CRISPR-Based KCC2 Upregulation Attenuates Drug-Resistant Seizure in Mouse Models of Epilepsy.

OBJECTIVE: The precise intervention of K-Cl cotransporter isoform 2 (KCC2) as a promising target for drug-resistant epilepsy remains elusive. METHODS: Here, we used a CRISPRa system delivered by adeno-associated viruses to specifically upregulate KCC2 in the subiculum to confirm its therapeutic potential in various in vivo epilepsy models. Calcium fiber photometry was used to reveal the role of KCC2 in the restoration of impaired GABAergic inhibition. RESULTS: CRISPRa system effectively upregulated KCC2 expression both in in vitro cell culture and in vivo brain region. Delivery of CRISPRa with adeno-associated viruses resulted in upregulating the subicular KCC2 level, contributing to alleviating the severity of hippocampal seizure and facilitating the anti-seizure effect of diazepam in a hippocampal kindling model. In a kainic acid-induced epilepticus status model, KCC2 upregulation greatly increased the termination percentage of diazepam-resistant epilepticus status with the broadened therapeutic window. More importantly, KCC2 upregulation attenuated valproate-resistant spontaneous seizure in a kainic acid-induced chronic epilepsy model. Finally, calcium fiber photometry showed CRISPRa-mediated KCC2 upregulation partially restored the impaired GABAA -mediated inhibition in epilepsy. INTERPRETATION: These results showed the translational potential of adeno-associated viruses-mediated delivery of CRISPRa for treating neurological disorders by modulating abnormal gene expression that is directly associated with neuronal excitability, validating KCC2 as a promising therapeutic target for treating drug-resistant epilepsy. ANN NEUROL 2023;94:91-105.

Inflachromene attenuates seizure severity in mouse epilepsy models via inhibiting HMGB1 translocation.

Epilepsy is not well controlled by current anti-seizure drugs (ASDs). High mobility group box 1 (HMGB1) is a DNA-binding protein in the nucleus regulating transcriptional activity and maintaining chromatin structure and DNA repair. In epileptic brains, HMGB1 is released by activated glia and neurons, interacting with various receptors like Toll-like receptor 4 (TLR4) and downstream glutamatergic NMDA receptor, thus enhancing neural excitability. But there is a lack of small-molecule drugs targeting the HMGB1-related pathways. In this study we evaluated the therapeutic potential of inflachromene (ICM), an HMGB-targeting small-molecule inhibitor, in mouse epilepsy models. Pentylenetetrazol-, kainic acid- and kindling-induced epilepsy models were established in mice. The mice were pre-treated with ICM (3, 10 mg/kg, i.p.). We showed that ICM pretreatment significantly reduced the severity of epileptic seizures in all the three epilepsy models. ICM (10 mg/kg) exerted the most apparent anti-seizure effect in kainic acid-induced epileptic status (SE) model. By immunohistochemical analysis of brain sections from kainic acid-induced SE mice, we found that kainic acid greatly enhanced HMGB1 translocation in the hippocampus, which was attenuated by ICM pretreatment in subregion- and cell type-dependent manners. Notably, in CA1 region, the seizure focus, ICM pretreatment mainly inhibited HMGB1 translocation in microglia. Furthermore, the anti-seizure effect of ICM was related to HMGB1 targeting, as pre-injection of anti-HMGB1 monoclonal antibody (5 mg/kg, i.p.) blocked the seizure-suppressing effect of ICM in kainic acid-induced SE model. In addition, ICM pretreatment significantly alleviated pyramidal neuronal loss and granule cell dispersion in kainic acid-induced SE model. These results demonstrate that ICM is an HMGB-targeting small molecule with anti-seizure potential, which may help develop a potential drug for treating epilepsy.

Compact and broadband silicon-based polarization beam splitter using asymmetric directional couplers embedded with subwavelength gratings and slots.

A compact and broadband silicon-based polarization beam splitter (PBS) is proposed and investigated in detail, where the two arms of the directional coupler (DC) are, respectively, embedded with subwavelength gratings (SWGs) and vertical slots so that field distributions for the TE mode are significantly changed, effectively weakening coupling strength, whereas those for the TM mode are almost unaffected, nearly analogous to the DC with strip waveguides. By carefully optimizing structural parameters, efficient coupling will emerge between the two waveguides for the TM mode, while TE mode will be confined in the SWG-assisted strip waveguide. Consequently, the two modes can be effectively separated, and thus the realization of a PBS is accomplished. Results show that a compact PBS with a coupling length of 6.45 µm is achieved, together with the extinction ratio (ER) of 27.54/31.88 dB, the insertion loss of 0.12/0.14 dB, and the reflection loss of -43.67/-30.50dB, respectively, for TE/TM mode at the wavelength of 1.55 µm. The bandwidth, for both modes, is up to 230/100 nm when ER is larger than 15/20 dB. In addition, fabrication tolerances to the critical structural parameters and field evolution through the proposed device are analyzed.

Excitatory somatostatin interneurons in the dentate gyrus drive a widespread seizure network in cortical dysplasia.

