Predicting the successful generation and suppression of postictal discharges with fmri and mapping their seizure networks.

Abstract

Successful generation of seizures or shortening of seizure duration was associated with seizure onset time, while corresponding suppression of afterdischarges with earlier onset times was less effective. To identify targets for intervention, seizure networks originating from afterdischarges in the dorsal and ventral hippocampus were mapped using fMRI or simultaneous fMRI-LFP.

Description

[0001] Cross-references to related applications

[0002] This patent document claims priority to U.S. Provisional Application No. 62 / 980,964, filed on February 24, 2020, the entire contents of which are incorporated herein by reference for all purposes.

[0003] introduce

[0004] Targeted therapies such as deep brain stimulation (DBS) or, more recently, optogenetics, promise to revolutionize the treatment of refractory epilepsy by disrupting specific neuronal circuits involved in the generation and propagation of seizures. However, while this is considered the goal of many experimental epilepsy therapies, it remains unclear whether local inhibition at the seizure origin is sufficient to shorten seizure duration, or whether a multi-site approach is needed to explore the rapid propagation of seizures across a broader network. Furthermore, the development of such approaches is limited due to our limited understanding of seizure networks in animal models. A better understanding of seizure propagation pathways is a necessary step towards enabling targeted therapies for refractory patients.

[0005] Optogenetics, employing gene-targeting techniques, can be used to investigate the roles of different cell types and networks in the persistence of epileptic seizures. Most studies using optogenetics to control seizures focus on spontaneous seizure models, which provides an opportunity to study the pathogenesis of epilepsy and the relationship between different brain states and the effectiveness of interventions. However, without temporal control of seizures, it is difficult to determine the exact origin of seizures or to employ advanced imaging methods such as optogenetic functional MRI (fMRI) or calcium imaging.

[0006] Developing methods for suppressing epileptic seizures and mapping seizure networks has significant clinical implications. This invention addresses this problem. Summary of the Invention

[0007] This invention provides methods for mapping seizure patterns and reducing seizure frequency. In some embodiments, seizures are locally suppressed, for example by local optogenetic suppression. In some embodiments, the seizures are caused by optogenetically induced afterdischarges (AD). The invention also provides methods for generating seizure network maps that can be used to identify intervention targets. Locally suppressed AD exhibits a similar seizure network to unsuppressible AD, but with a less widespread distribution. The invention demonstrates that successful seizure induction or shortening of seizure duration is related to the seizure onset time, and that suppression of afterdischarges with earlier onset times is less effective. To identify downstream intervention targets, simultaneous fMRI and LFP can be used to map seizure networks originating from afterdischarges in the dorsal and ventral hippocampi.

[0008] This invention provides a model for the induction and inhibition of epileptic seizures, which can be analyzed by simultaneous electrophysiology, including local field potentials (LFP) and functional MRI (fMRI). In some embodiments, specific neurons or overlapping groups of neurons contain inhibitory and excitatory photoresponsive peptides. The model provides a means of mapping the induction and inhibition of epileptic seizures. For example, whole-brain fMRI imaging can be used to identify regions involved in atrial fibrillation (AD) whose duration cannot be shortened by local inhibition. Detailed analysis of the imaging of induced seizure events is possible. For example, different networks are identified for AD originating from the vegetative hemisphere (VH) compared to AD originating from the delta (DH). The model includes an animal brain, such as a living animal, which can be a mammal (e.g., rodents such as rats, mice, etc.), a non-human primate, etc. In some embodiments, in order to image induced seizures in an animal model using simultaneous LFP-fMRI, the animal can be sedated and given a short-acting neuromuscular blocking agent to prevent the animal from moving during seizure imaging; exemplary agents for this purpose include, but are not limited to, dexmedetomidine sedatives and vecuronium bromide.

[0009] In some embodiments, seizures are generated by depolarizing target neurons in an individual. Neurons that can be used in this invention include, but are not limited to, neurons in the ventral hippocampus, dorsal hippocampus, medial prefrontal cortex, and medial temporal lobe, including CaMKII-positive excitatory neurons in the dorsal and ventral hippocampuses. Various methods can be used to depolarize neurons, including but not limited to optogenetic stimulation, chemical treatment, and electrical stimulation. When using optogenetic stimulation to induce seizure networks, various photoresponsive peptides can be used. The photoactivated peptide used for stimulation can be, for example, channel rhodopsin, including but not limited to ChR2, ChR1, and VChR1. The photoresponsive peptide can be operatively linked to a promoter expressed in excitatory hippocampal neurons. Promoters used in the methods of this invention include, but are not limited to, the hSyn promoter, the CAMKII promoter, and the thy1 promoter.

[0010] In some embodiments, the second photoreactive polypeptide is used to hyperpolarize the target neuron after depolarization, thereby inhibiting epileptic seizures, i.e., inhibiting Alzheimer's disease (AD). In some embodiments, optogenetics is used to hyperpolarize the neuron. When using optogenetics to hyperpolarize (inhibit) the target neuron, various photoreactive proteins can be used. Target photoreactive polypeptides include, but are not limited to, NpHr, eNpHr2.0, eNpHr3.0, eNpHr3.1, GtR3, etc. The second photoreactive polypeptide can be operatively linked to a promoter expressed in excitatory hippocampal neurons. Promoters that can be used in the methods of the present invention include, but are not limited to, the hSyn promoter, the CaMKII promoter, the thy1 promoter, etc. In some embodiments, the first photoreactive polynucleotide is operatively linked to a promoter different from the second photoreactive polynucleotide.

[0011] In some embodiments, excitatory and inhibitory photoresponsive peptides are co-expressed in cells or overlapping cell populations. For example, ChR2 and eNpHR3.0 can be expressed in cells via the same or different promoters. In some embodiments, the promoter is hSyn.

[0012] In some embodiments, a minimum light intensity is required to suppress AD induced by optogenetic stimulation. For example, at least 10 mW may be required to suppress AD induced by optogenetic stimulation. In some embodiments, the light intensity may be, but is not limited to, 1000-1500 mW / mm². 2 1500-2000mW / mm 2 2000-2500mW / mm 2 2500-3000mW / mm 2 or greater than 3000mW / mm 2 .

[0013] When using optogenetics for stimulation and / or inhibition, light is applied to neurons in an individual. Light can be delivered to the region of interest using any suitable method, including but not limited to percutaneous, implanted devices, etc.

[0014] Early spikes have been shown to be precursors to successful seizure suppression. Seizure onset time can be used to predict seizures in the dorsal and ventral hippocampi. Shorter-duration dorsal and ventral seizures, respectively, are bilateral and unilateral, and their fMRI networks are similar but more extensive when optogenetic suppression is ineffective.

[0015] The data presented in this paper suggest that Alzheimer's disease (AD) with faster progression and earlier seizure onset is more likely to persist. While optogenetic inhibition did not block faster-progressing AD, local optogenetic inhibition shortened the duration of seizure-like AD. Voxel-based activation time maps were generated, comparing the spread of seizure activity between AD originating from the dorsal hippocampus and AD originating from the ventral hippocampus. fMRI activation maps of AD originating from the ventral hippocampus showed that non-progressing AD was present only in the hypothalamus, amygdala, and prefrontal cortex. fMRI activation maps showing dorsal hippocampal stimulation and inhibition indicated that AD was non-progressing and present only in the anterior dorsal hippocampus. Opogenetic inhibition applied during optogenetic stimulation significantly attenuated the fMRI response, while optogenetic inhibition alone did not alter the CBV-weighted fMRI signal.

[0016] During afterdischarge induction using optogenetics, LFP recordings showed that stimulation with earlier seizure onset was more likely to induce afterdischarge and was more difficult to shorten with optogenetic suppression. These results profiled the two seizure onset sites (dorsolateral and ventral hippocampus). fMRI showed that afterdischarges originating from the dorsal and ventral hippocampuses exhibited distinct networks. Shorter-duration seizures originating from the dorsal and ventral hippocampuses were unilateral and bilateral, respectively, while longer-duration afterdischarges showed a more extensive bilateral network. When optogenetic suppression was ineffective in preventing seizures, the spread of network activity was more extensive, but largely overlapped with network activity associated with seizures whose duration could be shortened. These results provide insights into how to suppress seizures, which is significant for targeted seizure interventions.

[0017] These results reveal how optogenetic local inhibition of neuronal populations reduces the probability of induced autistic ileus (AD) originating from either the delta (DH) or the vegetative hepatic horn (VH), but this inhibition typically fails to shorten the duration of AD. The results indicate that seizure onset time can predict both the development of AD and the success of optogenetic inhibition in shortening AD duration, suggesting that local inhibition cannot shorten the duration of faster-progressing AD. Simultaneous LFP fMRI was used to map the seizure network responsible for sustaining seizure activity in the presence of local inhibition. Different networks were identified for VH-originating AD compared to AD originating from the DH. Interestingly, these networks were largely similar in AD with shortenable duration compared to AD with non-shortable duration, while the activity in non-shortable duration AD was more diffuse.

[0018] The effectiveness of local optogenetic inhibition in shortening the duration of Alzheimer's disease (AD) is associated with AD progression; AD with earlier seizure onset is less sensitive to local inhibition. Focal seizures originating from the hippocampus (DH) that can be reliably blocked locally involve both hippocampi. In cases where the duration of AD cannot be shortened, the AD does not necessarily involve extrahippocampal regions but may spread further towards the temporal pole. AD originating from the vena cava (VH) and which can be interrupted optogenetically often involves ipsilateral limited activation of the septum, amygdala, percutaneous fossa (PFC), and hypothalamus. Shorter-duration AD that cannot be shortened by local optogenetic inhibition spreads extensively in these regions and the thalamus. Therefore, the network activities involved in shortenable and non-shortenable AD are largely similar but differ in degree, with significant differences between AD originating from the DH and AD originating from the VH.

[0019] It has been confirmed that CBV-weighted fMRI is highly advantageous compared to blood oxygen level-dependent (BOLD) fMRI because activation can be easily detected in a single trial without averaging. Furthermore, CBV-weighted fMRI typically improves detection sensitivity and contrast-to-noise ratio by at least 2-fold compared to BOLD fMRI, and enhances confidence in the accuracy of activation maps in representing seizure activity. Another advantage of our protocol is that it uses shorter duration (5 s) optogenetic stimulation to induce relatively short duration AD (median = 10.1 s), with an AD duration of approximately Q11 minutes, typically shorter than the duration of AD or spontaneous seizures in SE models. This advantage manifests in two ways. First, shorter duration AD may induce a weaker ignition effect. Evidence suggests that even after multiple AD episodes in a single animal, we have not observed motor seizures, making it possible to study AD in a reproducible manner in the same animal, even if the AD originates from VH (where the ignition rate is known to be relatively fast). Second, relatively mild and short-lived AD that causes seizure activity occurs only in a few areas outside the site of seizure onset. This makes it possible to use fMRI to map the areas involved in the early stages of seizure activity, which would otherwise lack the temporal resolution required to study the rapid spread of seizure activity. Attached Figure Description

[0020] Figure 1AD with faster progression and earlier seizure onset is more likely to persist. (A) Photoelectrode implantation into VH for electrophysiological and optogenetic excitation. (B) Example LFP traces from a single subject and a single round, showing that AD was not induced by lower light intensities (e.g., 2 and 3 mW, see upper and middle plots, respectively), but by stimulation of ChR2-positive neurons with higher light intensities (e.g., 3.5 mW, see lower plot). The estimated seizure onset time for persistent AD is earlier. The lower subplot shows how seizure onset time can be calculated by subtracting the sliding window evoked potential. (C) Relationship between AD probability and seizure onset time, modeled using a hierarchical Bayesian logistic regression model, which allows for different corresponding intercepts for different subjects and excludes trials in rounds where the seizure threshold is known (i.e., fMRI scan rounds, n = 13; AD, 206 trials; no AD, 192 trials). Solid lines and shaded areas represent the mean ± 95% quantile confidence interval of the posterior predicted distribution, and single points represent observational data. (D) Left plot: Forest plot of estimated parameter distribution, showing the interquartile range and 5th and 95th percentiles of two independently running Monte Carlo chains. Since the 95% HPD parameter distribution does not contain zeros, determining the onset time is an important predictor of AD. Right plot: Gelman-Rubin convergence statistic (r-hat) indicating Monte Carlo chain convergence. A value less than 1.1 here indicates good convergence. (E) 10-fold cross-validation, which shows that the hierarchical model has good predictive performance compared to the merged model.

[0021] Figure 2Local optogenetic inhibition shortens seizure-like AD duration. (A) VH is the target of electrophysiological and optogenetic excitation and inhibition. (B) Experimental design. Individual ChR2 excitation followed by ChR2 inhibition of the NpHR3.0 block was randomly assigned at 10-minute intervals. (C) Example LFP traces from rats expressing hSyn-eNpHR3.0 show examples where optogenetic inhibition was considered ineffective in shortening AD duration (top) and where optogenetic inhibition was considered successful in shortening AD duration (bottom). Traces in the left and right images are from the same recording round. The left image is from stimulation including the control condition (ChR2 only), and the right image is from a case where optogenetic inhibition was applied immediately after ChR2 blue light stimulation. These examples suggest that AD from rounds where optogenetic inhibition failed to shorten AD duration appears to be longer and more severe. (D) The proportion of AD lasting longer than 2.5 / 5 s under the following two conditions: control condition (ChR2 only) and optogenetic suppression condition (ChR2+eNpHR3). (E) Histograms of AD duration under the two stimulation conditions. This indicator was included because AD duration can vary greatly between different rounds and between different subjects. *p<0.05 indicates a paired t-test based on n=6 subjects.

[0022] Figure 3Optogenetic suppression failed to halt faster-progressing AD. Considering the relationship between seizure onset time and AD probability, seizure onset time was used as a covariate in subsequent regression analysis. (A) VH is the target of electrophysiological and optogenetic activation and suppression. (B) Example LFP traces indicating stimulation conditions and seizure onset time in a single round. Top: ChR2 only and late-onset seizure activity with persistent AD. Middle: ChR2+eNpHR3.0 and early-onset seizure activity with persistent AD. Bottom: ChR2+eNpHR3.0 and late-onset seizure activity (without AD). (C) Modeled using a Bayesian hierarchical (random intercept) logistic regression model; i.e., the log odds of AD probability logit(p) is interpreted as a linear combination of stimulation conditions and seizure onset time, along with a subject-specific intercept term. Solid lines and shaded areas represent the mean ± 95% quantile confidence interval of the posterior predicted distribution, and single points represent observed data. (D) Left plot: Forest plot of estimated parameter distribution, showing the interquartile range and 5th and 95th percentiles of two independently running Monte Carlo chains. Identifying the onset time and stimulus condition (ChR2 vs. ChR2+eNpHR3.0) are important predictors of AD because their 95% HPD parameter distribution does not contain zeros. Right plot: Gelman-Rubin convergence statistic (r-hat) indicating Monte Carlo chain convergence. A value less than 1.1 here indicates good convergence. (E) 10-fold cross-validation, showing that the hierarchical model has good predictive performance compared to the pooled model. All plots include n = 6 rats.

[0023] Figure 4Voxel-based activation time maps were used to compare the spread of seizure activity between AD originating from DH and AD originating from VH. (A) Logistic function fitting of each trial (blue dots) progressing to AD, with inflection points used as estimates of AD duration, where 50% of the trials showed activation of each voxel. (B and C) Voxel relationship between AD duration and fMRI activation in (B) focal VHAD and (C) focal DH AD. A different pattern was observed in seizures originating from VH compared to DH. For shorter-duration AD, ipsilateral PFC was activated, while the inflection points for contralateral VH and PFC appeared slightly later. However, ipsilateral and contralateral DH were activated only in longer-duration AD. In seizures originating from DH, ipsilateral and contralateral DH and VH were activated in shorter-duration AD, while longer-duration AD may begin to involve cortical areas. (D) ROI-based analysis. If the activation level of each subject exceeded 10% of the maximum, the corresponding area was considered activated. Observed data are represented as binary response variables, where 1 indicates an active region and 0 indicates an inactive region. The responses predicted by random intercept logistic regression are shown as mean ± 95% bootstrap confidence intervals. * indicates that the inflection points differed significantly at the p<0.05 level based on hypothesis testing using parametric bootstrapping (VH, n = 6 rats; DH, n = 3 rats). The stratified model demonstrated better predictive performance compared to the pooled model. All figures include n = 6 rats.

[0024] Figure 5 fMRI activation maps of AD originating in the VH show that non-progressing AD is only present in the hypothalamus, amygdala, and PFC. (A) Photoelectrode implantation in the VH for electrophysiological and optogenetic activation and inhibition. (B) Random alternation of fMRI stimulation patterns within rounds involving different trials (i.e., trials involving eNpHR3.0 and trials not involving eNpHR3.0). (C and D) Examples of LFP and fMRI signals acquired simultaneously from different ROIs. (E and F) Fixed-effects group-level analysis (n=3) of trials that did not progress to (E) and progressed to (F) AD under optogenetic inhibition. (G) Proportion of activated regions in cases where optogenetic inhibition successfully or unsuccessfully inhibited AD. Thresholds for single-trial t-statistics were p<0.001 (uncorrected), and thresholds for group-level activation maps were p<0.0001 (uncorrected). Data are presented as mean ± SEM. Amyg, amygdala; Cpu, caudate putamen; Ent, entorhinal cortex; RSG, posterior granular cortex; ThalDL, dorsomedial thalamus; ThalMD, medial dorsolateral thalamus; ThalVM, ventromedial thalamus. * indicates a fixed-effect difference between having AD and not having AD at a significance level of 0.05.

[0025] Figure 6 fMRI activation maps showing DH stimulation and inhibition indicate that AD did not progress and was only present in the anterior dorsal hippocampus. (A) Photopole implantation of DH for electrophysiological and optogenetic stimulation and inhibition. (B) Randomized alternation of fMRI stimulation patterns within rounds between different trials (i.e., trials involving eNpHR and those not involving eNpHR). (C and D) Simultaneous acquisition of LFP and fMRI signals from different ROIs. (E and F) Fixed-effects group-level (t-statistic) analysis (n=3) of trials that did not progress to (E) and progressed to (F) AD under optogenetic inhibition. (G) Proportion of activated regions in cases where optogenetic inhibition successfully or unsuccessfully inhibited AD. The t-statistic threshold was p<0.0001 (uncorrected). Data are presented as mean ± SEM. Amyg, amygdala; Cpu, caudate putamen; Ent, entorhinal cortex; RSG, posterior granular cortex; ThalDL, dorsomedial thalamus; ThalMD, medial dorsolateral thalamus; ThalVM, ventromedial thalamus. * indicates a fixed-effect difference between having AD and not having AD at a significance level of 0.05.

