1. Reactivation of the Same Synapses during Spontaneous Up States and Sensory Stimuli 
Xiaowei Chen, Nathalie L. Rochefort, Bert Sakmann, Arthur Konnerth 
Cell Reports 
Volume 4, Issue 1, Pages (July 2013)
DOI: /j.celrep
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2. Cell Reports 2013 4, 31-39DOI: (10.1016/j.celrep.2013.05.042)
Copyright © 2013 The Authors Terms and Conditions

3. Figure 1 Single Spine Calcium Signals during Spontaneous Up States
(A) Spontaneous up and down states recorded in the soma of a L2 cortical pyramidal neuron in the primary auditory cortex. Left: examples of electrical traces; right: distribution of membrane potentials of this neuron during a 32 s recording period.
(B) Distribution of up-state frequency, calculated from three trials for each neuron (n = 10 neurons).
(C) Two-photon image of a dendritic segment (average of 6,250 frames, frame rate = 1,000 frames/s−1).
(D) Subthreshold calcium transients in the dendritic spines (S1–S4) indicated in (C) during five up states. Last column: action potential-evoked calcium transients. Note that for displaying subthreshold up states on a large scale, spikes in the last column are truncated in amplitude. The neuron was held at resting membrane potential.
(E) Classification of spines based on their response rates. Left: cartoon showing three classes of spines: spines with “high” response rates marked in red, spines with “low” response rates marked in green, and silent spines marked in blue. This is the same dendrite as shown in (C) and (D). Right: distribution of the response rate of spine calcium signals. Data were from 207 imaged spines.
(F) Spatial distribution of active spines during up states in the dendritic field of a L2 cortical pyramidal neuron. Reconstruction of a neuron with dendritic recording sites marked and numbered, surrounded by corresponding insets indicating spine activity. Insets show the two-photon image of the corresponding dendritic segments, indicating with colored dots the spines that had high response rates (red), low response rates (green), and no response (blue).
See also Figure S1.
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4. Figure 2
Synaptic Origin and Temporal Distribution of Spine Calcium Signals during Slow Oscillations
(A) Left: two-photon image of a dendritic segment (average of 6,250 frames). Right: calcium signals recorded in the spines (S1–S5) indicated in the left panel, during spontaneous up states and action potentials in the presence of intracellular MK-801 (1 mM). Note that for clarity of the subthreshold up states, spikes in last column are truncated.
(B) Percentage of active spines during up states in control condition (n = 30 dendritic segments from ten neurons) and in the presence of intracellular MK-801 (n = 12 dendritic segments from four neurons). Unpaired t test, ∗∗∗p < Error bars show SEM.
(C) Example of spine calcium signals during up states. Note the variable latencies (pointed out by blue arrows) of the onset of calcium transients with respect to the onset of the up states.
(D) Distribution of the latency of the onset of calcium transients during up states. Δt is the time from the onset of the up state to the onset of the spine calcium transient. Δt was normalized to the duration of each corresponding up state.
(E) Spine calcium signals during up states (S1, marked in red) and during a down state (S2, marked in blue). Inset: expanded scale of the somatic EPSP marked with an asterisk, temporally corresponding to the calcium response in spine S2.
See also Figure S2.
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5. Figure 3 Number of Active Spines during Spontaneous Up States
(A and B) Left panels: two-photon images of dendritic segments, corresponding to the dendrites 1 and 2 in Figure 1F. Right panels: cartoons of these two dendritic segments with red dots indicating the spines that were activated during their corresponding up states.
(C) Schematic representation showing the number of active spines (marked in red) during a single up state.
(D) Distribution of the duration of up states (n = 150 up states from ten neurons).
(E) Distribution of the density of active spines (number of active spines per dendritic length) during single up states. The length of each imaged dendritic segment was normalized to 100 μm (n = 150 up states from ten neurons). The red line is an exponential fit to the data.
(F) Dendritic length of L2 cortical pyramidal neurons in the primary auditory cortex. Left: post hoc reconstruction of a biocytin-filled neuron (projection along the anteroposterior axis) with dendrites in black and the axons in red (the total dendritic length of this neuron = 4.1 mm). Note that for clarity, axon is truncated. Right: summary of dendritic lengths of three neurons.
See also Figure S3.
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6. Figure 4
Similar Activity Profile of Spines during Sensory-Evoked and Spontaneous Up States
(A) Two-photon image of a dendritic segment (average of 6,250 frames) used for calcium imaging in (B).
(B) Calcium responses in spines (S1–S3) during sound stimulation (six consecutive trials). Gray bars indicate sound stimulation (broadband noise, 100 ms duration, 0 dB attenuation).
