Multibranch activity in basal and tuft dendrites during firing of layer 5 cortical neurons in vivo

Abstract

Layer 5 pyramidal neurons process information from multiple cortical layers to provide a major output of cortex. Because of technical limitations it has remained unclear how these cells integrate widespread synaptic inputs located in distantly separated basal and tuft dendrites. Here, we obtained in vivo two-photon calcium imaging recordings from the entire dendritic field of layer 5 motor cortex neurons. We demonstrate that during subthreshold activity, basal and tuft dendrites exhibit spatially localized, small-amplitude calcium transients reflecting afferent synaptic inputs. During action potential firing, calcium signals in basal dendrites are linearly related to spike activity, whereas calcium signals in the tuft occur unreliably. However, in both dendritic compartments, spike-associated calcium signals were uniformly distributed throughout all branches. Thus, our data support a model of widespread, multibranch integration with a direct impact by basal dendrites and only a partial contribution on output signaling by the tuft.

Keywords: dendritic integration; excitatory synapses; mouse motor cortex.

Fig. 1.

Small-amplitude subthreshold calcium signals are observed in all compartments. (A) Example data from a spontaneously active tuft dendrite of a L5 pyramidal neuron filled with 100 μM OGB-1 via a whole-cell recording pipette. Upper trace is the calcium fluorescence signal recorded at 40 frames per second. Lower trace is the simultaneously recorded membrane potential. Horizontal lines indicate baseline fluorescence for the calcium signal and mean down-state voltage for membrane potential. Gray boxes indicate epochs where up-states and significant calcium transients occur concurrently. Pink boxes indicate epochs where up-states occurred without a calcium transient. (B) Two-photon images and example data from different compartments of the same L5 pyramidal neuron as in A. (Left) Max projection of a Z-stack along the XZ plane obtained at the end of the experiment. Soma was located at ∼450 μm below the cortical surface. (Center) Imaging planes from example dendrites from the tuft (Top), apical trunk (Middle), and basal dendrites (Bottom). Solid lines indicate the ROI that was processed to form a calcium signal. Dashed red lines are placed at imaging depth for the indicated dendrite. At right of each imaging plane is an example calcium transient and the concurrently recorded membrane potential. (C) Histograms of the amplitude of subthreshold calcium transients for all dendritic branches recorded from the labeled compartment across all experiments (n = 43 apical tuft dendrites, 35 apical trunks, and 23 basal dendrites). Bin width is 2.5% ΔF/F.

Fig. 2.

Subthreshold calcium signals are not uniform within a dendritic branch. (A) Example local calcium signals in tuft dendritic branch from a L5 pyramidal cell. Soma was located at 520 μm below the pia. At left is a two-photon fluorescence image of a tuft dendritic branch. Colored lines show ROIs segmented at 10 μm intervals along the length of the branch. At right are traces for four example subthreshold events. Colored traces are calcium signals recorded from the ROI of the same color as in the fluorescence image. Dashed circles indicate the largest calcium transient associated with each subthreshold event. Rightmost example shows a contiguous subthreshold calcium event. Black trace is the membrane potential recorded in the whole-cell patch configuration. Bottom row shows amplitude for each calcium trace as given by the height of a bar of the same color. (B) Same as A but for a basal dendritic branch from a L5 pyramidal cell with soma located at 480 μm below the pia. A vertical line was placed in one example to facilitate comparison of differences in timing of signals in different ROIs. An arrow indicates a transient that begins later than in other ROIs from the same branch. (C) Histogram of the amplitude of categorized subthreshold calcium transients. Events were defined as contiguous if there was significant concurrent activity in at least three contiguous 10 μm ROIs, and otherwise were classified as distributed. Data were combined from different types of branches (n = 2 tuft dendrites, 1 oblique dendrite, and 4 basal dendrites). (D) Example traces from an experiment where 100 μM NBQX and 500 μM APV were injected below the cortical surface by applying positive pressure through a micropipette.

Fig. 3.

