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SUMMARYPurpose: Our earlier findings of the modulation of cholinergic neurotransmission by an early life generalized seizure and the reported interaction between muscarinic and N-methyl-D-aspartate (NMDA) receptors prompted us to investigate the effects of endogenous acetylcholine (ACh) on the frequency (Hz) of the epileptiform discharges following NMDA-receptor activation in the hippocampal slice. Methods: A sustained (>20 min) generalized convulsion was induced in Sprague-Dawley juvenile rats by intraperitoneal injection with pentylenetetrazole (PTZ, 70-90 mg/ kg) at postnatal day (P) 20. Temporal and septal hippocampal slices were prepared of normal (N) and PTZ-treated (PTZ) adult ( ‡P60) rats, and CA3 field potentials were recorded during perfusion with Mg 2+ -free artificial cerebrospinal fluid (ACSF) or with ACSF containing 50 lM 4-aminopyridine (4-AP). Key Findings: In Mg 2+ -free ACSF, spontaneous interictallike epileptiform discharges (IEDs) were recorded in all slices, with significantly higher frequencies in temporal (0.46 ± 0.03 Hz, n = 85) versus septal slices (0.20 ± 0.02 Hz, n = 47, p < 0.000001) but no consistent differences in any other group (i.e., male vs. female or N vs. PTZ). The anticholinesterase eserine (10 lM) increased their frequencies by 150-200% in N-septal and in all temporal slices and by 300% in PTZ-septal slices (p = 0.0028). In 60% of the slices the excitatory effect persisted throughout drug perfusion, whereas in the remaining ones it was distinguished in two phases: an early ''transient'' and a late ''steady state.'' The steady-state frequencies resembled the predrug ones in N slices but remained significantly elevated in PTZ slices, especially in the septal group. The muscarinic antagonist atropine (1 lM) decreased IED frequency in all slices (n = 36, p = 0.005) and also fully reversed the eserine effect (n = 38, p < 0.0001). In 4-AP ACSF, eserine increased spontaneous IED frequency (n = 21) in N and PTZ slices alike; IEDs were subsequently abolished by addition of the NMDA-receptor antagonist D())-2-amino-5-phosphonopentanoic acid (AP5; 50 lM, n = 6).Significance: These results demonstrate an intrinsic tonic positive muscarinic acetylcholine receptor (mAChR) contribution to the frequency of NMDA receptor-dependent epileptiform discharges that is amplified following an elevation of endogenous ACh and is more pronounced in the septal hippocampus. Moreover, this positive mAChR contribution to the frequency of IEDs is even more pronounced and persistent in the septal extremity after an early life generalized sustained convulsion. This cholinergic enhancement of the excitatory septal hippocampal output may influence cognitive function and performance, and possibly the adult seizure threshold.

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Gamma-aminobutyric acid (GABA)-releasing interneurons modulate neuronal network activity in the brain by inhibiting other neurons. The alteration or absence of these cells disrupts the balance between excitatory and inhibitory processes, leading to neurological disorders such as epilepsy. In this regard, cell-based therapy may be an alternative therapeutic approach. We generated light-sensitive human embryonic stem cell (hESC)-derived GABAergic interneurons (hdIN) and tested their functionality. After 35 days in vitro (DIV), hdINs showed electrophysiological properties and spontaneous synaptic currents comparable to mature neurons. In co-culture with human cortical neurons and after transplantation (AT) into human brain tissue resected from patients with drug-resistant epilepsy, light-activated channelrhodopsin-2 (ChR2) expressing hdINs induced postsynaptic currents in human neurons, strongly suggesting functional efferent synapse formation. These results provide a proof-of-concept that hESC-derived neurons can integrate and modulate the activity of a human host neuronal network. Therefore, this study supports the possibility of precise temporal control of network excitability by transplantation of light-sensitive interneurons.

