Dynamic representation of whisker deflection by synaptic potentials in spiny stellate and pyramidal cells in the barrels and septa of layer 4 rat somatosensory cortex

Abstract

Whole-cell voltage recordings were made in vivo from excitatory neurons (n = 23) in layer 4 of the barrel cortex in urethane-anaesthetised rats. Their receptive fields (RFs) for a brief whisker deflection were mapped, the position of the cell soma relative to barrel borders was determined for 15 cells and dendritic and axonal arbors were reconstructed for all cells. Three classes of neurons were identified: spiny stellate cells and pyramidal cells located in barrels and pyramidal cells located in septa. Dendritic and, with some exceptions, axonal arborisations of barrel cells were mostly restricted to the borders of a column with a cross sectional area of a barrel, defining a cytoarchitectonic barrel-column. Dendrites and axons of septum cells, in contrast, mostly extended across barrel borders. The subthreshold RFs measured by evoked postsynaptic potentials (PSPs) comprised a principal whisker (PW) and several surround whiskers (SuWs) indicating that deflection of a single whisker is represented in multiple barrels and septa. Barrel cells responded with larger depolarisation to stimulation of the PW (13.7 +/- 4.6 mV (mean +/- S.D.), n = 10) than septum cells (5.7 +/- 2.4 mV, n = 5), the gradient between peak responses to PW and SuW deflection was steeper and the latency of depolarisation onset was shorter (8 +/- 1.4 ms vs. 11 +/- 2 ms). In barrel cells the response onset and the peak to SuW deflection was delayed depending on the distance to the PW thus indicating that the spatial representation of a single whisker deflection in the barrel map is dynamic and varies on the scale of milliseconds to tens of milliseconds. Septum cells responded later and with comparable latencies to PW and SuW stimulation. Spontaneous (0.053 +/- 0.12 action potentials (APs) s(-1)) and evoked APs (0.14 +/- 0.29 APs per principal whisker (PW) stimulus) were sparse. We conclude that PSPs in ensembles of barrel cells represent dynamically the deflection of a single whisker with high temporal and spatial acuity, initially by the excitation in a single PW-barrel followed by multi-barrel excitation. This presumably reflects the divergence of thalamocortical projections to different barrels. Septum cell PSPs preferably represent multiple whisker deflections, but less dynamically and with less spatial acuity.

Figure 1. Position, morphology and sensory responses of a barrel spiny stellate cell

A, left panel shows a schematic representation of the whisker arrangement in the rat's right face. Right panel, schematic representation of the whisker barrel field of left hemisphere: A anterior, L lateral. The soma of reconstructed cell is located in the C2 barrel. Projection of dendritic (red) and axonal arbors (blue) onto the horizontal plane is shown relative to position of barrel borders (dashed lines); thickness of the axonal tracing has been slightly increased to improve visibility. B, left panel shows location of principal whisker (PW, wC2). Right panel illustrates two successive responses (V_m) to PW deflection evoking a PSP followed by an AP (upper trace) or a PSP (middle trace) at the deflection onset and offset respectively. The average response (20 trials) is shown in the lower trace. Time course of whisker deflection (wC2) is shown schematically below the records of membrane potential. C, stimulation of surround whisker (SuW, wD2): Two successive responses (V_m) to a surround whisker (wD2) deflection and averaged response (20 trials). Onset and offset stimulus artefacts are seen in all records as small upward deflections. The calibration bars apply to both B and C.

Figure 2. Subthreshold RF maps of a barrel spiny stellate cell

A, position of mapped whiskers. The box delineates those whiskers, whose responses were quantified. B, subthreshold peak response amplitude to onset of the deflection of different whiskers. Length of each bar represents amplitude of response. Position of the whisker is identified by the intersection of arc and row lines, respectively. C, dendritic and axonal arbors of the cell recorded from, when projected onto the horizontal plane, shown relative to barrel borders in the horizontal plane. D, same reconstruction projected onto a vertical plane, parallel to arc-2 (the projection plane is indicated in C by the dotted line).

