Functionally Independent Columns of Rat Somatosensory Barrel Cortex Revealed with Voltage-Sensitive Dye Imaging

Morphology of excitatory neurons of layer 2/3 and layer 4 within a barrel column. A, Excitatory neurons were filled with biocytin during whole-cell recordings and subsequently stained. Axonal and dendritic arbors were reconstructed in three dimensions for 18 layer 2/3 and 18 layer 4 excitatory neurons. Two-dimensional projections into the original plane of the slice are illustrated from the pia to a depth including layer 5A. The dimensions of the reconstruction of each neuron are normalized with respect to the pia, bottom of layer 4, and the lateral width of the layer 4 barrel, thus generating a representation of a normalized barrel column. Layer 2/3 pyramidal neurons are shown with red dendrite andblue axon. Layer 4 spiny stellate neurons are shown withblack dendrite and green axon. The scale bar represents the mean normalization length. B, The same reconstructed neurons are shown with the dendritic and axonal compartments separated. The layer 4 dendrites are primarily confined to the layer 4 barrel. The layer 4 axon forms an ascending column of input to the layer 2/3 pyramidal neurons. The major lateral spread of neuronal arborization derives from the axons of the layer 2/3 pyramids.

Correlation of voltage-sensitive dye signal with dual whole-cell recordings of membrane potential. A, Bright-field image of somatosensory barrel cortex from a slice stained with RH155. Dark regions correspond to the barrels. Two whole-cell recording pipettes are visible, one in layer 2 (blue box) and one in layer 4 (red box). The extracellular stimulation electrode is visible at thebottom of the micrograph at the border of layer 4 and 5 in the middle of a barrel. The time series of pictures indicates the optical response of the voltage-sensitive dye after stimulation at 0 msec. The images were collected with 0.6 msec exposure time and are presented without filtering or averaging. Excitation can be observed to spread gradually from layer 4 to layer 2/3, maintaining a strict columnar excitation. Scale bar, 100 μm. B, Comparison of optical responses quantified from the 60 × 60 μmboxes indicated in A.Blue, layer 2/3; red, layer 4, with the membrane potential response of two individual neurons located within the boxes from which simultaneous whole-cell recordings were made. Three stimulus strengths are compared. As stimulus strength is increased, both the optical and the whole-cell responses increase in amplitude in a similar manner. Equally, the latency of responses in both layer 2/3 and layer 4 is similar in optical and whole-cell recordings. The decay kinetics are also similar, although in this example the voltage-sensitive dye signal decays somewhat slower.C, The amplitude of responses to the highest stimulation strength in an experiment was used to normalize the response to less intense stimuli and the relative response amplitude detected optically in a region 60 × 60 μm surrounding the soma of neurons from which the whole-cell recordings were made. The linear fit suggests a good correlation between the amplitudes of voltage signals detected optically and by whole-cell recordings. D, The latency to half-maximal response of recordings is greater in layer 2/3 (blue squares) than in layer 4 (red circles), as measured both optically and by whole-cell recordings. The dye latency, however, is somewhat slower than the latency determined by whole-cell recordings.

Voltage-sensitive dye responses within layer 4 are primarily confined to the stimulated barrel and associated cortical column. A, Bright-field micrograph (top left) of the region of barrel cortex imaged with the voltage-sensitive dye. Cyan boxes indicate extent of individual barrels, and the stimulation electrode is visible in the center of the barrel at the layer 4/5 border. Images of the peak voltage-sensitive dye signal (12 msec after stimulation) without (top right) and with (bottom left) barrel demarcation. The optical responses at the locations indicated bydots in the top left panel are plotted (bottom right), with responses from within the stimulated barrel colored blue, from the septum ingreen, and from the neighboring barrel inred. The spatial extent of the response decays rapidly outside of the stimulated barrel, with very little response at short latencies detected in neighboring barrels. A small response can, however, be detected late in the neighboring barrels. Scale bar, 100 μm. B, The normalized spatial extent of the optical dye signal within layer 4 (red curve) measured at 12 msec after stimulation is confined to the stimulated barrel (barrel septum shown in black, with half-maximal barrel boundary at 0 μm). The black superimposed curve is a sigmoidal fit, with half-maximal value at −1.09 μm and a length constant of 36.4 μm. The spatial extent of the associated signals in layer 2/3 is plotted in blue, indicating that, although the response is more diffuse, it also shows a marked decay over similar distances with half-maximal value at 9.85 μm and a transition length of 47.3 μm. However, >10% of the signal remains 200 μm outside of the stimulated barrel. C, The full-width at half-maximum of the voltage-sensitive dye signal shows a close correlation with the anatomically defined width of the barrels stimulated.

