Maltais, Daniela D.

Abstract Rationale Optogenetics offers unmatched cellular specificity and control over cellular activity. Various opsins have been tested in animal models of epilepsy, each contributing to our understanding of seizure circuit dynamics. However, inhibitory optogenetic tools based on microbial rhodopsins have low light sensitivity and, thus, are less suitable for applications involving larger brains. We evaluated eOPN3, a red-shifted, highly sensitive inhibitory G-protein coupled receptor opsin in a porcine seizure model using integrated electro-optical sensing and modulation. The results demonstrated the feasibility of eOPN3 circuit modulation in a large animal epilepsy model. Methods MRI-guided stereotactic surgery was used to deliver 20–60 µL of AAV-eOPN3 (AAV5-/AAV9-CaMKII-eOPN3-mScarlet) into the hippocampus (HPC) of three Göttingen minipigs. Each hemisphere received either an active or a control viral vector (AAV5/9-CaMKII-mScarlet) with gadolinium to visualize the injection sites and diffusion volume via post-operative MRI. Two to three months post-injection, bilateral deep brain stimulation electrodes integrated with optic fibers were stereotactically implanted into the anterior nucleus of the thalamus (ANT) and HPC to assess: 1) opsin expression using fiber photometry, 2) optogenetic modulation of stimulation evoked response potentials (SERPs), 3) induction and propagation of seizure-like activity via intrahippocampal kainic acid (KA) injection, and 4) optogenetic modulation of KA-induced seizure activity. After the electrophysiology recording, brains were harvested for histological analysis to evaluate injection target precision, eOPN3 expression, and estimate eOPN3-modulated volume. Results eOPN3 expression was confirmed during surgery via fiber photometry. ANT electrical stimulation elicited robust SERPs in the HPCs, which were attenuated by HPC light illumination. HPC stimulation similarly induced SERPs in the ipsilateral ANT and the contralateral HPC. The HPC stimulation-induced SERPs were significantly reduced by illuminating the site of the recording areas, the ipsilateral ANT and the contralateral HPC, demonstrating the optogenetic inhibition of the synaptic release from the HPC. KA injection into the HPC induced 20-30 Hz seizure-like activity. The ANT and HPC light illumination suppressed the localized KA-induced seizure activity in the early stage. However, after the generalization of KA-induced seizures, the ANT-HPC illumination lost efficacy for the control of seizures. Histological analysis confirmed eOPN3 expression in the HPC, ANT and other Papez circuit nodes. Conclusion Our pilot study highlights that eOPN3-mediated inhibition alters SERP and the latency and spread of KA-induced seizure-like activity. We developed a platform incorporating pre- and postoperative MRI for precise viral vector delivery, real-time fiber photometry for quantifying opsin expression, and integrated electro-optical sensing and stimulation to assess optogenetic efficacy in a large animal model. The large animal model provides a solid foundation for future translational research to develop electro-optical devices and cellular therapies for human epilepsy.

Abstract Translational large-animal models that can accommodate human-scale implantable devices are essential for advancing neuromodulation therapies in epilepsy. This study establishes a kainic acid (KA)-induced porcine model of mesial temporal lobe epilepsy (mTLE) using clinical imaging, stereotactic surgery, and a fully implantable neural stimulation and recording (INSR) device designed for humans. Seven pigs (six KA-treated and one saline control) underwent MRI-guided stereotactic implantation of electrodes targeting bilateral hippocampus (HPC) and anterior thalamus (ANT), followed by intra-hippocampal KA or saline infusion. Local field potentials (LFP) were recorded continuously with synchronized video monitoring. Seizures and LFP interictal epileptiform-like discharges (IEDs) were quantified using validated automated detectors. Histology was performed in the saline control and the longest surviving KA-treated pig. Intra-hippocampal KA infusion induced acute status epilepticus in all (6/6) treated pigs. Four animals survived to chronic monitoring with spontaneous seizures observed in three pigs (2,733 seizures; mean duration of 27.2 ± 17.6 seconds). IEDs were observed in bilateral HPC of all animals, including saline control, with higher rates in the lesioned HPC (p <0.0001). While the IED morphology is consistent with epileptiform activity; IEDs alone are not specific for epilepsy and physiological transients (e.g. sharp-wave ripples) and injury-related hyperexcitability or strain-specific hyperexcitability cannot be excluded. Histological analysis revealed patchy neuronal loss and cytoarchitectural changes in HPC. This porcine model reproduces electrophysiological features of human mTLE. This approach provides a powerful translational bridge for developing and testing next-generation INSR and neuromodulation strategies in freely behaving large animals.

