Supplemental Data Increased Ethanol Resistance and Consumption in Eps8 Knockout Mice Correlates with Altered Actin Dynamics

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Cell, Volume 127

Supplemental Data

Increased Ethanol Resistance and

Consumption in Eps8 Knockout Mice

Correlates with Altered Actin Dynamics Nina Offenhäuser, Daniela Castelletti, Lisa Mapelli, Blanche Ekalle Soppo, Maria Cristina Regondi, Paola Rossi, Egidio D’Angelo, Carolina Frassoni, Alida Amadeo, Arianna Tocchetti, Benedetta Pozzi, Andrea Disanza, Douglas Guarnieri, Christer Betsholtz, Giorgio Scita, Ulrike Heberlein, and Pier Paolo Di Fiore Supplemental Experimental Procedures Animal Experiments

Ethanol Induced Loss-of Righting Reflex Threshold. The threshold concentration of ethanol required to induce the loss of righting reflex was determined using the “up and down” method (Dixon, 1965; Wallace et al., 2006). Ethanol was injected intraperitoneally as a 10% v/v solution in PBS; after 5 min the ability of the mouse to right itself within 1 min was assessed. If the mouse lost the ability to right within 5 min, then the next mouse was given a lower dose (log dose) of ethanol; if it did not lose the righting reflex, then the next mouse was given a higher dose ethanol. The average log dose interval was 0.0129, corresponding to 0.1 g/kg differences between doses. The ED50 value was determined as described, with 95% Ci = dosing increment x square root of (2/n) x 1.96, where n is the last n trials (6) and 1.96 reflects the 0.05 α level (Wallace et al., 2006). ED50 values are considered significantly different if their 95% Ci do not overlap.

Rotarod. A mouse rotarod treadmill (Ugo Basile, accelerating from 7-30 rpm in 300 sec) was used. Mice were given two trial sessions of 5 consecutive trials on 2 consecutive days for training (see Figure S1A). In rare cases (less than 1 trial in fifty), mice were observed to passively hang onto the turning rod. If they remained passive for two consecutive turns, they were considered to have fallen off the rod. On the third day, mice were given one trial session and then received an i.p. injection of PBS (mock treatment) before being tested again, after a 30 min recovery time. On the fourth day, PBS was substituted by ethanol (20% in PBS, at the indicated concentrations). No significant effect of PBS injection was detected for either genotype. The same groups of mice were used for the 1. 0 and 1.5 g/kg ethanol experiments; mice were allowed to rest for 4 days after the first experiment. Naïve mice were used for the 2.0 and 2.5 g/kg ethanol experiments.

Ethanol-Stimulated Locomotor Activity. Ethanol stimulated locomotor activity was assessed recording the walking activity of the mice every 5 sec for 10 min in an open-field of 60 x 60 cm. Mice were habituated to the open field apparatus for two

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consecutive days. On the third day mice were injected i.p. with PBS immediately before being placed in the center of the open-field. On the fourth day ethanol (20% in PBS) was substituted for PBS . Walking activity was registered every 5 sec as either “yes” or “no”, and the sum of walking [maximal 12, expressed as arbitrary unit (a.u.) in Figure 1 D of the main text] within each min of the 10 min time frame was plotted against time.

Two-Bottle Choice Test. Mice were single-housed in cages supplied with two water bottles, for one week, to allow them to habituate to the test environment. Mice were then offered increasing ethanol concentrations, as shown, for 8 days each. Tastants were tested sequentially using increasing concentrations of the test solutions each for 4 days. Bottle position was changed daily (every other day in ethanol experiments) to prevent side effects, and liquid and food consumption was registered. No significant difference in body weight was observed at the beginning and at the end of the experiments. Nicotine consumption was tested for 4 days, at each concentration, with the identical scheme used to test for tastants.