Figure 3. Subthreshold RF map of a barrel spiny stellate cell

A, position of mapped whiskers. B, subthreshold peak responses to deflection onset. C, left panel: dendritic arbor of the cell recorded from, when projected onto the horizontal plane, shown relative to barrel borders in the horizontal plane; right panel: axonal arbor of the cell recorded from, when projected onto the horizontal plane, shown relative to barrel borders. D, dendritic and axonal arbor projected to a vertical (coronal) plane shown relative to position of barrel borders (the projection plane used for C is indicated by the dotted line).

Figure 4. Subthreshold RF map of a barrel pyramidal cell

A, position of mapped whiskers. B, subthreshold peak responses to deflection onset. C, dendritic and axonal arbors of the cell recorded from, when projected onto the horizontal plane, shown relative to barrel borders in the horizontal plane. D, dendritic and axonal arbor projected to a vertical (coronal) plane shown relative to position of barrel borders (the projection plane used for C is indicated by the dotted line). The axon extends to the white matter, but reconstruction is not completely shown.

Figure 5. Subthreshold RF map of a barrel pyramidal cell

A, position of mapped whiskers. The box delineates those whiskers, whose responses were quantified. B, subthreshold peak response amplitude to onset of the deflection of different whiskers. Length of each bar represents amplitude of response. Position of the whisker is identified by the intersection of arc and row lines, respectively. C, dendritic and axonal arbors of the cell recorded from, when projected onto the horizontal plane, shown relative to barrel borders in the horizontal plane. D, same reconstruction projected onto a vertical plane, parallel to arc-2 (the projection plane is indicated in C by the dotted line).

Figure 6. Subthreshold RF maps of a pyramidal a cell located in septum

A, position of mapped whiskers. The box delineates those whiskers, whose responses are illustrated. B, subthreshold peak response amplitude to onset of the deflection of different whiskers. Length of each bar represents amplitude of response. Position of the whisker is identified by the intersection of arc and row lines, respectively. C, dendritic and axonal arbors of the cell recorded from, when projected onto the horizontal plane, shown relative to barrel borders in the horizontal plane. D, same reconstruction projected onto a vertical plane, parallel to arc-2 (the projection plane is indicated in C by the dotted line).

Figure 7. Comparison of averaged subthreshold RF map and dendritic and axon segment 2D density plots of barrel cells

A, average subthreshold RF map for barrel cells. The black grid indicates whisker positions, given with respect to the PW. Surround whisker positions are given by intersection of horizontal and vertical lines. The number −2 refers to SuW located in the second arc away from the PW in the caudal direction. The number +2 indicates whiskers in the second arc away from the PW in the rostral direction. The response amplitude is encoded by the brightness normalised to the peak of the PW deflection amplitude as determined in each individual experiment. The black contour line delineates an area on the RF map responding with ≥ 50 % of the PW peak response amplitude. An average cytoarchitectonic barrel field pattern (white, dashed lines) in the horizontal plane is superimposed for comparison of the average RF structure of a barrel cell with the cytoarchitectonic barrel structure. Outlines above the PW barrel correspond to barrels located laterally of the PW barrel. Outlines right to the PW barrel outline correspond to barrels located anterior to the PW barrel. B, 2D map of dendritic ‘length density’ (red, n = 6) projected onto a horizontal plane. An average cytoarchitectonic barrel field projected into the horizontal plane (white, dashed lines) is superimposed for comparison with dendritic density map and barrel structure. Convention on location of barrels as in A. The inner white contour line delineates an area that contains densities that are ≥ 50 % of the maximal density of dendrite segments. The outer white contour line delineates an area that includes 80 % of all dendritic segments. C, 2D map of axon ‘length density’ (n = 6, blue) projected onto a horizontal plane. Barrel field structure and white contour lines as described in B for dendritic segments. D, 2D map of dendritic length density (n = 12, red) projected onto a vertical plane. The map includes three spiny stellate cells whose barrel position was inferred from their asymmetric orientation of dendrites. The dashed lines represent outlines of averaged barrels. The outlines to the right of the PW barrel correspond to barrels located lateral to the PW barrel. White lines are contour lines as described in B. E, 2D map of axon length density (n = 12, blue). Projection onto a vertical, coronal plane along the arcs. Barrel outlines as in D, white lines are contour lines as described in B. Data from barrel cells include both spiny stellate and pyramidal neurons.

