Confocal imaging and 3D reconstructions of pairs and triplets of layer V pyramidal neurons. (A) Flattened representation from 3D image stacks of a pair of pyramidal neurons in layer V, as acquired by confocal microscopy. The entire span of the neurons is captured, allowing for a detailed investigation of all apposition points between axons and dendrites. (B) The high resolution of confocal imaging allowed for unambiguous tracing of axons and dendrites to create detailed 3D reconstructions. The higher neuron is colored blue for soma and dendrites and red for the axon. The lower neuron is colored green for soma and dendrites and black for the axon. (C) Geometrical connectivity between pyramidal neurons of layer V can be predicted from their characteristic morphology. We assumed a setting of two layer V pyramidal neurons with any spatial separation between their somata. The contour plot shows the expected number of touch sites as a function of the relative somatic location of the two neurons when one of them is located at the center. The plot was calculated from the neuronal morphologies obtained from the 3D reconstructions (18) and corrected for neuropil volume.

Geometrical connectivity. (A) Three-dimensional rendering of confocal microscope images of a close apposition between an axon and dendritic shaft in which no presynaptic bouton is observed. (B) Three-dimensional rendering showing a touch without synaptic bouton between an axon and a dendritic spine. (C) Three-dimensional rendering showing a putative synaptic contact between an axon and a dendritic shaft. (D) Three-dimensional rendering showing a putative synaptic contact between an axon and a dendritic spine. (Scale bar, 2 μm.)

Functional connectivity. (A) Synaptic boutons found between pairs of neurons after analysis of high-magnification confocal images and classified according to the functional connectivity of the neuronal pairs. C, connected; A, autapses; U, unconnected. (B) Example of functional connectivity between layer V pyramidal neurons. The presynaptic action potential train (Top) elicited synaptic responses in the first postsynaptic neuron (Middle) but not in the second (Bottom). The postsynaptic traces represent an average of 30 individual trials. (C) Correlation between EPSP amplitudes and the numbers of boutons in connected pairs. Amplitudes of unitary EPSPs (first EPSP in a train) are plotted against the number of boutons, as identified by confocal microscopy. The correlation coefficient is 0.77, and the slope of the linear fit is 0.41 ± 0.12 mV per bouton. (D) Negative correlation between the CV of EPSP amplitudes and the number of boutons. The CV was calculated as SD divided by the mean of EPSP amplitudes from at least 30 individual responses. The continuous line represents a fit to the inverse square function, which is predicted from models of CV analysis of quantal synaptic transmission.