Ethanol Metabolism. Blood ethanol concentrations were determined on naïve mice, using a diagnostic kit (Diagnostic chemicals Limited), and they are reported in Figure S1B-C. For the time curve at 3.5 g/kg (Figure S1B), mice were injected intraperitoneally with ethanol (20% in PBS) and blood was collected from the tail vein at the indicated time points. For blood ethanol concentrations at 1.5 g/kg (Figure S1C), blood was collected from the retro-orbital sinus after the open field experiment (30 min time point) and after the rotarod experiment (90 min time point). Retro-orbital blood samples at 2.5 g/kg ethanol (Figure S1C) were obtained on resting mice (30 min time point) and after the rotarod experiment (90 min time point). Blood samples were collected using heparinized tips. Plasma was obtained by centrifugation, immediately frozen on dry ice and stored at -80 °C until analyzed.

Statistical Analysis. Simple comparisons were made using Student’s t-test, multiple comparisons were made using two-way, repeated measures ANOVA. Histology and Immunoelectron Microscopy Animals were perfused transcardially with 4% paraformaldehyde, brains were dissected out and cut in 50 µm-thick serial sections. Free-floating sections from wild-type and Eps8-KO mice were processed in the same vial to standardize conditions. Sections adjacent to those processed for immunocytochemistry were stained with Thionin (0,1% in distilled water) for cytoarchitectonic control. Immunoperoxidase staining was performed using the ABC kit from Vector. Antibodies (Ab) used were: mouse monoclonal anti-Eps8 (Transduction Laboratories), rabbit polyclonal anti-Eps8, anti-parvalbumin (Swant, Bellinzona, Switzerland). For electron microscopy, sections were processed as for immunohistochemistry, except that permeabilization of the tissue was achieved by graded ethanol treatment. After the immunoperoxidase reaction, sections were postfixed in 2.5% glutaraldehyde, and then in 1% OsO4. Sections were dehydrated, cleared in propylene oxide, flat-embedded in Epon-Spurr and glued to resin blocks. Ultrathin

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sections were cut with a Reichert ultramicrotome and examined with a Jeol T8 electron microscope. For double immunofluorescence, sections were incubated with anti-Eps8 and either anti-NeuN (Chemicon, Temecula, CA) or phalloidin-Oregon green (Molecular probes), and appropriate secondary Ab (Cy2- or Cy3-conjugated, Jackson), and examined with a confocal laser scanning microscope (BIO-RAD, Hemel Hempstead, UK) equipped with an argon/krypton-mixed gas laser with excitation peaks at 488 (for Oregon green), 510 nm (for Cy2), and 550 nm (for Cy3). Confocal image series were recorded through separate channels to avoid crosstalk and merged with BIO-RAD Lasersharp 2000 software. Cultivation of Cerebellar Granule Neurons CGN, from cerebella dissected from P6/P7 mice, were prepared using the Papain dissociation system (Worthington). Briefly, cerebella were digested with papain 20 U/mL and Dnase-I 5U/mL in Hank’s buffered salt solution (HBSS, Sigma) for 20 min at 37°C in an Eppendorf thermomixer at 500 rpm, passed repeatedly through a 1 mL plastic pipette in HBSS, and centrifuged (180 rpm, 5 min). Cells were resuspended in HBSS and loaded on a DnaseI-albumin-ovomucoid inhibitor gradient to eliminate debris. 5 x 105 neurons were cultured on poly-D-lysine coated glass coverslips (12 mm diameter), in DMEM-F10 1:1, 5 µg/mL insulin, 2 mM L-glutamine, 2% bovine serum albumin, 100 µg/mL apotrasferrin, 0.04 µg/mL sodium selenite, 0.062 µg/mL progesterone, 16 µg/mL putrescein and 25 mM KCl. Cultures were used at 8-12 days. Quantitative PCR RNA from cultured CGN was extracted with the TRIzol® reagent (Invitrogen), followed by DNase treatment. Two µg of RNA were reverse-transcribed using SuperScript First-Strand Synthesis System (Invitrogen). RNA from CGN obtained by laser-capture (Leica AS LMD) from frozen cerebellar sections was isolated using the PicoPure RNA Isolation Kit (Arcturus), reverse transcribed and amplified by three repeated cycles of cDNA synthesis (Superscript ds cDNA synthesis kit, Invitrogen) and in vitro transcription reactions (RiboMax T7 kit, Promega for the first cycle and MEGAscript T7 kit, Ambion for the last two cycles); cRNA was then processed with the RNeasy Mini Kit (Quiagen). Two µg of cRNA were reverse-transcribed as described above and expression levels were determined using Taqman chemistry on an ABI 7900HT sequence detection system (Applied Biosystems). Antibodies Used in this Study Primary Antibodies • Mouse monoclonal anti-Eps8 (610144), BD Biosciences, Franklin Lakes, NJ, USA. • Rabbit polyclonal anti-Eps8, (Fazioli et al., 1993). • Rabbit polyclonal anti-Parvalbumin (PV28), Swant, Bellinzona/Switzerland. • Mouse monoclonal anti-NeuN (MAB377), Chemicon, Temecula, CA, USA. • Rabbit polyclonal anti-GluR1 (AB1504), Chemicon, Temecula, CA, USA. • Rabbit polyclonal anti-cofilin (C8736), Sigma-Aldrich.