[0026] Figure 7 Optogenetic inhibition applied during optogenetic stimulation significantly reduced the fMRI response, while optogenetic inhibition alone did not alter the CBV-weighted fMRI signal. (A) Photoelectrode implantation of DH for electrophysiological and optogenetic excitation and inhibition. (B) Comparison of three different stimulation modalities for each animal using LFP-ofMRI simultaneously. (i) Optogenetic inhibition alone (eNpHR3 only). (ii) Optogenetic stimulation of ChR2 using only blue light (ChR2 only). (iii) Simultaneous application of optogenetic stimulation and inhibition (ChR2 + eNpHR3 together). (C) Typical activation maps for a single trial and a single subject for the above three conditions. (D) fMRI and LFP timelines for the example data shown in (C). Local optogenetic inhibition applied during ChR2 activation almost completely eliminated the fMRI response, while the LFP response to ChR2 stimulation remained unchanged. (E) Activation maps generated by fixed-effects fMRI analysis for all trials and all three subjects. (F) The activation levels of the four activated regions are plotted in three subjects. When halophilic rhodopsin and ChR2 were simultaneously activated using blue and orange light, limited activation occurred in only one rat. DH I, ipsilateral dorsal hippocampus; DH C, contralateral dorsal hippocampus; VHI, ipsilateral ventral hippocampus; contralateral ventral hippocampus. The threshold for all activation maps was p<0.001 (uncorrected). (E) Change to orange, then blue, then blue + orange.

[0027] Figure 8The expression of CaMKII-ChR2-YFP and eNpHR3.0-mCherry in the dorsal and ventral hippocampuses after co-injection of two different viral vectors. Figure 1 and Figure 2 Related. (A) and (B) are wide-field fluorescence images of the ipsilateral dorsal and ventral hippocampuses, respectively. (C) A confocal image within the ventral hippocampus shows that ChR2-YFP expression is localized in the cell body and dendrites, while eNPHR3.0 expression is mainly localized in the cell body. NpHR3.0-mCherry cells co-localize with CaMKII-positive and CaMKII-negative cells. Blue arrows indicate co-localization of ChR2 and NpHR3.0, while yellow arrows indicate co-localization of NpHR3.0 and repressive GAD67-positive cells. NpHR3.0-positive cells that do not co-localize with ChR2-YFP-positive cells tend to co-localize with repressive GAD67-positive cells.

[0028] Figure 9 This section shows an example of a procedure for estimating the onset time of an attack, along with examples of correctly classified false negatives and false positives. Figure 1 and Figure 3 Related. Each example is displayed in four rows (from top to bottom) and consists of the following: (i) raw LFP data, (ii) LFP during stimulation after centering the average of each stimulation period, (iii) average evoked potentials over a sliding window of eight stimulation periods, and (iv) LFP after subtracting the average evoked potentials from the sliding window. The last row indicates the threshold for detecting spike events (dashed line), calculated as 4.5 × the standard deviation of the baseline, where the baseline period is represented by the purple shaded area and defined as 500–2500 ms after the start of stimulation. (A)–(D) represent trial examples classified as true negative, false positive, false negative, and true positive, respectively.

[0029] Figure 10 If hSyn-eNpHR3.0 optogenetic inhibition is applied concurrently during optogenetic stimulation, local spike activity can be suppressed and after-discharge can be prevented. CAMKII-eNpHR3.0 is not effective in suppressing spontaneous discharge and shortening AD duration. Figure 2 and Figure 3Related. (A) Implantation of photoelectrodes in the ventral hippocampus for electrophysiological and optogenetic excitation and inhibition. (B) Photograph of a tungsten photoelectrode used for multi-unit recording under an optical microscope. The electrode tip was positioned approximately 0.65 mm from the fiber tip to ensure that multi-unit recording occurred in the illumination cone originating from the fiber tip. (C) Multi-unit firing rate during stimulation, expressed as a ratio to the baseline firing rate of rats expressing hSyn-eNpHR3.0 (n = 2 animals, 21–30 trials for each light power). Data are presented as mean ± standard deviation from different trials. (D) An example of a single-trial multi-unit recording trace from rats expressing eNpHR3.0 driven by the hSyn promoter at the highest light power (18 mW) used during recording. (E) Higher light intensity during simultaneous stimulation of ChR2 and eNpHR3.0 also more effectively prevents AD. The figure above illustrates an example illustrating the fact that lower light power (e.g., <10 mW) may be insufficient to prevent afterfire when neurons are simultaneously optogenetically excited and inhibited. The figure below shows that optogenetic inhibition can prevent AD if the power is increased to, for example, 18 mW in the same round. * indicates that the mean firing rate was lower than baseline based on a one-sample t-test and a significance level of p < 0.05. (F) Example of a single-trial multi-unit recording trace from rats expressing eNpHR3.0 driven by the CAMKII promoter at the highest light power (30 mW). (G) Multi-unit firing rate during stimulation, expressed as a ratio to the baseline firing rate of CAMKII rats (n = 2 animals, 15–30 trials for each light power, except for the cases of 0.5, 6, and 20 mW, in which cases only data from a single animal are included). Data are expressed as mean ± SD across different trials. (H) Example LFP traces from rats expressing CAMKII-eNpHR3.0, showing examples where optogenetic suppression was considered ineffective in shortening AD time (top panel) and where optogenetic suppression was considered successful in shortening AD time (bottom panel). Traces in the left and right panels are from the same recording round. The left panel is from stimulation including the control condition (ChR2 only), and the right panel is from the case where optogenetic suppression was applied immediately after ChR2 blue light stimulation. (I) Histograms of AD duration under two different stimulation conditions. (J) Percentage of AD with a duration exceeding 2.5 under both different conditions. Based on paired t-test of n = 7 subjects, p < 0.05. (K) Confocal immunofluorescence examination of rats injected with CaMKIIeNpHR3.0-mCherry, showing robust eNpHR3.0 expression.

[0030] Figure 11Local optogenetic inhibition shortened the after-discharge time in the dorsal hippocampus under stimulation lasting 5 seconds, but did not shorten the corresponding after-discharge time under stimulation lasting longer. Figure 2 and Figure 3 Related. (A) Implantation of photoelectrodes into the dorsal hippocampus for electrophysiological and optogenetic excitation and inhibition. (B) LFP traces from hSyn-eNpHR3.0 rats, showing AD generated by 5-second stimulation without inhibition (top panel) and failed attempts to reduce AD ​​with optogenetic inhibition (bottom panel). (C) Representative LFP traces from hSyneNpHR3.0 rats, showing AD generated by 6-7.5s stimulation without inhibition (top panel) and successful AD reduction achieved through optogenetic inhibition (bottom panel). (D) Histograms of AD duration under 5-second stimulation alone (blue) and subsequent inhibitory stimulation (orange). (E) Histograms of AD duration under 6-7.5s stimulation alone (blue) and subsequent inhibitory stimulation (orange). (F) Percentage of ADs lasting longer than 2.5s under 5s ChR2 stimulation with and without inhibition. (G) The proportion of AD cases lasting longer than 2.5 s when ChR2 stimulation is applied for 6-7.5 s with or without inhibition. (H) Estimate the relationship between seizure onset time and stimulation duration. (I) Estimate the relationship between seizure onset time and whether AD duration exceeds 2.5 s.

[0031] Figure 12 Voxel-based activation time plots in the presence and absence of rhodopsin inhibition by halophilic bacteria. Figure 4 Correlation. No significant differences were found in the activation timemaps of afterfire originating from the dorsal or ventral hippocampus in the presence or absence of optogenetic suppression; therefore, these data were combined for... Figure 4 The analysis in the text.

[0032] Figure 13 Example of optogenetic fMRI activation maps of the ventral and dorsal hippocampus in an individual trial. Figure 5 and Figure 6 Related. Activation graphs correspond to... Figure 4 c, d and Figure 5Experiments with the same time series in c and d. Dorsal hippocampus: (A) fMRI activation map (t-statistic) of a single trial of short-duration AD whose duration can be shortened by optogenetic inhibition. (B) fMRI activation map of short-duration AD that still progresses under optogenetic inhibition. Ventral hippocampus: (C) fMRI activation map of short-duration AD that does not progress to AD under optogenetic inhibition. (D) fMRI activation map (t-statistic) of a single trial of short-duration AD whose duration cannot be shortened by optogenetic inhibition. The threshold for the t-statistic is p < 0.001 (uncorrected).

[0033] Figure 14 Compared to typical HRF or the more flexible 5th-order gamma-ray matrix, the gamma distribution function with custom parameters more accurately describes the CBV-weighted fMRI response to optogenetic stimulation in the dorsal hippocampus because it has a faster onset and a slower decay. Figure 4-7 Related. (A) Example of a gamma function with best-fit parameters compared to typical HRF and SPM 1st-order gamma. (B) 5th-order gamma basis set. Higher-order functions are able to account for significantly slower responses compared to the function shown in (A). (C) Mean sum of squared errors in fitting all voxels in the ipsilateral dorsal hippocampus for different gamma shape and scale parameters, indicating estimated best-fit results obtained using shape and scale parameters of 1.3 and 0.09, respectively. (D) Example of an HRF for fitting block-designed stimulus data. Measurements and fitted responses for nine stimulation periods are shown here, each consisting of a 5-s stimulus block (40 Hz) and a 55-s resting block. Data were collected from at least three six-cycle acquisitions from each subject (three subjects in total). Activation amounts are expressed as fractions representing the proportion of the total ROI in the contralateral (E) and ipsilateral (F) dorsal hippocampus. Compared to the 5th-order gamma model, the gamma custom parameter model has a larger activation volume because the single custom gamma function uses fewer parameters and is therefore more concise. (G) Example segmentation atlas for regional analysis of fMRI data. All structural and functional images are spatially aligned with the same target, which is first segmented by registration with an atlas (Valdés-Hernández et al., 2011) and then manually corrected. Abbreviations: Amyg – amygdala, Cpu – caudate putamen, DH – dorsal hippocampus, Ent – entorhinal cortex, Hypothal – hypothalamus, PFC – prefrontal cortex, RSG – posterior compression cortex, S1 – primary somatosensory cortex, Thal Ant – anterior thalamus, ThalDL – dorsolateral thalamus, ThalMD – dorsomedial thalamus, ThalVM – ventromedial thalamus, VH – ventral hippocampus.

[0034] Implementation

[0035] Before further describing the invention, it should be understood that the invention is not limited to the specific embodiments described, as differences will certainly exist in actual implementation. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to limit the inventive concept; the scope of the invention will be defined only by the appended claims.

[0036] When a numerical range is provided, it should be understood that every intermediate value between the upper and lower limits of the range, as well as any other specified value or intermediate value within that range, is included within the scope of this invention. Unless the context explicitly specifies otherwise, each intermediate value should be as low as one-tenth of the lower limit unit. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also within this invention, subject to the requirements of any specifically excluded limits within the range. Where the range includes one or two limits, the range excluding any one or both of the included limits is also included within this invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although similar or equivalent methods and materials may be used in the implementation or testing of this invention, preferred methods and materials are described below. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials relating to the cited publications.

[0038] It should be noted that the singular forms “a,” “an,” and “the” used herein and in the appended claims include plural references unless the context clearly indicates otherwise. Thus, for example, “a rhodopsin” refers to a variety of such rhodopsins, while “the carbon fiber” refers to one or more carbon fibers and their equivalents known to those skilled in the art, and so on. It should also be noted that claims can be drafted to exclude any optional elements. Therefore, this statement is intended as a precondition for the use of specialized terms such as “alone,” “only,” or the use of the limiting word “negative” in relation to elements of a claim.

[0039] It should be understood that, for clarity, certain features of the invention described in the context of a single embodiment may also be presented in combination in a single embodiment. Conversely, for brevity, various features of the invention described in the context of a single embodiment may also be presented individually or in any suitable sub-combination. All combinations of embodiments relating to the invention are expressly covered by the invention and disclosed herein as if each combination were individually and expressly disclosed. Furthermore, all sub-combinations of various embodiments and their constituent parts are also expressly covered by the invention and disclosed herein as if each sub-combination were individually and expressly disclosed.

[0040] The publications discussed herein are only those disclosed prior to the filing date of this patent. Nothing herein is to be construed as an admission that this invention is not entitled to any earlier publication due to prior inventions. Furthermore, the publication dates provided may differ from the actual publication dates and may require separate verification.

[0041] The phrases “for example,” “for instance,” “such as,” or “including” as used herein are intended to introduce examples that further illustrate a more general subject matter. These examples are provided only to aid in understanding the invention and are not intended to be limiting in any way.

[0042] The terms “activator,” “antagonist,” “inhibitor,” “drug,” and “pharmaceutical activator” are used interchangeably in this document to refer to a chemical substance or compound that, when applied to a living organism (human or animal), induces a desired pharmacological and / or physiological effect through local and / or systemic action.

[0043] As used herein, the terms “therapeutic” and the like refer to achieving the desired pharmacological and / or physiological effects. A preventive effect refers to complete or partial prevention of a disease or its symptoms, and a therapeutic effect refers to the partial or complete cure of a disease and / or adverse reactions caused by it. As used herein, “therapeutic” encompasses any treatment of diseases in mammals, particularly humans, including: (a) preventing the occurrence of a disease or disease symptoms in a subject who may be susceptible to the disease or symptoms but have not yet been diagnosed (e.g., a disease that may be related to or caused by a primary disease); (b) suppressing the disease, i.e., preventing its development; and (c) alleviating the disease, even if the disease subsides.

[0044] "Therapeutic effective amount" or "effective amount" refers to the amount of a compound administered to a mammal or other subject in a manner sufficient to treat a disease, symptom, or condition. Therapeutic effective amount will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0045] As used herein, the term "unit dosage form" refers to a physically dispersed, single dosage form suitable for human and animal subjects, each unit containing a predetermined amount of the compound, calculated in an amount sufficient to produce the desired effect when mixed with a pharmaceutically acceptable diluent, carrier, or solvent. The strength of a unit dosage form depends on the specific compound used, the desired effect, and the pharmacokinetic properties of the compound in the host.

[0046] "Pharmaceutically acceptable excipients," "pharmaceutically acceptable diluents," "pharmaceutically acceptable carriers," and "pharmaceutical acceptable adjuvants" refer to excipients, diluents, carriers, and adjuvants that can be used to prepare pharmaceutical compositions that are generally safe, non-toxic, and biologically or otherwise undesirable, including excipients, diluents, carriers, and adjuvants acceptable for veterinary and human pharmaceutical uses. As used in this specification and claims, "pharmaceuticalally acceptable excipients, diluents, carriers, and adjuvants" includes one or more of such excipients, diluents, carriers, and adjuvants.

[0047] As used herein, "pharmaceutical composition" is intended to encompass compositions suitable for administration to subjects (e.g., mammals, particularly humans). Typically, "pharmaceutical compositions" are sterile and, preferably, free of contaminants that could cause unintended reactions in a subject (e.g., compounds in the pharmaceutical composition are pharmaceutical-grade compounds). Pharmaceutical compositions can be designed for administration to subjects or patients in need via a variety of different routes of administration, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, and subcutaneous administration.

[0048] The terms “individual,” “host,” “subject,” and “patient” used herein are used interchangeably and refer to animals, including but not limited to humans and non-human primates, including apes and humans; rodents, including rats and mice; cattle; horses; sheep; cats; dogs; birds, etc. “Mammal” refers to any one or more mammals, including, for example, dogs; cats; horses; cattle; sheep; rodents, etc., and primates, for example, non-human primates and humans. Non-human animal models (e.g., mammals, such as non-human primates, mice, rabbits, etc.) can be used for experimental research. Suitable animal models include, in particular, rodents, such as rats and mice.

[0049] The terms “determine,” “measure,” “evaluate,” and “determine” used in this article are used interchangeably and include both quantitative and qualitative determinations.

[0050] Aspects of the invention include the use of functional magnetic resonance imaging (fMRI) of a subject, including simultaneous fMRI-LFP analysis. In some embodiments, the method is a compressed sensing (CS) high-resolution fMRI method. Compressed sensing is a signal processing method in which an image can be reconstructed from a series of sampled measurements obtained at a sampling rate lower than the Nyquist sampling rate. Typically, the method may include acquiring one or more fMRI images of a target region of the subject. For example, typically, the method may include applying a magnetic field to the target region of the subject using an MRI system (e.g., a permanent magnet or electromagnet of an MRI system). In some cases, the method further includes applying an excitation waveform (e.g., an RF excitation waveform) to the target region of the subject using an MRI system (e.g., an RF coil of an MRI system) to obtain detectable image data (e.g., a magnetic resonance (MR) signal) of the target region of the subject. The MRI system may also apply one or more additional fields, such as (but not limited to) one or more shimming fields using one or more shimming coils, one or more gradient fields using one or more gradient coils, etc. Furthermore, the method includes acquiring image data (e.g., using a receiver of an MRI system) and obtaining an image of the target region of the subject based on the acquired image data.

[0051] The acquired image data may be stored in a computer-readable storage device and analyzed at a later time (also referred to herein as “offline” processing or “offline MRI”). In other cases, the acquired image data may be analyzed in real time to obtain an image of the subject’s target region. “Real time” means analyzing the acquired signal immediately after acquisition and / or during acquisition using an MRI system (e.g., using a processor within the MRI system).

[0052] In some embodiments of offline fMRI, the method may include applying an excitation waveform to a target region of the subject to obtain MR image data. In some embodiments, the method includes applying a pulse sequence to the target region of the subject. The pulse sequence may be a balanced steady-state free precession (b-SSFP) sequence applied to the target region of the subject. In some cases of offline MRI, the method includes acquiring image data (MR signal) of the target region of the subject. In some cases, the method includes using a sampling trajectory. The sampling trajectory may be a random sampling trajectory. For example, the method may include acquiring image data of the target region of the subject using a random undersampling trajectory (e.g., a random undersampling variable density spiral (VDS) trajectory).

