Suppression of cortical seizures by optic stimulation of the reticular thalamus in PV-mhChR2-YFP BAC transgenic mice
© The Author(s). 2017
Received: 15 March 2017
Accepted: 16 August 2017
Published: 2 September 2017
Deep brain stimulation in thalamic regions has been proposed as a treatment for epilepsy. The electrical current excites thalamocortical activity which is controlled by γ-aminobutyric acid (GABA)ergic interneurons in the reticular thalamic nucleus (nRT). Previous studies showed that enhancing GABAergic inhibitory strength in the nRT reduces the duration and power of seizures, indicating that the thalamus plays an important role in modulating cortical seizures. The aim of the present study was to apply optogenetics to study the role of the nRT in modulating cortical seizures. We used PV-ChR2-EYFP transgenic mice from Jackson Laboratories, in which only Channelrhodopsin-2 (ChR2) is expressed in parvalbumin-expressing interneurons. Cortical seizure-like activity was induced by electrical stimulation of the corpus callosum after applying 4-aminopyridine. ChR2 expression was abundant in the nRT and cerebellum in PV-ChR2-EYFP transgenic mice. Light stimulation in the nRT caused burst firing in regions of the thalamus and nRT in vitro. Multi-unit activity increased during high-frequency (100 and 50 Hz) light stimulation in the S1 region and thalamus in vivo. Corpus callosum stimulation-induced seizure-like activity was effectively suppressed by high-frequency (100 Hz) and long-duration (10 s) light stimulation. The suppressive effects were reversed by applying a GABAB receptor antagonist but not a GABAA receptor antagonist in the cortex. The results indicated that light stimulation affected thalamocortical relay neurons by activating ChR2-expression neurons in the nRT. High-frequency and long-duration light stimulation was more effective in suppressing cortical seizure-like activity. GABAB receptors may participate in suppressing seizure-like activity.
Epilepsy is a neurological disorder that is characterized by seizure activity associated with abnormal synchronous neural overexcitation . Epilepsy affects 50 million people worldwide, and nearly 0.1% die from seizure-related disorders every year in the United States. More than 30% of patients suffer from drug-resistant epilepsy . For these patients, several alternative therapeutic approaches have been employed, such as transcranial direct current stimulation, transcranial magnetic stimulation, and deep brain stimulation (DBS). Deep brain stimulation has been adapted for both clinical treatment and animal studies. Different brain regions have been targeted to treat such neuronal disorders as epilepsy, Alzheimer’s disease, Parkinson’s disease, essential tremor, dystonia, cluster headache, and chronic pain . Deep brain stimulation has been applied to several brain nuclei, including the cerebellum, hippocampus, amygdala, and thalamus, among others. The thalamus plays an important role in seizure development and inhibition [4, 5]. The centromedial and anterior nuclei of the thalamus have been targeted for the treatment of epilepsy. These two nuclei can suppress refractory epilepsy and generalized seizure disorder [3, 6]. Despite the successful application of DBS for the treatment of epileptic disorders, the mechanism by which the DBS acts on the nervous system is still unclear.
The reticular thalamic nucleus (nRT) is an important region that lies between thalamocortical circuits . Most of the nRT contains GABAergic neurons that innervate the thalamus and produce inhibitory postsynaptic potentials (IPSPs) and provide synaptic inhibition of excitatory thalamocortical relay neurons . Previous studies found that nRT stimulation can suppress limbic motor seizures . Enhancing GABAergic inhibitory strength in the nRT reduced the duration and power of absence seizures . Furthermore, several studies have shown that nRT plays a critical role in the modulation of cortical activity during sleep and arousal through their strong impact on thalamocortical neurons [11–14]. The aforementioned studies used electrical stimulation approaches that might lead to unwanted side effects. The nRT also receives inputs from the cortex, and thalamocortical and thalamocortical pathways lie in the vicinity of the nRT. Electrical or pharmacological stimulation of the nRT can excite these passing fibers. Optogenetic methods have recently been developed, in which light-sensitive channels are engineered to be expressed in target cells, which may provide a better method of DBS . Cortical injury that was induced by thalamocortical seizures was interrupted by suppressing the activity of thalamic nuclei using a closed-loop optogenetic strategy . However, the involvement of nRT in cortical seizures requires further investigation. The present study investigated whether optic stimulation of the nRT suppresses cortical seizure activity. We use transgenic mice in which Channelrhodopsin-2 (ChR2) is expressed in parvalbumin (PV)-expressing neurons in the nRT. These neurons can be specifically activated by a blue light laser (473 nm) . This optogenetic approach can selectively activate the nRT and control its activity in real time, while not affecting adjacent neurons or having side effects that typically occur with traditional pharmacological or electrical stimulation methods. The present study may be critically important in laying the groundwork for understanding the role of the nRT in thalamocortical seizures.