Seizures due to cortical dysplasia are notorious for their poor prognosis even with medications and surgery, likely due to the widespread seizure network. Previous studies have primarily focused on the disruption of dysplastic lesions, rather than remote regions such as the hippocampus. Here, we first quantified the epileptogenicity of the hippocampus in patients with late-stage cortical dysplasia. We further investigated the cellular substrates leading to the epileptic hippocampus, using multiscale tools including calcium imaging, optogenetics, immunohistochemistry and electrophysiology. For the first time, we revealed the role of hippocampal somatostatin-positive interneurons in cortical dysplasia-related seizures. Somatostatin-positive were recruited during cortical dysplasia-related seizures. Interestingly, optogenetic studies suggested that somatostatin-positive interneurons paradoxically facilitated seizure generalization. By contrast, parvalbumin-positive interneurons retained an inhibitory role as in controls. Electrophysiological recordings and immunohistochemical studies revealed glutamate-mediated excitatory transmission from somatostatin-positive interneurons in the dentate gyrus. Taken together, our study reveals a novel role of excitatory somatostatin-positive neurons in the seizure network and brings new insights into the cellular basis of cortical dysplasia.

Ultracompact Polarization Splitter-Rotator Based on Shallowly Etched Subwavelength Gratings and Anisotropic Metasurfaces.

Polarization splitter-rotators (PSRs) are an essential component in on-chip polarization-sensitive and polarization-division multiplexing systems. In this work, we propose an ultracompact and high-performance silicon-based polarization splitter-rotator utilizing anisotropic metasurfaces, which is the first to combine the two, to our knowledge. The tilted periodic metasurface structure has different modulation effects on different polarized light fields, such as the transverse-electric (TE) mode and the transverse-magnetic (TM) mode, which are beneficial for designing polarization management devices. According to the results, the entire length of the silicon PSR was ~13.5 µm. The TE-to-TM conversion loss and polarization conversion ratio ere -0.154 dB and 96.5% at 1.55 µm, respectively. In the meanwhile, the cross talk and reflection loss were -27.0 dB and -37.3 dB, when the fundamental TE mode was input. The insertion loss and cross talk were -0.19 dB and -25.01 dB at the central wavelength when the fundamental TM mode was input. In addition, the bandwidth reached up to ~112 nm with polarization conversion loss and insertion loss higher than -0.46 dB and -0.36 dB. The simulations also show that the designed devices had good fabrication tolerance.

Interictal-period-activated neuronal ensemble in piriform cortex retards further seizure development.

Epileptic networks are characterized as having two states, seizures or more prolonged interictal periods. However, cellular mechanisms underlying the contribution of interictal periods to ictal events remain unclear. Here, we use an activity-dependent labeling technique combined with genetically encoded effectors to characterize and manipulate neuronal ensembles recruited by focal seizures (FS-Ens) and interictal periods (IP-Ens) in piriform cortex, a region that plays a key role in seizure generation. Ca2+ activities and histological evidence reveal a disjointed correlation between the two ensembles during FS dynamics. Optogenetic activation of FS-Ens promotes further seizure development, while IP-Ens protects against it. Interestingly, both ensembles are functionally involved in generalized seizures (GS) due to circuit rearrangement. IP-Ens bidirectionally modulates FS but not GS by controlling coherence with hippocampus. This study indicates that the interictal state may represent a seizure-preventing environment, and the interictal-activated ensemble may serve as a potential therapeutic target for epilepsy.

Conductivity Anisotropy Influence on Acoustic Sources for Magnetoacoustic Tomography With Magnetic Induction.

OBJECTIVE: As the multi-physics imaging approach, magnetoacoustic tomography with magnetic induction (MAT-MI) attracts more and more attentions, focusing on image reconstruction for conductivity isotropic tissues. METHODS: By introducing vector analyses to electromagnetic stimulation and magnetoacoustic excitation for a single-layer cylindrical conductivity anisotropic model, the acoustic source strength (ASS) of MAT-MI is derived in explicit formula and the influence of the anisotropic conductivity tensor is also analyzed. RESULTS: Theoretical and numerical studies demonstrate that the ASS generated at the tissue boundary is composed of an alternating current (AC) fluctuation and a direct current (DC) bias, where the distribution of the AC fluctuation with respect to the spatial angle exhibits a double-period cosine function, and the DC bias remains constant at each angle. The dependences of the AC fluctuation and the DC bias on the anisotropic component ratio (ACR) and the conductivity tensor are proved by numerical results, which are also verified by the special cases of the zero AC fluctuation for the conductivity isotropic medium and the negative DC bias of the low-conductivity medium. CONCLUSION: With the measurements of the ASS around the model, the anisotropic conductivity tensor can be reconstructed by the amplitudes of the AC fluctuation and the DC bias with the conductivity of the isotropic surrounding medium. SIGNIFICANCE: The favorable results provide a new method for anisotropic conductivity measurement, and suggest the application potential of MAT-MI in biomedical imaging and nondestructive testing for conductivity anisotropic tissues.