Enhanced infragranular and supragranular synaptic input onto layer 5 pyramidal neurons in a rat model of cortical dysplasia

Abstract

Cortical dysplasias frequently underlie neurodevelopmental disorders and epilepsy. Rats with a neonatally induced cortical microgyrus [freeze-lesion (FL)], a model of human polymicrogyria, display epileptiform discharges in vitro. We probed excitatory and inhibitory connectivity onto neocortical pyramidal neurons in layers 2/3 and 5 of postnatal day 16-22 rats, approximately 1-2 mm lateral of the lesion, using laser scanning photostimulation (LSPS)/glutamate uncaging. Excitatory input from deep and supragranular layers to layer 5 pyramidal cells was greater in FL cortex, while no significant differences were seen in layer 2/3 cells. The increased input was due to a greater number of LSPS-evoked excitatory postsynaptic currents (EPSCs), without differences in amplitude or kinetics. Inhibitory input was increased in a region-specific manner in pyramidal cells in FL cortex, due to an increased inhibitory postsynaptic current (IPSC) amplitude. Connectivity within layer 5, parts of which are destroyed during lesioning, was more severely affected than connectivity in layer 2/3. Thus, we observed 2 distinct mechanisms of altered synaptic input: 1) increased EPSC frequency suggesting an increased number of excitatory synapses and 2) higher IPSC amplitude, suggesting an increased strength of inhibitory synapses. These increases in both excitatory and inhibitory connectivity may limit the extent of circuit hyperexcitability.

Figure 1.

Generation of monosynaptic input maps using LSPS. (A) Nissl-stained sections from control (upper) and FL (lower) cortex. The lesion is marked with an asterisk. Black rectangles mark area shown in (B). (B) Enlarged area indicated by rectangles in (A) from control (left) and FL (right) cortex. Approximate layer borders are indicated by dashed lines. Numbers indicate layers; wm, white matter. Cortical lamination does not differ systematically between control and FL. (C) Examples of cortex as view in live slices in the recording chamber. Midline is toward the left. Note that layers 1, 2/3, 4 and underlying white matter are well resolved in the Nissl-stained sections and can also be clearly distinguished in the live slice. The border between 5 and 6 is ambiguous in the live slices, and therefore, the laminar thickness analysis in (D) combines them and represents the composite depth as layer 5/6. Scale bar in (B) applies to (C). (D) Thickness of cortical layers as estimated from overview images such as the ones shown in (A) for 25 slices each in control and FL cortex 1.5–2.5 mm from the central sulcus. There were no significant differences between FL and control cortex. (E) Example input map grid of a layer 5 cell in FL cortex. White circles indicate the LSPS grid; some spots are marked with lower case letters. The pipette tip indicates the position of the soma, approximately at spot “h”. (F) Example traces recorded at −65 mV (left) and −5 mV (right) at the locations indicated by lowercase in (E). The gray line corresponds to the time of LSPS stimuli; circles indicate evoked PSCs. Boxes delineate detection windows for EPSCs (box 1 on left, 10–50 ms), box 2 (right) outlines the detection window used to detect slIPSCs (2–10 ms), box 3 adjacent to box 2 outlines the detection window for rlIPSCs (10–50 ms). Selected traces are ordered to have minimum overlap and to show a progression from no to high LSPS-evoked activity. (G) Input maps generated from the cell shown in (E and F). (G1) EPSCs (box 1 from D); (G2) slIPSCs (box 2 from D); and (G3) rlIPSCs (box 3 from D). x- and y-axes correspond to the grid in (C); grayscale values represent the cumulative amplitude of evoked events (number of events times their average amplitude), going from white to black (low to high cumulative amplitude). The triangle indicates the location of the recorded cell's soma.

Figure 2.

Direct excitation profiles do not differ in FL and control cells. (A) Example map of LSPS-evoked action potentials recorded in cell-attached mode in a layer 5 pyramidal cells in sham-control cortex. Traces are superimposed onto the image of the slice to indicate the approximate location of LSPS and the location of the recorded cell's soma (indicated by the pipette tip). (B) Comparison of spike probability after LSPS in control and FL cortex in layer 2/3 cells (left panel; control: n = 10; FL: n = 7) and 12 layer 5 cells (right panel; control: n = 10; FL: n = 8). The probability to evoke a spike for each cell was averaged into 50 μm bins according to the distance between stimulation site and the soma. There were no significant differences between cells from sham-control and FL cortex. (C) Current responses (bottom traces) to equivalent stimuli producing just subthreshold and just suprathreshold depolarizations (top traces) in a layer 2/3 and a layer 5 pyramidal cells in control cortex. The layer 2/3 cell requires a much larger amplitude equivalent current to reach action potential threshold. (D) Summary of direct excitation parameters. (D1) Average equivalent currents for suprathreshold depolarization in layer 2/3 (control: n = 9, FL: n = 11) and layer 5 pyramidal cells (control: n = 10, FL: n = 6). Although “threshold currents” differed significantly between layers 2/3 and 5, there were no statistical differences between cells from FL and control cortex in either layer. (D2) Average spikes per hotspot (i.e., LSPS site from which action potentials were evoked) for the same set of cells shown in (B). There were no significant differences between cells from FL and control cortex. (D3) Median distance of hotspots from the recorded cells' soma, same set of cells as those shown in (B). There were no significant differences between cells from FL and control cortex.