Prv-mhChR2-YFP BAC transgenic mice were purchased from Jackson Laboratories (stock no. 012355, Bar Harbor, ME, USA). These mice express channelrhodopsin-2/EYFP fusion protein (mhChR2::YFP) that is directed to neuronal populations by the mouse PV promoter on the BAC transgene. Prv-mhChR2-YFP mice were genotyped using the Jackson Laboratories genotyping protocol. Polymerase chain reaction (PCR)-positive mice were mated with C57BL/6 J mice to establish transgenic lines. All of the mouse experiments were performed according to the guidelines established by the Academia Sinica Institutional Animal Care and Utilization Committee. Efforts were made to minimize animal suffering and reduce the number of animals used.
To determine ChR2 gene expression in individual mice, we examined each mouse based on tail DNA. Tail samples were clipped about 0.3 cm per mice for DNA extraction, the tail samples were incubated for approximately 16 h in tail extraction buffer (50 mM Tris [pH 8.0], 100 mM ethylenediaminetetraacetic acid [pH 0.8], and 10% sodium dodecyl sulfate) that contained 20 mg/ml proteinase K (Qiagen, Hilden, Germany). Proteinase K was then inactivated by incubation for 5 min at 95 °C. The samples were then centrifuged at 20,000 × g for 1 min, and the supernatant was collected. Polymerase chain reaction amplification was used to detect the Prv-mhChR2-YFP transgene in somatic DNA. DNA polymerase was purchased from Thermo Scientific (Waltham, USA) and used according to the manufacturer’s instructions. The PCR conditions were the following: 95 °C for 2 min, followed by 30 × [95 °C for 30 s, 56.8 °C for 30 s, and 72 °C for 30 s], with final extension at 72 °C for 5 min. The PCR products were run on 2% agarose gels with ethidium bromide, visualized using a gel imaging system.
Functional assessment in brain slices in vitro
Brains from 6 to 8 week old PV-ChR2 mice were sliced into 400 μm coronal sections using a vibratome (Microslicers, DTK-1000). The slices were kept fresh by artificial cerebrospinal fluid (aCSF) that contained the following: 124 mM NaCl, 25 mM NaHCO3, 10 mM D-glucose, 4.4 mM KCl, 1 mM NaH2PO4, 2 mM MgSO4, and 2 mM CaCl2 (pH 7.4). The slices were incubated in oxygenated aCSF (95% O2 and 5% CO2) for at least 1 h for recovery.
The recording pipettes were constructed from borosilicate glass with a pipette puller (P-97, Sutter Instruments (Novato, USA). The pipettes were filled with aCSF and inserted into the nRT or thalamic regions to record local field potentials. Blue light was generated by a blue light laser (473 nm, Sol-473-050MFL, Shanghai Dreamlaser, Shanghai, China). A 62.5 µm diameter optic fiber (GIF625, Thorlabs, New Jersey, USA) that was connected to the blue light laser was placed in the nRT region for light stimulation. Light power (422.4 mW/mm2) was measured by LaserCheck (LaserCheck, Coherent, California, USA) to activate expressing ChR2 neurons. Analog signals were amplified by Axon MultiClamp 700B and processed using the Power 1401 Mk II CED data acquisition system (Cambridge Electronic Design, Cambridge, England). Spike2 software (Cambridge Electronic Design, Cambridge, England) was used for offline analysis.