[0053] In some embodiments of offline MRI, the method includes obtaining an image of the subject's target region based on the acquired image data. For example, the method may include analyzing (also referred to herein as processing) the image data to obtain an image of the target region. Therefore, in some cases, the method includes reconstructing an image using the acquired image data. In some cases, the method includes reconstructing the image using a cost function (e.g., an L1 regularized cost function).

[0054] Local field potentials (LFPs) are potentials recorded in the extracellular space of brain tissue using microelectrodes (metallic, silicon, or glass micropipettes). Deep recording of LFPs occurs within cortical tissue (or other deep brain structures). LFP signals in the mammalian cortex reflect the activity of thousands of neurons and are commonly used to study underlying network dynamics such as sensory processing, motor planning, attention, memory, and perception. In recent decades, the importance of LFP signals has been further enhanced by the development of high-density silicon-based microelectrodes capable of simultaneously recording LFPs at thousands of locations across entire brain regions. LFPs can be used to guide neuroprosthetic devices because they are easier and more stable to record in a chronic environment compared to the spike activity of a single neuron.

[0055] Magnetic resonance imaging (MRI) is used to analyze neurophysical events. Specifically, MRI can be used to analyze functionally associated regions (anatomical neural networks) in the brain related to neurophysical events. Association patterns can represent the temporal and / or spatial correlations of neurophysical events. The target MRI technique is functional MRI (fMRI). With fMRI, temporal changes in image contrast are displayed using appropriate MR imaging scan sequences. Functional MRI (fMRI) measures signal changes in the brain caused by variations in neural activity. The brain is scanned rapidly (typically every 2-3 seconds) at low resolution. Increased neural activity can cause changes in the MR signal through variations in T*.sub.2. This mechanism is known as the blood oxygen level dependent (BOLD) effect. Increased neural activity leads to an increased demand for oxygen, and the vascular system actually overcompensates for this, resulting in an increase in the amount of oxygenated hemoglobin relative to deoxygenated hemoglobin. Since deoxygenated hemoglobin attenuates the MR signal, the vascular response leads to an increase in the signal associated with neural activity. The BOLD effect also allows for the generation of high-resolution 3D maps of the venous vascular system within neural tissue.

[0056] While the BOLD signal is the most commonly used method in neuroscience research on human subjects, the flexibility of MR imaging offers means to make the signal more sensitive to other aspects of blood supply. Alternative techniques employ arterial spin labeling (ASL) or weight the MRI signal via cerebral blood flow (CBF) and cerebral blood volume (CBV). The CBV method requires the injection of an MRI contrast agent currently undergoing human clinical trials. Because this method has been shown to be significantly more sensitive than BOLD in preclinical studies, it may expand the clinical application of fMRI. The CBF method provides more quantitative information than the BOLD signal, but its detection sensitivity is significantly reduced.

[0057] Epilepsy. Epilepsy is a brain disorder characterized by recurrent seizures over time. Types of epilepsy can include, but are not limited to, generalized epilepsy (e.g., childhood absence epilepsy, juvenile myoclonic epilepsy, grand mal seizures during wakefulness, West syndrome, Lennox-Gasto syndrome) and partial epilepsy (e.g., temporal lobe epilepsy, frontal lobe epilepsy, benign focal epilepsy in children).

[0058] Status epilepticus (SE). Status epilepticus (SE) can include, for example, convulsive status epilepticus, such as early status epilepticus, definitive status epilepticus, refractory status epilepticus, and ultra-refractory status epilepticus; non-convulsive status epilepticus, such as generalized status epilepticus and complex partial status epilepticus; generalized periodic epileptiform discharges; and periodic unilateral epileptiform discharges. Convulsive status epilepticus is characterized by the presence of a convulsive seizure state and can include early status epilepticus, definitive status epilepticus, refractory status epilepticus, and ultra-refractory status epilepticus. Early status epilepticus is treated with first-line therapy. Definitive status epilepticus is characterized by the persistence of the seizure state despite treatment with first-line therapy and second-line therapy. Refractory status epilepticus is characterized by the persistence of the seizure state despite treatment with first-line and second-line therapy and usually general anesthesia. Ultra-refractory status epilepticus is characterized by the persistence of the seizure state despite treatment with first-line therapy, second-line therapy, and general anesthesia for 24 hours or longer.

[0059] Nonconvulsive status epilepticus can include, for example, focal nonconvulsive status epilepticus, such as complex partial nonconvulsive status epilepticus, simple partial nonconvulsive status epilepticus, and mild nonconvulsive status epilepticus; and generalized nonconvulsive status epilepticus, such as delayed absence nonconvulsive status epilepticus, atypical absence nonconvulsive status epilepticus, or typical absence nonconvulsive status epilepticus.

[0060] A seizure is a physiological event or behavioral change that occurs following an event of abnormal electrical activity in the brain. The term "seizure" is often used interchangeably with "convulsion." A convulsion is a rapid and uncontrollable shaking of the body. During a convulsion, the body's muscles repeatedly contract and relax. Based on behavioral type and brain activity, seizures are classified into two main categories: generalized and partial (also known as focal or regional). Classifying the type of seizure helps doctors diagnose whether a patient has epilepsy.

[0061] A generalized seizure is caused by electrical impulses originating from the entire brain, while a partial seizure is caused by electrical impulses originating from a relatively small part of the brain (at least initially). The part of the brain from which a seizure occurs is sometimes referred to as a lesion.

[0062] There are several types of generalized seizures. The most common and most severe (and therefore the most well-known) is a generalized tonic-clonic seizure, also known as a grand mal seizure. In this type of seizure, the patient loses consciousness and usually collapses. After loss of consciousness, the patient's body stiffens (called the "tonic" phase of a seizure) for 30 to 60 seconds, followed by violent muscle twitching (the "clonic" phase) for 30 to 60 seconds, after which the patient enters a deep sleep (the "postictal" or post-ictal phase). During a grand mal seizure, injuries and accidents can occur, such as tongue biting and urinary incontinence.

[0063] Absence seizures cause a brief loss of consciousness (lasting only a few seconds) with little or no symptoms. Patients (most commonly children) typically stop what they are doing and stare blankly. These seizures occur and end suddenly and can happen several times a day. Patients are usually unaware that they are having a seizure, only knowing that "time has been lost."

[0064] Myoclonic seizures are characterized by occasional muscle twitching, usually occurring on both sides of the body. Patients sometimes describe these muscle twitches as brief electric shocks. In severe cases, these seizures may cause objects to fall or the person to involuntarily throw objects.

[0065] A clonic seizure is a repetitive, rhythmic muscle spasm that affects both sides of the body simultaneously.

[0066] The characteristic of a tonic seizure is muscle stiffness.

[0067] Atonic attacks are characterized by a sudden loss of muscle tone throughout the body, especially in the arms and legs, which often leads to falls.

[0068] Focal progression to bilateral tonic-clonic (FBTC) seizures originate in one area of ​​the brain and then spread to both sides of the brain as tonic-clonic seizures.

[0069] When assessing the severity of epileptic seizures, scales are commonly used to classify seizure-related behaviors. The Racine scale is the most widely used scale to describe these behaviors. The Racine scale consists of 5 levels, each classified as follows: Level 1: Mouth and facial clonic movements; Level 2: Level 1 + head nodding; Level 3: Level 2 + forelimb clonic movements; Level 4: Level 3 + standing; Level 5: Level 4 + repetitive standing and falling.

[0070] Drawing

[0071] In some embodiments of the invention, the method is a method for generating seizure maps of an individual using functional MRI (fMRI). For example, the invention generally provides a method for monitoring seizure activity and responses in an individual (also referred to herein as a “subject”). In some cases, the target organ or tissue is an excitable organ or tissue in the subject. As used herein, “excitable” refers to electrically excitable cells in an organ or tissue, such as neurons and muscle cells. Excitable cells typically transmit signals within the cell using changes in their membrane potential. Thus, excitable cells are characterized by having a resting state and an excited state, in which the membrane potential is the resting membrane potential, and in the excited state, the rapidly depolarizing membrane potential is transmitted within the cell as an action potential. The “cellular electrical activity” of an excitable cell can refer to changes in membrane potential, or any indirect measurement of changes in membrane potential, such as changes in intracellular calcium concentration, or any other biochemical change that is a functional indicator of changes in membrane potential.

[0072] In some embodiments, the method includes surgically implanting the device of the present invention into or near an individual's organ or tissue and monitoring the activity of the organ or tissue using fMRI. In some cases, surgical implantation of the device includes creating a pathway in the subject and inserting at least a portion of the device through the pathway. The pathway may be a pathway through the subject's skin, bone, muscle, and / or other tissues. For example, the pathway may include a pathway through the subject's bones (e.g., skull) to place at least a portion of the device (e.g., an optical electrode) near a target neuron of the subject.

[0073] In some cases, monitoring the activity of the organ or tissue involves functional magnetic resonance imaging (fMRI) of the organ or tissue. In some cases, the organ or tissue includes excitable cells (e.g., cells expressing one or more photoresponsive peptides). The terms “photoactivated” and “photoresponsive” refer to having photoresponsive peptides or proteins; they are used interchangeably and include photoresponsive ion channels or opsins and ion pumps as described herein. Such photoresponsive proteins may have depolarizing or hyperpolarizing effects on cells expressing the protein on their plasma membrane, depending on the ion permeability of the activating protein and the electrochemical gradient present across the plasma membrane.

[0074] In one embodiment, a combination of electrophysiological techniques (e.g., local field potential (LFP) and functional magnetic resonance imaging (fMRI) scanning different brain regions) is used to stimulate specific areas of an individual's brain to determine functional connectivity between the seizure propagation zone and other brain regions, and to image the motor activity of the seizure. Suitable analytical protocols include electrophysiology; photoinducible modulation of neural activity; electroencephalography (EEG) recording; functional imaging; and behavioral analysis. Electrophysiology may include single-electrode, multi-electrode, and / or field potential recording. As further described herein, photoinducible modulation of neural activity may include any suitable optogenetic approach. Functional imaging may include fMRI, as well as any functional imaging protocol using gene-encoded indicators (e.g., calcium indicators, voltage indicators, etc.). Behavioral analysis may include any suitable behavioral assays, such as those relating to arousal, memory (e.g., water maze tests), conditioned phenomena (e.g., fear conditioning), and sensory responses (responses to visual, somatosensory, auditory, gustatory, and / or olfactory cues).

[0075] Some protocols (e.g., fMRI) provide non-invasive, whole-brain measurements representing neural activity. Other protocols (e.g., electrophysiology) provide rapid measurements of neural activity at cellular resolution, as well as rapid control over these measurements. Still others (e.g., optogenetics) provide spatial localization and temporal control over the firing of action potentials within defined groups of neurons.

[0076] In some cases, the one or more photoresponsive peptides include hyperpolarized photoresponsive peptides. In some cases, the one or more photoresponsive peptides include depolarized photoresponsive peptides. Therefore, in some cases, the method includes obtaining images of the target organ or tissue using fMRI. In some cases, the organ or tissue can be imaged using fMRI before light is delivered to the target organ or tissue using the photopolarizer. In some cases, the organ or tissue can be imaged using fMRI during the process of delivering light to the target organ or tissue using the photopolarizer. In some cases, the organ or tissue can be imaged using fMRI after light is delivered to the target organ or tissue using the photopolarizer.

[0077] The method may further include using the device (e.g., an optical electrode) to detect and / or record detectable parameters of the organ or tissue. The optical electrode may be configured to detect electrical signals, such as local field potentials generated by changes in the membrane potential of the excitable cells. Therefore, in some cases, the method includes using carbon fiber electrodes of the optical electrode to detect and / or record detectable parameters of the organ or tissue.

[0078] The device (e.g., an optical electrode) may include a light source. In these embodiments, the method includes delivering light to the target organ or tissue using the light source. For example, the method may include stimulating the excitable cells in the target organ or tissue with light from the light source. In some cases, the light source includes an optical fiber as described herein. Therefore, in these embodiments, the method includes delivering light to the target organ or tissue using the optical fiber (e.g., stimulating the excitable cells with light delivered through the optical fiber). In some cases, the light source includes a laser. Therefore, in some embodiments, the method includes delivering light to the target organ or tissue using the laser. For example, the method may include generating light using the laser and directing the light from the laser to the target organ or tissue using the optical fiber (e.g., for stimulating the excitable cells in the target organ or tissue with light from the laser). In some cases, the light source includes a light-emitting diode (LED). Therefore, in some embodiments, the method includes delivering light to the target organ or tissue using the LED. For example, the method may include using the LED to generate light and using the optical fiber to direct the light from the LED to the target organ or tissue (e.g., to stimulate the excitable cells in the target organ or tissue with light from the LED).

[0079] In some embodiments, the detectable parameter of the target organ or tissue includes a local field potential, such as a local field potential generated by changes in the membrane potential of the excitable cell. The local field potential can be generated by stimulating the excitable cell with light from the light source. In some cases, the detectable parameter is a single-unit activity, such as detectable activity from a single target region (i.e., single-measurement). In some cases, the detectable parameter is a multi-unit activity, such as detectable activity from two or more target regions (i.e., multiplex measurement).

[0080] In some cases, monitoring of the activity of the organ or tissue is performed once. In other cases, monitoring of the activity of the organ or tissue is performed two or more times. In some cases, monitoring of the activity of the organ or tissue is performed several times over a period of time; for example, the method includes long-term monitoring of the activity of the organ or tissue. In some cases, the activity of the organ or tissue can be monitored for a longer period of time, such as 1 day or longer, 2 days or longer, 3 days or longer, 4 days or longer, 5 days or longer, 6 days or longer, 7 days or longer, 8 days or longer, 9 days or longer, 10 days or longer, for example, 1 week or longer, 2 weeks or longer, 3 weeks or longer, 1 month or longer, 2 months or longer, 3 months or longer, 4 months or longer, 5 months or longer, 6 months or longer, 7 months or longer, 8 months or longer, 9 months or longer, 10 months or longer, 11 months or longer, 1 year or longer, or even longer.

[0081] In some cases, the individual is a human. In some cases, the individual is a non-human primate. In some cases, the individual is a rodent (e.g., rat, mouse, etc.). The tissue or organ (e.g., "target tissue" or "target organ") can be in vivo neuronal tissue, tissue sections, nerve fiber bundles, neuromuscular junctions, etc. The in vivo neuronal tissue can be neuronal tissue from an anesthetized or unanesthetized and restrained or unrestrained animal. Target tissues include, but are not limited to, the neocortex, hypothalamus, entorhinal and hippocampal cortex, mammillary bodies, septa, bed nuclei of the stria terminalis, dorsal and ventral striatum, thalamus, amygdala, nucleus accumbens, brainstem, general subcortical structures, muscles, spinal cord, and cardiac tissue.

[0082] In some embodiments, the excitable cells (e.g., neurons) in a target tissue or organ are genetically modified to express a photoresponsive peptide that, upon stimulation with appropriate light stimulation, causes the stimulated excitable cells to hyperpolarize or depolarize. The term "genetic modification" refers to a permanent or transient genetic alteration induced in the cell after the introduction of a heterologous nucleic acid (i.e., a nucleic acid foreign to the cell). The genetic alteration ("modification") can be achieved by integrating the heterologous nucleic acid into the host cell's genome or by transiently or stably maintaining the heterologous nucleic acid as an extrachromosomal element. When the cell is a eukaryotic cell, permanent genetic alterations can be achieved by introducing the nucleic acid into the cell's genome. Suitable genetic modification methods include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microscopic injection, etc.

[0083] Photoresponsive peptides

[0084] In some cases, the target polypeptide contains an amino acid sequence encoding a photoreactive polypeptide. In some cases, the photoreactive polypeptide is a photoreactive cation channel. In some cases, target cells expressing a photoreactive polypeptide can be activated or inhibited upon exposure to light of different wavelengths. In some cases, the target cells expressing a photoreactive polypeptide are neurons expressing the photoreactive polypeptide, and exposure to light of different wavelengths causes these neurons to depolarize or polarize.

[0085] In some cases, the photoresponsive peptide is a photoactivated ion channel peptide. When the peptide is irradiated with light of an activation wavelength, the photoactivated ion channel peptide is adapted to allow one or more ions to cross the plasma membrane of a target cell. Photoactivated proteins can be characterized as ion pump proteins (capable of facilitating the passage of small numbers of ions through the plasma membrane based on photons) or as ion channel proteins (allowing a free flow of ions through the plasma membrane when the channel is open). In some embodiments, when the photoresponsive peptide is activated by light of an activation wavelength, it depolarizes the excitable cell. In some embodiments, when the photoresponsive peptide is activated by light of an activation wavelength, it hyperpolarizes the excitable cell.

[0086] In some cases, when cells are irradiated with light, photoresponsive peptides mediate hyperpolarization currents in their target cells. Non-limiting examples of photoresponsive peptides capable of mediating hyperpolarization currents can be found in International Patent Application No. PCT / US2011 / 028893, U.S. Patent No. 9,175,095, etc. Non-limiting examples of hyperpolarizing photoresponsive peptides include NpHr, eNpHr2.0, eNpHr3.0, eNpHr3.1, or GtR3. In some cases, when cells are irradiated with light, photoresponsive peptides mediate depolarization currents in their target cells. Non-limiting examples of depolarizing photoresponsive peptides include “C1V1”, ChR1, VChR1, and ChR2. Further information on other photoactivated cation channels, anion pumps, and proton pumps can be found in U.S. Patent Application Publication No. 2009 / 0093403 and International Patent Application No. PCT / US2011 / 028893.

[0087] In one embodiment, the photoreactive peptide can be activated by light with a wavelength of approximately 560 nm. In another embodiment, the photoreactive peptide can be activated by red light. In another embodiment, the photoreactive peptide can be activated by light with a wavelength of approximately 630 nm. In other embodiments, the photoreactive peptide can be activated by violet light. In one embodiment, the photoreactive peptide can be activated by light with a wavelength of approximately 405 nm. In other embodiments, the photoreactive peptide can be activated by green light. Those skilled in the art will recognize that each photoreactive peptide has its own activation wavelength range.

