Functionally independent columns of rat somatosensory barrel cortex revealed with voltage-sensitive dye imaging

Abstract

Whisker movement is somatotopically represented in rodent neocortex by electrical activity in clearly defined barrels, which can be visualized in living brain slices. The functional architecture of this part of the cortex can thus be mapped in vitro with respect to its physiological input and compared with its anatomical architecture. The spatial extent of excitation was measured at high temporal resolution by imaging optical signals from voltage-sensitive dye evoked by stimulation of individual barrels in layer 4. The optical signals correlated closely with subthreshold EPSPs recorded simultaneously from excitatory neurons in layer 4 and layer 2/3, respectively. Excitation was initially (<2 msec) limited to the stimulated barrel and subsequently (>3 msec) spread in a columnar manner into layer 2/3 and then subsided in both layers after approximately 50 msec. The lateral extent of the response was limited to the cortical column defined structurally by the barrel in layer 4. Two experimental interventions increased the spread of excitation. First, blocking GABA(A) receptor-mediated synaptic inhibition caused excitation to spread laterally throughout wide regions of layer 2/3 and layer 5 but not into neighboring barrels, suggesting that the local excitatory connections within layer 4 are restricted to single barrels and that inhibitory neurons control spread in supragranular and infragranular layers. Second, NMDA receptor-dependent increase of the spread of excitation was induced by pairing repetitive stimulation of a barrel column with coincident stimulation of layer 2/3 in a neighboring column. Such plasticity in the spatial extent of excitation in a barrel column could underlie changes in cortical map structure induced by alterations of sensory experience.

Fig. 1.

Experimental setup for simultaneous whole-cell and voltage-sensitive dye recording. A, Bright-field image of somatosensory barrel cortex from a slice stained with RH155.Dark regions correspond to the layer 4 barrels, which are outlined in cyan. Two whole-cell recording pipettes are visible, one in layer 2/3 and the other in layer 4. A large-diameter patch pipette filled with extracellular solution is used for field stimulation, with the tip positioned at the bottom of the central barrel. B, The excitatory neurons are filled with biocytin during the whole-cell recordings, allowing their structure to be visualized after fixation and staining.C, Reconstruction of the biocytin-filled layer 2/3 pyramidal neuron (reddendrite andblueaxon) and the layer 4 spiny stellate (blackdendrite and greenaxon). D, A single unfiltered differential image normalized to the bright-field transmitted light (ΔI/I₀) of the voltage-sensitive dye signal taken 12 msec after stimulation.E, Time course of voltage-sensitive dye responses (top traces) compared with the simultaneously obtained whole-cell recordings (bottom traces). The voltage-sensitive dye signals were quantified from an area of 50 × 50 μm around the location of the neurons from which the whole-cell recordings are made (layer 2/3 red traces and layer 4blue traces). F, Optical arrangement for simultaneous whole-cell recordings and voltage-sensitive dye recordings. The slice is illuminated with 700 nm light from a halogen lamp and viewed with a 10× water immersion lens. The slice is stained with voltage-sensitive dye RH155, which increases its absorption of 700 nm light when membranes are depolarized. Small changes in the amount of light transmitted through the slice can be recorded with a Fuji Deltaron camera. With a square field of view of 775 μm obtained under these conditions, each pixel covers a region of 6 × 6 μm, and each frame has 0.6 msec duration.

Fig. 2.

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.

Fig. 3.

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.

Fig. 4.

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.

Fig. 5.

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.

Fig. 6.

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.

Fig. 7.

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.

Fig. 8.

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.

Fig. 9.

Pairing stimulation of a barrel column with a neighboring region of layer 2/3 induces an NMDA receptor-dependent expansion of excitation. A, The experimental arrangement and stimulation protocol used to induce a spatial expansion of responses (top left). Stimulation of the electrode in layer 2/3 placed to the right of the barrel column evoked a local response (top right). Stimulation of the electrode at the lower center of a barrel evoked a columnar response (bottom left). Both electrodes were then stimulated simultaneously five times at 50 msec intervals, and this was repeated at 5 sec intervals for a total of 20 pairing bursts. Responses were then tested 15–20 min after this pairing protocol, and the area within layer 2/3 excited by stimulating a barrel was found to have expanded toward the layer 2/3 stimulating electrode located on the right (bottom right). The voltage-sensitive dye images are shown at 12 msec after stimulus. Scale bar, 200 μm. B, The voltage-sensitive dye signal across the lateral extent of layer 2/3 after the pairing is selectively enhanced on the right of the barrel column at which the layer 2/3 stimulating electrode was located. A region at equal distance from the peak response but to the left shows no potentiation. The difference between left and right regions shows that the spatial extent of the layer 2/3 signal can be asymmetrically changed. Same experiment as shown in A.C, Subtracting the lateral voltage-sensitive dye signal after pairing from the control signal shows that the signal is increased on the right after pairing within layer 2/3, but within layer 4 there is no asymmetrical change. Same experiment as shown inA and B. D, Across all experiments, the changes at equal distances to the left and to the right of the peak are plotted. On average, only a small rundown of responses is observed after pairing in the presence of APV, but under control conditions, there is a significant selective enhancement of responses to the right of the peak, which is the region close to the layer 2/3 stimulating electrode. E, A schematic drawing of the changes induced by pairing the response of a barrel column with excitation of a neighboring region of layer 2/3 on the right.