Surgery for in vivo electrophysiological measurements
PV-Chr2 mice were initially anesthetized with 4% isoflurane mixed in oxygenated air and then placed in a stereotaxic apparatus. The animals were maintained under anesthesia with 1.75% isoflurane in oxygen during surgery. Body temperature was maintained at 36.5–37.5 °C with a homeothermic blanket system (Model 50–7079, Harvard Apparatus, Holliston, MA, ISA). Craniotomy was performed over the skull regions on top of the primary somatosensory cortex (S1). Small parts of the dura over S1 were carefully removed using a 23-gauge needle. After surgical preparation, the animals were anesthetized with 1.6% isoflurane during the recording session. The depth of anesthesia was periodically monitored and maintained by pinching the paw or tail so that no overt body movement occurred.
Recording evoked field potentials in the S1 and thalamus in vivo
Extracellular field potentials that were evoked by electrical pulses were first mapped by a silver-sliver chloride (Ag-AgCl) tip electrode in the S1 region (~1.5 mm posterior and 3.5 mm lateral to bregma). To induce a cortical response, a pair of stainless-steel needles was inserted into the mouse whisker region or hind paw and used to deliver bipolar electrical stimulation (5 mA, 0.5 ms duration, model 2100, AM Systems, Washington, USA). The position in S1 that resulted in a maximal local field potential that was evoked by electrical stimulation was identified and designated as the insertion point for the multichannel recording probe (16 contact points, 100 μm interval spacing). The probe was inserted perpendicular to the S1 region. Another multichannel recording probe was used to record extracellular field potentials in the thalamus (~3.5 mm posterior and ~1.8 mm lateral to bregma; probe inserted 40° from vertical). In some of the experiments, an optoelectrode that combined an optic fiber and electrode (OAx16-10 mm-100-177, Neuronexus, Michigan, USA) was inserted into thalamic regions for simultaneous light stimulation and field potential recording. For optic stimulation, the optic fiber was inserted into the nRT for light stimulation (10–1000 ms continuous light or 10–100 Hz stimulation) by a blue light laser. An Ag-AgCl reference electrode was placed in the olfactory bulb. The sampling rate of recorded analog signals was 6 kHz, and data were processed using a multichannel data acquisition system (TDT, Florida USA) and computer.
Cortical seizure induction
Spontaneous cortical seizure activity was induced by local application of the potassium channel blocker 4-aminopyridine (4-AP; 50 mM) on the cortical surface. A tungsten electrode was inserted into contralateral side of the corpus callosum. Electrical stimulation was performed with a 500 μA pulse current intensity, 0.5 ms pulse duration, and 10 Hz in 4 s pulse width using a constant-current pulse generator (Model 2100, A-M Systems, Washington, USA). Cortical seizure activity was induced every 10 s electrical stimulation. The induction of cortical seizure activity by electrical stimulation occurred simultaneously with optic stimulation of the nRT to evaluate possible suppressive effects.
Chronic electroencephalographic recording and behavioral assessment in freely moving mice
A silver-ball electrode with an omnetic connector (A79014, Omnetics Connectors, Minnesota, USA) was fixed over the S1 region for electroencephalographic (EEG) recording. An optic fiber (BFL37–200, Thorlabs, New Jersey, USA) was inserted into the nRT for optic stimulation. The mice were allowed to recover from the surgical procedures at least for 1 week. EEG signals over the S1 region were recording and calculated by short-time Fourier transform (STFT). EEG signals in the S1 region were divided into shorter segments (1 s) of equal length, and the Fourier transform was computed. Computations were performed using the stft function in Matlab software (MathWorks, Natick, MA, USA). The optic fiber was inserted in the nRT and connected with a LC Ferrule (MM-FER2007C-1270, Precision Fiber Products, California, USA) and ceramic split sleeve (SM-CS, Precision Fiber Products, California, USA) that were fixed on the head by dental cement. Another optic fiber with a fiber optic rotary joint (FRJ_1X1 FC-FC, Doric Lenses, Quebec, Canada) was connected to the ferrule and sleeve to avoid optic fiber interruption when the mice were freely moving.
