Cholinergic modulation of multivesicular release regulates striatal synaptic potency and integration.

Slice preparation and pharmacology All animal handling was performed in accordance with the Harvard Institutional Animal Care and Use Committee and federal guidelines. Recordings were made from MSNs in striatal slices taken from postnatal day 15–18 Sprague-Dawley rats. Sagittal ( Fig. 8 ) or coronal ( Fig. 1 – Fig. 7 ) slices (300 µm thick) were cut in ice-cold external solution containing (in mM): 110 choline, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 KCl, 7 MgCl 2 , 0.5 CaCl 2 , 25 glucose, 11.6 Na-ascorbate, and 3.1 Na-pyruvate, bubbled with 95 % O 2 and 5 % CO 2 . Slices were then transferred to artificial cerebrospinal fluid (ACSF) containing (in mM): 127 NaCl, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 KCl, 1 MgCl 2 , 2 CaCl 2 , and 25 glucose, bubbled with 95 % O 2 and 5 % CO 2 . After an incubation period of 30–40 min at 34° C, slices were stored at room temperature. All experiments were conducted at 32° C. In all experiments, 10 µM bicuculline, was present in the ACSF to block GABA A/C receptor-mediated inhibition. For all glutamate uncaging experiments, 10 µM serine was included in the ACSF to reduce NMDAR desensitization and VGCCs were blocked with a cocktail of (in µM): 1 ω-conotoxin-MVIIC (N/P/Q-types), 20 nimodipine (L-types), 10 mibefradil (R- and T-types). For some experiments (see text), extracellular MgCl 2 was reduced to nominally 0 µM. In experiments in which extracellular Ca was reduced to 1 mM, Mg was increased to 2 mM in order to maintain a constant concentration of divalent ions. Finally, in some experiments (see text), one or more of the following drugs were added to the ACSF, unless otherwise stated, at the following concentrations (in µM): 10 muscarine, 10 NBQX, 10 CPP, 50 APV, 1 TTX, 1 ω-conotoxin-GVIA, 2500 γ-DGG. In order to block ∼50% of AMPARs and NMDARs ( Fig. 8 ), 0.1 and 1 µM of NBQX and CPP were used, respectively. All chemicals were from Sigma or Tocris, with the exception of ω-conotoxin-GVIA and ω-conotoxin-MVIIC (Peptides International, Inc.).

Electrophysiology and imaging

Whole-cell recordings were obtained from MSNs identified with video-IR/DIC and 2-photon laser scanning microscopy (2PLSM) based on their small cell bodies and prominent dendritic spines. For current clamp recordings, glass electrodes (2–4 MΩ) were filled with internal solution containing (in mM): 135 KMeSO 3 , 10 HEPES, 4 MgCl 2 , 4 Na 2 ATP, 0.4 NaGTP, and 10 Na 2 CreatinePO 4 , adjusted to pH 7.4 with KOH. For voltage clamp recordings, cesium was substituted for potassium to improve space clamping. For physiology-only experiments, 1 mM EGTA and 20 µM Alexa Fluor-594 (to image neuronal morphology) were added to the internal solution. For Ca imaging experiments, 300 µM of the Ca sensitive indicator Fluo-5F and 20 µM Alexa Fluor-594 were added. Current and voltage recordings were made using a Multiclamp 700B amplifier. Data was filtered at 5 kHz and digitized at 10 kHz. Excitatory input fibers were stimulated with a small glass electrode (tip diameter 2 – 4 µm) filled with ACSF using brief (0.2 ms) current injections. For paired-pulse stimulation experiments, the electrode was placed at the border between the striatum and the overlying white matter. For optical quantal analysis experiments, the electrode was placed ∼10 µm from the dendritic spine of interest. Intracellular Ca imaging and glutamate uncaging were accomplished with a custom microscope combining 2PLSM and 2PLU, as previously described 33 , 35 . Neurons were filled via the patch electrode for 10–15 minutes before imaging. Fluo-5F (green) and Alexa Fluor-594 (red) were excited using 840 nm light to monitor Ca signals and spine morphology, respectively. To measure Ca signals, green and red fluorescence were collected during 500 Hz line scans across a spine and a neighboring dendrite. Ca signals were quantified as increases in green fluorescence from baseline normalized to the red fluorescence (ΔG/R). Reference frame scans were taken between each acquisition in order to correct for small spatial drift of the preparation over time. For 2PLU experiments, MNI-glutamate was bath applied at 2.5 mM, and glutamate uncaging was achieved using a 0.5 ms pulse of 720 nm light. In order to achieve standard uncaging power, (which translates into a constant amount of glutamate uncaged on each trial) we used photobleaching of Alexa Fluo-594 in the spine of interest as previously described 33 . Bleaching is a function of the laser power and thus provides readout of power delivery that is independent of spine depth and electrophysiological responses.