AbstractBackgroundIsolated REM Sleep Behavior Disorder (iRBD) patients are known to be at high risk for developing an overt synucleinopathy – particularly Dementia with Lewy Bodies (DLB) and Parkinson’s Disease (PD). Reduced nigrostriatal uptake on 123I‐FP‐CIT SPECT reflects dopamine deficiency, which is common in the overt synucleinopathies. We sought to assess how 123I‐FP‐CIT SPECT imaging in iRBD changes longitudinally and whether an abnormal 123I‐FP‐CIT SPECT scan precedes and predicts future transition to an overt synucleinopathy.MethodParticipants with polysomnography‐confirmed iRBD from the Mayo Clinic Alzheimer’s Disease Research Center database +/‐ North American Prodromal Synucleinopathy (NAPS) Consortium who had two or more longitudinal 123I‐FP‐CIT SPECT scans and been followed prospectively for at least 24 months were included in this analysis. Striatonigral dopamine transporter uptake was evaluated semi‐quantitatively with 123I‐FP‐CIT SPECT using DaTQUANT software (GE Healthcare) to calculate z‐scores of putamen uptake.ResultThirty‐five iRBD participants (86% male) were evaluated prospectively (mean 62.5 ± 19.4 months) and underwent two or more 123I‐FP‐CIT SPECT scans at a minimum of 12 months intervals. Thirteen participants (37%) developed an overt synucleinopathy (11 DLB and 2 PD; phenoconverters) and 22 participants (63%) remained as iRBD (stable iRBD). Phenoconverters were older (mean 70.8 ± 7.0 vs 63.4 ± 6.0 years old) and had more negative DaTQUANT putamen z‐scores (mean ‐1.68 ± 1.42 vs ‐0.33 ± 1.52) than stable iRBD participants at baseline. Among phenoconverters, nine participants (69%) had one or more 123I‐FP‐CIT SPECT scans with DaTQUANT putamen z‐score of <‐2.0 prior to phenoconversion, and mean time from initial DaTQUANT putamen z‐score of <‐2.0 to phenoconversion was 36.8 ± 28.7 months. In stable RBD participants, eight participants (36%) showed a decline in DaTQUANT putamen z‐score of >1 between the most recent and the baseline scan, and four participants (18%) presented DaTQUANT putamen z‐score of <‐2.0 at the most recent scan.ConclusionThese findings suggest that a decline in putamen uptake on 123I‐FP‐CIT SPECT in iRBD serves as a predictor and a surrogate biomarker for progression of iRBD to an overt synucleinopathy.

AbstractBackgroundMultiple imaging modalities have been individually shown to be useful in diagnosing Dementia with Lewy Bodies (DLB). Reduced striatonigral uptake on ioflupane‐SPECT reflects striatonigral dopamine dysfunction observed in DLB, while other imaging modalities assess different aspects of DLB, which potentially work complementarily when used together. We assessed how well ioflupane‐SPECT differentiates DLB from Alzheimer’s Disease Dementia (ADem) and whether adding another imaging modality to ioflupane‐SPECT would provide additional value.MethodIoflupane‐SPECT, MRI, FDG‐PET, and PiB‐PET were assessed on 35 DLB and 14 ADem patients (including 12 patients with eventual autopsy confirmation). Striatonigral dopamine transporter uptake was evaluated semi‐quantitatively with ioflupane‐SPECT using DaTQUANT software (GE Healthcare) to calculate z‐scores of putamen uptake. Hippocampal volume was calculated with structural MRI, cingulate island sign (CIS) ratio with FDG‐PET, and global cortical PiB retention with PiB‐PET.ResultLower DaTQUANT z‐scores of putamen were observed in DLB patients compared to ADem patients (c‐statistic 0.896, p<0.001). Ioflupane‐SPECT showed higher c‐statistic in differentiating DLB from ADem than hippocampal volume on MRI (c‐statistic 0.718, p=0.034), CIS on FDG‐PET (c‐statistic 0.869, p=0.003), or global cortical PiB retention on PiB‐PET (c‐statistic 0.869, p=0.002), but some overlap between two diagnostic groups was still observed with the single imaging modality. Among these imaging modalities, ioflupane‐SPECT was the only modality correlated with the Unified Parkinson Disease Rating Scale (UPDRS) part III, while only PiB‐PET correlated with Montreal Cognitive Assessment (MoCA) test score. Adding another imaging modality to ioflupane‐SPECT enhanced c‐statistics (+MRI c‐statistic 0.931, p<0.001; +FDG‐PET c‐statistic 0.957, p=0.005; +PiB‐PET c‐statistic 0.955, p=0.007), and ioflupane‐SPECT in combination with both FDG‐PET and PiB‐PET showed the highest c‐statistic of 0.974 (p=0.043).ConclusionIoflupane‐SPECT is an excellent imaging modality to identify DLB by detecting striatonigral dopamine dysfunction which is strongly associated with motor impairment in DLB. Correlative neuroimaging with MRI, FDG‐PET, or PiB‐PET may add small incremental value in differentiation of DLB and ADem. Supported by NIH grants (AG016574, AG006786, AG015866, NS100620, AG062677), grant from GE Healthcare, the Mayo Clinic Dorothy and Harry T. Mangurian Jr. Lewy Body Dementia Program, the Deal Family Foundation, and the Little Family Foundation.