Article Figures & Data
Figures
- Figure 1.
The slow stepwise propagation of 0 Mg2+ epileptiform events reveals prominent feedforward inhibition. A, Schematic of a coronal slice showing the recording electrode (labeled e) and the location of a line scan through layer 5. Imaging was done at 30 Hz using a 10× objective. L, Lateral; D, dorsal. Bi, Line scan showing the neuropil Ca2+ fluorescence as an epileptiform event progresses from top to bottom; distance is plotted on the ordinate, and time is on the abscissa. Each row of pixels represents the neuropil Ca2+ fluorescence from a bin 40 × 4 μm (165 bins is equivalent to 660 μm total length). The location of the recording electrode (labeled e) is shown. Bii, The Vclamp recording from the layer 5 pyramidal cell plotted below on the same timescale. The cell was held at −30 mV, approximately halfway between the reversal potentials for GABA and glutamate, to distinguish inhibitory drives (upward deflections) and excitatory drives (downward deflections). Note the prominent inhibitory volleys (arrowed) corresponding with upstream activity in the line scan.
- Figure 2.
Correlated synaptic barrages in layer 5 pyramidal cells. A, Vclamp recordings from a pair of layer 5 pyramids (Pyr 1, Pyr 2) located 395 μm apart. B, An expanded view of same recording. The arrows highlight the correspondence between excitatory drive in the earlier activated cell and inhibition of later activated cell. C, D, Enlarged view of one of the early synaptic volleys in the event, showing the tight temporal correlation between the excitation in the earlier activated cell with the inhibition in the later activated one. The correlation coefficient achieves its maximal negative value close to 0 time difference. E, F, The late barrages also show a high temporal correlation, and, because they are both predominantly excitatory, there is a peak in the correlation coefficient close to 0.
- Figure 3.
Input–output functions from layer 5 pyramid cells during epileptiform events indicate an inhibitory veto. A, Paired Vclamp recordings from adjacent layer 5 pyramidal cells show that adjacent cells experience almost identical synaptic volleys during the epileptiform events. The bottom panels show an expanded view of the same recordings. B, Paired Vclamp and Iclamp recordings from adjacent layer 5 pyramidal cells. Early in the event, pyramid 2 fires only occasionally despite the Vclamp recording showing relatively intense excitatory barrages. The derivative of the Vclamp recordings (dI/dt) highlights the role of the large-amplitude inhibitory volleys, showing that the development of intense firing during the paroxysmal depolarizations occurs only as the inhibitory volleys wane (arrow). C, Paired recordings from adjacent layer 5 pyramids, with one cell being held in Vclamp mode, whereas the second cell is recorded in cell-attached mode. This preserves the physiological integrity of the cell better than whole-cell current clamp yet still permits a high quality recording of the action potential trains. Like the Vclamp/Iclamp pairs, these recordings show an abrupt transition to firing synchronous with the cessation of the intense inhibitory restraint, as indicated by the sudden change in the dI/dt trace (arrow). Pyr 1, Pyramidal cell 1; Pyr 2, pyramidal cell 2.
- Figure 4.
The transition coincides with a sharp drop in the index of inhibition. Analysis of the excitatory barrages either side of the transition from paradoxical quiescence to intense bursting for three recordings from adjacent pyramids, in which there was a long delay between the first excitatory barrages and the first action potential burst. We analyzed 200 ms bins around the peaks of the inward currents in the Vclamp recordings, to derive the number of action potentials in the Iclamp/cell-attached recorded cell in that bin (i), the peak excitatory shift in the holding current in the Vclamp cell (ii), and the degree of inhibition as assessed by the dI/dt plots (iii) (for more details, see Results). Bi, The mean ± SEM firing rate (normalized) for the 20 excitatory bursts immediately preceding the transition and the 10 immediately after. Bii, Equivalent plot for the peak excitatory shift in the holding current for the Vclamp cell. Biii, Same plot showing the drop in inhibition at the time of the transition.
- Figure 5.