Data acquisition and analysis

Imaging and physiology data were acquired using National Instruments boards and custom software written in MATLAB (Mathworks) 50 . Off-line analysis was performed using custom routines written in MATLAB and Igor Pro (Wavemetrics). Peak amplitudes of electrically evoked EPSPs were calculated by averaging a 3 ms window around the peak. The amplitudes of electrically evoked EPSCs and uncaging-evoked EPSCs mediated by AMPARs were calculated by averaging over a 2 ms window, whereas a 10 ms window was used to calculate peaks of NMDAR-mediated EPSCs. EPSP and EPSC widths were calculated as the interval between points at half-maximal amplitude. For imaging experiments, measurements of ΔG/R were calculated by taking the average of the signal over a 150 ms post-stimulus window. For paired-pulse experiments, we measured the response to a single stimulation or paired stimulation at an interstimulus interval of 50 or 20 ms. The paired-pulse ratio (PPR) was calculated by subtracting the response to the single stimulus from that to the paired stimulus and then calculating the ratio of the peak of the remaining response to the single response. For optical quantal analysis, successes were distinguished from failures by setting a threshold equal to two standard deviations above baseline noise. To determine the effect of muscarine and conotoxin-GVIA on the amplitude of NMDAR-mediated Ca signals, the average ΔG/R on success trials during the baseline period were compared to the average ΔG/R after application of each drug. The probability of success was calculated by dividing the number of success trials by the total number of trials during either baseline or after bath application of each drug. To ensure that the dendrite was not stimulated directly, analysis was limited to those experiments in which Ca entry was confined to a single spine. In sections describing optical or uncaging responses measured from individual spines, the stated n indicates the number of spines analyzed. In sections describing electrically-evoked synaptic responses, the stated n indicates the number of cells analyzed. All statistics are expressed as mean±SEM and comparisons were made using a two-tailed Student’s t-test. Differences were judged statistically significant for p = λ q where q is the quantal postsynaptic amplitude for a single vesicle. Furthermore, the mean spine head Ca transient amplitude of only success trials (the synaptic potency, Pot NMDA ) is: Pot NMDA = λ q / P s = − ln ( 1 − P s ) q / P s . If the quantal amplitude q is the same in two conditions (i.e., before and after drug application), the ratio of synaptic potencies in the two conditions is purely a function of the probability of successful vesicular release: (eq. 1) Pot 1 / Pot 2 = [ ln ( 1 − P s 1 ) / P s 1 ] / [ ln ( 1 − P s 2 ) / P s 2 ] ( eq. 1 ) Figure 6e shows the results of using this equation to predict changes in synaptic potency for all experiments involving either muscarine or conotoxin-GVIA application. We plotted the expected fractional change in Pot NMDA as a function of the observed change in P s . The model ignores possible supralinearities in the activation of NMDARs due to a Hill coefficient of greater than 1 for activation by glutamate and possible sublinearities due to saturation of Ca indicator. Despite these simplifications and the lack of free parameters, eq. 1 fits the experimental data with a residual error of