Optogenetics in Epilepsy
Optogenetics in Epilepsy
Optogenetics has now been applied to numerous neurological diseases, as well as some nonneurological applications, including stimulation of heart muscle. Other cell types being targeted include medium spiny neurons in the basal ganglia for studying Parkinson disease, midbrain ventral tegmental area dopaminergic neurons in depression models, and dopamine D1-expressing neurons of the prefrontal cortex to investigate a circuit underlying temporal control of behavior. Probing various physiological mechanisms has also become a focus of optogenetic manipulation, with recent studies elucidating a role of ventral surface astrocytes in control of breathing. In epilepsy, optogenetics has become an attractive option to silence hyperactive neurons, with targeting of those believed to initiate or propagate seizures.
Epilepsy presents a difficult entity to study because of the large number of network pathologies that initiate or propagate seizures. However, there are several recent studies that aim to leverage the selectivity of optogenetics to analyze and control network dynamics in several animal models of seizures. Epilepsy is characterized by overactivity of various circuits, and consequently, many of the current investigations have focused on ways to silence or inhibit overactive circuits. The ability to selectively target mammalian hippocampal neurons was a first step toward probing this disease pathway, and was achieved using the inhibitory opsin halorhodopsin in organotypic cultures of mouse hippocampi. Following this achievement, halorhodopsin was used in selected principal glutamatergic neurons in the hippocampus. In this mouse model, the injection of a lentiviral vector into CA1 and CA3 pyramidal cells resulted in hyperpolarization in response to orange light (wavelength 573–613 nm). Action potentials were attenuated and paroxysmal depolarizing shifts were curbed, suppressing epileptiform activity. Importantly, the presence of the virally transfected opsins into neurons did not affect normal physiological properties of cells. To test this, organotypic hippocampal slices were subjected to complete darkness, and various properties of the cells were tested. Parameters including input resistance, resting membrane potential, action potential threshold and duration, amplitudes in response to hyperpolarizing and depolarizing pulses, and accommodation of cells to action potentials were similar in the halorhodopsin neurons in comparison with the controls.
Recently, this technology has been demonstrated in 3 different in vivo rodent models of epilepsy. In the rat tetanus toxin injection model, principal neurons were transfected with a lentiviral vector carrying halorhodopsin 2.0. Illuminating the transfected cells with 561-nm wavelength light resulted in decreased epileptiform EEG activity. Decreased high-frequency power was also observed, which correlate to bursts of activity in human epilepsy, and event frequency also diminished.
The other two examples used a closed-loop strategy to abort seizures. In the first, a cortical stroke model was used in rats to promote seizures induced by photothrombosis, in which animals were first sensitized by injection of Rose Bengal dye, after which illumination resulted in increased light absorption and focal thrombosis. Intracranial EEG electrodes confirmed spontaneous epileptiform oscillations in the thalamus and cortex of injured rats but not in controls, correlating with thalamic network hyperexcitability. To determine whether these hyperactive thalamic cells could respond to light and silence seizure activity, a viral vector was injected consisting of the cell-type specific promoter Camk2a, the halorhodopsin eNpHR3.0, optimized with membrane trafficking signal motifs, and a reporter (eYFP, yellow fluorescent protein). The promoter ensured that only excitatory thalamic cells would be subject to viral transduction. Injections were performed stereotactically into the ventrobasal thalamus, followed by slice preparation for in vitro whole-cell recordings, as well as chronic recordings and stimulation in freely moving animals. To perform the chronic recordings, a device containing multiple EEG electrodes and chronic multisite optrode was implanted into the injected thalamus and peristroke cortex. Yellow light of a wavelength 594 nm was delivered, which resulted in silencing of epileptic EEG activity in thalamus and cortex and correlated with abortion of behavioral seizures as well. Lower intensity light was not effective, and the 594-nm light did not affect interictal EEG tracings or behaviors. The authors also developed a method of detecting and interrupting seizures online by routing to a real-time processor capable of determining seizure onset, which triggered light stimulation and resulted in seizure abortion within 1 second.
