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. 2009 Feb 26;457(7233):1142-5.
doi: 10.1038/nature07709.

The Subcellular Organization of Neocortical Excitatory Connections

Free PMC article

The Subcellular Organization of Neocortical Excitatory Connections

Leopoldo Petreanu et al. Nature. .
Free PMC article


Understanding cortical circuits will require mapping the connections between specific populations of neurons, as well as determining the dendritic locations where the synapses occur. The dendrites of individual cortical neurons overlap with numerous types of local and long-range excitatory axons, but axodendritic overlap is not always a good predictor of actual connection strength. Here we developed an efficient channelrhodopsin-2 (ChR2)-assisted method to map the spatial distribution of synaptic inputs, defined by presynaptic ChR2 expression, within the dendritic arborizations of recorded neurons. We expressed ChR2 in two thalamic nuclei, the whisker motor cortex and local excitatory neurons and mapped their synapses with pyramidal neurons in layers 3, 5A and 5B (L3, L5A and L5B) in the mouse barrel cortex. Within the dendritic arborizations of L3 cells, individual inputs impinged onto distinct single domains. These domains were arrayed in an orderly, monotonic pattern along the apical axis: axons from more central origins targeted progressively higher regions of the apical dendrites. In L5 arborizations, different inputs targeted separate basal and apical domains. Input to L3 and L5 dendrites in L1 was related to whisker movement and position, suggesting that these signals have a role in controlling the gain of their target neurons. Our experiments reveal high specificity in the subcellular organization of excitatory circuits.


Figure 1
Figure 1. Subcellular Channelrhodopsin-2-Assisted Circuit Mapping (sCRACM)
a, Confocal image showing L2/3 neurons expressing mCherry (red) and ChR2-Venus (green) in the barrel cortex. b, Left, Schematic of the photostimulation geometry. Right, EPSCs evoked by photostimuli corresponding to the locations indicated in the schematic. Blue ticks indicate the laser pulse. Laser power is indicated on top. c, EPSCsCRACM evoked by photostimulation with increasing laser powers (right). d, Brightfield image of a brain slice showing the recording pipette and the photostimulation grid (blue dots). e, sCRACM map overlayed on a fluorescence image, showing ChR2-positive neurons, and the reconstructed dendrite of the recorded neuron (same experiment as d). Non-zero EPSCsCRACM are color-coded to represent mean amplitude. f, EPSCsCRACM recorded in the boxed regions in Panel e.
Figure 2
Figure 2. Subcellular distribution of inputs onto L3 pyramidal neurons
a, Examples of sCRACM maps overlaid on reconstructed dendrites and fluorescence images showing ChR2-positive axons (VPM, M1 and POm) or axons and dendrites (L2/3 and L4). White arrow head, bundle of ascending axons from M1. b, Group averages aligned by pia position (White triangles, soma position). c, top: Group averages aligned by soma position ; bottom: vertical profiles of the distribution of synaptic input (red) and the dendritic length density (green; from d). d, Average normalized dendritic length density of L3 pyramidal neurons. Error bars, s.e.m.
Figure 3
Figure 3. Subcellular distribution of inputs onto L5B pyramidal neurons
a, Examples of sCRACM maps overlaid on reconstructed dendritic arbors and fluorescence images. b, Group averages aligned by pia position. White triangles, soma position. c, sCRACM map of POm input onto a L5A pyramidal cell (blue). No responses were detected on the L5B neuron (green). d, EPSCsCRACM recorded on the L5A neuron (blue) or the L5B neuron (green) when photostimuating the boxed regions in Panel c . The stimulus occurred at the beginning of each trace.
Figure 4
Figure 4. The laminar position of L5 pyramidal neurons determines the dendritic location of L2/3 inputs
a, Examples of the subcellular distribution of L2/3 input on superficial (L5A) (left) or deep (L5B) (right) pyramidal neurons. b, Vertical profiles of the subcellular distribution of L2/3→L5 inputs. Each column represents one cell, ordered by cortical depth. Cells were aligned by pia position. The relative density of L2/3 axons in the deep layers is indicated to the left of the panel. c, Average subcellular location of L2/3 input (aligned by soma position) of L5 cells grouped by increasing distance from the pia (groups correspond to the white lines in b).d, Plot of the vertical distance from the soma to the center of mass of L2/3 input on the perisomatic region ( < 285 μm from the soma) of L5 pyramidal neurons vs. cortical depth. The line is a regression fit.

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