Initiation of network bursts by Ca2+-dependent intrinsic bursting in the rat pilocarpine model of temporal lobe epilepsy

Abstract

Chronically epileptic rats, produced by prior injection of pilocarpine, were used to investigate whether changes in intrinsic neuronal excitability may contribute to the epileptogenicity of the hippocampus in experimental temporal lobe epilepsy (TLE). Paired extra-/intracellular electrophysiological recordings were made in the CA1 pyramidal layer in acute hippocampal slices prepared from control and epileptic rats and perfused with artificial cerebrospinal fluid (ACSF). Whereas orthodromic activation of CA1 neurons evoked only a single, stimulus-graded population spike in control slices, it produced an all-or-none burst of population spikes in epileptic slices. The intrinsic firing patterns of CA1 pyramidal cells were determined by intrasomatic positive current injection. In control slices, the vast majority (97%) of the neurons were regular firing cells. In epileptic slices, only 53% the pyramidal cells fired in a regular mode. The remaining 47% of the pyramidal cells were intrinsic bursters. These neurons generated high-frequency bursts of three to six spikes in response to threshold depolarizations. A subgroup of these neurons (10.1% of all cells) also burst fired spontaneously even after suppression of synaptic activity. In epileptic slices, burst firing in most cases (ca 70%) was completely blocked by adding the Ca2+ channel blocker Ni2+ (1 mM) to, or removing Ca2+ from, the ACSF, but not by intracellular application of the Ca2+ chelater 1,2-bis(o-aminophenoxy)ethane-N,N,N ',N '-tetra-acetic acid (BAPTA), suggesting it was driven by a Ca2+ current. Spontaneously recurring population bursts were observed in a subset of epileptic slices. They were abolished by adding 2 M 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) to the ACSF, indicating that synaptic excitation is critical for the generation of these events. All sampled pyramidal cells fired repetitively during each population burst. The firing of spontaneously active bursters anteceded the population discharge, whereas most other pyramidal cells began to fire conjointly with the first population spike. Thus, spontaneous bursters are likely to be the initiators of spontaneous population bursts in epileptic slices. The dramatic up-regulation of intrinsic bursting in CA1 pyramidal cells, particularly the de novo appearance of Ca2+-dependent bursting, may contribute to the epileptogenicity of the hippocampus in the pilocarpine model of TLE. These findings have important implications for the pharmacological treatment of medically refractory human TLE.

Figure 1. Neuronal hyperexcitability and hypersynchrony in CA1 slices from epileptic rats

Comparative recordings were made in three CA1 slices obtained from naive-control (A), sham-control (B) and epileptic rats (C). Each pair of traces shows intra-/extracellular responses (upper/lower traces) of CA1 pyramidal cells to orthodromic stimuli of increasing intensity (from 2 to 12 V). Resting potentials (in mV) of the pyramidal cells in this and in all other figures are indicated to the left of the uppermost traces. Subthreshold stimulation evoked an EPSP-IPSP sequence in both control slices (A and B, 4 V), but a protracted EPSP in the epileptic slice (C, 2 V). Further increasing stimulus intensity evoked maximally a single spike in control tissue (as seen at both single cell and population levels; A and B, 8 and 12 V), but an all-or-none spike burst in epileptic tissue (C, 4, 8 and 12 V).

Figure 2. Variant intrinsic firing patterns in CA1 pyramidal cells in epileptic tissue

A-D, recordings from four different neurons arranged according to a gradient of increasing propensity to burst fire. The neurons were stimulated with threshold-straddling 200 ms (a) and 4 ms (b) depolarizing current pulses injected through the recording microelectrode. In each panel, upper and lower traces depict the neuronal response and the current stimulus, respectively. A, regular firing cell, responding with two separate spikes to the long stimulus (a) and with a single spike to the brief stimulus (b). B, grade I burster, responding with a burst to the long stimulus (a), but with a single spike to the brief stimulus (b). C, grade II burster, responding with bursts to both long (a) and brief stimuli (b). D, grade III burster, which in addition to bursting in response to long (a) and brief stimuli (b), also burst fired spontaneously at a frequency of 0.3 Hz (c, superposition of four sequential traces; one of the spontaneous bursts is shown on an expanded time scale in d).

Figure 6. Early recruitment of a grade III burster during spontaneous epileptiform events in epileptic tissue

A, paired intra-/extracellular (upper/lower traces) recordings of spontaneous activity in CA1 in an epileptic slice. The intracellular recordings were obtained first at resting membrane potential (-67 mV, a), then at a hyperpolarized membrane potential (-85 mV; b) and finally at resting membrane potential again, but 30 min after addition of 2 μm CNQX to block polysynaptic activity (c). In b and c, two sequential responses are superimposed. The neuron displayed two types of spontaneous bursts, namely, intrinsic (left event in a) and network-driven (right event in a) bursts. Intrinsic bursts were not associated with a population burst (a) and were blocked by hyperpolarization (b) but not by CNQX (c). Network-driven bursts were associated with a population burst (a) and were blocked by CNQX (c) but not by hyperpolarization (b). B, temporal relation between the firing of the neuron and the population. In the three consecutive examples (a, b and c) the neuron burst fired shortly before the onset of population discharge (marked with dashed line). C, time histogram of burst probability of the neuron with respect to the onset of population discharge. Each bin depicts the probability that the neuron will start bursting during that 10 ms. The continuous line represents a single exponential function fitted to the experimental data. The time constant of exponential increase was 108.1 ms.

Figure 3. Suppression of Ca²⁺ currents blocks intrinsic bursting in CA1 pyramidal cells in epileptic tissue

In each panel, upper and lower traces depict the neuronal response and the current stimulus, respectively. A, intrinsic bursting in a CA1 pyramidal cell perfused with standard ACSF (a) was blocked 22 min after changing to Ni²⁺-ACSF (b). The effect reversed after a 30 min wash with standard ACSF (c). B, intrinsic bursting in another CA1 pyramidal cell perfused with standard ACSF (a) was blocked 20 min after changing to Ca²⁺-free ACSF (b). The effect reversed after 30 min wash with standard ACSF (c).

Figure 4. Intracellular BAPTA injection does not suppress intrinsic bursting

A, before injecting BAPTA the neuron burst fired when depolarized by a threshold-straddling 200 ms stimulus (a). Strong 400 ms depolarization of the neuron evoked many action potentials, and was followed by a medium and slow after-hyperpolarization (b). B, injecting BAPTA for 30 min did not affect the burst response (a), but suppressed the slow after-hyperpolarization (b).

Figure 5. Spontaneous epileptiform events in epileptic tissue

A, paired intra-/extracellular (upper/lower traces) recordings of spontaneous activity in CA1 in an epileptic slice. The intracellular recordings were obtained at resting membrane potential (-65 mV, a), at a hyperpolarized membrane potential (-85 mV; b) and at resting membrane potential 30 min after addition of 2 μm CNQX (c). The neuron displayed only network-driven burst activity (a), which was blocked by CNQX (c) but not by hyperpolarization (b). B, temporal relation between the firing of the neuron and the population. In the three sequential examples (a, b and c) the neuron fired shortly after the onset of population discharge (marked with dashed line). C, the responses of the neuron (upper traces) to brief (b) and long (a) depolarizing current pulses (lower traces) identify it as a regular firing cell.