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• Rabbit polyclonal anti-P-cofilin (3311), Cell Signaling, Danvers, MA, USA. • Rabbit polyclonal anti-NR1 (sc-9058), Santa Cruz Biotechnology, Santa Cruz,

CA, USA. • Goat polyclonal anti-NR2A (sc-1468), Santa Cruz Biotechnology, Santa Cruz,

CA, USA. • Rabbit polyclonal anti-NR2B (sc-9057), Santa Cruz Biotechnology, Santa Cruz,

CA, USA. • Goat polyclonal anti-NR2C (sc-1470), Santa Cruz Biotechnology, Santa Cruz, CA,

USA. • Rabbit polyclonal anti-synapsin (A6442), Molecular Probes, Eugene, OR, USA. • Mouse monoclonal anti-PSD-95 (MA1-046), Affinity Bioreagents, Golden, CO,

USA. • Mouse monoclonal anti-synaptophysin, (A-BM2470) ListarFISH, Milano, Italy. Phalloidin and Secondary Antibodies • Phalloidin- Oregon green labeled (O-7466),Molecular Probes, Eugene, OR, USA. • Phalloidin- FITC labeled (P5282), Sigma-Aldrich. • Rodamine-phalloidin (R415), Molecular Probes. • Phalloidin-TRITC labeled (P1951), Sigma-Aldrich. • Secondary antibodies, FITC, CY2, or CY3 labeled, Jackson, Soham, UK. Quantitative Analyses in Cerebellar Granule Neurons For the quantitative analyses shown in Figures 5 and 7 of the main text, neurons were randomly selected from areas of the coverslip were cultures were less dense (5-25 cells/100 µm2), and an average of 10-30 neurites/condition (40 µm each neurites) were analyzed, using Image J (http://rsb.info.nih.gov/ij/). Results were expressed as pixel fluorescence intensity per unit length (40 µm). Intensity lower than a threshold value was cut off. Threshold levels were determined to maximize the signal due to synaptic clusters and were optimized for each antibody or phalloidin used. Settings were then maintained for each data set. Each experimental condition was repeated at least thrice on independent neuronal preparations. Results are presented as average ± standard error mean, and significance was determined using Student’s t-test. P values less than 0.05 were considered significant. Electrophysiological Recordings

Slice Preparation and Solutions. Whole-cell patch recordings were performed from granule cells in the internal granular layer of acute cerebellar slices. Slices were obtained from 18 to 24-day-old (day of birth=1) wild-type or Eps8-KO mice. Briefly, mice were anesthetized with halothane (Aldrich) and killed by decapitation. Two hundred µm-thick slices were cut in the parasagittal plane from the cerebellar vermis in cold Krebs’ solution and maintained at room temperature for at least 30 min before being transferred to a recording chamber mounted on the stage of an upright microscope (D'Angelo et al., 1999; Rossi et al., 2002). The preparations were superfused with Krebs’ solution and maintained at 30 °C with a Peltier feedback device (HCC-100A, Dagan Corp., Minneapolis, MN, USA).

