Dysfunction of the dentate basket cell circuit in a rat model of temporal lobe epilepsy

Abstract

Temporal lobe epilepsy is common and difficult to treat. Reduced inhibition of dentate granule cells may contribute. Basket cells are important inhibitors of granule cells. Excitatory synaptic input to basket cells and unitary IPSCs (uIPSCs) from basket cells to granule cells were evaluated in hippocampal slices from a rat model of temporal lobe epilepsy. Basket cells were identified by electrophysiological and morphological criteria. Excitatory synaptic drive to basket cells, measured by mean charge transfer and frequency of miniature EPSCs, was significantly reduced after pilocarpine-induced status epilepticus and remained low in epileptic rats, despite mossy fiber sprouting. Paired recordings revealed higher failure rates and a trend toward lower amplitude uIPSCs at basket cell-to-granule cell synapses in epileptic rats. Higher failure rates were not attributable to excessive presynaptic inhibition of GABA release by activation of muscarinic acetylcholine or GABA(B) receptors. High-frequency trains of action potentials in basket cells generated uIPSCs in granule cells to evaluate readily releasable pool (RRP) size and resupply rate of recycling vesicles. Recycling rate was similar in control and epileptic rats. However, quantal size at basket cell-to-granule cell synapses was larger and RRP size smaller in epileptic rats. Therefore, in epileptic animals, basket cells receive less excitatory synaptic drive, their pools of readily releasable vesicles are smaller, and transmission failure at basket cell-to-granule cell synapses is increased. These findings suggest dysfunction of the dentate basket cell circuit could contribute to hyperexcitability and seizures.

Figure 1.

Hilar neuron loss and mossy fiber sprouting in epileptic rats. A–C, Nissl-stained sections of dentate gyrus from a naive control (A), a rat 4 d after status epilepticus (B), and an epileptic rat (C). m, Molecular layer; g, granule cell layer; h, hilus, CA3, CA3 pyramidal cell layer. D, E, Timm-stained sections of dentate gyrus from a pilocarpine-treated control (D) and an epileptic rat (E). Arrow indicates black, aberrant Timm-staining in the inner molecular layer of the epileptic rat.

Figure 2.

Identification of dentate basket cells. A, Dentate basket cells were identified initially based on electrophysiological characteristics, including action potential duration <1 ms, non-adapting high-frequency spike firing, and frequent spontaneous EPSPs (arrows). Responses to injected current, −100 pA and +800 pA. Subsequently, basket cell identity was verified based on the presence of biocytin-labeled axon concentrated in the granule cell layer (g) (arrowheads). m, Molecular layer; h, hilus. B, Other interneurons (HICAP, HIPP, and regular-spiking interneurons) were found at the border of the granule cell layer and hilus but concentrated their axon projections in the molecular layer (arrowheads) and were excluded from further analysis.

Figure 3.

Reduced excitatory synaptic input to dentate basket cells in pilocarpine-treated epileptic rats. A, B, Spontaneous (A) and miniature EPSCs (B) recorded in basket cells from controls, rats 3–7 d after status epilepticus, and epileptics. Expanded views indicated by bars. C, Amplitude (number of recorded basket cells indicated), rise time (10–90%), τ (100–37% decay time), mean charge transfer per minute, and frequency of spontaneous and miniature EPSCs. Error bars indicate SEM. *p < 0.05, compared with control and epileptic sEPSCs; **p < 0.05, compared with control mEPSCs (ANOVA).

Figure 4.

Similar dentate basket cell somatic and dendritic morphology and synaptophysin-immunoreactive punctae density in control and epileptic rats. A, Examples of basket cells recorded and biocytin-labeled in control and epileptic rats. m, Molecular layer; g, granule cell layer; h, hilus. B, Reconstructions of basket cells from control (left) and epileptic rats (right). *Same basket cells shown in A. C, Biocytin-labeled basket cells (red) and synaptophysin immunoreactivity (green) in a control (left) and epileptic rat (right). D, Soma area and total dendritic length per basket cell were similar in control and epileptic rats. E, Densities of synaptophysin-positive punctae were higher on distal dendrites versus somata and proximal dendrites. There were no significant differences between control and epileptic rats.

Figure 5.

Higher failure rate of unitary IPSCs at dentate basket cell-to-granule cell synapses in epileptic rats. A–D, uIPSCs were evoked in granule cells by discharging action potentials in presynaptic basket cells in controls (A, C), rats 3–7 d after status epilepticus, and epileptics (B, D). Biocytin labeling revealed recorded granule cells (arrowheads) surrounded by axons concentrated in the granule cell layer (g) from recorded basket cells (arrows). There are two closely apposed labeled granule cells in the control rat. m, Molecular layer; h, hilus. C, D, Failures of uIPSCs indicated by red traces. E, Failure rate, amplitude, 10–90% rise time, 100–37% decay time, and charge transfer/event of uIPSCs. Failures were excluded for all parameters except failure rate. Error bars indicate SEM. *p < 0.05 (Kruskal–Wallis ANOVA on ranks).

Figure 6.

High failure rate of unitary IPSCs in epileptic rats is independent of presynaptic mACh and GABA_B receptor activation. A, Unitary IPSCs in granule cells evoked by single action potentials in basket cells of epileptic rats in normal ACSF (top traces), 5 μm atropine (left bottom traces), and 10 μm CGP 55845 (right bottom traces). Red traces indicate failures. B, There were no significant changes in failure rates in the presence of atropine or CGP 55845. Black bars indicate average failure rates of six pairs tested with atropine and three pairs tested with CGP 55845. Gray symbols indicate values of individual pairs.

Figure 7.

High-frequency transmission and quantal size at basket cell-to-granule cell synapses reveal smaller RRP in epileptic rats. A, Trains (20 pulses at 50 Hz) of uIPSCs in a control (left) and epileptic rat (right). Averages of at least 10 traces. B, Corresponding plots of cumulative amplitude versus time. Regression lines were calculated from last 10 responses. Regression line y-intercepts indicate RRP cumulative amplitude, and slopes indicate steady-state vesicle resupply rate. C, Putative quantal uIPSCs (blue traces) recorded in bathing medium that included 0.5 mm Ca²⁺/3.5 mm Mg²⁺. Quantal averages shown in black, failures in red. D, Group averages of regression line y-intercepts (RRP cumulative amplitude) and slopes (vesicle recycling rate). No significant differences. E, In a subset of recordings, quantal amplitude was measured. In those cases, number of vesicles per RRP was calculated by dividing cumulative amplitude intercept by quantal size. Error bars indicate SEM. *p = 0.04, **p = 0.002, t test.