Figure 8. Comparison of averaged subthreshold RF maps and dendritic and axonal density plots of septum cells

A, average subthreshold RF map for septum cells. The black grid matches whisker positions given with respect to the PW as shown in Fig. 7. The central black contour line delineates the area responding to a response amplitude of 50 % or more of the PW deflection response. An average cytoarchitectonic barrel pattern in the tangential plane (white dashed lines) is superimposed for comparison of RF structure and cytoarchitectonic structure of the barrel field (n = 5 cells). Location of whiskers and barrels as described in Fig. 7A. B, 2D map of dendritic density (red, n = 5) projected onto a tangential plane. Cell reconstructions (n = 5) were aligned by superimposing the centres of their home septum prior to averaging. An average barrel pattern in the tangential plane (white dashed line) is superimposed for comparison between the dendrite density map and the dimensions of septa in the horizontal plane. The white contour lines delineate the areas that contain more than 50 % (inner line) of the maximal density of dendrite segments or more than 80 % of the total dendrite length (outer line). C, 2D map of axon length density (blue, n = 5) projected onto a horizontal plane. Barrel field structure and white contour lines as described in B for dendritic segments. D, 2D map of dendrite length density (red, n = 2), projected onto a vertical (coronal) plane along the arcs. The dashed lines represent outlines of averaged barrels, whereby outlines to the right from the septum correspond to barrels located laterally. White contour lines are as described in B. E, 2D map of axon length density (blue, n = 2), projected onto vertical (coronal) plane along the arcs. Barrel outlines as in D, white lines are contour lines as described in B.

Figure 9. Comparison of averaged sub- and suprathreshold RF maps

A, average suprathreshold (AP) RF map for cells (n = 10) located in barrels. Prior to averaging RFs were aligned to the PW position determined by the subthreshold RF map. The position of a whisker is given by the intersection of horizontal and vertical lines. APs refers to number of APs per deflection. B, average subthreshold RF map for cells (n = 10) located in barrels. C, average suprathreshold (AP) RF map for cells (n = 5) located in septa. D, average subthreshold RF map for cells (n = 5) located in septa. To reveal the differential responsiveness of barrel and septum cells, the top two (A and C) and the bottom two graphs (B and D) have the same scale. B-D, conventions as in A.

Figure 10. Direction sensitivity

A, subthreshold average responses of a barrel pyramidal cell to whisker deflection in different directions. Clockwise the direction is upward, forward, downward and backward as indicated by arrows. Stimulation artefacts are indicated by the small arrow in the top trace. Average responses from 20 stimuli. Calibration bars refer to all traces. B, direction tuning of PSPs and APs, same cell as shown in A. The four axes indicate (clockwise) upward, forward, downward and backward deflections. The shaded area refers to APs per stimulus. The lines indicate peak EPSP amplitudes. Same scaling for all directions as indicated for the forward direction. C, directionality indices for PSPs of different types of L4 cells tested (n = 13). A value of 1 would represent responses in only one direction whereas a value of close to 0 represents responses that are independent of direction of deflection. Error bars represent ± 1 s.d. Means are not statistically different. D, direction preferences of all recorded cells, convention for direction as in B.

Figure 11. Single- and multi-whisker stimulus responses

Plots of multi-whisker stimulus depolarisation amplitudes against single-whisker PW deflection depolarisation amplitudes in barrel cells (left) and septum cells (right). Symbols represent spiny stellate cell (open circles), barrel pyramidal cells (filled triangles), septum pyramidal cells (open triangles). Crosses indicate means ± s.e.m.

