1. Volume 91, Issue 4, Pages 837-850 (August 2016)
Differential Dendritic Integration of Synaptic Potentials and Calcium in Cerebellar Interneurons 
Alexandra Tran-Van-Minh, Therése Abrahamsson, Laurence Cathala, David A. DiGregorio 
Neuron 
Volume 91, Issue 4, Pages (August 2016)
DOI: /j.neuron
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2. Figure 1 Large Amplitude and Rapid Time Course of Local EPSPs
(A) Schematic diagram showing that a large local dendritic depolarization (Vlocal) from a resting membrane potential (Vrest) could significantly reduce the synaptic driving force (ΔV = Esyn − Vlocal). Esyn is the reversal potential for the total synaptic current.
(B) Top: 2PLSM image (maximum-intensity projection) of an SC patch loaded with DiO. Bottom: dendritic region corresponding to the blue box in top image with a schematic diagram of extracellular electrode to indicate PF stimulation location for recordings shown in (C) and (D) (60 μm from the soma). The blue line indicates line-scan location.
(C) EPSPs recorded using somatic patch-clamp (black) and optical recording of dendritic voltage (green) using DiO/DPA in response to a single (top) or 50 Hz extracellular train stimuli (bottom) at location indicated in (B). Optical traces are spatial averages of all pixels along the line scan. All traces represent averages of 15 trials. Black arrows indicate stimulus time.
(D) Averaged somatic EPSCs (black) and dendritic local voltage (green) in response to a single (top) or 50 Hz train of extracellular stimuli (bottom) at same location as (C).
(E and F) Summary plots showing the local dendritic EPSP amplitude (E) and decay time constant (F) as a function of the distance of the synapse from the soma (n = 29 dendrites from 27 cells) in voltage-clamp mode. Dashed lines are linear fits. See also Figure S1.
Neuron  , DOI: ( /j.neuron )
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3. Figure 2
Spatial Distribution of Synapse-Evoked Dendritic Depolarization
(A) 2PLSM maximum-intensity projection image of part of an SC (DiO fluorescence). The cell is also filled with 200 μM XRhod-5F. The location of the stimulation electrode is indicated in white, close to segment 3 (37 μm from soma). Blue lines indicate location of 5-μm-long dendritic segments imaged in line-scan mode, used to calculate the fluorescence profiles in (B).
(B) Simultaneous somatic recording of PF-evoked EPSCs (black trace, top), optical recording of dendritic EPSPs (green traces, left), and dendritic [Ca2+] (red traces, right) for each segment. Traces are averages of five to ten trials.
(C) Peak amplitude of the dendritic EPSP (green curve) and Ca2+ transient (red curve) for each segment imaged in (A). Error bars indicate mean ± SEM. The dotted line is a Gaussian fit of the spatial profile of the Ca2+ transient obtained in segment 3, averaged over a 50 ms time window after stimulation.
(D) Summary plot of the dendritic EPSP (green squares) and Ca2+ transient (open red circles) peak amplitude over a 20 μm region around the activated synapses (n = 5 cells). 0 μm corresponds to the dendritic segment centered on the extracellular stimulation electrode. Negative distance values represent imaging locations closer to the soma and positive distance values represent locations more distal to stimulus electrode location (40 ± 9 μm from the soma).
(E) Numerical simulations of the voltage spread along an SC dendrite (morphology with three dendrites having respectively 0, 2, or 4 branch orders) using a ball-and-stick passive model of SC under voltage clamp in response to synaptic conductance (gsyn) equivalent to 5.3 quanta and located 47 μm from the soma. The voltage profiles are averaged over the time window from 240 to 980 μs after the onset to match imaging acquisition rate. See also Figure S2.
Neuron  , DOI: ( /j.neuron )
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4. Figure 3
Concomitant Sublinear Voltage and Supralinear [Ca2+] Integration in SC Dendrites
(A) Top: 2PLSM maximum-intensity projection image of an SC (Alexa Fluor 594 fluorescence). Bottom: zoomed image of the dendrite (blue box, top) showing glutamate uncaging locations (blue circles). Most proximal location is 55 μm from the soma. 20 mM MNI-glutamate was locally perfused.
