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Enhancement of Asynchronous Release from Fast-Spiking Interneuron in Human and Rat Epileptic Neocortex

  • Man Jiang,

    Affiliation Institute of Neuroscience, State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

  • Jie Zhu,

    Affiliation Institute of Neuroscience, State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

  • Yaping Liu,

    Affiliation Institute of Neuroscience, State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

  • Mingpo Yang,

    Affiliation Institute of Neuroscience, State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

  • Cuiping Tian,

    Affiliation Institute of Neuroscience, State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

  • Shan Jiang,

    Affiliation Institute of Neuroscience, State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

  • Yonghong Wang,

    Affiliation Institute of Neuroscience, State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

  • Hui Guo,

    Affiliation Department of Neurosurgery, Shanghai Quyang Hospital, Tongji University, Shanghai, China

  • Kaiyan Wang ,

    shu@ion.ac.cn (YS); wang_kaiyan@yahoo.com.cn (KW)

    Affiliation Department of Neurology, Huashan Hospital, Fudan University, Shanghai, China

  • Yousheng Shu

    shu@ion.ac.cn (YS); wang_kaiyan@yahoo.com.cn (KW)

    Affiliation Institute of Neuroscience, State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

Enhancement of Asynchronous Release from Fast-Spiking Interneuron in Human and Rat Epileptic Neocortex

  • Man Jiang, 
  • Jie Zhu, 
  • Yaping Liu, 
  • Mingpo Yang, 
  • Cuiping Tian, 
  • Shan Jiang, 
  • Yonghong Wang, 
  • Hui Guo, 
  • Kaiyan Wang, 
  • Yousheng Shu
PLOS
x

Abstract

Down-regulation of GABAergic inhibition may result in the generation of epileptiform activities. Besides spike-triggered synchronous GABA release, changes in asynchronous release (AR) following high-frequency discharges may further regulate epileptiform activities. In brain slices obtained from surgically removed human neocortical tissues of patients with intractable epilepsy and brain tumor, we found that AR occurred at GABAergic output synapses of fast-spiking (FS) neurons and its strength depended on the type of connections, with FS autapses showing the strongest AR. In addition, we found that AR depended on residual Ca2+ at presynaptic terminals but was independent of postsynaptic firing. Furthermore, AR at FS autapses was markedly elevated in human epileptic tissue as compared to non-epileptic tissue. In a rat model of epilepsy, we found similar elevation of AR at both FS autapses and synapses onto excitatory neurons. Further experiments and analysis showed that AR elevation in epileptic tissue may result from an increase in action potential amplitude in the FS neurons and elevation of residual Ca2+ concentration. Together, these results revealed that GABAergic AR occurred at both human and rat neocortex, and its elevation in epileptic tissue may contribute to the regulation of epileptiform activities.

Author Summary

The balance between excitation and inhibition in the cerebral cortex is important for multiple brain functions. Down-regulation of GABA-induced inhibition disrupts this balance and may lead to epileptic seizures. Asynchronous release of GABA is known to occur at certain GABAergic synapses and represents release of inhibitory neurotransmitter that is not precisely timed to presynaptic action potentials. Whether asynchronous release is subject to change after the induction of epilepsy remains unclear. In this study, using simultaneous recordings from inhibitory fast-spiking neurons and excitatory pyramidal cells, we found that asynchronous release occurred at the output synapses of fast-spiking neurons in both human and rat neocortex. The occurrence of asynchronous release depended on the level of residual calcium at the presynaptic terminals but not on postsynaptic spiking. Further experiments using cortical tissue derived from human patients with intractable epilepsy and from a rat model of the disorder revealed an elevation of asynchronous release in epileptic cortex, possibly resulting from an increase in action potential amplitude of fast-spiking neurons and changes in calcium dynamics in their axon terminals. Taken together, these results demonstrate that asynchronous release is a fundamental property shared by neocortical fast-spiking neurons regardless of species, and the enhancement of asynchronous release in epileptic tissue suggests a role for it in regulating epileptic activities.

Introduction

During active states in the cerebral cortex, cortical neurons receive both excitatory and inhibitory synaptic inputs. Proper balance of these inputs [1],[2] is important for neuronal responsiveness to incoming inputs [3],[4] and for sensory processing [5],[6]. Disruption of this balance may cause malfunctioning of the network, leading to various brain disorders such as epileptic seizures [7],[8]. The main inhibitory neurotransmitter in the cortex is GABA, which is normally released from axonal terminals of inhibitory interneurons and mainly activates GABAA and GABAB receptors, leading to cortical inhibition [9]. The balance between excitation and inhibition largely depends on proper regulation of the activities of these interneurons and the excitatory pyramidal cells (PCs) [10][13].