Figure 12. AP responses of barrel and septum cells

A, left: records of 10 superimposed responses of a spiny cell to PW deflection; right: peri-stimulus time histograms (PSTHs) of six spiny stellate cells. B, left: ten superimposed responses to PW deflection of a barrel pyramidal cell, right: PSTHs of seven barrel pyramidal cells. Four cells did not respond with APs. C, left: ten superimposed responses to PW deflection of a septum pyramidal cell; right: PSTHs of five septum cells, two cells did not respond. D, population PSTH of all L4 cells. AP responses of five pyramidal cells of undetermined position within a barrel have also been included. Bin width of PSTHs is 0.5 ms. The peak AP response to stimulus onset is at 11.5 ms.

Figure 13. Response latency in barrel and septum cells

A, response onset latencies in a barrel cell in response to deflection of whiskers at different RF positions. The labels on each trace refer to the whisker deflected. E1 is the PW. Records with median latencies are selected for each whisker position. B, response onset latencies in a barrel cell deflections of whiskers at different RF positions Stimulus artefacts are partially blanked in A and B. Calibration in A applies to A and B. C, population statistics for response onset latencies of barrel (open bars) and septum (filled bars) cells, error bars indicate ± 1 s.e.m. Mean (± 1 s.d.) onset latencies for barrel and septum cells are respectively: PW 8.0 ± 1.4 ms and 11 ± 2 ms, 1st order SuW 9.9 ± 2.1 ms and 11.6 ± 2 ms, 2nd order SuW 11.0 ± 4.2 ms and 13.5 ± 2.8 ms. The differences between barrel and septum cell onset latencies are significant for all whisker positions. D, population statistics for response peak latencies of barrel (open bars) and septum (filled bars) cells, error bars indicate ± 1 s.e.m. Mean (± 1 s.d.) peak latencies are for barrel and septum cells, respectively: PW 15.0 ± 6.8 ms and 22.3 ± 6.8 ms, 1st order SuW 19.6 ± 6.8 ms and 15.8 ± 8.6 ms, 2nd order SuW 19.9 ± 5.8 ms and 27.4 ± 8.9 ms. The differences between barrel and septum cell onset latencies are significant for PW and 2nd order SuW responses.

Figure 14. Time dependence of subthreshold RF structure of barrel and septum cells

The grid of white lines represents the localisation of a whisker with respect to the PW as intersection between horizontal and vertical lines. Response amplitudes are normalised with respect to the response to PW stimulation. Top, averaged and smoothed subthreshold RFs of barrel cells (n = 5 cells) at different times after whisker deflection. The same grey scaling applies to all RF plots. The white lines delineate the area of ≥ 80 % and ≥ 50 % of the maximal response to PW stimulation (maximal response at 15–20 ms for barrel cells and at 40 ms for septum cells). For clarity, the contour lines are shown only in those RF plots where the response reaches at least 50 % of the maximal amplitude. The dashed white lines delineate the outlines of average anatomical barrels arranged around the PW barrel. Barrel identification as in Figs 7 and 8. Bottom, same plots for four septum cells. Same conventions as in Figs 7A and 8A.

Figure 15. Responses of barrel and septum cells to repetitive PW stimuli

Stimuli were brief (2 ms) backward and forward deflections (6 deg) in the horizontal direction applied at 10 Hz. All but the first needlelike stimulation artefacts in the respective traces (V_m) have been blanked. Bottom trace labelled ‘w’ indicates sequence of whisker deflections. A, response average of three spiny stellate cells. Peak response amplitudes to each deflection in the train is maintained. B, response average of eight barrel pyramidal cells with decreasing peak response amplitude during the train. C, response average of four septum pyramidal cells with strongly decreasing peak response amplitude. D, dependence of PW evoked subthreshold peak responses normalised to the response evoked by the first deflection in a train. Spiny stellate cell (open circles) maintain their response, barrel pyramidal cells (filled triangles) and septum pyramidal cells (open triangles) show smaller responses with successive deflections in the train.