The inhibitory barrages occlude very powerful excitatory synaptic drives. Ai, GABAA postsynaptic currents recorded at six different holding potentials (−100 to 0 mV; 20 mV steps) in a layer 5 pyramidal cell, evoked by extracellular stimulation. Aii, Current–voltage plot showing reversal of the GABAA current at −59.3 mV. B, Paired voltage-clamp recordings from two adjacent layer 5 pyramidal cells (P1, P2). One cell is held at −30 mV, whereas the second is held at the experimentally determined GABAA reversal potential. The bottom traces show an expanded view of the first two barrages. The −60 mV recording shows a very large inward current simultaneous with the inhibitory barrages in the other cell. C, We used an average of the −60 mV preictal excitatory current to determine how much this excitatory drive exceeds the normal threshold for triggering action potentials. Ci, Average excitatory current during a preictal barrage derived from the somatic voltage-clamp recording at −60 mV of five preictal barrages (maximum inward current, 1.12 pA). Cii, Five consecutive current-clamp recordings from a different layer 5 pyramidal cell when 6% of the excitatory drive shown in Bi is injected at the soma (firing in 3 traces and failures in 2). This level of excitatory drive gave approximately equal numbers of single action potentials or failures in the traces. Action potentials tended to be aligned closely to the peak current, justifying our decision to concentrate on 200 ms windows around the peak inward current for our previous analyses depicted in Figure 4. Ciii, The excitatory drive induces intense, ictal-like firing when injected at the soma in the absence of any inhibition.
- Figure 6.
Inhibitory barrages are effectively nullified by low doses of picrotoxin. A, B, Voltage-clamp recordings from layer 5 pyramidal cells held at −30 mV when 2.5 μm PTX is bath applied simultaneously with the washout of Mg2+ ions. A shows the single example in which the inhibitory barrages were reasonably preserved at this PTX concentration. The inset shows an expanded view of a preictal burst. B shows four preictal bursts from a more typical example, with the first burst shown at an expanded timescale in the inset. The characteristic upward deflections (see arrows in A) in the voltage-clamp trace are absent, being replaced by episodic, massive excitatory barrages (arrows). C, Synaptic barrages in 5 μm PTX; several preictal events are shown as well as the first full ictal event recorded. Again, the upward deflections are absent. Inhibitory barrages are never seen with 5 or 10 μm (data not shown) picrotoxin, despite the occurrence of isolated IPSC being readily apparent during quiescent periods in all recordings (inset). D, Summary plot showing the proportion of experiments at the different levels of picrotoxin that displayed inhibitory barrages. These preictal inhibitory barrages were always seen in 0 Mg2+ in the absence of GABAA blockers.
- Figure 7.
Somatic Ca2+ fluxes reveal episodic localized recruitment. A, The derivation of the cellular Ca2+ signal. We recorded from adjacent pyramids (Pyr 1, Pyr 2). Pyr 1 was recorded in Vclamp mode to monitor the local synaptic activity. Pyr 2 was a labeled cell that we recorded in cell-attached mode to allow the Ca2+ signal to be related to the action potential train. The simple somatic Ca2+ signal shows several prominent deflections that reflect the synaptic barrage (dashed line arrows) but importantly do not reflect the action potential train. Subtracting the neuropil signal removes these artifactual deflections (third trace). The derivative of the signal highlights the sudden increases in fluorescent signal, which reflect the sudden recruitment of neurons. The bottom panel shows how this derivative trace is transformed into the grayscale plots shown in B, which shows grayscale representations from 152 identified neurons. Bi, Grayscale plot of the temporal derivatives (dCS/dt) of the center–surround signals from 152 neurons ordered by the time of the maximum for each trace. Each row of pixels represents a dCS/dt trace: high rate of change of fluorescence is depicted as lighter gray. Each trace has several peaks. Bii, The same plot, with the single maximum rate of change for each cell marked (red). This ordering clearly shows three main clusters of cells. They are color-coded as shown, and their positions are indicated in the photomicrograph in Biii. Note the tight and generally non-overlapping spatial distributions of the yellow and red groups in layer 4. The lamina borders were determined from the location of the filled cells after post hoc staining for biocytin and matching this up to the electrode location in the field of view (the faint triangular shadow of one of the electrodes can just be seen at the bottom right corner of the field of view). Movie was taken using a 20× objective.
Additional Files
Supplemental Data
Files in this Data Supplement:
- supplemental material - Supplemental material legends
- supplemental material - Movie 1
- supplemental material - Figure 1
- supplemental material - Figure 2
- supplemental material - Figure 3
- supplemental material - Figure 4
- supplemental material - Figure 5
- supplemental material - Figure 6