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Electrophysiological Recordings. The Krebs’ solution for slice cutting and recovery contained (mM): NaCl 120, KCl 2, MgSO4 1.2, NaHCO3 26, KH2PO4 1.2, CaCl2 2, glucose 11, and was equilibrated with 95% O2 and 5% CO2 (pH 7.4). For recordings, Krebs’ control solution was supplemented with the GABA-A receptor antagonist bicuculline (10 µM, SIGMA). Local perfusion through a multi-barrel pipette was used to apply various solutions to the preparation. Perfusion with control solution was commenced before seal formation and was maintained until switching to the test solutions. The patch-clamp pipette solution contained (mM): Cs2SO4 81, KCl 2, MgSO4 1.2, CaCl2 0.02, BAPTA 0.1, glucose 10, ATP-Mg 3, GTP 10-4, HEPES 15 (pH adjusted to 7.2 with CsOH). BAPTA tetrapotassium salt was from Molecular Probes (Eugene, OR, USA). Ethanol was obtained from Fluka (Switzerland). Recombinant Eps8 was prepared as described (Disanza et al., 2004) and dialyzed against a buffer containing 10 mM Tris pH 8, 100 mM KCl, 5% glycerol and 1 mM DTT. Recombinant Eps8 was used at 0.3 µM and the dialysis buffer was used as the buffer control. Latrunculin A (LTA) was from Sigma. LTA was applied at 20 or 40 µM (n= 6 each), and data were pooled as no significant differences were observed. Buffer control (0.1% DMSO) was applied both to wild-type and Eps8-KO slices (n=4 each). Whole-cell patch-clamp recordings were obtained by conventional methods (Hamill et al., 1981). Electrical signals were recorded with an Axopatch 200-A amplifier (-3dB fc=5 kHz), sampled with a Digidata-1200 interface, and analyzed off-line with P-Clamp software (Molecular Devices). Membrane potential was measured relative to an Ag-AgCl reference electrode (Clark Instruments, Pangbourne, UK). The mossy fiber bundle was stimulated using a bipolar tungsten electrode (Clark Instruments, Pangbourne, UK) positioned in the internal granular layer. EPSCs were elicited by current pulses (200 µsec duration) applied every 10 s (0.1 Hz) via a stimulus isolation unit. Passive granule cell parameters were monitored throughout the recordings. Current transients elicited by 10 mV hyperpolarizing steps (holding potential –70 mV; 20 kHz sampling rate) decayed monoexponentially and were used to estimate series resistance (Rs), membrane capacitance (Cm), membrane time constant (τm), membrane input resistance (Rm) and voltage-clamp rate (fVC) as shown in Table S2 [a full description of the procedure can be found in (D'Angelo et al., 1999)]. EPSCs were measured at different holding potentials to investigate their voltage-dependence using a sampling rate of 20 kHz. EPSC peak amplitude and EPSC amplitude 25 ms after stimulation (average of 20 contiguous data points) were taken to measure the non-NMDA and the NMDA current amplitude respectively. Non-NMDA EPSC kinetics were measured at the holding potential of –60 mV, at which the NMDA current is almost completely blocked by Mg2+. To measure the non-NMDA current decay, the first 25 msec after the EPSC peak were fitted with a double exponential function: (1) y(t)=A1 exp-t/τ1 + A2 exp-t/τ2 NMDA EPSC kinetics were measured at +60 mV, where the NMDA current is unblocked and well separated from the non-NMDA current. NMDA decay was measured starting 25 msec after stimulation and NMDA current decay was fitted

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according to equation 1. The weighted decay time constant (Rumbaugh and Vicini, 1999) for the NMDA current was estimated as: (2) τw= τ1•[A1/(A1+A2)]+ τ2•[A2/(A1+A2)]. Minis account for the whole spontaneous activity in granule cells in acute cerebellar slices (Sola et al., 2004), so that minis could be unambiguously detected in the absence of TTX. Minis were recorded during the pause between subsequent EPSCs, as reported (Sola et al., 2004). Results are reported as mean ± SEM. Statistical comparisons were performed using Student’s t test (a difference was considered not significant at p>0.05).