Relation between AP activity and dendritic calcium signaling. (A) Two-photon images of a spontaneously firing L5 pyramidal neuron filled with 100 μM OGB-1 via a whole-cell recording pipette. The soma was located ∼550 μm below the pia. Shown are imaging planes from example dendrites from the tuft (A, a), apical trunk (A, c), and basal dendrites (A, d). Solid lines indicate the ROI that was processed to form a calcium signal. Also shown is a max projection of a Z-stack along the XZ plane obtained at the end of the experiment (A, b). Dashed red lines indicate the imaging depth for imaging planes A, a; A, c; and A, d. (B) Example data from a spontaneously active dendrite indicated in A, a. Upper trace is calcium signal recorded at 40 frames per second. Lower trace is the simultaneously recorded membrane potential. Gray boxes indicate epochs where calcium activity and APs occur concurrently. Pink boxes indicate epochs where APs fired without calcium activity. (C) Same as B but using data from apical trunk shown in A, c. (D) Same as B but using data from basal dendrite shown in A, d. (E) Example data comparing the amplitude of calcium transients associated with subthreshold and superthreshold electrical activity. (Left) A two-photon fluorescence image of a basal dendrite from a L5 pyramidal cell with soma located at 520 μm below the pia. (Right) Two calcium traces above the concurrently recorded membrane potential. Example at left shows spike activity and at right shows subthreshold activity. The number of APs in the burst is indicated. Conventions are otherwise same as in A–C. (F) Scatterplot of the largest calcium transient associated with suprathreshold (empty squares) and subthreshold (filled circles) activity for all dendrites recorded in our study (n = 101 branches). Relative sparseness of subthreshold data points is due to exclusion of dendritic branches that lacked any detectable calcium transients in nonspiking intervals.

Fig. 4.

Basal and trunk dendrites but not tuft dendrites show strong linear correlation between AP firing and calcium activity. (A) Analysis of relation between spike activity and calcium activity in tuft dendrites. (A, a) Examples of spontaneous calcium activity in a single tuft dendrite when different numbers of spikes are fired. Upper trace is calcium signal, and lower trace is concurrently recorded membrane potential. The number of spikes fired in a burst is indicated at right in red. (A, b) Scatterplot of amplitude of calcium transients as a function of the number of spikes fired for a single dendritic branch. Spiking that was unaccompanied by a calcium transient is not included in this analysis. Red line is a best linear fit to the data. (A, c) Best linear fit of calcium amplitude versus number of spikes for all analyzed tuft branches. Gray lines show data from individual tuft dendrites. Dendrites that had less than 10 calcium transients or burst events were excluded from analysis. Red line indicates best linear fit from averaging all data. (B) Same as A but for data recorded from apical trunks. (C) Same as A but for data recorded from basal dendrites. (D) Bar graph of percent of spike events that were accompanied by a detectable calcium transient for different classes of dendrites. Black vertical lines indicate 95% confidence interval. (E) Scatterplot of correlation coefficient between number of spikes fired and amplitude of calcium signal as a function of the depth of the dendritic branch below the pia. Color of data point indicates class of dendrite recorded. All spike events were included in this analysis regardless of whether there was a detectable calcium transient in the imaged dendrite.

Fig. 5.

Suprathreshold calcium activity is global within the tuft and basal dendritic compartments. (A) Example data recorded simultaneously from multiple tuft dendrites. (Left) Fluorescence image of tuft dendrites from a L5 pyramidal cell with soma located 520 μm below the pia. Yellow dashed lines represent the path of dendrites located just outside the imaging plane. Colored solid lines are ROIs used to extract calcium signals. (Right) Example traces from this imaging plane. Colored traces are calcium signals that correspond to dendritic branches in ROIs at left. Vertical gray boxes denote a significant transient on at least one of the three traces. Black trace is the simultaneous membrane potential recorded in the whole-cell patch configuration. Bottom trace shows the amplitude (% ΔF/F) of the three calcium signals at each transient. Asterisk indicates a transient that appeared significantly larger than in the other ROIs. (B) Histograms of the difference in calcium amplitude between each pair of branches shown in A for all spike events (n = 133). (C) Same as A but for an imaging plane containing multiple basal dendrites. Soma is located at a depth of 460 μm. (D) Same as B but for data from dendrites shown in C (n = 36 events). (E) Data representing the observed differences in amplitude of calcium activity measured between pairs of simultaneously imaged tuft branches during spike activity (n = 13,791 events from 72 pairs of tuft dendrites). Data have been smoothed with a boxcar filter of width 1%. (F) Same as E but for basal dendritic branches (n = 40,657 events from 251 pairs of basal dendrites).