Verification of electrode placement and immunohistochemistry
At the end of the experiment, a small lesion was made by passing an anodal current (25 μA, 5 s) through the deepest electrode of the electrode probe. Another lesion was made at the same lead after the electrode probe was withdrawn by 500 μm. The brains were fixed by perfusion with normal saline followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS; pH 7.4). The brains were removed, postfixed for 72 h, sectioned at 60 μm on a cryostat, and processed for immunohistochemistry. The brain slices were incubated with 1% bovine serum albumin, 0.02% sodium azide, and 0.3% Triton in PBS for 1 h. The sections were incubated with rabbit polyclonal anti-PV (GTX11427, GeneTex, California, USA) and anti-DAPI at dilutions of 1:500 for 24 h at 4 °C. Immunohistochemistry was performed using rabbit secondary IgG (H + L) antibody (pre-adsorbed, DyLight 594) and rabbit IgG (H + L) antibody (pre-adsorbed, DyLight 405, GTX 76756, GeneTex, California, USA).
4-Aminopyridine (100 mM), the GABAA receptor antagonist bicuculline (100 μM), and the GABAB antagonist CGP52432 (100 μM) were purchased from Tocris Cookson (Ellisville, MO, USA), dissolved in physiological saline, and administered by local application on the S1 region. For seizure induction in freely moving animals, pentylenetetrazol (PTZ; 50 mg/kg), which causes alterations in excitatory and inhibitory neurotransmitter systems, was dissolved in saline and intraperitoneally injected.
Field potential data were recorded by Spike2 and Signal software (Cambridge Electronic Design, Cambridge, England), and multi-unit activity in the behavioral and electrophysiological experiments was analyzed by Matlab software (MathWorks, Natick, MA, USA). Multi-unit activity was counted when the amplitude of the field response was two-times greater than the background threshold. Average EEG discharges were based on the average field potential after CC stimulation for 1 min. The EEG signals and field potentials were first averaged every 5 s (one bin) for the entire period (12 bins). The average discharges were based on the average of all 12 bins during the 1 min period. Correlations between seizure-like activity in the S1 and thalamus, recorded at different locations, were evaluated by cross-correlation analysis. Computations were performed using the xcorr function in Matlab software. If data follow normal distribution in normality test, significant changes after drug application were determined using one-way analysis of variance (ANOVA) and Systat (SPSS) software. The data were plotted using Microsoft Excel software. One-way ANOVA with repeated test was used to analyze the data of neuronal discharges in multiple groups and FFT power of EEG recordings in freely moving animals. Values of p < 0.05 were considered statistically significant. Tukey’s post hoc test was used to detect the sources of group differences in the ANOVA.
Expression of channelrhodopsin
Functional verification of ChR2
Cortical and thalamic responses evoked by different light stimulation durations and frequencies
Closed loop of cortical and thalamic seizure activity
Suppressive effects of the nRT on electrically evoked cortical seizures
Effects of GABA antagonism on cortical seizure suppression
Evaluation of optic stimulation of the nRT in awake freely moving mice
The present study found that 4-AP-induced seizures in the cortex and thalamus were suppressed by optogenetically activating the nRT. We also found that the suppressive effect of light stimulation on cortical seizures was related to GABAB receptor transmission. Overall, cortical seizures were effectively suppressed by light stimulation in freely moving mice.
ChR2-EYFP was abundantly expressed in PV-expressing neurons in the nRT in Prv-mhChR2-YFP BAC transgenic mice, and these neurons responded to light stimulation. ChrR2 has been shown to have low expression in the thalamus, striatum, cortex, and hippocampus [17, 20]. The low transgene expression in the cortex may be caused by transcription regulatory elements that are inherent to the BAC construct or post-translational processes. Low expression can be rescued by reconstructing PV-expressing neurons with tdTomato, and these neurons were shown to be expressed throughout the nRT and other areas . We further found innervation of thalamocortical projection neurons by these nRT neurons, confirmed by the observation of connections between extended processes of PV-ChR2 neurons and the retrogradely labeled thalamic neurons. The nRT has two types of interneurons: projection neurons that innervate thalamic relay neurons and local interneurons. ChrR2 is expressed in both types of neurons, and the optical stimulation likely indiscriminately excited both types of neurons. The effects of excitation would be conveyed by projection neurons and exert effects on thalamic relay neurons. The functional role of GABAergic interneurons in the nRT was demonstrated in a recent study in which deletion of the GABAA receptor α1 subunit increased inhibition in the nRT and reduced absence seizures, in which abnormal oscillations are believed to develop in thalamocortical pathways [22–24].