(B) Top: two-photon uncaging-evoked somatic EPSPs (uEPSPs) in response to 200 μs illumination at locations indicated in (A) (gray traces) or in response to increasing number of spot locations (blue traces). Bottom: arithmetic sum of individual uEPSPs. Vh was −60 mV.
(C) Subthreshold input-output relationship (sI/O) of the arithmetic sum of uEPSPs recorded at individual sites (expected EPSP) versus the observed compound uEPSP for increasing number of spot locations for the cell shown in (B). The dotted line is the linear relationship.
(D and E) Same as (B) and (C) for uncaging-evoked Ca2+ transients (Fluo-5F). (D) Individual transients were fitted and added together to produce traces labeled expected sum. (E) Plot of integral of fitted traces over a 590 ms window after the uncaging pulse. Units are ΔF/F × s. The dashed trace is the plot of the arithmetic sum of Ca2+ transient integrals versus the expected EPSP.
(F) Average sI/O for uEPSP from Vh = −60 mV (blue circles, n = 9, proximal uncaging location 55 ± 10 μm from soma) or −77 mV (magenta circles, n = 6, proximal location 67 ± 19 μm from soma), fitted with single exponential functions. Dashed trace is unity line.
(G and H) Average sI/O for the integral of Ca2+ transients as a function of EPSP amplitude after normalization to the maximal expected response from Vh = −60 mV (G) or −77 mV (H). Square symbols show the arithmetic sum of Ca2+ integrals versus its corresponding expected EPSP. The dashed line is a linear fit to the arithmetic prediction. The sI/O for Ca2+ transients are fitted with sigmoid functions. ∗p < 0.05, paired test between observed and expected Ca2+ integral.
(I–K) Effect of VGCC or NMDAR antagonists on the average subthreshold input/output relationships for uEPSP (I), for the integral of Ca2+ transients as a function of EPSP amplitude (J), or for the integral of Ca2+ as a function of the number of photolysis sites (K) (control, n = 9 cells, blue, 50 μM AP5, n = 12, at 51 ± 21 μm from soma, dark gray, 5 μM Nifedipine, n = 6, light gray, at 44 ± 8 μm from soma, 5 μM TTA-P2, n = 6, at 51 ± 11 μm from soma, light blue, AP5 + TTA-P2 + Nifedipine, n = 5, at 50 ± 9 μm from soma, purple). Error bars represent ±SEM. See also Figure S3.
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5. Figure 4 Linear Integration of Synapse-Evoked Dendritic [Ca2+]
(A) Schematic diagram showing a smaller fractional reduction in the driving force for Ca2+ influx (ΔVCa, red) than for synaptic current (ΔVsyn, green).
(B) 2PLSM image (maximum-intensity projection) of an SC (Alexa Fluor 594 fluorescence). The blue box indicates stimulation and Ca2+ imaging location (56 μm from the soma).
(C) Left: 2PLSM image of stimulated and imaged dendrite. The blue line indicates location of line scan. Right: ΔF/F line-scan image (average of 17 trials) of OGB-5N fluorescence profile in response to synaptic stimulation (black arrows).
(D) Left: average somatic EPSC measured in response to a paired-pulse extracellular stimulation of PFs at 50 Hz. Right: Ca2+ transients (single trials, gray, n = 17; average, red) obtained from the line scan in (C). Individual traces are shown in gray and their average in red. The black trace is a fit of the first Ca2+ transient, used to estimate peak amplitude of the second response.
(E) Summary plot of the PPR of EPSCs amplitude and of the matching Ca2+ transient (p = 0.001, paired, n = 12). The center of the fluorescence profile was located, on average, at 51 ± 10 μm from the soma.
(F) Average EPSCs and Ca2+ transients recorded in voltage clamp Vh = −81 mV (black traces, average of eight trials) or −1 mV (yellow traces, average of nine trials).
(G) Summary plot of the PPR of EPSCs recorded from Vh = −81 mV and of Ca2+ transients at −81 mV and −1 mV. The center of the fluorescence profile was, on average, 48 ± 11 μm from the soma.