Supplemental References

D'Angelo, E., Rossi, P., Armano, S., and Taglietti, V. (1999). Evidence for NMDA and mGlu receptor-dependent long-term potentiation of mossy fiber-granule cell transmission in rat cerebellum. J Neurophysiol 81, 277-287.

Disanza, A., Carlier, M. F., Stradal, T. E., Didry, D., Frittoli, E., Confalonieri, S., Croce, A., Wehland, J., Di Fiore, P. P., and Scita, G. (2004). Eps8 controls actin-based motility by capping the barbed ends of actin filaments. Nat Cell Biol 6, 1180-1188.

Dixon, W. J. (1965). The Up-and-Down method for small samples. JAMA 60, 967-978.

Fazioli, F., Minichiello, L., Matoska, V., Castagnino, P., Miki, T., Wong, W. T., and Di Fiore, P. P. (1993). Eps8, a substrate for the epidermal growth factor receptor kinase, enhances EGF-dependent mitogenic signals. Embo J 12, 3799-3808.

Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391, 85-100.

Rossi, P., Sola, E., Taglietti, V., Borchardt, T., Steigerwald, F., Utvik, J. K., Ottersen, O. P., Kohr, G., and D'Angelo, E. (2002). NMDA receptor 2 (NR2) C-terminal control of NR open probability regulates synaptic transmission and plasticity at a cerebellar synapse. J Neurosci 22, 9687-9697.

Rumbaugh, G., and Vicini, S. (1999). Distinct synaptic and extrasynaptic NMDA receptors in developing cerebellar granule neurons. J Neurosci 19, 10603-10610.

Sola, E., Prestori, F., Rossi, P., Taglietti, V., and D'Angelo, E. (2004). Increased neurotransmitter release during long-term potentiation at mossy fibre-granule cell synapses in rat cerebellum. J Physiol 557, 843-861.

Wallace, M. J., Newton, P. M., Oyasu, M., McMahon, T., Chou, W. H., Connolly, J., and Messing, R. O. (2006). Acute Functional Tolerance to Ethanol Mediated by Protein Kinase C varepsilon. Neuropsychopharmacology.

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Figure S1. Motor Learning and Ethanol Metabolism Are Normal in Eps8-KO Mice

(A) Mice were trained on an accelerating rotarod on two sequential days for five consecutive trials (T1-T5, n=10 per genotype). No significant difference in rotarod performance was observed between Eps8-KO (closed circles) and wild-type (open circles) mice.

(B and C) Blood ethanol concentrations (BEC on y axes) were determined spectrophotometrically using NADH conversion.

(B) Time-course between 30 min and 3 h after i.p. injection of 3.5 g/kg ethanol (n=10 per genotype).

(C) Single point measurements at 30 and 90 min after i.p. injection of 1.5 and 2.5 g/kg ethanol (naïve mice were used per time point, ethanol dose and genotype, n=6-10). In all panels: open symbols, wild-type mice; closed symbols, Eps8-KO mice.

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Figure S2. General Taste Perception and Nicotine Consumption Are Not Altered in Eps8-KO Mice

Taste perception (A‐E) was tested sequentially using the two‐bottle choice test (n=8 per genotype).

(A) Eps8-KO mice consume significantly (p<0.05) less of a 4% sucrose solution respect to wild‐type mice. Consumption of a less sweet solution (1.7% sucrose) is not altered in Eps8-KO mice.

(B‐E) Eps8-KO mice show normal consumption of bitter (B, 0.01 mM and 0.03 mM quinine), salty (C, 0.3 M and 1M NaCl), acid (D, 15 mM and 150 mM citric acid) and umami (E, 3 mM and 10 mM glutamic acid) tasting solutions.

(F) Nicotine consumption was determined using the two‐bottle choice test. Eps8-KO mice show normal consumption of 10 and 50 mg/ml nicotine‐containing solutions (n=12 per genotype, female mice). In all panels: open bars, wild‐type mice; closed bars, Eps8-KO mice.