Following this report, optogenetic silencing of seizures was tested in a model of temporal lobe epilepsy. Here, mice were injected with kainic acid unilaterally into the dorsal hippocampus, and 2 methods of seizure control were explored: inhibition of excitatory principal cells and stimulation of inhibitory GABAergic cells. Opsins were expressed by using transgenic animals, crossing mice expressing the Cre-Iox recombinase in either principal cells or parvalbumin-containing GABAergic interneurons with those expressing halorhodopsin or channelrhodopsin-2, respectively. With this method, only cells containing Cre were able to express the opsin of interest, ensuring specificity. As in previous studies, a reporter fluorescent protein was also expressed. Seizure detection was achieved via depth electrodes and tuning of seizure "signatures" (magnitude, amplitude, width, rate, and frequency) with custom software. For inhibition of principal neurons with halorhodopsin, amber light of wavelength 589 nm was delivered via an optical fiber implanted in the ipsilateral hippocampus upon seizure activity detection. All animals (n = 6) responded with silencing of electrographic seizures, with a majority (57% ± 12%) aborted within 1 second. The percentage of seizures aborting within 5 seconds was approximately 70%, and was unaffected by light duration (30 seconds vs 10 seconds). Further interrogation was made into the effect of red-shifted light, which has increased tissue penetrance, but is suboptimal for channelrhodopsin-2. This 635-nm light aborted 46% ± 12% seizures within 1 second of illumination. One animal in particular responded with 100% of seizures aborting within 1 second. The results from this portion of the experiment are shown in Fig. 3.
(Enlarge Image)
Figure 3.
Seizure control in mice expressing halorhodopsin (HR) in principal cells in a model of temporal lobe epilepsy. a: Crossing CamK-Cre and Cre-dependent HR mouse lines generated mice expressing the inhibitory opsin HR in excitatory cells (Cam-HR mice). b: Experimental timeline. c–e: Example electrographic seizures detected (vertical green bars), activating amber light (589 nm) randomly for 50% of events (light: amber line, example in d; no light example in e). f: Typical example distribution of postdetection seizure durations (5-second bin size) during light (solid amber) and no-light internal control conditions (hatched gray). Inset: First 5-second bin expanded, 1-second bin size. Note that most seizures stop within 1 second of light delivery. Adapted from Nat Commun 4: Krook-Magnuson E, Armstrong C, Oijala M, Soltesz I, On-demand optogenetic control of spontaneous seizures in temporal lobe epilepsy, p. 1376, 2013, with permission from Elsevier. KA = kainic acid.
Activation of parvalbumin-GABAergic neurons using channelrhodopsin-2 also resulted in reduced seizure duration. Blue light delivery (wavelength 473 nm) aborted 59% ± 11% of seizures within 5 seconds. This was a significant finding, as these cells make up less than 5% of hippocampal neurons, suggesting that even in a small population of targeted cells, optical stimulation of transgenic cells may be efficacious.
Optogenetics in Epilepsy
Optogenetics has now been applied to numerous neurological diseases, as well as some nonneurological applications, including stimulation of heart muscle. Other cell types being targeted include medium spiny neurons in the basal ganglia for studying Parkinson disease, midbrain ventral tegmental area dopaminergic neurons in depression models, and dopamine D1-expressing neurons of the prefrontal cortex to investigate a circuit underlying temporal control of behavior. Probing various physiological mechanisms has also become a focus of optogenetic manipulation, with recent studies elucidating a role of ventral surface astrocytes in control of breathing. In epilepsy, optogenetics has become an attractive option to silence hyperactive neurons, with targeting of those believed to initiate or propagate seizures.
Epilepsy presents a difficult entity to study because of the large number of network pathologies that initiate or propagate seizures. However, there are several recent studies that aim to leverage the selectivity of optogenetics to analyze and control network dynamics in several animal models of seizures. Epilepsy is characterized by overactivity of various circuits, and consequently, many of the current investigations have focused on ways to silence or inhibit overactive circuits. The ability to selectively target mammalian hippocampal neurons was a first step toward probing this disease pathway, and was achieved using the inhibitory opsin halorhodopsin in organotypic cultures of mouse hippocampi. Following this achievement, halorhodopsin was used in selected principal glutamatergic neurons in the hippocampus. In this mouse model, the injection of a lentiviral vector into CA1 and CA3 pyramidal cells resulted in hyperpolarization in response to orange light (wavelength 573–613 nm). Action potentials were attenuated and paroxysmal depolarizing shifts were curbed, suppressing epileptiform activity. Importantly, the presence of the virally transfected opsins into neurons did not affect normal physiological properties of cells. To test this, organotypic hippocampal slices were subjected to complete darkness, and various properties of the cells were tested. Parameters including input resistance, resting membrane potential, action potential threshold and duration, amplitudes in response to hyperpolarizing and depolarizing pulses, and accommodation of cells to action potentials were similar in the halorhodopsin neurons in comparison with the controls.
Recently, this technology has been demonstrated in 3 different in vivo rodent models of epilepsy. In the rat tetanus toxin injection model, principal neurons were transfected with a lentiviral vector carrying halorhodopsin 2.0. Illuminating the transfected cells with 561-nm wavelength light resulted in decreased epileptiform EEG activity. Decreased high-frequency power was also observed, which correlate to bursts of activity in human epilepsy, and event frequency also diminished.