Molecular and functional changes in GABA receptors [14],[15] or selective loss [16][20] or dormancy [21][23] of inhibitory interneurons may result in hyperexcitability of neuronal networks and contribute to epileptogenesis. However, there are also several lines of evidence showing no substantial change in the basal GABAergic transmission in epileptic tissues [24][27]. It is possible that other changes in the properties of inhibitory synapses associated with high-frequency discharges may be involved in generating and regulating the network activities, including the epileptiform activity.

Under most circumstances, action potential (AP) is initiated at the axon initial segment and propagates to the presynaptic terminals, triggering neurotransmitter release within milliseconds [28]. This tightly coupled or synchronized transmitter release with presynaptic AP generation ensures precise signaling in the complex neural network. However, prolonged asynchronous release (AR) for hundreds of milliseconds following presynaptic AP burst has been observed at some excitatory and inhibitory synapses, particularly after high-frequency firing of presynaptic neurons [29][32]. At GABAergic synapses, AR may provide long-lasting inhibition and reduce the discharge probability and precision in postsynaptic neurons, leading to desynchronization of network activities. A recent study demonstrated that, after a burst of APs, fast-spiking (FS) interneurons in the rat neocortex show AR at their output synapses, including FS autapses and FS-PC synapses [33]. AR at FS autapses results in self-inhibition and consequently excitation of its target cells, while that at FS-PC synapses causes inhibition of target PCs. Therefore, regulation of the AR-induced self-inhibition in FS neurons and inhibition in PCs may contribute to the proper excitation-inhibition balance in the cerebral cortex. In this study, we examine whether AR occurs in human epileptic neocortical tissue and whether AR is subjected to change after the induction of epileptic seizures.

We obtained human cortical tissues from small brain blocks that were surgically removed to cure intractable epileptic patients and brain tumor patients. Since the surgery is considered a therapy of the last resort for patients that had frequently suffered severe epileptic seizures, the cortical tissue should have experienced chronic epileptiform activities. We found that although AP burst-evoked AR occurred in all GABAergic synapses of FS interneurons (including FS autapses, FS-FS and FS-PC synapses) in these human epileptic tissues, FS autapses exhibited the strongest AR among these synapses. Further experiments in rats revealed similar differences in AR at different synapses. Importantly, as compared with control tissues, AR is significantly stronger in epileptic tissues, indicating that AR at GABAergic synapses might be subjected to modulation by epileptic seizures and involved in regulating epileptiform activities.

Results

AR at FS Autapses and FS-FS Synapses in Human Cortical Slices

Human neocortical tissues from 52 patients (aged 5–42 y) with frontal or temporal lobe epilepsy were sliced and examined by electrophysiology within 2–10 h after surgical removal. Whole-cell recording was performed on single FS neurons or synaptically connected FS-FS and FS-PC pairs in layer 5.

We first examined the properties of asynchronous release (AR) of GABA at autapses made by single FS neurons on themselves. In about 22% of FS neurons tested (n = 85/392), we consistently observed elevated spontaneous synaptic events immediately after high-frequency firing evoked by DC current injection through the recording pipette in current-clamp mode (Figure 1A). By using a high-Cl pipette solution (75 mM Cl), inhibitory postsynaptic potentials (IPSPs) were depolarizing events at the resting membrane potential (∼−70 mV). Consistent with previous findings in rodents [34], we found that in voltage-clamp mode (Vhold = −70 mV) single AP could trigger an inward current in the same recorded cell that peaked within 2 ms and could be completely blocked by the bath application of picrotoxin (PTX, 50 µM; n = 15), a GABAA receptor antagonist (Figure 1B). This indicates the existence of monosynaptic autaptic connections in these human FS interneurons. These unitary inhibitory postsynaptic currents (IPSCs) had a failure rate of 0.3±0.3% and an onset latency of 0.84±0.07 ms; the rise time and decay time constant were 0.59±0.08 and 3.9±0.5 ms, respectively (n = 12 FS neurons). The amplitudes of these IPSCs were relatively large (255.8±53.6 pA) because we selectively examined the effects of PTX on FS neurons with obvious autaptic unitary IPSCs, ensuring accurate measurements of the IPSC kinetics after subtraction (control – PTX, Figure 1B). In another set of recordings