone BCs to a more hyperpolarized level (Hull et al. 2006). This explanation was further strengthened by the finding that perfusion of bicuculline and strychnine not only eliminated mIPSCs, but also reduced the mEPSC frequency (Fig. 10). Based on these data, it seems likely that an increased number of T-type Ca2? channels could be activated in the presence of the inhibitory inputs. A puzzle is that the increase may be slight, given the small change in membrane potential (6.9 mV) caused by the inhibitory

Fig. 10 Blockade of inhibitory inputs suppresses the frequency of mEPSC of RGCs. a Sample traces showing that blockade of GABAAand GlyR-mediated inhibitory inputs by bicuculline (10 lM) and strychnine (10 lM) not only suppressed mIPSCs (black), but also reduced the frequency of mEPSCs (red) recorded from an RGC in a rat retinal slice. Summary data showing that blockade of inhibitory inputs reduced the frequency (b), but not the amplitude of mEPSCs (c) (n = 5). All data were normalized to control and are presented as mean ± SEM. ***P \ 0.001 vs. control

inputs. In this context, it should be noted that membrane potential at the terminals of cone BCs may be more negative than that of the cell bodies at which recordings were made because the terminals may receive much more inhibitory inputs from ACs (Du and Yang 2000). It is also possible that there is a change in number of activated T-type channels produced by a small hyperpolarization of membrane potential, which could be critical for WINinduced regulation. Injection of depolarizing currents into RGCs suppressed the frequency of mIPSCs of these cells and the effect could be abolished by SR. This result provides an indirect evidence for the release of eCBs from RGCs. Our results further show that inhibition of eCB transporter by AM404 (Du et al. 2013) and chelation of intracellular Ca2? by BAPTA obstructed the depolarization-induced effects, but inhibition of 2-AG hydrolyzing enzyme by URB602 enhanced it. It seems likely that rat RGCs may release eCBs (mainly 2-AG) in a Ca2?-dependent manner. These eCBS, when transported to synaptic clefts, may inhibit neurotransmitter release by acting on presynaptic CB1Rs. The depolarization-induced suppression of inhibitory synaptic transmission was first reported in cerebellar Purkinje cells (Llano et al. 1991). Later, this effect was also found in various brain areas (hippocampus, striatum, substantia nigra, etc.). There is evidence,

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showing that the effect was mediated by eCBs released from postsynaptic neurons in a calcium-dependent manner (Ohno-Shosaku et al. 2001, 2002; Maejima et al. 2005; Isokawa and Alger 2005; Hofmann et al. 2006; Narushima et al. 2006; Szabo et al. 2006; Trattner et al. 2013). In the inner retina eCB released from RGCs may serve as a negative modulator of the release of glutamate from cone BCs and of GABA and glycine from ACs, thus directly influencing retinal information processing. Such putative role in gain control has been reported in other brain regions (Chevaleyre et al. 2006; Galve-Poperh et al. 2007; Hashimotodani et al. 2007). In addition, activation of presynaptic CB1Rs can act as a high-pass filter that renders afferent inputs of high frequencies to evoke postsynaptic responses (Gerdeman 2008). Specifically, the CB1R-mediated retrograde regulation of glutamate release starts to work only when GABAergic and glycinergic signaling is generated, thus providing a unique retinal mechanism for integrating and processing signals from BCs and ACs at the RGC level. Acknowledgments This work was supported by grants from the National Program of Basic Research sponsored by the Ministry of Science and Technology of China (2013CB835100; 2011CB504602), the National Natural Science Foundation of China (31271173; 31470054), the Key Research Program of Science and Technology Commissions of Shanghai Municipality (11JC1401200; 13DJ1400302). Conflict of interest peting interests.

The authors declare that they have no com-

Ethical standard The authors declare that the manuscript does not contain clinical studies or patient data.

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Cannabinoid CB1 receptor signaling dichotomously modulates inhibitory and excitatory synaptic transmission in rat inner retina.

In the inner retina, ganglion cells (RGCs) integrate and process excitatory signal from bipolar cells (BCs) and inhibitory signal from amacrine cells ...
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