Epilepsy is a complex disorder affecting the central nervous system and is characterised by spontaneously recurring seizures (SRSs). Epileptic patients undergo symptomatic pharmacological treatments, however, in 30% of cases, they are ineffective, mostly in patients with temporal lobe epilepsy. Therefore, there is a need for developing novel treatment strategies. Transplantation of cells releasing γ-aminobutyric acid (GABA) could be used to counteract the imbalance between excitation and inhibition within epileptic neuronal networks. We generated GABAergic interneuron precursors from human embryonic stem cells (hESCs) and grafted them in the hippocampi of rats developing chronic SRSs after kainic acid-induced status epilepticus. Using whole-cell patch-clamp recordings, we characterised the maturation of the grafted cells into functional GABAergic interneurons in the host brain, and we confirmed the presence of functional inhibitory synaptic connections from grafted cells onto the host neurons. Moreover, optogenetic stimulation of grafted hESC-derived interneurons reduced the rate of epileptiform discharges in vitro. We also observed decreased SRS frequency and total time spent in SRSs in these animals in vivo as compared to non-grafted controls. These data represent a proof-of-concept that hESC-derived GABAergic neurons can exert a therapeutic effect on epileptic animals presumably through establishing inhibitory synapses with host neurons.

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Epilepsy is a severe neurological disease manifested by spontaneous recurrent seizures due to abnormal hyper‐synchronization of neuronal activity. Epilepsy affects about 1% of the population and up to 40% of patients experience seizures that are resistant to currently available drugs, thus highlighting an urgent need for novel treatments. In this regard, anti‐inflammatory drugs emerged as potential therapeutic candidates. In particular, specific molecules apt to resolve the neuroinflammatory response occurring in acquired epilepsies have been proven to counteract seizures in experimental models, and humans. One candidate investigational molecule has been recently identified as the lipid mediator n‐3 docosapentaenoic acid‐derived protectin D1 (PD1n‐3DPA) which significantly reduced seizures, cell loss, and cognitive deficit in a mouse model of acquired epilepsy. However, the mechanisms that mediate the PD1n‐3DPA effect remain elusive. We here addressed whether PD1n‐3DPA has direct effects on neuronal activity independent of its anti‐inflammatory action. We incubated, therefore, hippocampal slices with PD1n‐3DPA and investigated its effect on excitatory and inhibitory synaptic inputs to the CA1 pyramidal neurons. We demonstrate that inhibitory drive onto the perisomatic region of the pyramidal neurons is increased by PD1n‐3DPA, and this effect is mediated by pertussis toxin‐sensitive G‐protein coupled receptors. Our data indicate that PD1n‐3DPA acts directly on inhibitory transmission, most likely at the presynaptic site of inhibitory synapses as also supported by Xenopus oocytes and immunohistochemical experiments. Thus, in addition to its anti‐inflammatory effects, PD1n‐3DPA anti‐seizure and neuroprotective effects may be mediated by its direct action on neuronal excitability by modulating their synaptic inputs.

Convulsive status epilepticus (SE) in immature life is often associated with lasting neurobiological changes. We provoked SE by pentylenetetrazole in postnatal day 20 rat pups and examined communication modalities between the temporal hippocampus and medial entorhinal cortex (mEC) in vitro. After a minimum of 40 days post-SE, we prepared combined temporal hippocampal - medial entorhinal cortex (mEC) slices from conditioned (SE) and naive (N) adult rats and recorded 4-aminopyridine-induced spontaneous epileptiform interictal-like discharges (IED) simultaneously from CA3 and mEC layer V-VI. We analyzed IED frequency and high frequency oscillations (HFOs) in intact slices and after surgical separation of hippocampus from mEC, by two successive incisions (Schaffer collateral cut, Parasubiculum cut). In all slices, IED frequency was higher in CA3 vs mEC and Raster plots indicated no temporal coincidence between them either in intact or in CA1-cut slices. IED frequency was significantly higher in SE mEC, but similar in SE and N CA3, independently of connectivity state. Ripples (R) and Fast Ripples (FR) coincided with IEDs and their power differed between SE and N intact slices, both in CA3 and mEC. CA3 FR/R ratios were higher in the absence of mEC. Moreover, SE (vs N) slices showed significantly higher FR/R ratios independently of the presence of mEC. Taken together, these findings suggest lasting effects of immature SE in network dynamics governing hippocampal-entorhinal communication which may impact adult cognitive, behavioral and/or seizure threshold sequalae.