[0088] In some embodiments, the photoresponsive peptide is activated by blue light. In some embodiments, the photoresponsive peptide is activated by green light. In some embodiments, the photoresponsive peptide is activated by yellow light. In some embodiments, the photoresponsive peptide is activated by orange light. In some embodiments, the photoresponsive peptide is activated by red light.

[0089] In some cases, one or more optical fibers are used to irradiate the brain region containing neurons with photoresponsive peptides. The optical fiber can be configured in any suitable manner to guide light emitted from a suitable light source (e.g., a laser or light-emitting diode (LED) light source) to the brain region. The optical fiber can be any suitable optical fiber. In some cases, the optical fiber is a multimode optical fiber. The optical fiber may include a core defining a core diameter through which light from the light source passes. The optical fiber can have any suitable core diameter. In some cases, the core diameter of the optical fiber is 10 mm or greater, for example, 20 mm or greater, 30 mm or greater, 40 mm or greater, 50 mm or greater, 60 mm or greater, including 80 mm or greater, and is 1,000 mm or less, for example, 500 mm or less, 200 mm or less, 100 mm or less, including 70 mm or less. In some embodiments, the core diameter of the optical fiber ranges from 10 to 1,000 mm, for example, 20 to 500 mm, 30 to 200 mm, including 40 to 100 mm.

[0090] The fiber optic tip implanted in the target brain region can have any suitable configuration suitable for irradiating the brain region with light stimulation delivered through the fiber optic cable. In some cases, the fiber optic cable includes an attachment device located at or near the distal end of the fiber optic cable, wherein the distal end of the fiber optic cable corresponds to the tip inserted into the subject. In some cases, the attachment device is configured to connect to the fiber optic cable and facilitate attachment of the fiber optic cable to the subject, such as to the subject's skull. Any suitable attachment device can be used. In some cases, the attachment device includes a collar, such as a metal, ceramic, or plastic collar. The collar can have any suitable size for securing and attaching the fiber optic cable.

[0091] In some embodiments, any suitable electronic components can be used to implement the method of the invention to control and / or coordinate various optical components used to irradiate the brain region. The optical components (e.g., light sources, optical fibers, lenses, objectives, mirrors, etc.) can be controlled by a controller, for example, to coordinate the irradiation of the brain region with light pulses. The controller may include a driver for the light source capable of controlling one or more parameters associated with the light pulses, such as (but not limited to) the frequency, pulse width, duty cycle, wavelength, intensity, etc. of the light pulses. The controller can communicate with components of the light source (e.g., collimators, shutters, filter wheels, moving mirrors, lenses, etc.).

[0092] In some embodiments, the photoreactive peptide is activated by a light pulse, the duration of which can be any of the following: about 1 millisecond (ms), about 2 ms, about 3 ms, about 4 ms, about 5 ms, about 6 ms, about 7 ms, about 8 ms, about 9 ms, about 10 ms, about 15 ms, about 20 ms, about 25 ms, about 30 ms, about 35 ms, about 40 ms, about 45 ms, about 50 ms, about 60 ms, about 70 ms, about 80 ms, about 90 ms, about 100 ms, about 200 ms, about 300 ms, about 400 ms, about 500 ms, about 600 ms, about 700 ms, about 800 ms, about 900 ms, about 1 second, about 1.25 seconds, about 1.5 seconds, or about 2 seconds (inclusive), including any time between these numbers. In some embodiments, the photoreactive peptide is activated by a light pulse, the light power density of which can be any of the following: approximately 0.05 mW / mm². 2 Approximately 0.1 mW / mm 2 Approximately 0.25mW / mm 2 Approximately 0.5mW / mm 2 Approximately 0.75mW / mm 2 Approximately 1mW / mm 2 Approximately 2mW / mm 2 Approximately 3mW / mm 2 Approximately 4mW / mm 2 Approximately 5mW / mm 2 Approximately 6mW / mm 2 Approximately 7mW / mm 2 Approximately 8mW / mm 2 Approximately 9mW / mm 2 Approximately 10mW / mm 2 Approximately 20mW / mm 2 Approximately 50mW / mm 2 Approximately 100mW / mm 2 Approximately 250mW / mm 2 Approximately 500mW / mm 2 Approximately 750mW / mm 2 Approximately 1000mW / mm 2 Approximately 1100mW / mm 2 Approximately 1200mW / mm 2 Approximately 1300mW / mm 2 Approximately 1400mW / mm 2 Approximately 1500mW / mm 2 Approximately 1600mW / mm 2 Approximately 1700mW / mm 2 Approximately 1800mW / mm 2Approximately 1900mW / mm 2 Approximately 2000mW / mm 2 Approximately 2100mW / mm 2 Approximately 2200mW / mm 2 Approximately 2300mW / mm 2 Approximately 2400mW / mm 2 Approximately 2500mW / mm 2 Approximately 2600mW / mm 2 Approximately 2700mW / mm 2 Approximately 2800mW / mm 2 Approximately 2900mW / mm 2 Approximately 3000mW / mm 2 Approximately 3100mW / mm 2 Approximately 3100mW / mm 2 Approximately 3300mW / mm 2 Approximately 3400mW / mm 2 Or approximately 3500mW / mm 2 (inclusive), including any value between these numbers.

[0093] The photostimulation used to activate the photoresponsive peptide may include light pulses characterized by parameters such as frequency, pulse width, duty cycle, wavelength, and intensity. In some cases, the photostimulation includes two or more sets of different light pulses, each set characterized by a different temporal pattern. The temporal pattern may be characterized by any suitable parameter, including but not limited to frequency, period (i.e., the total duration of the photostimulation), pulse width, duty cycle, etc.

[0094] The light pulses can have any suitable frequency. In some cases, the group of light pulses contains a single light pulse that continues throughout the entire duration of light stimulation. In some cases, the frequency of the group of light pulses is 0.1 Hz or higher, for example, 0.5 Hz or higher, 1 Hz or higher, 5 Hz or higher, 10 Hz or higher, 20 Hz or higher, 30 Hz or higher, 40 Hz or higher, including 50 Hz or higher, 60 Hz or higher, 70 Hz or higher, 80 Hz or higher, 90 Hz or higher, 100 Hz or higher, and the frequency is 100,000 Hz or lower, for example, 10,000 Hz or lower, 1,000 Hz or lower, 500 Hz or lower, 400 Hz or lower, 300 Hz or lower, 200 Hz or lower, including 100 Hz or lower. In some embodiments, the frequency range of the optical pulse is 0.1 to 100,000 Hz, for example, 1 to 10,000 Hz, 1 to 1,000 Hz, including 5 to 500 Hz or 10 to 100 Hz.

[0095] In some cases, the two sets of optical pulses are characterized by having different parameter values, such as different pulse widths, for example, short or long. The optical pulses can have any suitable pulse width. In some cases, the pulse width is 0.1 ms or longer, for example 0.5 ms or longer, 1 ms or longer, 3 ms or longer, 5 ms or longer, 7.5 ms or longer, 10 ms or longer, including 15 ms or longer, 20 ms or longer, 25 ms or longer, 30 ms or longer, 35 ms or longer, 40 ms or longer, 45 ms or longer, 50 ms or longer, and 500 ms or shorter, for example 100 ms or shorter, 90 ms or shorter, 80 ms or shorter, 70 ms or shorter, 60 ms or shorter, 50 ms or shorter, 45 ms or shorter, 40 ms or shorter, 35 ms or shorter, 30 ms or shorter, 25 ms or shorter, including 20 ms or shorter. In some embodiments, the pulse width ranges from 0.1 to 500 ms, for example, 0.5 to 100 ms, 1 to 80 ms, including 1 to 60 ms, 1 to 50 ms, or 1 to 30 ms.

[0096] The average power of the light pulse (measured at the tip of the optical fiber that delivers the light pulse to the brain region) can be any suitable power. In some cases, the power is 0.1 mW or more, for example, 0.5 mW or more, 1 mW or more, 1.5 mW or more, including 2 mW or more, 2.5 mW or more, 3 mW or more, 3.5 mW or more, 4 mW or more, 4.5 mW or more, 5 mW or more, and can be 1,000 mW or less, for example, 500 mW or less, 250 mW or less, 100 mW or less, 50 mW or less, 40 mW or less, 30 mW or less, 20 mW or less, 15 mW or less, including 10 mW or less or 5 mW or less. In some embodiments, the power ranges from 0.1 to 1,000 mW, for example, 0.5 to 100 mW, 0.5 to 50 mW, 1 to 20 mW, including 1 to 10 mW or 1 to 5 mW.

[0097] The wavelength and intensity of the light pulse can vary and can depend on the activation wavelength of the photoresponsive peptide, the optical transparency of the brain region, the expected brain volume to be irradiated, etc.

[0098] The volume of the brain region irradiated by the light pulse can be any suitable volume. In some cases, the irradiated volume is 0.001 mm. 3 Or larger, for example, 0.005mm 3 Or larger, 0.001mm 3 Or larger, 0.005mm3 Or larger, 0.01mm 3 Or larger, 0.05mm 3 Or larger, including 0.1mm 3 Or larger, and 100mm 3 Or smaller, for example, 50mm 3 Or smaller, 20mm 3 or smaller, 10mm 3 or smaller, 5mm 3 or smaller, 1mm 3 Or smaller, including 0.1mm 3 Or smaller. In some cases, the irradiated volume ranges from 0.001 to 100 mm. 3 For example, 0.005 to 20 mm 3 0.01 to 10 mm 3 0.01 to 5 mm 3 Including 0.05 to 1 mm 3 .

[0099] In some embodiments, the photoresponsive polypeptide expressed in cells may be fused to one or more amino acid sequence motifs selected from the group consisting of signal peptides, endoplasmic reticulum (ER) output signals, membrane transport signals, and / or N-terminal Golgi output signals. The one or more amino acid sequence motifs that enhance the transport of the photoresponsive protein to the mammalian cell membrane may be fused to the N-terminus, C-terminus, or both. In some cases, the one or more amino acid sequence motifs that enhance the transport of the photoresponsive protein to the mammalian cell membrane are fused internally to the photoresponsive polypeptide. Optionally, the photoresponsive polypeptide and the one or more amino acid sequence motifs may be separated by linkers. In some embodiments, the photoresponsive polypeptide may be modified by adding a transport signal (ts) that enhances the transport of the protein to the cell membrane. In some embodiments, the transport signal may be derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In some embodiments, the signal peptide sequence in the protein may be deleted or replaced with a signal peptide sequence from a different protein.

[0100] Exemplary photoresponsive peptides and amino acid sequence motifs that can be used in the systems and methods of this invention can be found in PCT patent applications numbered PCT / US2011 / 028893 and PCT / US2015 / 23087, etc.

[0101] The target photoresponsive peptide includes, for example, step-function opsin (SFO)6 protein or stable step-function opsin (SSFO) protein, which may have specific amino acid substitutions at key positions in the retinaldehyde binding pouch of the protein. See, for example, WO 2010 / 056970, the contents of which are incorporated herein by reference in their entirety. The peptide may be a cation channel derived from Volvox (VChR1) and optionally contain one or more amino acid substitutions, such as C123A; C123S; D151A, etc. The photoresponsive cation channel protein may be a C1V1 chimeric protein of the VChR1 protein derived from Volvox and the ChR1 protein derived from Chlamydomonas reinhardtii, wherein the protein comprises the amino acid sequence of VChR1 having at least the first and second transmembrane helices replaced by the first and second transmembrane helices of ChR1, optionally having an amino acid substitution at amino acid residue E122 or E162. In other embodiments, the photoresponsive cation channel protein is a C1C2 chimeric protein derived from the ChR1 and ChR2 proteins of *Chlamydomonas reinhardtii*, wherein the protein is light-responsive and mediates depolarization currents in the cell when the cell is illuminated. In some embodiments, the depolarization photoresponsive peptide is a redshifted variant of the depolarization photoresponsive peptide derived from *Chlamydomonas reinhardtii*; referred to as "ReaChR peptide" or "ReaChR protein" or "ReaChR". In some embodiments, the depolarization photoresponsive peptide is the SdChR peptide derived from *Scherffelia dubia*, wherein the SdChR peptide is capable of transporting cations across the cell membrane when the cell is illuminated. In some embodiments, the depolarization photoresponsive peptide is CnChR1 derived from *Chlamydomonas noctiluca*, wherein the CnChR1 peptide is capable of transporting cations across the cell membrane when the cell is illuminated. In some embodiments, the photoresponsive cation channel protein is a CsChrimson chimeric protein derived from the CsChR protein of *Chloromonas subdivisa* and the CnChR1 protein of *Chloromonas natans*, wherein the N-terminus of the protein comprises amino acid residues 1-73 of CsChR, followed by amino acid residues 79-350 of CnChR1; it is photoresponsive; and when the cell is irradiated with light, it mediates a depolarization current in the cell. In some embodiments, the depolarization photoresponsive peptide may be, for example, ShChR1 derived from *Trichoderma pulvinata*, wherein the ShChR1 peptide is capable of transporting cations across the cell membrane when the cell is irradiated with light.

[0102] In some embodiments, the depolarization-responsive peptide is derived from *Chlamydomonas reinhardtii* (CHR1, particularly CHR2), wherein the peptide is capable of transporting cations across the cell membrane when the cells are irradiated with light, and mediates depolarization currents within the cells when the cells are irradiated with light. In some embodiments, a humanized rhodopsin CHR2 H134R mutant fused to EYFP and driven by CaMKIIa is used for optogenetic activation. The wavelength of the light used to activate the photoresponsive cation channel protein derived from *Chlamydomonas reinhardtii* can be between about 460 and about 495 nm, or the wavelength can be about 480 nm. The photoresponsive cation channel protein may additionally include substitutions, deletions, and / or insertions introduced into the native amino acid sequence to increase or decrease sensitivity to light, increase or decrease sensitivity to light of a specific wavelength, and / or increase or decrease the ability of the photoresponsive cation channel protein to regulate the polarization state of the cytoplasmic membrane. Furthermore, the photoresponsive cation channel protein may contain one or more conserved amino acid substitutions and / or one or more non-conserved amino acid substitutions. The light-responsive proton pump protein containing substitutions, deletions, and / or insertions introduced into the natural amino acid sequence appropriately retains the ability to transport cations across the cell membrane. The protein may contain a variety of amino acid substitutions, such as one or more of H134R, T159C, L132C, E123A, etc.; the protein may also contain fluorescent proteins, such as (but not limited to) yellow fluorescent protein, red fluorescent protein, green fluorescent protein, or cyan fluorescent protein.

[0103] The individual can be any suitable individual used to analyze data on the individual's brain functional activity. In some cases, the individual is a single person. In some cases, the person is either physically healthy or has a neurological disorder. The neurological disorder can be any suitable neurological disorder. In some cases, the neurological disorder is caused by a disease such as a neurological disorder. The neurological disorder can be any suitable disease related to the pathological activity of the neuronal network. Suitable neurological disorders include, but are not limited to, Parkinson's disease, Alzheimer's disease, dementia, epilepsy, autism, bipolar disorder, schizophrenia, Tourette syndrome, obsessive-compulsive disorder, attention deficit hyperactivity disorder, Huntington's disease, multiple sclerosis, or migraine. In some embodiments, the neurological disorder is an age-related brain dysfunction.

[0104] In some embodiments, the method may be used to treat a disease or condition (e.g., a neurological disorder) in a subject, wherein the subject is fit to be treated with the targeted method. As used herein, “treatment” and the like refer to achieving the desired pharmacological and / or physiological effect. A preventive effect refers to complete or partial prevention of a disease or its symptoms, and a therapeutic effect refers to partial or complete cure of a disease and / or adverse reactions caused by the disease. As used herein, “treatment” encompasses any treatment of diseases in mammals, particularly humans, including: (a) preventing a subject from developing a disease, wherein the subject may be susceptible to the disease but has not yet been diagnosed; (b) inhibiting the disease, i.e., preventing its development; or (c) alleviating the disease, e.g., causing the disease to regress, e.g., completely or partially eliminating the symptoms of the disease.

[0105] Neurons can be selectively activated using any suitable method to measure subtype-specific functional activity. Suitable methods for selectively activating neurons include, but are not limited to, optogenetic stimulation, single-unit electrophysiology, etc. When the neuron is selectively activated by optogenetic stimulation, the neuron may express one or more photoactivating peptides configured to hyperpolarize or depolarize the neuron. Suitable photoactivating peptides and methods of their use are further described below.

[0106] The photoactivated polypeptide used in this invention can be any suitable photoactivated polypeptide for selectively activating neuronal subtypes by irradiating the neurons with activating light. In some cases, the photoactivated polypeptide is a photoactivated ion channel polypeptide. When the polypeptide is irradiated with light of an activation wavelength, the photoactivated ion channel polypeptide is adapted to allow one or more ions to cross the plasma membrane of the target cell. The photoactivated protein can be characterized as an ion pump protein (capable of facilitating the passage of a small number of ions through the plasma membrane based on photons) or as an ion channel protein (allowing ion flow to freely pass through the plasma membrane when the channel is open). In some embodiments, when the photoactivated polypeptide is activated by light of an activation wavelength, it depolarizes the cell. In some embodiments, when the photoactivated polypeptide is activated by light of an activation wavelength, it hyperpolarizes the cell. Suitable hyperpolarizing and depolarizing peptides are peptides known in the art, including, for example, channel rhodopsin (e.g., ChR2), variants of ChR2 (e.g., C128S, D156A, C128S+D156A, E123A, E123T), iC1C2, C1C2, GtACR2, NpHR, eNpHR3.0, C1V1, VChR1, VChR2, SwiChR, Arch, ArchT, KR2, ReaChR, ChiEF, Chronos, ChRGR, CsChrimson, etc. In some cases, the photoactivated peptide includes bReaCh-ES as described herein and further described in the following publications: e.g., Rajasethupathy et al., Nature, October 29, 2015, 526(7575):653, which is incorporated herein by reference. For hyperpolarized and depolarized opsins, see various publications; see, for example, Berndt and Deisseroth (2015), Science, 349:590; Berndt et al. (2014), Science, 344:420; and Guru et al. (July 25, 2015), International Journal of Neuropsychopharmacology, pp. 1–8 (PMID 26209858).