Figure 3.

Direct glutamatergic currents do not differ in FL and control cells. (A) Example traces from a layer 2/3 (A1) and a layer 5 (A2) pyramidal cells in sham-operated control cortex recorded at −65 mV, focusing on the time immediately following the stimulus (indicated by arrow). Currents rising within 3 ms of the stimulus, as indicated by the gray rectangles, were evoked by direct activation of glutamate receptors on the recorded neurons. (B) Maps representing the amplitude of direct currents generated from the cells shown in (A). The open white triangles indicate position of the soma in layer 2/3 (B1) and layer 5 (B2). Direct currents were evoked perisomatically and their amplitude decreased with increasing distance from the soma. (C) Comparison of direct currents evoked in 13 layer 2/3 cells (C1) and 12 layer 5 cells (C2) each in control and FL cortex. Current amplitudes for each cell were averaged into 50 μm bins according to the distance between stimulation site and the soma. There were no significant differences between direct glutamatergic currents in cells from control and FL cortex.

Figure 4.

Short-term plasticity of EPSCs is similar in FL and control cells. (A) Example EPSC responses to trains of 8 electrical stimuli (100 ms interstimulus interval, −65 mV holding potential) in layer 5 pyramidal cells from sham-control (black trace) and FL cortex (gray trace). (B) Paired pulse ratios (EPSC 2:EPSC 1 in each 8-pulse train) measured at interstimulus intervals of 25, 50, 75, 100, 250, 500, and 1000 ms in layer 5 (B1) and layer 2/3 (B2) pyramidal cells of control (filled circles; layer 2/3, n = 9; layer 5, n = 7) and FL (open circles; layer 2/3, n = 9; layer 5, n = 8) cortex. There were no significant differences between cells from control and FL cortex. (C) Ratios of the eighth to the first EPSC in the same 8-pulse trains analyzed in (B). The EPSC 8:EPSC 1 ratios at interstimulus intervals between 25 and 100 ms show larger depression than the EPSC 2:EPSC 1 ratios (B), but there were no significant differences between cells from control and FL cortex.

Figure 5.

Increased excitatory input onto layer 5, but not layer 2/3 cells in FL cortex. (A) Excitatory input maps for 13 layer 2/3 (A1) and 12 layer 5 (A2) pyramidal cells each in sham-operated control (left) and FL (right) cortex. Cumulative EPSC amplitudes in individual input maps were averaged and contour lines were derived by linear interpolation. Individual maps were rotated to be parallel to the pia and aligned to the position of the soma on the x-axis, and to the border between layer 1 and 2 on the y-axis. The color scale represents weak to strong inputs (blue to red), and purple represents activation above the highest level of the regular color scale to emphasize the weaker input patterns, especially in layer 5 control cells. The location of the soma (precise in x-axis, approximate in y-axis) is indicated by black solid triangles. The numbers and lines at the very left indicate the approximate position of the 6 cortical layers. (B) Averaged cumulative inputs for 50 μm rows for cells from control (black) and FL (red) cortex in layer 2/3 (B1) and layer 5 (B2). (C) Average cumulative EPSCs per stimulation site onto layer 2/3 (C1) and layer 5 (C2) cells originating from layers 2/3, 4/5a, and 5b/6. Input from layer 2/3 and from layer 5b/6 onto layer 5 cells was increased in FL cortex (P < 0.05 vs. control). There were no significant changes in layer 2/3 cells from FL cortex, although there was a trend toward increased inputs from layers 2/3 and 4/5a. (D) Average activation per stimulation site for each cell. EPSC input was significantly higher in layer 5 cells from FL cortex (P < 0.01; D2) and did not differ between control and FL cortex in layer 2/3 cells (D1). (E) Averaged cumulative inputs for 50 μm columns for cells from control (black) and FL (red) cortex in layer 2/3 (E1) and layer 5 (E2) fitted to Gaussian distributions. The half width of the Gaussian curves did not differ significantly between cells from control and FL cortex. (F) Average frequency of sEPSCs and eEPSCs (number of evoked events in the LSPS detection window/window duration, corrected for sEPSC frequency) in layer 2/3 (F1) and layer 5 (F2) pyramidal cells in control (black) and FL (red) cortex. The frequency of spontaneous and evoked EPSC is significantly higher in layer 5 pyramidal cells of FL cortex (P < 0.05), but the smaller increase in layer 2/3 cells is not statistically significant. To facilitate comparison of the number of spontaneous versus LSPS-evoked EPSCs, we have converted the number of eEPSCs/stimulated spot (Table 2) into frequency, given the detection window of 45 ms. (G) Average amplitudes of spontaneous EPSCs (sEPSCs detected outside of the LSPS window) and evoked EPSCs (eEPSCs, within 5–50 ms of the stimulus) in layer 2/3 (G1) and layer 5 (G2) pyramidal cells in control (black) and FL (red) cortex. Amplitudes of spontaneous and evoked EPSC do not differ in pyramidal cells of FL cortex.