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Figure S3. Ethanol Induces Rapid Actin Dynamics and Delocalization of Eps8 in CGN, Is Reversible, and the Effect of Ethanol Is Independent of NMDAR Activation

(A) Cultured CGN were treated with 200 mM ethanol for the indicated lengths of time and then stained for Eps8 or F-actin (phalloidin). As shown, effects were already visible after 5 min of treatment.

(B) Cultured CGN were treated with the indicated concentrations of ethanol (30 min), and then stained for Eps8 or F-actin (phalloidin). As shown, concentrations as low as 50 mM already had strong effects on F-actin.

(C) Cultured CGN were treated with 200 mM ethanol (EtOH, 30 min), followed by a 30 min recovery period in normal medium (recovery) and then stained for F-actin (phalloidin). As shown, the ethanol effect is reversible.

(D and E) Cultured CGN were treated with 200 mM ethanol (EtOH, 30 min) or 50 µM glutamate (Glu, 20 min) with or without prior incubation with the NMDAR antagonist MK-801 (0.1 µM, 20 min) and then stained for F-actin (A, phalloidin) or Eps8 (B). In both panels, a quantitative assessment is also shown, and expressed as relative pixel intensity ± SD (n=15-19 random neurites/condition). Bar, 2 µm in all panels.

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Table S1. Basic Neurological and Behavioral Parameters Are Normal in Eps8-KO Mice Activity Score WT Score KO t Test Body position N.S.

Active 10 10 Inactive 0 0

Tremor Absent 10 10 Present 0 0

N.S.

Palpebral Closure Eyes open 10 10 Eyes closed 0 0

N.S.

Coat Appearance Tidy and well groomed 10 10 Irregularities 0 0

N.S.

Whiskers Present 10 10 Absent 0 0

N.S.

Lacrimation Absent 4 5 Present 6 5

N.S.

Defecation Present 4 5 Absent 6 5

N.S.

Transfer Arousal Immediate movement 1 2 Brief freeze 9 8 Extended freeze (> 5 sec) 0 0

N.S.

Gait Fluid movement and ca. 3mm pelvic elevation 10 10 Lack of fluidity in movement 0 0

N.S.

Tail Elevation Horizontal extension 10 10 Dragging/Elevated 0 0

N.S.

Touch Escape Response to touch 10 10 Flees prior to touch 0 0 No response 0 0

N.S.

Positional passivity Struggles when held by the tail 10 10 No struggle 0 0

N.S.

Skin Color Pink 10 10 Blanched, Bright or deep red flush 0 0

N.S.

Trunk Curl Absent 10 10 Present 0 0

N.S.

Limb Grasping Present 10 10 Absent 0 0

N.S.

Pinna Reflex Present 10 10 Absent 0 0

N.S.

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Corneal Reflex Present 10 10 Absent 0 0

N.S.

Contact Righting Reflex Present 10 10 Absent 0 0

N.S.

Evidence of Biting

None 10 10 Biting in response to handling 0 0

N.S.

Vocalization None 10 10 Vocal 0 0

N.S.

Locomotor Activity Total number of squares the animal enters in 30 sec 27 ± 6 23 ± 6

N.S.