[0107] The photoactivated polypeptide can be introduced into the neuron using any suitable method. In some cases, the target neuronal subtype is genetically modified to express the photoactivated polypeptide. In other cases, the neuron can be genetically modified using a viral vector, such as an adeno-associated virus vector containing a nucleic acid having a nucleotide sequence encoding the photoactivated polypeptide. The viral vector may include any suitable control elements (e.g., promoters, enhancers, recombination sites, etc.) to control the expression of the photoactivated polypeptide based on neuronal subtype, timing, the presence of inducers, etc.

[0108] "Operationally linked" means juxtaposed, in which the components described so far are in a relationship that allows them to function in the intended manner. For example, a promoter is operably linked to a nucleotide sequence (e.g., a protein-coding sequence, such as a sequence encoding mRNA; a non-protein-coding sequence, such as a sequence encoding a light-reactive protein; etc.), provided that the promoter is capable of influencing the transcription and / or expression of the nucleotide sequence.

[0109] Neuron-specific promoters and other control elements (e.g., enhancers) are promoters and control elements known in the art. Suitable neuron-specific control sequences include, but are not limited to, neuron-specific enolase (NSE) promoters (see, e.g., EMBL HSENO2, X51956; see also, e.g., U.S. Patent No. 6,649,811 and U.S. Patent No. 5,387,742); aromatic amino acid decarboxylase (AADC) promoters; neurofilament promoters (see, e.g., GenBank HUMNFL, L04147); synaptic protein promoters (see, e.g., GenBank HUMSYNIB, M55301); thy-1 promoters (see, e.g., Chen et al. (1987), Cell, 51:7-19; and Llewellyn et al. (2010), Nature: Medicine, 16:1161); serotonin receptor promoters (see, e.g., GenBank...). S62283); Tyrosine hydroxylase promoter (TH) (see, for example, Nucleic Acid Research, 15: 2363-2384 (1987) and Neuron, 6: 583-594 (1991)); GnRH promoter (see, for example, Radovick et al., Proceedings of the National Academy of Sciences, 88: 3402-3406 (1991)); L7 promoter (see, for example, Oberdick et al., Science, 248: 223-226 (1990)); DNMT promoter (see, for example, Bartge et al., Proceedings of the National Academy of Sciences, 85) : 3648-3652 (1988)); Enkephalin promoter (see, e.g., Comb et al., EMBO Journal, 17: 3793-3805 (1988)); Myelin basic protein (MBP) promoter; CMV enhancer / platelet-derived growth factor-β promoter (see, e.g., Liu et al. (2620), Gene Therapy, 11: 52-60); Motor neuron-specific gene Hb9 promoter (see, e.g., U.S. Patent No. 7,632,679; and Lee et al. (2620), Development, 131: 3295-3306); Ca 2+- The α subunit of the calmodulin-dependent protein kinase II (CaMKIIa) promoter (see, for example, Mayford et al. (1996), Proceedings of the National Academy of Sciences, 93:13250). Other suitable promoters include elongation factor (EF)1 and dopamine transporter (DAT) promoters.

[0110] In some cases, neuronal subtype-specific expression of the photoactivated peptide can be achieved using recombination systems, such as Cre-Lox recombination, Flp-FRT recombination, etc. Cell type-specific expression of genes achieved using recombination can be found in the following publications: for example, Fenno et al., Nature: Methods, July 2014; 11(7): 763; and Gompf et al., Frontiers in Behavioral Neuroscience, July 2, 2015; 9: 152, which are incorporated herein by reference.

[0111] In some embodiments, the vector is a recombinant adeno-associated virus (AAV) vector. AAV vectors are relatively small DNA viruses that can integrate into the genome of the cells they infect in a stable and site-specific manner. They can infect a range of cells without affecting cell growth, morphology, or differentiation, and they appear not to be involved in human pathogenesis. The AAV genome has been cloned, sequenced, and characterized. It comprises approximately 4700 bases and contains approximately 145 bases at each end of an inverted terminal repeat (ITR) region, serving as the origin of viral replication. The remainder of the genome is divided into two basic regions with capsid functions: the left-hand portion of the genome, containing the rep gene involved in viral replication and viral gene expression; and the right-hand portion of the genome, containing the cap gene encoding viral capsid proteins.

[0112] In recent years, the application of AAV as a gene therapy vector has developed rapidly. Wild-type AAV can infect dividing or non-dividing cells or tissues of mammals (including humans) at high titers, and can also integrate into human cells at specific sites (on the long arm of chromosome 19) (Kotin et al., Proceedings of the National Academy of Sciences, 1990, 87: 2211-2215; Samulski et al., EMBO Journal, 1991, 10: 3941-3950, the contents of which are incorporated herein by reference in full). AAV vectors without the rep and cap genes do not have the specificity to achieve site-specific integration, but can still mediate the long-term stable expression of exogenous genes. AAV vectors exist in cells in two forms: one is an extrachromosomal episome; the other is integrated into the chromosome, with the former being the predominant form. Furthermore, to date, no human diseases have been found to be associated with AAV, nor have any changes in biological characteristics caused by integration been observed. The literature reports 16 AAV serotypes, namely AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16. AAV5 was first isolated from humans (Bantel-Schaal and H. zur Hausen, Virology, 1984, 134: 52-63), while AAV1-4 and AAV6 were discovered in adenovirus studies (Ursula Bantel-Schaal, HajoDelius, and Harald zur Hausen, Journal of Virology, 1999, 73: 939-947).

[0113] AAV vectors can be prepared by any suitable method. Any serotype of adeno-associated virus is suitable (see, for example, Blacklow, “Parvoviruses and Human Diseases”, JR.R. Attison (ed.), pp. 165–174 (1988); Rose, Synthetic Virology, 3:1, 1974; P. Tattersall, “Evolution of the Taxonomic Criteria for Parvoviruses” (JR. Kerr, S. F. C. Otmore, ME. Bloom, RM. Linden, CR. Parrish, ed.), Hudder Arnold, London, UK, pp. 5–14 (2006); and DE. Bowles, J.E. Rabinowitz, R.J. Samulski, “Dependent Viruses” (JR. Kerr, S. F. C. Otmore, ME. Bloom, RM. Linden, CR. Parrish, ed.), Hudder Arnold, London, UK, pp. 15–23 (2006), the contents of which are incorporated herein by reference in their entirety). Methods for purifying the vector are described in the following publications: e.g., U.S. Patents Nos. 6,566,118, 6,989,264, and 6,995,006; WO / 1999 / 011764, entitled “Method for producing a high-titer, helper-free viral formulation of a recombinant AAV vector,” the contents of which are incorporated herein by reference in their entirety. Details of the preparation of the hybridization vector are described in the following publications: e.g., PCT application No. PCT / US2005 / 027091, the contents of which are incorporated herein by reference in their entirety. The transfer of genes in vitro and in vivo using vectors derived from AAV is described (see, e.g., International Patent Application Publications Nos. 91 / 18088 and WO 93 / 09239; U.S. Patents Nos. 4,797,368, 6,596,535, and 5,139,941; and European Patent No. 0488528, the contents of which are incorporated herein by reference in their entirety). These publications describe various AAV-derived constructs (in which the rep and / or cap genes are deleted and replaced by the target gene) and the uses of these constructs for transferring the target gene in vitro (integrated into cultured cells) or in vivo (directly integrated into an organism). The replication-defective recombinant AAV according to the invention can be prepared by co-transfecting a plasmid containing a target nucleic acid sequence flanked by two AAV terminal inverted repeat (ITR) regions and a plasmid carrying AAV capsidation genes (rep and cap genes) into a cell line infected with a human helper virus (e.g., adenovirus). The resulting AAV recombinant is then purified using standard techniques.

[0114] In some embodiments, the vector used in the methods of the present invention is encapsulated in viral particles (e.g., AAV viral particles, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16). Therefore, the present invention includes recombinant viral particles (recombinants, because they contain recombinant polynucleotides) that comprise any of the vectors described herein. Methods for producing such particles are known in the art, and a related description is given in U.S. Patent No. 6,596,535.

[0115] Regarding the animal cells described herein, it should be understood that one or more vectors may be applied to nerve cells, cardiac cells, or stem cells. If more than one vector is used, it should be understood that these vectors may be applied to the animal cells simultaneously or at different times.

[0116] Examples of the methods and systems described herein can be used in a variety of MRI applications, such as MRI methods and systems that require high-resolution MRI images. In some cases, the target methods and systems can be used to generate high-resolution functional MRI (fMRI) images of individual target regions. For example, the target methods and systems can be used in fMRI techniques for measuring individual brain activity, such as by detecting changes in blood flow associated with one or more target regions in an individual's brain. In other cases, the target methods and systems can be used to generate high-resolution functional MRI (fMRI) images of individual target regions, where the activity of excitable cells in an individual target organ or tissue is assessed. As described herein, the target methods and systems can be used to detect the activity of light-responsive peptides (e.g., light-activated ion channels) in individual excitable cells (e.g., neurons). Therefore, the target methods and systems can be used for whole-brain and / or regional brain function studies, such as mapping the activity of one or more target regions in the brain at high resolution.

[0117] In some embodiments, the target methods and systems can be used to generate high-resolution fMRI images of individual target regions, including high-resolution fMRI images generated offline (i.e., processing image data at some point after image data acquisition) and high-resolution fMRI images generated in real time (i.e., processing image data immediately after image data acquisition and / or during image data acquisition).

[0118] In some embodiments, the methods and systems of the present invention can be used to screen for neuronal circuit elements suitable for the diagnosis or etiological determination of neuropsychiatric diseases in in vitro and / or in vivo animal models of disease. For example, the methods and systems of the present invention can be used for preoperative brain function diagnosis. Embodiments of the methods and systems of the present invention can also be used to plan brain-computer interfaces, for example, by mapping neuronal activity in brain regions to determine the appropriate location of the brain-computer interface in the brain.

[0119] In some embodiments, the methods and systems of the present invention can be used in methods for determining treatment regimens (e.g., therapeutic treatment regimens) that achieve desired activity in a group of neurons. If the desired outcome is known, the methods and systems of the present invention can be used to screen treatment regimens, including but not limited to optogenetic therapy, pharmaceuticals, non-chemical-based therapeutics, behavioral therapy, neuromodulation therapy based on electricity, magnetism, or light, etc., which will induce the desired neuronal activity pattern. The screening can be performed in any suitable animal model, which can be a normal model, a model of a neurological disorder (e.g., Alzheimer's and Parkinson's diseases, mild cognitive impairment, other dementias and Down syndrome, as well as schizophrenia, autism, mood disorders, affective disorders, anxiety disorders, and personality / developmental disorders), or other disease models described herein.

[0120] In some embodiments, the methods and systems of the present invention can be used to treat conditions or disorders, such as neurological or psychiatric disorders, using optogenetic control. When monitoring the real-time activity of neurons using the methods and systems of the present invention, a controller or processor can be configured to modulate the activity of neurons in response to imaged activity signals, thereby treating or alleviating the corresponding symptoms of the condition or disorder at a behavioral and / or physiological level.

[0121] epileptic seizure model

[0122] This paper provides methods and models for analyzing brain circuits and regional relationships involved in epileptic seizures in vivo, particularly by imaging individual seizures to finely differentiate their effects. The methods of this invention can image the effects of seizures using any number of combinations of appropriate neuronal stimulation and neuronal activity measurement protocols as needed. The methods and models may include, for example, one or more of the following: individual seizure analysis, focal progression to bilateral tonic-clonic (FBTC) seizure analysis, excitatory ventral hippocampal (VH) network analysis, etc.

[0123] To image a single seizure using simultaneous LFP-fMRI in an animal model, the animal may be sedated and given a short-acting neuromuscular blocking agent to prevent movement during seizure imaging. Exemplary agents for this purpose include, but are not limited to, dexmedetomidine sedatives and vecuronium bromide. Seizures can be induced by: optogenetic stimulation, electrical stimulation such as whole-brain electrical stimulation protocols, single-evoked post-epileptic discharges; chemoconvulsants such as pilocarpine, tetanus toxin, PTZ, phycocyanin, fluteil, etc.; hydraulic shock injury; and high-intensity acoustic stimulation. In some embodiments, optogenetic stimulation is preferred.

[0124] In one embodiment, a combination of electrophysiological techniques (e.g., local field potential (LFP) and functional magnetic resonance imaging (fMRI) scanning different brain regions) is used to stimulate specific areas of an individual's brain to determine functional connectivity between the seizure propagation zone and other brain regions, and to image the motor activity of the seizure. Suitable analytical protocols include electrophysiology; photoinducible modulation of neural activity; electroencephalography (EEG) recording; functional imaging; and behavioral analysis. Electrophysiology may include single-electrode, multi-electrode, and / or field potential recording. As further described herein, photoinducible modulation of neural activity may include any suitable optogenetic approach. Functional imaging may include fMRI, as well as any functional imaging protocol using gene-encoded indicators (e.g., calcium indicators, voltage indicators, etc.). Behavioral analysis may include any suitable behavioral assays, such as those relating to arousal, memory (e.g., water maze tests), conditioned phenomena (e.g., fear conditioning), and sensory responses (responses to visual, somatosensory, auditory, gustatory, and / or olfactory cues).

[0125] Some protocols (e.g., fMRI) provide non-invasive, whole-brain measurements representing neural activity. Other protocols (e.g., electrophysiology) provide rapid measurements of neural activity at cellular resolution, as well as rapid control over these measurements. Still others (e.g., optogenetics) provide spatial localization and temporal control over the firing of action potentials within defined groups of neurons.

[0126] In some embodiments, an optogenetic model of seizures is provided, wherein electrophysiological seizures are induced in an animal model via cell-type-specific optogenetic stimulation. Simultaneous electrophysiology and fMRI can be used to determine the inhibitory effect on targeted neurons. The animal model can be used to design and test therapeutic interventions, such as surgery, pharmacological therapies, etc., wherein the effect of the therapeutic intervention on the spread of seizures can be determined.

[0127] In some embodiments, if the duration or severity of a seizure decreases, it is determined that the medication can provide an effective targeted intervention for the seizure. In some embodiments, the severity of the seizure is determined using the Racine scale. In some embodiments, if the severity of a seizure decreases by at least one grade according to the Racine scale, it is determined that the medication can provide an effective targeted intervention. For example, reducing the severity of the seizure from Racine grade 5 to grade 4. In some embodiments, the severity of the seizure decreases by 2, 3, or 4 grades, or all seizure symptoms disappear.

[0128] Drug design

[0129] This invention provides a method for optimizing a therapy, wherein the optimization involves: analyzing the impact of epileptic seizures on brain regions, and based on the information, selecting appropriate candidate drugs and treatment modalities best suited to address seizure induction and spread, while minimizing unintended toxicity. The therapy is optimized by selecting treatment regimens that minimize unintended toxicity while providing effective activity.

[0130] The model presented in this paper can be used to design and test therapeutic interventions, such as surgery and pharmacological therapies, to determine the effects of these interventions on the induction and spread of epileptic seizures.

[0131] Parameters are quantifiable characteristics of cells, tissues, and organisms (especially those components that can be accurately measured). For example, parameters can be the location, intensity, duration, velocity, etc., of electrophysiological discharges, and can be imaged using fMRI, LFP, etc. Readings can include a single, definitive value, or can include a mean, median, or variance. Typically, for a parameter, multiple measurements are taken to obtain a series of parameter readouts. The corresponding values ​​are expected to vary, and standard statistical methods and common statistical methods used to provide individual values ​​are employed to obtain the range of values ​​for each parameter in the test parameter set.

[0132] The target candidate drugs are bioactive agents used in drug design, encompassing many chemical categories, primarily organic molecules (which may include organometallic molecules), inorganic molecules, gene sequences, etc. Also of interest are therapeutic interventions, such as surgery, deep brain stimulation, optogenetics, etc. An important aspect of this invention is evaluating candidate therapies with preferred biological responses.

[0133] This includes pharmacologically active drugs, genetically active molecules, etc. Target compounds include chemotherapeutic agents, anti-inflammatory agents, hormones or hormone antagonists, ion channel modifiers, and neuroactive agents. Examples of pharmaceutical formulations suitable for this invention are those described in the following publication: “Pharmacological Basis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, NY, (1996), Ninth Edition, Sections: Drugs Acting on Synaptic and Nerve Effector Connections; Drugs Acting on the Central Nervous System; Autologous Effective Substances: Pharmacological Treatment of Inflammation; Water, Salts, and Ions; etc.

[0134] Test compounds include all the types of molecules mentioned above, and may further include samples in unknown amounts. Complex mixtures of naturally occurring compounds derived from natural sources (e.g., plants) are of interest. While many samples contain compound solutions, solid samples soluble in suitable solvents can also be measured. Target samples include environmental samples, such as groundwater, seawater, mining waste, etc.; biological samples, such as lysates prepared from crop or tissue samples; manufacturing samples, such as timelines in drug development; and compound libraries prepared for analysis; etc. Target samples also include compounds for which potential therapeutic value is being evaluated, i.e., candidate drugs.

[0135] The term "sample" also includes the aforementioned fluid to which additional components have been added, such as those affecting ionic strength, pH, total protein concentration, etc. Furthermore, the sample can be processed to achieve at least partial fractionation or concentration. To minimize degradation of the compounds, the biological sample can be stored under nitrogen, cryopreserved, or a combination thereof. The volume of sample used should be sufficient for measurable detection; typically, approximately 0.1:1 to 1 ml of biological sample is adequate.

[0136] Compounds (including candidate agents) can be obtained from a variety of sources, including synthetic or natural compound libraries. For example, numerous methods are available for the random and directed synthesis of a wide range of organic compounds (including biomolecules), including the expression of random oligonucleotides and oligopeptides. Alternatively, natural compound libraries in the form of bacterial, fungal, plant, and animal extracts can be obtained or readily generated. Furthermore, naturally or synthetically produced libraries and compounds can be readily modified using conventional chemical, physical, and biochemical methods and can be used to generate combinatorial libraries. Agents are known to be capable of direct or random chemical modifications (e.g., acylation, alkylation, esterification, amidation, etc.) to generate structural analogs.