The other two examples used a closed-loop strategy to abort seizures. In the first, a cortical stroke model was used in rats to promote seizures induced by photothrombosis, in which animals were first sensitized by injection of Rose Bengal dye, after which illumination resulted in increased light absorption and focal thrombosis. Intracranial EEG electrodes confirmed spontaneous epileptiform oscillations in the thalamus and cortex of injured rats but not in controls, correlating with thalamic network hyperexcitability. To determine whether these hyperactive thalamic cells could respond to light and silence seizure activity, a viral vector was injected consisting of the cell-type specific promoter Camk2a, the halorhodopsin eNpHR3.0, optimized with membrane trafficking signal motifs, and a reporter (eYFP, yellow fluorescent protein). The promoter ensured that only excitatory thalamic cells would be subject to viral transduction. Injections were performed stereotactically into the ventrobasal thalamus, followed by slice preparation for in vitro whole-cell recordings, as well as chronic recordings and stimulation in freely moving animals. To perform the chronic recordings, a device containing multiple EEG electrodes and chronic multisite optrode was implanted into the injected thalamus and peristroke cortex. Yellow light of a wavelength 594 nm was delivered, which resulted in silencing of epileptic EEG activity in thalamus and cortex and correlated with abortion of behavioral seizures as well. Lower intensity light was not effective, and the 594-nm light did not affect interictal EEG tracings or behaviors. The authors also developed a method of detecting and interrupting seizures online by routing to a real-time processor capable of determining seizure onset, which triggered light stimulation and resulted in seizure abortion within 1 second.
Following this report, optogenetic silencing of seizures was tested in a model of temporal lobe epilepsy. Here, mice were injected with kainic acid unilaterally into the dorsal hippocampus, and 2 methods of seizure control were explored: inhibition of excitatory principal cells and stimulation of inhibitory GABAergic cells. Opsins were expressed by using transgenic animals, crossing mice expressing the Cre-Iox recombinase in either principal cells or parvalbumin-containing GABAergic interneurons with those expressing halorhodopsin or channelrhodopsin-2, respectively. With this method, only cells containing Cre were able to express the opsin of interest, ensuring specificity. As in previous studies, a reporter fluorescent protein was also expressed. Seizure detection was achieved via depth electrodes and tuning of seizure "signatures" (magnitude, amplitude, width, rate, and frequency) with custom software. For inhibition of principal neurons with halorhodopsin, amber light of wavelength 589 nm was delivered via an optical fiber implanted in the ipsilateral hippocampus upon seizure activity detection. All animals (n = 6) responded with silencing of electrographic seizures, with a majority (57% ± 12%) aborted within 1 second. The percentage of seizures aborting within 5 seconds was approximately 70%, and was unaffected by light duration (30 seconds vs 10 seconds). Further interrogation was made into the effect of red-shifted light, which has increased tissue penetrance, but is suboptimal for channelrhodopsin-2. This 635-nm light aborted 46% ± 12% seizures within 1 second of illumination. One animal in particular responded with 100% of seizures aborting within 1 second. The results from this portion of the experiment are shown in Fig. 3.
(Enlarge Image)
Figure 3.
Seizure control in mice expressing halorhodopsin (HR) in principal cells in a model of temporal lobe epilepsy. a: Crossing CamK-Cre and Cre-dependent HR mouse lines generated mice expressing the inhibitory opsin HR in excitatory cells (Cam-HR mice). b: Experimental timeline. c–e: Example electrographic seizures detected (vertical green bars), activating amber light (589 nm) randomly for 50% of events (light: amber line, example in d; no light example in e). f: Typical example distribution of postdetection seizure durations (5-second bin size) during light (solid amber) and no-light internal control conditions (hatched gray). Inset: First 5-second bin expanded, 1-second bin size. Note that most seizures stop within 1 second of light delivery. Adapted from Nat Commun 4: Krook-Magnuson E, Armstrong C, Oijala M, Soltesz I, On-demand optogenetic control of spontaneous seizures in temporal lobe epilepsy, p. 1376, 2013, with permission from Elsevier. KA = kainic acid.
Activation of parvalbumin-GABAergic neurons using channelrhodopsin-2 also resulted in reduced seizure duration. Blue light delivery (wavelength 473 nm) aborted 59% ± 11% of seizures within 5 seconds. This was a significant finding, as these cells make up less than 5% of hippocampal neurons, suggesting that even in a small population of targeted cells, optical stimulation of transgenic cells may be efficacious.
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