Basic neurological and behavioral parameters were assessed using a modified SHIRPA protocol (n=10 per genotype). No significant differences between wild‐type (WT) and Eps8-KO (KO) mice were observed. Table S2. Membrane Capacitance and Input Resistance Are Normal in Eps8-KO Neurons Cm (pF) Rs (MΩ) Ri (GΩ) fVC (kHz) n WT 4.9±0.3 14.0±1.5 3.3±0.4 2.5±0.2 9 KO 4.6±0.4 14.7±1.4 3.7±0.7 2.6±0.1 10 t-test N.S. N.S. N.S. N.S. Passive granule cell parameters were monitored throughout the recordings of EPSC elicited by current pulses (200 msec duration) applied every 10 s (0.1 Hz). Current transients elicited by 10 mV hyperpolarizing steps (holding potential –70 mV; 20 kHz sampling rate) decayed monoexponentially and were used to estimate series resistance (Rs), membrane capacitance (Cm), membrane input resistance (Ri), and voltage‐clamp rate (fVC). Results are reported as mean ± SEM. Comparisons were performed using Student’s t‐test (differences were considered not significant, N.S., at p>0.05). Table S3. Rise Time and Time to Peak of Non‐NMDA Currents Are Not Altered in Eps8 Null Neurons Rise Time (ms) Time to Peak (ms) n WT 0.43±0.08 0.66±0.12 11 KO 0.43±0.06 0.75±0.11 19 t-test N.S. N.S. The non-NMDA EPSC component was measured at peak at –60 mV and showed similar activation i.e. rise time and time to peak in mutant and wild-type mice. Results are reported as mean ± SEM. Statistical comparisons were performed using Student’s t test (differences were considered not significant, N.S., at p>0.05).

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Table S4. NMDA but Not Non‐NMDA Current Decay Time Constants Are Increased Eps8 Null Neurons Non-NMDA Currents NMDA Current τfast τslow n τfast τslow τw n WT 1.4±0.1 9.2±1.1 9 22.3±5.4 110.2±15.8 74.7±9.9 19 KO 1.2±0.1 9.5±1.6 16 44.4±2.9 323.9±37.4 163.8±13.4 27 t-test N.S. N.S. p<0.004 p<0.001 p<0.004 Non-NMDA EPSC kinetics were measured at the holding potential of –60 mV, at which the NMDA current is almost completely blocked by Mg2+. To measure the non-NMDA current decay, the first 25 msec after the EPSC peak were fitted with a double exponential function y(t)=A1 exp-t/τ1 + A2 exp-t/τ2. No statistical difference was found for either fast or slow decay time constants of non-NMDA currents from wild-type or KO mice. NMDA EPSC kinetics were measured at +60 mV, where the NMDA current is unblocked and well separated from the non-NMDA current. NMDA decay was measured starting 25 msec after stimulation and NMDA current decay was fitted according to the formula described above. The weighted decay time constant (Rumbaugh and Vicini, 1999) for the NMDA current was estimated as: τw= τ1•[A1/(A1+A2)]+ τ2•[A2/(A1+A2)]. The decay time constants of NMDA receptor currents were significantly increased in ep8 KO neurons with respect to wild-type neurons. Results are reported as mean ± SEM. Statistical comparisons were performed using Student’s t-test (differences were considered not significant, N.S., at p>0.05). Table S5. There Is No Difference in Neurotransmitter Release Parameters between WT and Eps8-KO Mice Mini Size (pA) Mini Frequency (Hz) n EPSC CV n WT -13.8±8 0.16±0.07 8 0.287±0.027 18 KO -13±1.1 0.15±0.06 10 0.289±0.027 23 t-test N.S. N.S. N.S. Spontaneous release of neurotransmitter quanta (minis) and EPSC coefficient of variation (CV, = SD/mean) were measured to assess whether changes in neurotransmitter release exist between Eps8-KO and wild-type mice. No change in mini amplitude or frequency was observed between genotypes. Similarly, EPSC CV was not significantly different between Eps8-KO and wild-type mice. Results are reported as mean ± SEM. Statistical comparisons were performed using Student’s t-test (differences were considered not significant, N.S., at p>0.05). Table S6. EPSC CV Is Not Affected by Ethanol EPSC CV Basal EPSC CV after EtOH n t-Test WT 0.238±0.042 0.244±0.0052 7 N.S. KO 0.237±0.034 0.211±0.030 7 N.S. t-test N.S. N.S. EPSC coefficient of variation (CV, = SD/mean) was measured to assess whether changes in neurotransmitter release exist after ethanol exposure in Eps8-KO or wild-type mice. EPSC CV was not significantly different between Eps8-KO and wild-type mice after exposure to 100 mM (n=4) or 400 mM (n=3) ethanol. Results are reported as mean ± SEM. Statistical comparisons were performed using Student’s t-test (differences were considered not significant, N.S., at p>0.05).