[0137] As used herein, the term "genetic factor" refers to a polynucleotide or analogue, which is tested in the screening assays of this invention by adding the genetic factor to cells. The introduction of the genetic factor alters the overall genetic composition of the cell. As used herein, the genetic factor can induce protein expression and its effects on one or more target pathways are being evaluated. The genetic factor (e.g., DNA) induces experimentally introduced changes in the cell genome, typically by integrating the sequence into a chromosome. Genetic changes can also be transient, where the exogenous sequence is not integrated but remains as an episome. RNA viruses containing the target gene and which are reverse transcribed and inserted into the host cell genome can be used. Genetic factors (peptides or polynucleotides) can also be synthesized in vitro and delivered into cells by conjugation with a portion that induces the transfer of the agent into the target cell (e.g., the 16-amino acid "Penetratin-1 peptide" from the antennae, available from Qbiogene). The genetic factor acts to increase the expression of a specific gene product in the cell and may increase and / or decrease other products in the cell.

[0138] In some cases, chemical agents with known or unknown activity are administered to animals, and their effects on seizure induction, spread, and movement are evaluated. These chemical agents can be used to activate pathways, inhibit pathways, etc., with a focus on regulating pathways other than the target pathway, and chemical agents may be more suitable than natural factors. The chemical agents are suitably added in solution or readily soluble form and can be administered to animals in various ways, such as oral, subcutaneous, and intubation methods known in the art. Preferred chemical agent formulations consist essentially of a biologically active compound and a physiologically acceptable carrier (e.g., water, saline, etc.).

[0139] In one embodiment, the induction and inhibition of AD are combined with a combination of electrophysiological techniques (e.g., local field potentials (LFP) and functional magnetic resonance imaging (fMRI) scans) to stimulate specific regions of the individual's brain. The animals may be sedated, for example with dexmedetomidine; and given a short-acting neuromuscular blocking agent, such as vecuronium bromide, to prevent movement of the animals during seizure imaging with simultaneous LFP-fMRI.

[0140] Suitable analytical protocols include electrophysiology; photoinducible modulation of neural activity; electroencephalography (EEG) recording; functional imaging; and behavioral analysis. Electrophysiology may include single-electrode, multi-electrode, and / or field potential recording. As further described herein, photoinducible modulation of neural activity may include any suitable optogenetic approach. Functional imaging may include fMRI, as well as any functional imaging protocol that uses gene-encoded indicators (e.g., calcium indicators, voltage indicators, etc.). Behavioral analysis may include any suitable behavioral assays, such as those relating to arousal, memory (e.g., water maze tests), conditioning (e.g., fear conditioning), and sensory responses (responses to visual, somatosensory, auditory, gustatory, and / or olfactory cues).

[0141] The model presented in this paper can be used to design and test therapeutic interventions, such as surgery and pharmacological interventions (drug therapy), to determine their impact on seizure induction and spread. It can be compared with known antiepileptic drugs, such as gabapentin, topiramate, lamotrigine, levetiracetam, staefenol and rufinamide, oxcarbazepine, lacosamide, and perampanel.

[0142] Comparison of measurement results obtained from the test and reference reagents can be accomplished using appropriate derivation schemes, AI systems, statistical comparisons, etc. The data is compared with a reference results database. A reference results database can be compiled. For each reference and test pattern, a data matrix is ​​typically generated, where each point in the data matrix corresponds to a reading of a parameter. The data for each parameter can come from repeated measurements, such as multiple individual seizures of the same type. Data points can be quantitative, semi-quantitative, or qualitative, depending on the nature of the parameter. Readings can be the mean, average, median, variance, or other statistically or mathematically derived values ​​related to the measurement. Parameter reading information can be further refined by directly comparing the corresponding reference readouts. The absolute values ​​obtained for each parameter under the same conditions will show the variability inherent in living organisms, and can also reflect individual cellular variability and inherent variability between individuals.

[0143] Classification rules are constructed based on a training dataset (i.e., a data matrix) obtained from multiple repeated experiments. Classification rules are selected to correctly identify repeated reference patterns and successfully distinguish different reference patterns. Classification rule learning algorithms can include decision tree methods, statistical methods, Naive Bayes algorithms, etc. The knowledge database will be sufficiently complex to effectively identify and classify new test drugs. Several methods for generating a sufficiently comprehensive set of classification patterns, along with sufficiently powerful mathematical / statistical methods for distinguishing them, can achieve this.

[0144] Computer

[0145] The method of the present invention may use a computing system (e.g., a computer) to control and / or coordinate stimuli via one or more controllers and analyze data from the brain region scans. The computing unit may include any suitable components to analyze the measurement images. Therefore, the computing unit may include one or more of the following: a processor; a non-transient computer-readable storage device, such as a computer-readable medium; an input device, such as a keyboard, mouse, touchscreen, etc.; an output device, such as a display, screen, speaker, etc.; a network interface, such as a wired or wireless network interface; and so on.

[0146] Raw measurement data, such as fMRI and LFP, can be analyzed and stored on a computer-based system. As used herein, "computer-based system" refers to the hardware, software, and data storage devices used to analyze the information of this invention. The minimum hardware of the computer-based system of this invention includes a central processing unit (CPU), input devices, output devices, and a data storage device. It will be readily understood by those skilled in the art that this invention is applicable to any currently available computer-based system. The data storage device may include any manufacturer from which this information is recorded as described above, or a memory access device from which such manufacturer's information can be accessed.

[0147] Various structural formats of input and output devices can be used to input and output information in computer-based systems. This provides technicians with a similarity ranking and determines the degree of similarity contained in the test data.

[0148] The analysis can be implemented in hardware or software, or a combination of both. In one embodiment of the invention, a machine-readable storage medium is provided, comprising data storage material encoded with machine-readable data, which, when used with a machine programmed with instructions of said data, can display a comparison of any dataset with the data of the present invention. This data can be used for a variety of purposes, such as drug discovery, analysis of interactions between cellular components, etc. In some embodiments, the invention is implemented by a computer program executing on a programmable computer, said computer including a processor, a data storage system (including volatile and non-volatile memory and / or storage elements), at least one input device, and at least one output device. Program code is applied to input data to perform the functions described above and generate output information. The output information is applied to one or more output devices in a known manner. The computer can be, for example, a conventionally designed personal computer, microcomputer, or workstation.

[0149] Each program can be implemented using a high-level programming or object-oriented programming language to communicate with the computer system. However, if necessary, the program can be implemented using assembly or machine language. In any case, the language can be a compiled or interpreted language. Each such computer program can be stored on a general-purpose or special-purpose programmable computer-readable storage medium or device (e.g., ROM or disk) for configuring and operating the computer when the computer reads the storage medium or device to execute the program described herein. The system can also be considered as being implemented as a computer-readable storage medium configured with a computer program, wherein the configured storage medium enables the computer to operate in a specific and predefined manner to perform the functions described herein. Various architectural formats for input and output devices can be used to input and output information in the computer-based system of the present invention.

[0150] This document further provides methods for storing and / or transmitting sequences and other data collected using the methods disclosed herein via a computer. Any computer or computer accessory (including, but not limited to, software and storage devices) can be used to implement this invention. Sequences or other data (e.g., results of immune repertoire analysis) can be input into the computer directly or indirectly by a user. Additionally, any device used for DNA sequencing or analysis of DNA or analysis of immune repertoire data can be connected to a computer to enable data transfer to the computer and / or a computer-compatible storage device. Data can be stored on a computer or a suitable storage device (e.g., a CD). Data can also be transmitted from the computer to another computer or data collection point via methods known in the art (e.g., the Internet, terrestrial mail, airmail). Therefore, data collected by the methods described herein can be collected at any point or geographical location and transmitted to any other geographical location.

[0151] Example

[0152] The following examples are provided to fully disclose and describe the methods for constructing and using the present invention to those skilled in the art, and are not intended to limit the scope of the inventors' invention as they see it, nor to represent that the following experiments are all or only the experiments performed. Efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but some experimental errors and biases should be taken into account. Unless otherwise stated, parts are by weight, molecular weight is by weight-average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, such as bp: base pair; kb: kilobase; pl: picoliter; s or sec: second; min: minute; h or hr: hour; aa: amino acid; kb: kilobase; bp: base pair; nt: nucleotide; im: intramuscular; ip: intraperitoneal; sc: subcutaneous injection; etc.

[0153] Example 1

[0154] fMRI was used to predict the successful generation and suppression of seizure-like postepileptic discharges and to map their seizure networks.

[0155] To understand the necessary conditions for initiating and terminating seizures, we investigated optogenetically induced hippocampal seizures using local field potentials (LFP), fMRI, and optogenetic suppression. During afterdischarge induction using optogenetics, LFP recordings indicated that stimuli with earlier seizure onset were more likely to induce afterdischarges and were more difficult to shorten with optogenetic suppression. These results profiled the two seizure onset sites: the dorsal and ventral hippocampi. fMRI showed that afterdischarges originating from the dorsal and ventral hippocampi exhibited distinct networks. Shorter-duration seizures originating from the dorsal and ventral hippocampi were unilateral and bilateral, respectively, while longer-duration afterdischarges showed a more extensive bilateral network. When optogenetic suppression was ineffective in stopping seizures, the spread of network activity was more extensive, but largely overlapped with network activity associated with seizures whose duration could be shortened. These results provide insights into how to suppress seizures, which is significant for targeted seizure interventions.

[0156] Using an optogenetically induced AD model, we first investigated whether LFP (Local Prescription Photometry) could be used to predict whether a trial would progress to AD by leveraging the non-stimulatory artifact properties of these recordings. Next, we explored the possibility of using spatially localized optogenetic suppression at the seizure focus to suppress AD, and whether it was possible to predict the success and failure of optogenetic suppression aimed at suppressing AD. Finally, we used simultaneous LFP and functional MRI (fMRI) to investigate the seizure networks of AD that could and could not be suppressed by local suppression in the dorsal hippocampus (DH) and ventral hippocampus (VH).

[0157] Optogenetic seizures in the VH. To induce AD, CaMKII-positive excitatory neurons in the VH were optogenetically stimulated. Initially, we calibrated the stimulation parameters required to induce AD ​​in each subject by progressively increasing the light power delivered to the VH (until a threshold for AD induction was determined). Figure 1 (A and 1B). The average optical power required to induce AD ​​under dexmedetomidine was 5.5 ± 1.1 mW. Optogenetics can acquire LFP recordings without stimulation artifacts, which allows us to estimate the seizure onset time, i.e., the time when the large peak activity begins during stimulation. We hypothesize that seizure onset time can predict the probability of developing AD, and therefore developed a method for estimating seizure onset time. An example of seizure onset time estimation can be found in [link to documentation]. Figure 9 The corresponding value of the sliding window for inducing the response is subtracted from the corresponding data to indicate the occurrence of epileptiform discharges, which is detected using a threshold method.

[0158] Prediction of successful AD development. To determine the relationship between seizure onset time and the severity of the resulting AD, we compared seizure onset time with the probability of successfully developing AD, defined in this paper as AD lasting longer than 2.5 s (measured from the end of the stimulus period). Visually, trials showing sustained AD were associated with earlier seizure onset time compared to stimuli lasting less than the duration threshold. Figure 1 B). To test the reliability of this observation, we first compared trials with persistent AD with subthreshold trials with durations shorter than the 2.5s threshold (no AD, 206 trials; 192 trials; n=13) ( Figure 1 C) It was found that trials progressing to AD had significantly earlier seizure onset times compared to trials not progressing to AD. This relationship was more pronounced in most subjects; however, in some subjects (e.g., subjects 6 and 8), a detectable spike was often not observed before the end of the stimulation period. Since different subjects are known to have different AD predispositions, a hierarchical Bayesian model was used to model the probability of AD, with seizure onset time as the explanatory variable, and the intercept (b0) was allowed to vary among different subjects. Figure 1 (C and 1D). Figure 1 C represents the observed data for individual subjects and the mean ± 95% confidence interval of the posterior predicted distribution. The data generated by the model reproduces the pattern of the observed data well, indicating a good model fit. An earlier onset time can largely predict the occurrence of AD, as evidenced by the fact that the regression coefficient for onset time and its 95% highest posterior density (HPD) uncertainty interval are greater than zero.

[0159] We evaluated the sensitivity and specificity of the variable intercept model for retention data using receiver operating characteristic (ROC) analysis. The area under the ROC curve (AUC) obtained using 10-fold cross-validation was 0.87. Figure 1 E), while the AUC obtained by pooling data was 0.79, indicating that using a stratified model is superior to a model that pools data from different subjects. False positive classifications occasionally occur, typically when an early spike signal appears in the LFP, but the duration of AD does not exceed the 2.5s threshold. Figure 9 B). False negatives are usually associated with an increase in amplitude and stimulation frequency over a period of time, but they do not produce a detectable spike (B). Figure 9 C). After determining that optogenetic stimulation could reliably induce AD ​​and identifying the onset time as a predictive marker of AD development, we investigated the possibility of blocking AD by silencing local neuronal populations at the site of AD initiation.

[0160] Expression of ChR2 and eNpHR3.0 in overlapping cell populations. To determine the effectiveness of inhibiting local neuronal populations in blocking AD in VH, we targeted the hSyn promoter to express eNpHR3.0 in all neuronal subtypes. Histological sections from rats co-injected with AAV5-ChR2 (driven by the CaMKII promoter) and AAV5-eNpHR3.0 (driven by the hSyn promoter) confirmed the co-expression of inhibitory and excitatory opsins. Although we expected transduction efficiency to be affected when both viruses were administered simultaneously, confocal data suggested that it is possible to express both opsins in overlapping cell populations (in a non-stoichiometric manner), where ChR2 was expressed primarily in axons and dendrites, while eNpHR3.0 was expressed primarily in the cell body. Figure 8 A and 8B). As expected, rats injected with hSyn-eNpHR3.0 virus expressed inhibitory opsin in CaMKII-positive excitatory neurons and GAD67-positive inhibitory neurons. Figure 8 C).

[0161] Optimization of light intensity required for optogenetic suppression. To determine the approximate light intensity required for suppressing local ventral hippocampal neurons using eNpHR3.0, we evaluated spikes at local sites using multi-unit activity (MUA) recording. The combined optical poles used for optical transmission and MUA recording were constructed such that the tip of the tungsten electrode was positioned approximately 0.65 mm below the fiber tip. Figure 10 A and 10B). As expected, transmission of continuous 589nm orange light to activate eNpHR3.0 was more effective in inhibiting spontaneous neuronal firing in rat VH expressing hSyn-eNpHR3.0. Figure 10 C and 10D).

[0162] We then tested the ability of eNpHR3.0 inhibition to suppress optogenetic stimulation by simultaneously activating eNpHR3.0 and ChR2. We found that at lower light intensities, such as below 10 mW (1,100 mW / mm²), the inhibition of eNpHR3.0 was effective. 2 While eNpHR3.0 inhibition has an inhibitory effect, it cannot prevent the occurrence of AD. Figure 10 E). When using higher light intensities, such as above 18mW (2,079mW / mm²),... 2 This effectively prevented the development of AD in rats expressing eNpHR3.0 via the hSyn promoter (9 trials in 3 animals); therefore, in the remaining experiments, a slightly higher intensity was used—21 mW (2,425 mW / mm). 2To maximize the possibility of seizure suppression. In addition to testing the effectiveness of suppression using hSyneNpHR3.0, we also tested CaMKII-eNpHR3.0, but found it to be ineffective in suppressing spontaneous discharges and AD. Figure 10 The low viral titer (F–10K) may be due to low available viral titers or selective targeting of excitatory neurons. Therefore, our research focuses on inhibiting hSyn-positive neurons to suppress seizures.

[0163] We used eNpHR3.0 to block seizure-like atrophic ileus (AD) in the ventricle hysterosalpingus (VH). First, we tested the possibility of blocking AD in the VH by applying local inhibition immediately after the stimulus that induces a seizure. Figure 2 (A and 2B). For example... Figure 2 As shown in the example in C, we found that in some cases, seizure-like activity could be blocked during the 5-second optogenetic suppression period, but a large spike of several seconds still occurred early in the process of achieving successful suppression. Therefore, to determine the relative success and failure of suppression, we binarized the data using AD thresholds of 2.5 seconds and 5 seconds. At these two thresholds, the suppression effect was robust and effectively reduced the probability of AD. Figure 2 D). For the distribution of AD duration with and without inhibition, please see [link / reference]. Figure 2 E. The median duration of AD was 10.1 ± 4.2 seconds without suppression, and decreased to 0.83 ± 8.6 seconds with suppression. After applying optogenetic suppression, the duration of AD was significantly shortened in 4 out of 6 animals (p < 0.05, paired t-test). Figure 2 E).

[0164] To test whether the failure of optogenetic suppression to shorten AD duration is related to the time of onset ( Figure 3 (A and 3B) We used a linear hierarchical Bayesian model to explore the relationship between suppression success and failure and the time of seizure onset. Figure 3 C), and used an intercept that allows for different values ​​among different subjects to illustrate intra-subject correlation and different subjects’ different tendencies toward AD. Figure 3 C and 3D).

[0165] The model results indicate that earlier seizure onset time again predicted AD occurrence, and optogenetic suppression significantly reduced the probability of AD (seizure onset time and stimulus conditions are important predictors because their regression coefficients do not contain zero in 95% of HPD cases). Samples drawn from the posterior distribution fit the observed data well. Figure 3D) indicates that the faster the progression of AD (early onset), the less likely its duration is to be shortened. Furthermore, the stratified variable intercept model demonstrates excellent ability to predict AD based on retained data, and in 10-fold cross-validation, the AUC obtained using it is 0.92 (…). Figure 3 E), in contrast, the AUC obtained by combining the logistic regression model is 0.9.