(H) Numerical simulations of total synaptic and Ca2+ current PPRs for synaptic conductances mimicking Ca2+-permeable AMPARs activated at the soma or at 47 μm from the soma in a model dendritic morphology with three dendrites displaying, respectively, 0, 2, or 4 branch orders. The total conductance ratio of the peak amplitude of the pair was AMPA receptors with a high Ca2+ permeability had a PCa/PNa,K of 2.3, while those with a low Ca2+ permeability had a PCa/PNa,K of 0.05 (GluA1/GluA2 versus GluA1 from Burnashev et al., 1996). The reversal potential for Ca2+ (Eca) was set at either +50 mV (open circles) or +100 mV (closed circles). Error bars represent variation in simulation result when changing Ri value ± 50 Ω × cm.
Box plots represent quartiles (minimum, 25%; median, 75%; and maximum values). See also Figure S4.
Neuron  , DOI: ( /j.neuron )
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6. Figure 5
Supralinear [Ca2+] Integration by Dendritic VGCCs Does Not Alter Dendritic EPSP Time Course
(A and B) Representative somatic EPSCs (top) and ΔF/F line-scan image of OGB-5N fluorescence (middle) from a dendrite stimulated 35 μm from the soma and recorded from Vh = −90 mV (black traces) or −50 mV in VC (blue traces). Bottom: Ca2+ transient traces averaged between red lines from the above line-scan images. Traces and images are averages of 10–15 trials.
(C) Population average of OGB-5N Ca2+ transients obtained during 50 Hz trains (left) or 200 Hz trains (right) recorded from Vh = −90 mV (black) or −50 mV (blue; n = 13 cells). Traces are normalized to the first peak. Line-scan center was 62 ± 22 μm from soma.
(D) Summary plot of the relative Ca2+ transient peak amplitudes normalized to the amplitude of the single response for Vh = −90 mV and −50 mV for control (p = 0.0002, paired, n = 13) in the presence of 50 μM AP5 (p < , paired, n = 15; 55 ± 15 μm from soma), in the presence of 5 μM TTA-P2 (p = 0.002, n = 11; 62 ± 19 μm from soma), and in the presence of 5 μM Nifedipine (p = 0.44, n = 6; 53 ± 13 μm from soma).
(E) Spatial profile of OGB-5N ΔF/F in SC dendrites during 50 Hz trains and averaged over 100 ms time windows (see colored bars under the fluorescence profiles in A). The profiles were fit with a Gaussian function (dashed lines) to measure the peak amplitude and width at 30% of peak (Vh = −90 mV, gray, or −50 mV, blue).
(F) Summary plot of spatial profile of [Ca2+] change during 50 Hz trains (p < , paired, n = 16).
(G) Somatic EPSCs (top) and optical (DiO/DPA) dendritic EPSPs (bottom) recorded in VC from Vh = −90 mV (black traces) or −50 mV (blue traces) in response to a 50 Hz (left) or 200 Hz (right) train. Traces are averages of 15 trials. Line-scan center was 47 ± 11 μm from soma.
(H) Average optical voltage traces from (G) normalized to the peak of the first EPSP.
(I) Summary plot of the time integral of optical dendritic EPSPs (n = 6). For each cell, the trace was normalized to the peak of the first EPSP.
Box plots represent quartiles (minimum, 25%; median, 75%; and maximum values). See also Figures S5 and S6.
Neuron  , DOI: ( /j.neuron )
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7. Figure 6
High-Frequency Synaptic Activation Induces Dendritic Branch-Specific Suppression of Synaptic Transmission and Requires NMDARs and VGCCs
(A) 2PLSM images of an SC (Alexa Fluor 594 fluorescence). Top: maximum-intensity projection of an SC. Middle: image of the stimulated dendrite (blue box from top image) superimposed with a Dodt contrast image of stimulation electrodes (bottom). Stimulation electrode location was 70 μm from soma.
(B) Representative traces of PF-evoked EPSCs using a 50 Hz paired-pulse stimulus delivered through electrode 1 before and after SSE induction evoked by electrode 2 (100 Hz train, 20 stimuli).