4-Aminopyridine has been widely used to induce focal epileptic discharges in different region in vitro, including the hippocampus , cingulate cortex [26–28], neocortex , amygdala , brain slice of guinea pig  and optical imaging study in Ferrets in vivo . The application of 4-AP in the somatosensory cortex induced seizure-like activity and affected the neighboring cortex . Our previous studies showed that the thalamus regulates 4-AP-induced cingulate cortex seizure-like activity by regulating gap junctions [28, 34]. Seizure-like activity that was induced by 4-AP application in the somatosensory cortex may have affected ventrobasal complex (VB) regions through the thalamocortical pathway, revealed by synchronous discharges in the cortex and thalamus (Fig. 4). Cortical stroke-induced seizure-like activity is known to produce long-distance synchronization in the cortex and thalamus . Long-distance synchronization is modulated by ascending cholinergic and serotonergic systems and thalamocortical feedback loops. When seizures occur, extracellular K+ concentrations increase and extracellular Ca2+ concentrations decrease, leading to changes in synaptic transmission, in which low Ca2+ concentrations inhibit the normal release of transmitters . 4-Aminopyridine induced seizure-like activity in the cortex, which may have altered synaptic neurotransmitter release and led to synchronous oscillatory activity through thalamocortical feedback loops.
Electrical stimulation of the nRT has been suggested for the treatment of temporal lobe epilepsy . However, electrical stimulation has side effects. Electrical stimulation can affect neurons that surround the target region, and all types of neurons are activated by such stimulation. Furthermore, damage or lesions can occur in the target region if the stimulation duration is too long. Optogenetics may solve these problems by activating specific neuronal groups. Previous studies used this new optogenetic method to control thalamic activity to suppress cortical injury-induced seizures  and to control different types of interneurons for the treatment of epilepsy . In the present study, we used different durations and frequencies of light stimulation of the nRT. Seizure activity and behavior were suppressed by nRT neuron activation (Figs. 5 and 8). These suppressive effects were more robust with high-frequency and long-duration light stimulation, which may have resulted from augmented GABA release when the nRT was activated. Previous studies found that a halorodopsin-expressing adeno-associated virus in the hippocampus suppressed kainic acid-induced temporal lobe epilepsy [36, 37]. Neocortical epilepsy is usually drug-resistant. A halorodopsin-expressing lentivirus also suppressed tetrodotoxin-induced focal cortical seizures . The inhibition of neuronal activity in the CA3 area of the hippocampus suppressed kainic acid-induced seizures [39, 40]. Seizures that were induced by cortical stroke through the thalamocortical pathway were suppressed by the halorodopsin-induced inhibition of VB neuron activity . High-frequency optic stimulation (20 and 50 Hz) of the CA3 area of the hippocampus suppressed 4-AP-induced seizures in Thy1-ChR2 transgenic mice . We also observed suppressive effects of high-frequency optic stimulation. Such high frequencies may thus be better for regulating GABA neurotransmitter release in the nRT to suppress cortical seizures.
