Structural plasticity within the barrel cortex during initial phases of whisker-dependent learning.

<p>We report learning-related structural plasticity in layer 1 branches of pyramidal neurons in the barrel cortex, a known site of sensorimotor integration. In mice learning an active, whisker-dependent object localization task, layer 2/3 neurons showed enhanced spine growth during initial skill acquisition that both preceded and predicted expert performance. Preexisting spines were stabilized and new persistent spines were formed. These findings suggest rapid changes in connectivity between motor centers and sensory cortex guide subsequent sensorimotor learning.</p

Recording backpropagating action potential responses from GECIs in hippocampal pyramidal cells.

<p>A, Schematic showing the linescan location at the base of the apical dendrite. B, Raw linescan images (top row) showing a dark period prior to shutter opening, followed by a shutter-open fluorescence baseline and action-potential (bottom row) evoked responses (left, 3 action potentials at 83 Hz; right, 10 action potentials at 83 Hz). Fluorescence time series (middle row) were obtained by averaging over the spatial extent of the dendrite (indicated by vertical white lines).</p

GCaMP2 and GCaMP2-actin uncaging responses in spines.

<p>A, Apical dendrite of a CA1 pyramidal cell (left) expressing EGFP-actin (green) and mCherry (red), showing an enrichment of EGFP-actin at spines (predominantly green) compared to dendrite (predominantly red). A GCaMP2-actin fusion (right) shows a similar spine enrichment in the apical dendrite of a different CA1 cell. B, Example images showing spine and dendrite fluorescence for GCaMP2 (left) and GCaMP2-actin (right). Each image shows the baseline frame prior to uncaging. Images are median filtered in a 3×3 pixel neighborhood. C, Traces show uncaging-evoked ΔF/F fluorescence signals for ROIs covering the spine (gray) and dendrite (black) for GCaMP2 (left) and GCaMP2-actin (right). Uncaging occurs at the start of the third frame. Traces correspond to the spines/dendrites shown in B. D, Traces showing NMDA-receptor currents in response to glutamate uncaging at the spines shown in B. Each trace shows an average of 6 trials. E, ΔF/F fluorescence change in spines versus peak NMDA-receptor current versus for GCaMP2 (gray circles) and GCaMP2-actin (black circles). Lines indicate measurements taken from the same spine at different uncaging powers.</p

Action-potential evoked responses in GCaMP-based GECIs.

<p>A, Amplitudes of GCaMP2 responses for individual hippocampal pyramidal cells (thin lines, left) in response to trains of action potentials given at 83 Hz, and the mean across cells (thick gray line). Dashed lines show perforated-patch recordings. Inset shows same data for 1–10 action potentials on a linear x-axis. Example single-trial responses (right) from four cells to 1, 5, 10 and 40 action potentials at 83 Hz (indicated by horizontal black lines). B, Responses of individual cortical layer 2/3 pyramidal cells (thin lines) and the group mean (thick gray line) expressing GCaMP2 after <i>in utero</i> electroporation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001796#s4" target="_blank">Materials and Methods</a>). C, GCaMP2 responses from hippocampal pyramidal cells at 34.5–35.5°C. D,E, Responses of previous versions of GCaMP family GECIs. B–E, Same conventions as in A.</p

Accuracy of action potential detection.

<p>Results of simulations (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001796#s4" target="_blank">Materials and Methods</a>) giving percentage of action potential trains (for the indicated numbers of action potentials at 83 Hz) that can be detected at a 5% false positive rate for 1 second of data (i.e., such that when a time series is divided up into subsequent intervals of 1 second length, 5% of these intervals will contain a false positive). Thin dotted lines show results for individual cells and the thick black line gives the group mean.</p

Action-potential evoked responses in GECIs targeted to subcellular locations.

<p>A, Amplitudes of the response to action potential trains at 83 Hz for the membrane-targeted GECI hCD4-GCaMP2 (left), for individual cells (thin black lines) and for the group mean (thick gray line). Insets show same data for 1–10 action potentials on a linear x-axis. Example traces (right) show single-trial responses to trains of action potentials at 83 Hz (indicated by horizontal black lines). B, Responses from the membrane-targeted construct MARCKS-GCaMP2. C, Actin-potential evoked responses from the GCaMP2-actin fusion. Conventions as in A. D–F, Action-potential responses measured simultaneously with GECIs (green curves, traces) and with the synthetic dye X-Rhod-5F (500 µM; red curves, traces). Cells were loaded with X-Rhod-5F for ≥20 min prior to data collection. Example traces show single-trial responses measured simultaneously from the green and red channels. Cells shown in D–F are different from those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001796#pone-0001796-g003" target="_blank">Figure 3A</a> and in panels A–C.</p

Domain structures of the GCaMP-family of genetically encoded calcium indicators (GECIs) and fusion constructs.

<p>A, Domain comparisons of GCaMP2 and GCaMP1.6; red labels indicate the differences. B, Constructs for subcellular targeting of the GECIs.</p

Analysis of curvature change on contact.

<p>(A–E) The sequence of steps used to extract detailed curvature measurements. (A) Whiskers in raw video frames are automatically traced and linked, yielding (B) an identified set of curves for each frame. (C) The raw curve is fit with a 5^th-degree parametric polynomial. (D) A mask is specified to determine where the curve intersects the face. Within a small interval (1–2.5 mm path length) about an interest point chosen for high signal to noise, the raw curve is re-fit to ensure measurements are not biased by whisker shape outside the interval. This new fit is to a 2^nd-degree polynomial. The curvature at the interest point is then measured as the curvature of this 2^nd fitted curve. (E) Follicle position is estimated by extrapolating a fixed distance into the face from the mask. Similarly, curves are extrapolated, when necessary, to contact points on the pole. Trajectories for curvature (F) and the angle of the whisker at its base (G) are shown for the first contacting whisker in 10 trials grouped by whether the first contact was during a retraction (top 5) or protraction (bottom 5). Trajectories are aligned to first contact. The intervals when the whisker is in whisker-pole contact are highlighted in red. (H) Histograms of peak contact curvature change (from resting) for the first whisker-pole contact in each trial (green) and all whisker-pole contacts prior to an answer-lick (red).</p

When linking <i>N</i> whiskers, each curve in a frame is assigned a label <i>W₁</i> to <i>W_N</i>, or <i>F₀</i> to <i>F_N</i>.

<p>Rules constrain the labeling to enforce consistent anterior-posterior ordering of whiskers. The most proximal point of curves labeled W_i or F_i must be posterior to that of curves labeled W_j or F_j when <i>i, and at most one curve may be labeled <i>W_i</i> for a given <i>i</i>. (A) A correct labeling is schematically illustrated. (B) These rules are encoded as transitions in a hidden Markov model. (C) Normalized feature histograms are used to compute the likelihood a curve is, or is not, a whisker.</i></p

Tracking whiskers from high-speed (500 Hz) videos during an object detection task.

<p>(A) A typical field of view. (B) Typical imaging configuration. (C–G) Automated results of tracing and linking. (C) Facial hairs and whiskers are traced in each video frame and then identified by a separate tracking step. (D) A whisker (blue) touches the pole. (E) Two whiskers (blue & green) are bent by the pole. The most posterior whisker is strongly retracted so that only a small segment is visible. (F) Tracking measures whisker orientation, such as the angle at base. (G) Tracking measures whisker shape, such as mean curvature, which can be observed over time. Changes in curvature allow the calculation of forces acting on the whisker follicle <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002591#pcbi.1002591-Birdwell1" target="_blank">[16]</a>.</p