Figure 6.

Increased regular latency inhibitory input onto layer 5, but not layer 2/3 cells in FL cortex. (A) Averaged input maps for rlIPSCs in 13 layer 2/3 (A1) and 12 layer 5 (A2) pyramidal cells each in sham-control (left) and FL (right) cortex. Maps were constructed as detailed for Figure 5. (B) Averaged inputs for 50 μm rows for cells from control (black) and FL (red) cortex in layer 2/3 (B1) and layer 5 (B2). The vertical inhibitory profile was altered in the FL for both cell types. Control layer 2/3 cells display a profile with clear peaks in layers 2/3 and 5, while FL 2/3 cells show a profile with a less prominent peak in layer 2/3 and a progressively decreased input in deeper layers. Layer 5 cells, by contrast, receive prominent inputs from layer 5b in control, which is shifted superficially to layer 4/5a in FL cortex. (C) Average cumulative rlIPSCs per stimulation site onto layer 2/3 (C1) and layer 5 (C2) cells originating in layers 2/3, 4/5a, and 5b/6. Input from layer 4/5a onto layer 2/3 and onto layer 5 cells was increased in FL cortex (P < 0.05 vs. control for both). (D) Average activation per stimulation site for each cell. rlIPSC input was significantly higher in layer 5 cells from FL cortex (P < 0.05; D2) and did not differ between control and FL cortex in layer 2/3 cells (D1). (E) Averaged cumulative inputs for 50 μm columns for cells from control (black) and FL (red) cortex in layer 2/3 (E1) and layer 5 (E2), for upper rows (to cortical depth of 400 μm, mainly layer 2/3) and lower rows. Gaussian fits suggest that input comes from a wider area within upper rows of layer 2/3 cells from FL cortex. (F) Average frequency of spontaneous and LSPS-evoked IPSCs in layer 2/3 (F1) and layer 5 (F2) pyramidal cells in control (black) and FL (red) cortex. The frequency of spontaneous and LSPS-evoked IPSC is not significantly altered in cells from FL cortex. The number of events was converted to frequency given a detection window of 40 ms. (G) Average amplitudes of spontaneous IPSCs and LSPS-evoked IPSCs (within 10–50 ms of the stimulus) in layer 2/3 (G1) and layer 5 (G2) pyramidal cells in control (black) and FL (red) cortex. Amplitudes of spontaneous and LSPS-evoked IPSC were significantly larger in layer 5 pyramidal cells of FL cortex (P < 0.05) but did not differ in layer 2/3 cells of sham-control and FL cortex.

Figure 7.

Increased short-latency inhibitory input onto layer 5 and 2/3 cells in FL cortex. (A) Input maps for slIPSCs in 13 layer 2/3 (A1) and 12 layer 5 (A2) pyramidal cells each in sham-control (left) and FL (right) cortex. Averaged maps were constructed as explained for Figure 5, except that maps were aligned to the position of the soma on both the x- and y-axes. (B) Comparison of slIPSC evoked in layer 2/3 (B1) and layer 5 cells (B2) in control (solid circles) and FL (open circles) cortex. Cumulative amplitudes for each cell were averaged into 50 μm bins according to the distance between stimulation site and the soma. Layer 2/3 and 5 cells received significantly more powerful short-latency PSC input, but it originated from similar cortical areas. (C) Average activation per stimulation site within 200 μm of the soma for each cell. slIPSC input was significantly higher in layer 2/3 and 5 cells from FL cortex (P < 0.05; C1 and C2, respectively). (D) Cumulative probability for amplitudes of slIPSC in layer 2/3 (D1) and layer 5 (D2) pyramidal cells in control (black) and FL (red) cortex. The curve for cells in FL cortex in both layers is shifted to the right, indicating higher IPSC amplitudes. (E) Left panel: average amplitudes of evoked slIPSCs (2–10 ms within stimulus) in layer 2/3 (E1) and layer 5 (E2) pyramidal cells in control (black) and FL (red) cortex. Right panel: average frequency of slIPSCs in layer 2/3 and 5 pyramidal cells in control (black) and FL (red) cortex. Number of events was converted to frequency given a detection window of 8 ms.

Figure 8.

Comparison of excitatory and inhibitory activation. (A) EPSC:rlIPSC ratios from each layer 2/3 cell mapped in FL (black diamonds) and sham-control (gray diamonds) cortex, for inputs originating in layer 2/3, 4/5a, and 5b/6. There were no significant differences between cells from FL and control cortex. (B) Same as (A), for layer 5 cells. Ratios representing inputs from layer 4/5a differed significantly between cells from FL and control cortex (P < 0.05), reflecting the increased rlIPSC input from that region.