(C) Calcium responses in the same spines as shown in (B) (S1–S3) during spontaneous up states. Gray bars indicate duration of up states. Color dots indicate the onset of calcium responses. The neuron was held at the resting membrane potential.
(D) Comparison of the response rate of spine calcium responses during sound stimulation (Evoked), spontaneous up states (Spont.), and action potentials. The number of spines is indicated.
(E) Proportion of active spines, defined by the number of active spines during individual up states divided by the total number of spines in each class, over eight consecutive up states (n = 21 spines with high response rates marked in red and 56 spines with low response rates marked in green).
See also Figure S4.
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7. Figure S1
Calcium Transient in a Silent Spine during Backpropagation of a Single-Action Potential, Related to Figure 1
Left: two-photon image of a dendritic segment (average of 6250 frames, frame rate = 1000 frames s-1). Right: calcium signals in the spine marked by a red dashed circle (top) and the corresponding electrical activity (bottom).
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8. Figure S2
Up and Down States Recorded in the Presence of MK-801 and Sparse Synaptic Activity during Down States, Related to Figure 2
(A) Electrical recording of up/down-state activity during intracellular application of MK-801.
(B) Distribution of membrane potentials of this neuron revealing two peaks corresponding to the down- and up-state, respectively.
(C) Analysis of excitatory postsynaptic potentials (EPSPs) during down-States. Upper, example of an EPSP during a down-state; Right, distribution of the amplitude of EPSPs during down-states (n = 100 EPSPs from 10 neurons).
(D) Whole-cell recording of excitatory postsynaptic currents obtained at a holding potential of −65 mV in a L2 pyramidal neuron in conditions that were similar to those used for calcium imaging (see Methods). The pipette solution contained: 134 mM Cs-gluconate, 10 mM HEPES, 6 mM CsCl, 4 mM MgATP, 0.3 mM Na2GTP, 10 mM Na2-Phosphocreatine, 100 μM QX314, titrated to pH Similar observations were made in three other neurons.
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9. Figure S3
Number of Active Spines during Spontaneous Up States, Related to Figure 3
(A) Distribution of distance of the imaged dendrites to soma.
(B) Median values of the density of active spines imaged in dendrites located < 100 μm,  μm and > 200 μm from soma, respectively. Note that there is no major difference in the median densities of active spines in different dendritic regions.
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10. Figure S4
Comparison of the Onset of Sound-Evoked and Spontaneous Up States, Related to Figure 4
(A) Membrane potential amplitude histogram obtained in the same neuron in conditions of spontaneous (left) and sound-evoked (right) up-states. The peak values of the up-states (−57 mV for both case) are indicated by red dotted lines.
(B) Plot of peak values of membrane potentials during spontaneous up-states versus sensory-evoked up-states. Each point indicates the values from a single neuron.
(C) Comparison of the durations of up-states during spontaneous activity and sound stimulation. n = 10 neurons.
(D) Example of sound-evoked activity and spontaneous up-states recorded from the soma of another neuron. Insets show the onset period of up-states, and two red dots indicate 40% of onsets.
(E) Comparison of the rate of 40% onset during sound-evoked activity and spontaneous up-states. ∗∗∗p <
(F) Distribution of the latency of spine calcium responses during sound-evoked (upper) and spontaneous (lower) up-states. The latency was measured from the onset of calcium transient to the corresponding onset of up-state. Note that approximately 2 times larger number of calcium transients occurred in the first 50 ms during sound stimulation than that during spontaneous up-states, which probably explains the faster rise time of sound-evoked up-states. n = 89 spines (14 dendritic segments from 10 neurons).
(G) and (H) Temporal distribution of activation of different spines with high response rates during spontaneous and sound-evoked up-states. (G) Temporal pattern of the spine indicated with a red dotted circle in the image. Six calcium transients were detected from this spine over 8 trials for each condition. (H) Summary of the temporal distribution of all spines with high response rates (n = 21 spines).
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11. Figure S5
Separation of Calcium Signals in the Spines and the Regions around Spines during Potential Firing, Related to Experimental Procedures
(A) Two-photon image of a dendritic segment (average of 6250 frames).
(B) Top: fluorescence changes (Δf, arbitrary units) from the sites indicated by colored dashed regions in (A) Bottom: the corresponding electrical recording.
(C) Plot of (Foutside – Fsd) versus the corresponding dendritic depths (n = 20 dendrites). Foutside is defined as the peak fluorescence values during synaptic activity and action potential firing in the regions around spines (outside), while Fsd is defined as baseline standard deviation from the same regions. The signals from the regions around spines fall to zero, indicating a clear separation of the signals in the spines from outside.
Cell Reports 2013 4, 31-39DOI: ( /j.celrep )
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