The suppression of cortical seizures by the activation of thalamocortical pathways likely occurred through a cortical feedforward inhibitory mechanism. Thalamocortical feedforward inhibition has been related to inhibitory GABAergic interneurons in the cortex [42, 43]. GABAA receptor-mediated IPSCs were abolished by the GABAA antagonist picrotoxin in thalamocortical slices . However, whether GABAB receptors mediate thalamocortical feedforward inhibition is still unclear. GABA receptors play an important role in mediating seizure activity. GABAA receptors mediate 4-AP-induced synchronous events in the hippocampus and entorhinal cortex. GABAA receptors also participate in the initiation, maintenance, and termination of ictal discharges [44, 45]. GABAB receptors mediate 4-AP-induced long-lasting depolarization and asynchronous excitatory and inhibitory potentials in the rat hippocampal region . CGP35348 blocks presynaptic GABAB receptors and leads to an increase in GABA release and elevation of [K+]o . In the present study, GABAergic interneurons in the nRT were activated by blue light, which strongly hyperpolarized VB neurons, results in the generation of Ca2+ spikes, and triggers rebound burst firing in the somatosensory cortex. . We hypothesize that 4-AP-induced seizures in the cortex are suppressed by cortical GABAergic neurons that receive inputs from the VB and release GABA. Ventrobasal inputs affect both cortical excitatory and inhibitory cells. Our results showed that the GABAB antagonist CGP46381 had no effect on seizure activity when it was applied alone (Fig. 7b), thus excluding the possible confounding effects of cortical disinhibition. The involvement of GABAB receptors in cortical feedforward inhibition has been reported previously, in which GABAB but not GABAA receptors regulate thalamocortical inputs of cortical neurons presynaptically and modulate the thalamocortical excitation of excitatory and inhibitory neurons in layer IV of the barrel cortex .
GABAergic interneurons and glutamatergic principal cells are excited by each other. Their interaction is reciprocal, and cortical interneuron excitation can directly suppress local networks through inhibitory feedback. Cortical interneurons can be excited by receiving long-range excitatory inputs from other subcortical nuclei, thus generating feedforward inhibition . Thalamocortical axons were afferents on cortical fast-spiking inhibitory neurons but not afferent in regular-spiking neurons which are pyramidal neurons in rodents . Thalamocortical relay neuron activation corresponds with synchronous discharges of S1 cortical neurons, leading GABAergic interneurons in the S1 region to release GABA and inhibit postsynaptic neurons . Spiny fast-spiking interneurons receive thalamocortical neuron inputs and mediate IPSCs. Thalamocortical relay neurons can precisely and reliably activate spiny fast-spiking interneurons in the S1 region and provide feedforward inhibition of postexcitatory neurons . Feedforward inhibition plays an important role in mature cortical function. GABAA receptor-mediated transmission regulates thalamocortical circuit function. GABAergic transmission was also shown to be strongly engaged in stellate cells that received strong thalamocortical inputs in mature mice, whereas this engagement was weaker in neonatal mice . These studies suggest that 4-AP-induced seizure activity was suppressed by the excitation of cortical interneurons through thalamocortical inputs and a mechanism of feedforward inhibition.
Our results showed that light stimulation affected thalamocortical relay neurons by activating ChR2-expressing neurons in the nRT. High-frequency and long-duration light stimulation may be more effective in suppressing cortical seizure-like activity. Histological examination verified that the stimulation of areas near the nRT and thalamus were more effective in suppressing cortical seizures, and GABAB receptors may participate in suppressing seizure-like activity.
The reticular thalamic nucleus (nRT) is a potential target for deep brain stimulation. It can affect ventrobasal thalamic regions, thus regulating the somatosensory cortex through the thalamocortical pathway. The present study found that evoking the activity of specifically nRT neurons by light stimulation of ChR2 protein attenuated cortical seizures. The mechanism of these suppressive effects involved γ-aminobutyric acid-B (GABAB) receptors in cortical regions. The findings suggest that neuronal firing in the nRT mediates cortical GABA neurons, which may be an effective target for deep brain stimulation to suppress cortical seizures.
We are thankful for technical support from the Neural Circuit Electrophysiology Core at Academia Sinica. This work was undertaken at the Institute of Biomedical Sciences, which received funding from Academia Sinica.
The present study was supported by Ministry of Science and Technology grants (NSC 99-2320-B-001-016-MY3 and NSC 100-2311-B-001-003-MY3) and a National Health Research Institutes grant (NHRI-EX106-10439NI).
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.
WJC conducted the experiments in vitro and in vivo, analyzed experimental data and wrote the manuscript. WPC participated the initial stage of experiments and help in experimental design. BCS conceived the experiments, and was a major contributor in writing the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All of the mouse experiments were performed under the guidance of National Institutes of Health and with the approval of the Academia Sinica Institutional Animal Care and Utilization Committee.
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