Barrel cortex slices cut tangential to the pia containing only layer 4 indicate that excitation is confined to the stimulated barrel. A, Bright-field micrograph (top left) of the region of tangentially sliced barrel cortex imaged with the voltage-sensitive dye. Cyan outline indicates the extent of the stimulated barrel with the stimulation electrode visible in the center. Images of the peak voltage-sensitive dye signal (12 msec after stimulation) without (top right) and with (bottom left) barrel demarcation. The optical responses at the locations indicated bydots in the top left panel are plotted (bottom right), with responses from within the stimulated barrel colored blue, from the septum ingreen, and from the neighboring barrel inred. The spatial extent of the response decays rapidly outside of the stimulated barrel with very little in neighboring barrels. The shape of the optical dye response matches closely the shape of the barrel viewed in bright-field microscopy. Scale bar, 100 μm. B, Quantification of the spatial extent of the voltage-sensitive dye signal with respect to the barrel border at 0 μm (red line indicates septum). The voltage-sensitive dye signal is fitted with a sigmoidal curve, with half-maximal value at 6 μm and length constant 32 μm. C, The full-width at half-maximum of the voltage-sensitive dye signal shows a close correlation with the width of the stimulated barrels, as defined by bright-field microscopy.

Block of inhibition by bicuculline evokes equivalent enhancement of whole-cell electrophysiological and voltage-sensitive dye responses. A, Time series of optical responses to stimulation of a layer 4 barrel under control conditions (top) or after application of 10 μm bicuculline to block GABA_A receptors (below). The same stimulation strength evoked a larger response with a longer duration in the presence of bicuculline. Scale bar, 100 μm. B, The effect of bicuculline on the response measured by voltage-sensitive dye in a region (60 × 60 μm) surrounding a whole-cell recording (black box inA). C, The effect of bicuculline on the responses measured with whole-cell recording. Thesetraces are averages of the same 16 consecutive sweeps as for the voltage-sensitive dye measurements in A andB. There is a close correspondence of the changes in amplitude and time course of responses with those of the voltage-sensitive dye. D, Consecutive individual sweeps of the whole-cell recordings under control conditions (left) and after application of bicuculline (right). No action potentials are evoked in control conditions, whereas several are evoked by each stimulus when inhibition is blocked. The responses to consecutive stimuli are similar.

Block of inhibition by bicuculline potentiates the spread of excitation in L2/3 and L5 but not layer 4. A, Schematic representation of the spread of excitation in paracoronal slices at 10 (red), 15 (green), and 20 (blue) msec after stimulation. The black dashed line indicates that excitation in layer 2/3, and layer 5 often spread beyond the field of view. The spread of voltage-sensitive dye signal within layer 4 increases only a little compared with the expansion of excitation within layer 2/3 and layer 5.B, Individual example showing columnar excitation under control conditions (image at 12 msec) but large lateral spread of excitation in layer 2/3 and layer 5 after 10 μmbicuculline application (image at 50 msec). Scale bar, 100 μm.C, In tangential layer 4 slices, application of bicuculline has no obvious effect on the spatial extent of excitation detected by optical imaging (control image at 12 msec; bicuculline image at 40 msec). Scale bar, 100 μm.

Activity evoked by the simultaneous stimulation of neighboring barrels is summated linearly. A, Bright-field micrograph showing stimulation electrodes placed in the lower center of neighboring barrels. Stimulation of single barrels (left or right) evoked responses in the barrel column. The response to simultaneous stimulation of neighboring barrels (labeled left and right) appears to be equivalent to the simple sum (labeled sum) of the responses evoked by stimulation of the barrels separately. The voltage-sensitive dye images are shown at 12 msec after stimulus. Scale bar, 200 μm. B, Comparison of the full-width at half-maximal for the voltage-sensitive dye signals between the experimental simultaneous stimulation of neighboring barrels and the linear sum of the individual responses. In layer 4, the width of excitation is unchanged and only a small increase of width is detected in layer 2/3.