[0166] We used eNpHR3.0 to block seizure-like AD in DH. To determine whether seizure onset time is also a predictive biomarker for AD suppression in DH, we applied the same experimental paradigm used in the VH experiment to AD originating from DH. Figure 11 A). Co-injection of the two viruses into DH resulted in robust expression of both opsins. Figure 8 A). For DH, we investigated the effect of ChR2 stimulation duration because halophilic rhodopsin appears to be more effective in DH than in VH, thus necessitating an increase in ChR2 stimulation duration to produce AD ​​whose duration could not be effectively shortened. AD induced by shorter stimulation (5 s) was more easily blocked than AD induced by stimulation of longer duration (6–7.5 s); that is, AD induced by ChR2 stimulation of longer duration (greater than 5 s) was difficult to shorten. Figure 11 A-11E). There is a strong linear relationship between the duration of stimulation and the time of onset (measured from the end of the stimulation period). Figure 11 H, p<0.05, t-test); therefore, as expected, in trials where optogenetic suppression failed to shorten the corresponding duration, the onset time was significantly earlier (H, p<0.05, t-test); Figure 11 I, p<0.05 (paired t test), highlight its utility as a marker for AD inhibition.

[0167] The spread of AD activity originating from the DH and VH. The failure to shorten the duration of AD with earlier seizure onset may be related to the spread of seizure activity; therefore, we describe how activity spreads from the DH and VH and explore the degree of difference in activity patterns between these two origin sites. We plotted the relationship between seizure network activity and AD duration on fMRI to explore the corresponding propagation patterns. Figure 4As shown in Figure A, binary variables were used to describe whether voxels were active (exceeding the statistical threshold of p < 0.001) or inactive, and a logistic function fit was performed for all subjects and trials (as a function of AD duration). The inflection point of this fitted curve provides an estimate of the mean activation time at each voxel level during AD, and is therefore used to visualize the spread of seizure activity during AD originating from different regions. In trials initiating AD, there was no difference in activation time plots between those with and without optogenetic suppression (…). Figure 12 Therefore, these data were combined. These figures, compared to VH, illustrate different patterns of seizures originating from DH. AD originating from VH indicates earlier activation of the ipsilateral VH, diaphragm, and ipsilateral prefrontal cortex (PFC) in shorter-duration AD. Figure 4 B). The average activation time in the contralateral VH was 17±2 s later than in the ipsilateral hippocampus, while the average activation time in the contralateral PFC was 18±6 s later than in the VH. However, the activation time of DH was significantly later (27±5 s), indicating that DH was only active in AD with longer durations.

[0168] Activation time map generated by AD originating from DH ( Figure 4 C) indicates that shorter-duration AD involves both the ipsilateral and contralateral hippocampus, with activation significantly spreading towards the temporal pole. Longer-duration AD spreads to the PFC and other cortical areas. These data suggest that, unlike AD originating from the VH, focal DH seizures rapidly spread bilaterally to the entire hippocampus, while focal VHAD involves the DH later and spreads to the amygdala and PFC. Quantitatively, a random intercept model was used to compare the inflection points of different regions of interest (ROIs). Figure 4 (D) In ​​AD originating from the VH, the activation times of the ipsilateral amygdala and both PFCs were significantly earlier than those in AD originating from the DH (27±5 s and 29±9 s, respectively), while the activation time of the contralateral hippocampus was 26±3 s earlier than that in AD originating from the DH. After mapping the network activation of AD originating from the DH and VH, we investigated the differences between two scenarios (i.e., the scenario where seizure activity persists despite local inhibition; and the scenario where seizure duration is shortened) to understand the seizure network that can sustain activity in the presence of local inhibition. Furthermore, only D cases with a duration <25 s were analyzed to understand the network that sustains seizure activity in the early stages of seizure onset and duration.

[0169] Simultaneous LFP and fMRI of AD in VH. Whole-brain fMRI imaging was used to investigate which areas are involved in AD where local inhibition could not shorten the corresponding duration. Figure 5A and 5B). Typical activation maps of experiments that cannot inhibit shortening via optogenetics only show localized activation and activation in limited regions within the septum and PFC. Figure 13 A). Despite optogenetic repression, persistent single-episode AD from the same cycle, lasting only 16 s, involves extensive activity in the anteroposterior direction of the VH, as well as in the lateral septum, hypothalamus, anterior thalamus, and PFC. Figure 13 B). Although the sensitivity artifacts produced by the air-tissue interface around the ear canal limit a more thorough assessment of the posterior amygdala, the anterior amygdala appears to be involved. Within the hippocampus, only the ventral region is active, and it does not spread to the DH. In the absence of AD persistence, LFP ( Figure 4 C) A high-amplitude peak appears after the stimulation period ends. In cases of persistent AD, the fMRI response in the ipsilateral PFC, amygdala, and PFC is also prolonged. Figure 5 D). Fixed-effects group-level activation map ( Figure 5 E and 5F) were consistent with the examples from a single trial, but in addition, the anterior, dorsomedial, ventromedial, and dorsolateral subregions of the thalamus were also activated. As indicated by the ROI activation levels, these results were consistent across different subjects. Figure 5 (F and 5G).

[0170] Simultaneous LFP and fMRI of AD in DH. When using optogenetics to shorten the duration of AD originating from DH, single-trial fMRI typically shows bilateral activation over a large area in the anteroposterior direction of DH. Figure 13 C). In the presence of persistent AD, activation spreads bilaterally to the hippocampus and dorsal septum (C). Figure 13 D), but the activity spreads more extensively to the temporal pole of the hippocampus. In trials with shortened duration, the duration of the large spike that begins near the end of ChR2 stimulation does not exceed the 2.5s threshold ( Figure 6 C). In cases of persistent AD, the fMRI response across the entire hippocampus is prolonged, with a delayed response in the VH compared to the DH. Figure 6 D). Fixed-effects group-level analysis supports the results of the trial example. Specifically, in the case where AD did not persist ( Figure 6 E), activity spreads to the ipsilateral hippocampal temporal pole, but remains highly restricted within the contralateral DH. In cases of persistent AD, activation typically spreads bilaterally throughout the hippocampus and the posterior flexor cortex (PVC). Figure 6 F). Quantitatively, in cases of persistent AD, 51% ± 15% of the contralateral DH ROIs were activated, while when AD duration could be shortened, this proportion was 10% ± 4%. Figure 6 G).

[0171] The effects of optogenetic suppression and high light intensity on fMRI signal. To eliminate fMRI artifacts associated with optogenetic suppression and demonstrate its effectiveness in suppressing local activation, we describe the effect of suppression on the fMRI response. For detection efficiency, a block-designed stimulation pattern was used (…). Figure 7 A and 7B), and rats with DH implanted photoelectrodes were used because in the VH group, the stimulation site was partially masked by sensitivity artifacts generated by the air-tissue interface of the ear canal.

[0172] First, to demonstrate that we were able to detect changes in cerebral blood volume (CBV)-weighted fMRI signal, we used 0.7 mW (81 mW / mm²). 2 Low-intensity light stimulation of CaMKII-positive neurons expressing ChR2 in the DH resulted in strong fMRI responses in both ipsilateral and contralateral DH. Figure 7 (C, ii). Assume the light intensity we use for the orange laser is 21 mW (2,420 mW / mm²). 2 Given that the light intensity is higher than that used for the blue laser, we anticipate that when both the blue and Q10 orange lasers are turned on simultaneously, substantial optogenetic suppression will eliminate the excitation level and CBV-weighted fMRI response. Our results support this hypothesis, such as... Figure 7 As shown in the example in Ciii, simultaneous optogenetic suppression resulted in almost complete elimination of the fMRI response to ChR2 stimulation. Furthermore, as we hypothesized, applying optogenetic suppression alone did not cause a significant change in the fMRI signal. Figure 7 D, i), but it does cause some deflection of the LFP at the beginning and end of the stimulation pulse. Despite optogenetic repression, ChR2 stimulation has a significant effect on the LFP trace ( Figure 7 D, iii), show the induced response at the stimulation frequency during stimulation. In all three rats, the significantly reduced activation was consistent upon application of synchronous optogenetic repression (D). Figure 7 E and 7F).

[0173] Our findings reveal how optogenetic local inhibition of neuronal populations reduces the probability of induced atopic ileus (AD) originating from DH or VH, but this inhibition typically fails to shorten AD duration. To understand why, we attempted to model the extent to which halophilic rhodopsin inhibits AD using multivariate, hierarchical Bayesian logistic regression (with seizure onset time and stimulus conditions as predictors). The results showed that seizure onset time predicts both the development of AD and the success of optogenetic inhibition in shortening AD duration, indicating that local inhibition cannot shorten the duration of faster-progressing AD. Simultaneous LFP fMRI was used to map the seizure network responsible for sustaining seizure activity in the presence of local inhibition. Different networks were identified for VH-originating AD compared to AD originating from DH. Interestingly, these networks were largely similar in AD with shortenable duration compared to AD with non-shortenable duration, while the activity in non-shortenable duration AD was more diffuse.

[0174] Our statistical model based on seizure onset time shows that the effectiveness of local optogenetic suppression in shortening AD duration is associated with AD progression, with AD from earlier onset being less sensitive to local suppression. The hypothesis that AD from earlier onset is more difficult to shorten was confirmed in both VH and DH, and this was further supported by the following observations: In DH, AD induced by shorter duration (5 s) stimuli was more easily blocked compared to AD induced by longer duration (6–7.5 s); this suggests that local suppression rapidly becomes ineffective as the seizure spreads beyond the lesion. LFP-fMRI was used to visualize the spread of seizure activity (involving AD that could not be blocked by eNpHR3.0 and AD whose duration could be locally shortened). Our data indicate that reliably localized blocking of focal seizures originating in DH involved both hippocampal regions. In cases where AD duration could not be shortened, the AD did not necessarily involve extrahippocampal regions but rather spread further towards the temporal pole.

[0175] AD originating from the VH and which can be interrupted by optogenetics often involves ipsilateral limited activation in the septum, amygdala, PFC, and hypothalamus. Shorter-duration AD that cannot be shortened by local optogenetic inhibition is widespread in these areas and in the thalamus. Therefore, the network activities involved in shortenable and non-shortenable AD are largely similar, but differ in degree, and AD originating from the DH differs significantly from AD originating from the VH.

[0176] By observing the relationship between fMRI activation and the duration of acute hypothalamic-pituitary-acute (AD), we found that the activity patterns of focal hypothalamic-pituitary-acute (DH) AD and focal vesicular-hypothalamic-pituitary-acute (VH) AD are significantly different. AD originating from the DH involves the contralateral DH from the outset and rapidly spreads to the VH, while focal VHAD typically maintains unilateral spread but gradually spreads to the contralateral VH and then to the septum. DH and VH are considered to be functionally and genetically distinct, but they exhibit significant associations with other regions. VH is primarily associated with the hypothalamus, amygdala, and percutaneous fossa (PFC), while DH mainly projects to the retro-hypophyseal cortex. This association is largely reflected in our data, which showed that seizures originating from the VH resulted in earlier activation of the hypothalamus, septum, PFC, and amygdala, while the retro-hypophyseal cortex is the primary extra-hippocampal region affected by focal DH AD. Surprisingly, short-duration (<25s) AD originating from the ventral hippocampus (VH) primarily spread unilaterally, while for seizures lasting longer, the VH and diaphragm in the contralateral hemisphere were clearly activated first, suggesting that seizure activity may spread to the contralateral hemisphere via the ventral hippocampal commissure. In all trials, AD originating from the hippocampus (DH) activated the contralateral DH from the outset. These data are consistent with other fMRI findings that demonstrate direct electrical stimulation of the DH leads to bilateral hippocampal activation, except for apparent short-term plasticity, which triggers extra-hippocampal spread.

[0177] Our findings are consistent with our previous optogenetic fMRI studies, which showed that stimulation of the hypothalamus (DH) resulted in activation patterns limited to the hippocampal structures and the subspinal cortex, while stimulation of the intermediate hippocampus elicited more widespread PFC activation. Consistent with our data, Motelow and colleagues found that in blank rats, electrically induced focal DH seizures resulted in fMRI activation limited to the hippocampus, septal nucleus, and anterior hypothalamus. However, contrary to their findings, we did not observe a widespread negative fMRI signal across the thalamus and cortex. This difference could be explained by different AD induction methods, including cell type-specific stimulation, different fMRI contrast mechanisms, and anesthesia protocols. Toyoda and colleagues' results indicated that spontaneous seizures in the pilocarpine model rapidly spread from the VH to the contralateral VH or ipsilateral DH. However, our results suggest that in the non-epileptic brain, AD originating from the VH continues to spread unilaterally, except for longer-lasting and more severe AD (which spreads to the contralateral VH and may persist there). Current research also provides evidence that, in the non-epileptic brain, short-duration aberrations originating from the vegetative-hemorrhagic (VH) do not spread to the delta-hysteria (DH). We intend to use LFP-fMRI in a chronic epilepsy model to compare these results to determine whether seizure activity spreads more rapidly in these circuits.

[0178] In our study, after determining the AD threshold, we achieved an AD induction success rate of approximately 80%, consistent with data reported by Khoshkhoo et al. (2017). A limitation of current research is the inability to achieve 100% success in inducing AD. This is particularly important because AD needs to be induced at levels just above the seizure induction threshold; however, this also poses a problem in other studies that require variations in light power and frequency to achieve consistent seizure induction using optogenetics. Excluding trials significantly below the seizure induction threshold ensured that most included trials progressed to AD without optogenetic intervention. However, more reproducible AD induction methods could not be used because stimulation significantly above the AD induction threshold also induced severe AD whose duration could not be shortened by local inhibition. Another limitation relates to the difficulty in separating the fMRI response during stimulation from the fMRI response during post-stimulation seizure activity. Separating these two responses would allow for more precise localization of the areas involved in the early stages of epileptic persistence. However, the fMRI response is known to be non-linear, especially during peak activity, which prevents us from employing this approach.

[0179] It has been confirmed that CBV-weighted fMRI is highly advantageous compared to blood oxygen level-dependent (BOLD) fMRI because activation can be easily detected in a single trial without averaging. Furthermore, CBV-weighted fMRI typically improves detection sensitivity and contrast-to-noise ratio by at least 2-fold compared to BOLD fMRI, and enhances confidence in the accuracy of activation maps in representing seizure activity. Another advantage of our protocol is that it uses shorter duration (5 s) optogenetic stimulation to induce relatively short duration AD (median = 10.1 s), with an AD duration of approximately Q11 minutes, typically shorter than the duration of AD or spontaneous seizures in SE models. This advantage manifests in two ways. First, shorter duration AD may induce a weaker ignition effect. Evidence suggests that even after multiple AD episodes in a single animal, we have not observed motor seizures, making it possible to study AD in a reproducible manner in the same animal, even if the AD originates from VH (where the ignition rate is known to be relatively fast). Second, relatively mild and short-lived AD that causes seizure activity occurs only in a few areas outside the site of seizure onset. This makes it possible to use fMRI to map the areas involved in the early stages of seizure activity, which would otherwise lack the temporal resolution required to study the rapid spread of seizure activity.

[0180] Finally, previous studies have found evidence of heating artifacts or light-induced vasodilation on fMRI. It is noteworthy that despite using a high intensity (2,420 mW / mm) wavelength of 589 nm... 2 We found no evidence of such artifacts even with continuous Q12 light (5 seconds). Unlike the studies mentioned above, our experiment was conducted several weeks after the initial surgery using long-term implanted fibers. This means we may have had time to remove blood that strongly absorbs light generated during the procedure. Nevertheless, we have previously shown that using long-term implanted fibers and 20 seconds of blue light stimulation (2,560 mW / mm²) is effective. 2 When blue light is used, heating-related artifacts may occur on fMRI. However, the stimulation duration is longer than that used in our current study, and blue light is absorbed significantly more in brain tissue than orange light, which leads to enhanced local heating.

[0181] In summary, our findings demonstrate that localized optogenetic suppression can shorten the duration of optogenetically induced Alzheimer's disease (AD), and the inability to shorten AD duration can be well explained by seizure onset time. Locally suppressed AD exhibits a similar seizure network to unsuppressible AD, but its distribution is less widespread. Our results provide important insights for future targeted epilepsy treatment research and further deepen our understanding of the spread of seizure activity originating from dementia (DH) and vegetative-hemorrhagic (VH).

[0182] method

[0183] A surgical procedure involving viral injection and photodynamic implantation was performed. This procedure aimed to induce the expression of eNpHR3.0 and ChR2 in a population of overlapping neurons in the ventral hippocampus by co-injecting two viruses with the same serotype. For AD induction, ChR2 expression was targeted at CAMKII neurons. For the inhibitory opsin eNpHR3.0, we tested the ability of the human synaptic protein (hSyn) promoter and the CAMKII promoter to block AD. Adult male Sprague-Dawley rats (n=18 in total) were obtained from Charles River Laboratories, and 13 rats were initially used for eNpHR3.0 expression driven by the hSyn promoter (n=6 rats) and the CAMKII promoter (n=5 rats). Under our experimental conditions, hSyn-eNpHR3.0 was more effective than CAMKII-eNpHR3.0 in shortening the duration of AD; therefore, only data from the hSyn group were analyzed. Figure 1 Except, because in Figure 1Only the case of ChR2 stimulation (without optogenetic suppression) was investigated, therefore data from both groups were included. Both groups received a virus encoding ChR2 (driven by the CaMKII promoter). 1.5 ml of AAV-5eYFP-CAMKII (titer = 8.5 x 10¹² vg / ml) and 1.5 ml of AAV-5-eNpHR3.0-mCherry virus were obtained from the Vector Core Institute at the University of North Carolina (UNC), at the highest titer available at the time (for hSyn targeting, titer = 6.7 x 10¹² vg / ml). 12 For CAMKII targeting, the titer is 4.7 x 10⁻⁶. 12 (vg / ml).