(C) Population average of SSE induction and recovery (n = 67 dendrites from 66 cells). The blue arrow indicates the time point of SSE induction. Error bars represent ±SEM.
(D) Population averages of SSE time course in the presence of 10 μM SR (within cell control, closed circles) and after washing of various combinations of antagonists (open circles). Each data point represents the average value from two consecutive sweeps.
(E) Summary plot of antagonist effects on EPSC evoked 4 s after SSE induction. SSE is blocked in the presence of 5 μM AM251 (p = 0.031, paired, n = 6 cells, electrode location 58 ± 16 μm from soma) and reduced in 50 μM AP5 (p = 0.048, paired, n = 10, at 64 ± 18 μm from soma) and not in 5 μM TTA-P2 (p = 0.275, paired, n = 10, at 55 ± 18 μm from soma). SSE was also reduced by addition of a combination of 50 μM AP5 and 5 μM TTA-P2 (p = 0.002, paired, n = 11, at 60 ± 10 μm from soma) and of 5 μM TTA-P2 and 5 μM Nifedipine (p = 0.029, paired, n = 14, at 55 ± 9 μm from soma).
Error bars indicate mean ± SEM. Box plots represent quartiles (minimum, 25%; median, 75%; and maximum values). See also Figure S6.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

8. Figure 7
Spatial Extent of Homo- and Heterosynaptic SSE at Parallel Fiber Synapses
(A) Spatial extent of SSE induction within the same dendrite. Left: schematic representation of the test electrode (electrode 1), the induction electrode (electrode 2), and SC dendrite. Middle: 2PLSM image of the stimulated dendrite (cyan) superimposed with a Dodt image of stimulation electrodes. Right: EPSCs recorded in response to stimulation of synapses at different locations before, during, and after the induction train. Electrode 2 was 52 μm from the soma.
(B) Effect of SSE induction on different dendritic branches. Top left: schematic representation and 2PLSM image (Alexa Fluor 594 fluorescence, cyan) superimposed with a Dodt image showing stimulation electrodes, test dendrite (dendrite A, green), and induction dendrite (dendrite B, magenta; 42 μm from the soma). Top right: somatic recording of EPSCs in response to stimulation of a neighboring branch (electrode 2). Bottom left: Ca2+ transients recorded from dendrite A (green trace) or from dendrite B (magenta trace) in response to paired-pulse stimulation through electrode 1. Bottom right: Ca2+ transients recorded from dendrite A (green trace) or from dendrite B (magenta trace) in response to paired-pulse stimulation through electrode 2.
(C) Summary of EPSC reduction at different locations in individual cells. EPSC amplitude was estimated from the first trial 4 s after induction. Locations are referenced as distance from electrode 2. Black circles, within branch SSE; light blue circles, across branch SSE. Electrode 2 was located 51 ± 24 μm from soma.
(D) Summary plot of test EPSC amplitudes at different times and locations after SSE induction (n = 5–9 locations from 11 cells). Error bars indicate mean ± SEM.
Neuron  , DOI: ( /j.neuron )
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9. Figure 8
Differential Input-Output Operations for Voltage and [Ca2+] Dynamically Regulate SC Computations
(A) Schematic diagram showing that sublinear summation of voltage (top) in SC dendrites is concomitant with linear or supralinear summation of [Ca2+] (bottom) in response to synchronous activation of synaptic inputs located on the same dendritic branch. Dendritic voltage operations mediate neuronal computations (e.g., sparse pattern detection), and [Ca2+] operations mediate induction of synaptic plasticity (LTD or SSE).
(B) Schematic diagram showing alterations of synaptic activation patterns by different synaptic plasticity mechanisms. Top: linear Ca2+ entry mediated by sustained, low-frequency synaptic inputs triggers long-term plasticity of synaptic weights at all active synapses (inputs 1, blue). Those inputs that are inactive or receive lower frequency activity remain unaltered (e.g., inputs 2, green). Bottom: supralinear Ca2+ entry mediated by high-frequency bursts of inputs transiently suppresses synaptic vesicle release probability within 10 μm of spatially clustered inputs (red and green inputs within magenta circle).
Neuron  , DOI: ( /j.neuron )
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