[0184] Rats were anesthetized with pure oxygen containing 5% isoflurane, and then isoflurane was maintained at a 2%–3% supply throughout the procedure. Viruses encoding the eNpHR3.0 and ChR2 genes were mixed at a 1:1 (v / v) ratio and injected into the right hippocampus (VH coordinates = AP: -5.6 mm, LR: 5.7 mm, DV: 6 mm, with the dura mater as the reference point) using a 33-gauge needle connected to a Hamilton syringe. A constant infusion rate (150 nl / min) was maintained using an infusion pump (Micro 4, World Precision Instruments, FL). MRI-compatible carbon fiber electrodes (constructed using a 0.22 numerical aperture, 105 mm diameter step-index multimode fiber (ThorLabs, Newton, NJ)) were inserted as previously described (Duffy et al., 2015) to position the electrode tip and fiber directly above the injection site. Prior to implantation, the electrodes were checked to ensure a light transmission percentage greater than 70% and that light transmission to the brain was estimated at 70% of the input intensity. Two brass screws were inserted above the cerebellum to secure the dental cement, which served as the ground and reference electrodes. Finally, the electrode wires were soldered to a DF13 connector (Hirose, Japan), and all components were secured to the skull using light-cured dental cement. Multi-unit recordings were performed on additional subjects (n=2 for the hSyn-eNpHR3.0 group and n=2 for the CAMKII-eNpHR3.0 group) who were not implanted with photoelectrodes. Buprenorphine extended-release (1 mg / kg, sc) was administered preoperatively to reduce surgical pain and discomfort. Lidocaine (4%) and bupivacaine (0.25%) were also administered topically preoperatively and postoperatively. To allow time for virus-induced protein expression, experiments were conducted at least 6 weeks postoperatively.

[0185] After the initial experiment demonstrating that eNpHR3.0 expressed via the hSyn promoter was more effective in shortening AD duration (compared to the CAMKII-targeted experiment), another group (n=3) of rats were included. These rats were co-injected with hSyn-eNpHR3.0 and hSyn-ChR2 virus in their dorsal hippocampus (DH coordinates = AP: -3.6 mm, LR: 3.2 mm, DV: 2.2 mm, with the dura mater as the reference point) for comparison with the VH-targeted experiment.

[0186] Electrophysiological recording. LFP recording was performed outside the MRI environment using a differential amplifier (DP304, Warner Instruments) and a data acquisition system (USB 6259, National Instruments). Multi-cell recording was performed using an OpenEphys system and a sharp tungsten electrode (impedance 0.5 kOhm, diameter 120 mm, AM system) connected to a 105 mm diameter fiber.

[0187] An anesthesia protocol for inducing seizure-like post-epileptic discharge (AD). Rats were sedated by bolus injection of dexmedetomidine (0.05 mg / kg) via a 24-gauge catheter inserted into a lateral caudal vein, followed by continuous intravenous (iv) infusion of dexmedetomidine (0.1 mg / kg / h). We have previously shown that this anesthesia protocol can potentially induce AD ​​in a reproducible manner (Duffy et al., 2015). At the end of each round, atipamazole (0.5 mg / kg, sc) was administered to partially reverse the effects of dexmedetomidine.

[0188] Inducing seizure-like post-epileptic discharge. To selectively stimulate ChR2-expressing cells to induce AD, we used a 473nm (blue light) diode-pumped solid-state laser (Laserglow Technologies, Toronto, Canada). A pulse train (200 pulses, 7.5ms duration) at a frequency of 40Hz was generated using a Master-9 (AMPI, Israel) pulse stimulator. The pulse train duration was 5s. To determine the appropriate parameter thresholds for AD induction, stimulation was performed every 2 minutes, starting from 0.7mW (80mW / mm²). 2 Starting with 0.35mW, gradually increase the light intensity in increments of 0.35mW (at the fiber tip, 40mW / mm). 2 / pulse). If at 1200mW / mm 2If AD was not induced at the specified level, the duration of the pulse train was increased to 7.5 s in 0.5 s increments during MRI imaging. After determining the threshold, an 8- to 10-minute stimulation interval was allowed between consecutive stimuli to mitigate any post-ictal refractory period effect. To selectively hyperpolarize cells expressing eNpHR3.0, a 589 nm (orange) diode-pumped solid-state laser (Laserglow Technologies, Toronto, Canada) was used. Orange light was transmitted to the same location as blue light (via the same fiber) for 5 s. Exposure and blocking of light were achieved using a Master-9 triggered mechanical shutter (Uniblitz, Vincent Associates, NY) to ensure stable light intensity and precise timing. Orange light was exposed immediately after blue light blocking. This condition was compared to a control condition without orange light. For each individual round, a randomized stimulation group containing 5 orange light stimuli and 5 control stimuli was used to generate the same number of conditioned stimuli within each round. If AD is not induced during the control stimulus period, increase the light intensity by 40 mW / mm. 2 Until AD can be induced.

[0189] fMRI data acquisition. All data acquisition was performed using a 7T horizontal drilling system (BrukerBioSpec 70 / 30) at the Stanford Center for In Vivo Imaging Innovation (SCi3). RF excitation was performed using a 2-channel volumetric coil with a diameter of 86 mm, and a 20 mm single-loop surface coil was used as the RF receiver. Similar to the recording rounds outside the scanner, rats were sedated during MRI acquisition by bolus injection of dexmedetomidine (0.05 mg / kg, sc) via a cannula inserted into the lateral caudal vein, followed by continuous infusion of dexmedetomidine (0.1 mg / kg, iv). A single bolus injection of feraheme (15 mg / kg, iv) was used for cerebral blood volume (CBV)-weighted imaging to improve the contrast-to-noise ratio (Mandeville et al., 1998) and microvascular sensitivity (Zhao et al., 2006), an advantage of this technique compared to BOLD fMRI. Approximately 15 minutes after contrast agent injection, fMRI was acquired using 4-segment spiral readout with the following acquisition parameters: TR = 0.75 ms, TE = 9 ms, flip angle = 30°, field of view = 32 x 32 mm, matrix = 70 370°, slice thickness = 0.6 mm, number of repetitions = 130, number of virtual scans = 4.

[0190] Simultaneous LFP recording and processing. LFP recording was performed concurrently with fMRI data acquisition using a 16-channel BrainAmpExG MR amplifier (Brain Products, Germany), equipped with a 1000 Hz low-pass filter and a sampling frequency of 5000 Hz. Gradient artifacts were removed using the mean artifact subtraction (AAS) method implemented by Niazy et al. (2005) (Allen et al., 2000; modified and adapted for segmented MRI sequences) to allow template estimation to exclude highly periodic stimulation periods or high-amplitude spikes. Furthermore, a 70 Hz low-pass filter threshold was used in the final stage of the process to reduce the influence of any residual artifacts.

[0191] Histological analysis. Rats were perfused and fixed with phosphate-buffered saline (PBS), followed by perfusion fixation with 4% paraformaldehyde (PFA). The tissues were placed in 4% PFA overnight, and then cryoprotected in 20% sucrose solution. Sections were cut into 50 mm pieces and fixed on glass slides for imaging under a confocal microscope (Zeiss LSM 880).

[0192] Quantitative and statistical analysis

[0193] Inclusion criteria for optogenetic suppression experiments. For experiments investigating the effects of optogenetic suppression, it is important to include only trials where the stimulation is sufficient to induce Alzheimer's disease (AD). To this end, strict quality control criteria are employed for optogenetic suppression experiments. If AD does not occur after a trial involving only blue light stimulation (trials without optogenetic suppression, i.e., AD duration less than 1 second), these trials are excluded along with all previous trials until a trial involving only blue light stimulation induces AD for a duration greater than 1 second. This criterion ensures that only trials with values ​​exceeding the seizure induction threshold are included.

[0194] Seizure onset time estimation. During ChR2 stimulation, the seizure onset time is estimated by subtracting the mean evoked potential to obtain the potential individual stimulation LFP estimate. Figure 9 First, the average evoked potential (LPP) for each 25ms stimulation period was calculated by averaging evoked potentials (LPPs) over a sliding window of eight consecutive stimulation periods, centered at zero. This average LFP was subtracted from the initial averaged centralized data, and the potential was used to detect potential seizure activity using a simple thresholding method. An event was defined each time the LFP obtained after subtracting the LFP exceeded an empirical threshold (4.5 times the standard deviation of the baseline signal, where the baseline was defined as a time interval between 500 and 2500 ms, as no significant seizure activity was observed within this interval). Finally, the seizure onset time was defined as the time it takes for five events to occur within a 500ms time interval.

[0195] fMRI data analysis and statistics. fMRI data were analyzed using SPM12. First, the data were smoothed at full width at half maximum (FWHM) using a 0.5 mm Gaussian kernel, followed by motion correction using 6-parameter rigid registration. Images were first manually masked, then aligned with a common space using 12-parameter affine registration implemented in the NiftyReg software package. This common template was segmented into regions of interest by atlas registration followed by manual correction. Activation maps were generated using a general linear model (GLM). This required searching for optimal parameters for a custom gamma distribution function. A single gamma distribution was used because there was no evidence of off-target effects when using CBVw fMRI (Mandeville, 2012); and we used Levenberg-Marquardt nonlinear least squares with grid search to estimate shape and scale parameters to ensure the absence of local minima. Figure 14 (A-14F). fMRI data for estimating gamma parameters were collected from the same animals from the block design experiment. This custom gamma distribution was convolved with the stimulation and post-discharge phases to model the activation of the two phases as a single rectangular function. The high-pass threshold was set to 400 s, and sequence correlation was modeled using an autoregressive AR(1) model and estimated using restricted maximum likelihood.

[0196] For single-subject, single-trial t-statistic activation plots, the threshold was set to p < 0.001 (uncorrected for multiple comparisons). For fixed-effects group-level analyses, a more stringent threshold of p < 0.0001 (uncorrected for multiple comparisons) was used. In ROI-based analyses, two-sample t-tests were used to compare AD with and without AD across different trials. For comparisons between the dorsal and ventral hippocampi, a voxel-based inflection point plot was generated using a (pooled) fixed-effects model and maximum likelihood estimation as a measure of mean onset time. There was no difference in activation time plots between those with and without optogenetic suppression. Figure 12 Therefore, targeting Figure 4 The results shown combine these data. For Figure 4 In ROI analysis, regions are considered active if the activation level of each subject is greater than 10% of the maximum activation level. A random intercept model is fitted using lme4 in R (treating subject effects as random effects). For each region, inflection points in VH are compared with those in DH using parameter bootstrapping (Bates et al., 2015) and a significance level of 0.05. For region-level analysis, segmentation is used as follows: Figure 14 As shown in G. Activation time is reported as median ± standard deviation, but... Figure 4 The values ​​are shown as median ± 5th and 95th percentiles.

[0197] Statistical analysis of after-discharge. Unless otherwise stated, paired t-tests were used for hypothesis testing between groups at a significance level of 0.05. After-discharge was considered sustained if its duration exceeded 2.5 s. This threshold was chosen to allow time for optogenetic suppression to take effect, as AD duration was not significantly shortened in trials including optogenetic suppression for AD durations exceeding 5 s, indicating that suppression is almost ineffective if AD is severe or progresses beyond the optogenetic suppression period. Without suppression, the median AD duration was 11 s, more than 4.3 s longer than the duration threshold.

[0198] Post-discharge probability ( Figure 1 and Figure 3 Hierarchical Bayesian logistic regression modeling is employed, which allows for different intercepts across different subjects. Hierarchical models are particularly useful when observations are grouped (e.g., by subject). Compared to pooling data and fitting a single regression model, or fitting a separate regression model for each subject, using a hierarchical model allows for the sharing of subject information; that is, each subject's estimate is partly based on data from other subjects, while subjects with smaller sample sizes are more influenced by the overall mean. In a hierarchical model, the AD probability (p) is modeled as: p = logit -1 (Xβ+β 0subject ), where it is assumed that the intercept for each subject originates from a common distribution: exist Figure 1 (Only data from ChR2 stimulation is shown) The input matrix (X) includes the onset time as the sole explanatory variable. Figure 3 Experiments involving the application of optogenetic suppression were also included, thus X was expanded to include the activation of eNpHR3.0 as a binary variable. Weakly informative priors (Gelman et al., 2008) were used because these are generally superior to uninformative priors, which can lead to incorrect posterior distributions. A zero-centered normal distribution with a variance of 1 was used as the prior distribution for the mean parameter, and a semi-Cauchy distribution with a scale factor of 5 was used as the variance parameter. PyMC3 and a U-turn-free sampler were used for back-sampling because it is known to be more efficient than the conventional Markov chain Monte Carlo method. The Gelman-Rubin convergence statistic (R-hat) for all parameters was less than 1.1, indicating good convergence of the sampling procedure. Figure 1 (D and 3D). Finally, the significance of the corresponding b-parameter values ​​for each input variable was determined using the 95% highest posterior density.

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[0250]

Claims

1. A method for analyzing epileptic seizure networks, the method comprising: a) Receiving functional magnetic resonance imaging (fMRI) data, wherein fMRI data is obtained from a subject who receives a first polynucleotide encoding a depolarizing light-reactive protein administered to a target neuron, wherein the first polynucleotide causes the target neuron to express the depolarizing light-reactive protein, and in, The functional magnetic resonance imaging data are obtained before, during, and / or after an effective amount of light is delivered to the subject, wherein the light is capable of specifically activating the depolarized photoreactive protein; as well as b) Using the functional magnetic resonance imaging data, an activation map is generated, wherein the activation map includes a voxel-based activation time map generated by comparing the distribution of seizure activity between afterdischarges, and the activation time map shows the location of local suppression of afterdischarges for therapeutic intervention.

2. The method according to claim 1, wherein, Further obtain functional magnetic resonance imaging data: After the second polynucleotide encoding a hyperpolarized photoreactive protein is administered to the target neuron; and The second effective amount of light is delivered before, during, and / or after the delivery of a second effective amount of light that specifically activates the hyperpolarized photoreactive protein, wherein the second effective amount of light is delivered after the effective amount of light that specifically activates the depolarized photoreactive protein has been delivered to the subject.

3. The method of claim 1, wherein the functional magnetic resonance imaging data used includes brain blood volume-weighted functional magnetic resonance imaging data.

4. The method of claim 1, wherein the first polynucleotide is operatively linked to a calmodulin-dependent kinase II or a human synaptic protein promoter.

5. The method of claim 2, wherein the second polynucleotide is operatively linked to a calmodulin-dependent kinase II or a human synaptic protein promoter.

6. The method of claim 2, wherein the promoter operatively linked to the first polynucleotide is different from the second polynucleotide.

7. The method of claim 1, wherein the functional magnetic resonance imaging is used in conjunction with local field potential recording.

8. The method according to claim 1, wherein, The post-discharge originates from the dorsal and ventral hippocampi.

9. The method of claim 1, wherein the early spike is confirmed to be a precursor to successful seizure suppression.

10. The method of claim 1, wherein the first polynucleotide encodes the ChR2 protein.

11. The method of claim 2, wherein the second polynucleotide encodes the eNpHR3.0 protein.

12. The method of claim 8, wherein the photoreactive protein is expressed in the ventral hippocampus.

13. The method of claim 8, wherein the photoreactive protein is expressed in the dorsal hippocampus.

14. The method of claim 2, wherein the intensity of the light used to achieve optogenetic suppression is 2000 mW / mm². 2 Up to 3000 mW / mm 2 .

15. An apparatus for analyzing seizure networks, said apparatus for use in the method of claim 1, the apparatus comprising: An optical electrode, surgically implanted in or near an individual's organ or tissue, is suitable for detecting and / or recording detectable parameters of the organ or tissue, wherein the optical electrode includes electrodes and optical fibers and is configured to detect electrical signals, and the detectable parameters of the organ or tissue include local field potentials generated by changes in the membrane potential of excitable cells in the organ or tissue; A light source, the light emitted from which is transmitted through the optical fiber to the organ or tissue to stimulate excitable cells in the organ or tissue to generate the local field potential; A functional magnetic resonance imaging system suitable for applying a magnetic field to the organ or tissue to acquire functional magnetic resonance imaging data and obtain an image of the organ or tissue; A local field potential system suitable for recording local field potentials alone or simultaneously with the functional magnetic resonance imaging (fMRI) data acquisition; and A computing system suitable for controlling and / or coordinating the stimuli via one or more controllers, and for analyzing and storing the aforementioned acquired and recorded data, wherein the computing system is configured to generate an activation map using the acquired and recorded data, wherein the activation map includes a voxel-based activation time map, compares the distribution of epileptic seizure activity between afterdischarges, and displays locations where afterdischarges can be locally suppressed for therapeutic intervention.

16. The device according to claim 15, wherein, The detectable parameters are single-unit activity and / or multi-unit activity.

17. The device according to claim 15, wherein, The light source includes a light source, which may be an optical fiber, a laser, or a light-emitting diode.

18. The device according to claim 15, wherein, The optical pole is configured as a combined optical pole for optical transmission and multi-unit active recording, wherein the tip of the electrode is located 0.65 mm below the fiber tip to ensure that multi-unit recording occurs in the illumination cone originating from the fiber tip.

19. The device according to claim 15, wherein, The excitable cells express photoresponsive peptides and can be activated or inhibited when exposed to light of different wavelengths.

20. The device according to claim 15, wherein, The light stimulation can activate the photoreactive polypeptides in the excitable cells, and the photoreactive polypeptides can depolarize or hyperpolarize the excitable cells when activated by light of the activation wavelength.

21. The device according to claim 19, wherein, The optical stimulation comprises one or more sets of optical pulses, the duration of which ranges from 1 millisecond to 2 seconds, and the optical power density ranges from 0.05 mW / mm². 2 Up to 3500mW / mm 2 .

22. The device according to claim 21, wherein, The light pulse group contains a single light pulse that continues throughout the entire duration of light stimulation; or The frequency of the light pulses in the light pulse group is 0.1 Hz or higher.

23. The device according to claim 21, wherein, The optical pulse width is 0.1 ms or longer.

24. The device according to claim 21, wherein, If measured at the tip of the optical fiber that delivers the light pulse to the brain region, the average power of the light pulse is 0.1 mW or more.

25. The application of the method of claim 1 in a seizure model, based on the activation map generated by the method of claim 1, for providing a model for in vivo analysis of brain circuits and regional relationships involved in seizures, including one or more of single seizure analysis, focal progression to bilateral tonic-clonic seizure analysis, and excitatory ventral hippocampal network analysis.

26. The application of the method according to claim 1 in whole-brain and / or regional brain function research, comprising: To generate an activation map of one or more target regions in the brain at high resolution, thereby producing the method of claim 1; as well as Assess the activity of excitable cells in an individual's target organ or tissue.

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