…

Functional verification of ChR2 in vitroA

Recording pipette Opticfiber stimulate

nRT

Thalamus

Results

IntroductionSeizure is a common neurological disorder which affects 1 % of population worldwide. Several alternative therapeutic approaches, such as, deep brain stimulation (DBS) in thalamus regions have been proposed as targets for treatment of epilepsy. DBS would excite thalamocortical activities which could be controlled by GABAergic interneuron in reticular thalamus nucleus (nRT). Previous studies showed that enhancing GABAergic inhibitory strength in nRT reduces the duration and power of seizure and indicated that thalamus play an important role in modulating cortical seizure. However, traditional electrical stimulation of nRT would excite passing fibers and cause controversial results. Recent develop optogenetic approach by excitation of light sensitive channel expressed in specific neuronal group could avoid this side effect. The aim of present study is to apply optogenetic method to study the role of nRT on modulation of cortical seizure. We use Pv-ChR2-EYFP transgenic mice from Jackson lab in which only Channelrhodopsin-2 (ChR2) is expressed in parvalbumin (Pv) positive interneurons. To study the effect of nRT activation to cortical seizure, cortical seizure-like activities were induced by electrical stimulation of corpus callosum (CC) after applying 4-Aminopyridine (4AP). Two Michigan electrodes were inserted into the primary somatosensory barrel field (S1BF) and ventral posteromedial nucleus (VPM) regions to record the seizure, and one optic fiber inserted into nRT to stimulate ChR2 expressed neurons. In this study, we have verified that ChR2 expression is abundant in nRT and cerebellum in Pv-ChR2-EYFP transgenic mice. Light stimulation in nRT could cause burst firing in thalamus and nRT regions in vitro. Unit activates were increased during high frequency (100Hz and 50Hz) or long light duration (1s) stimulation. The firing activities in thalamocortical relay regions (S1BF and thalamus) could respond to different duration of light in vivo. Unit activities were increased during high frequency (100Hz and 50Hz) light stimulation in S1BF and thalamus. CC-induced seizure-like activities were effectively suppressed during high frequency (100Hz) or long duration (10s) light stimulation. This suppression effects could be reversed by applying GABAB antagonist but not GABAA antagonist. Our results show that light stimulation can affect thalamocortical relay neurons by activating ChR2-expression neurons in nRT. High frequency and long duration light stimulation is more effective in suppressing cortical seizure-like activities. Histological examination verified that those areas near the nRT and thalamus are more likely to suppress cortical seizure. GABAB receptor may participate in suppressing seizure-like activities.

Suppression of seizure-like activity by optogenetic stimulation in thalamic reticular nucleus

Wei Jen Chang Wei Pang Chang, and Bai Chuang Shyu Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC

Material & MethodPreparation of animalsTransgenic mice in which only Channelrhodopsin-2 (ChR2) will be expressed in parvalbumin (Pv) positive interneurons. No functional ChR2 expression was found except in nRT and cerebellum of these Pv-ChR2-EYFP transgenic mice. PV-Chr2 mice (20-30g) were housed in an air-conditioned room (21–23°C, humidity 50%, 12-h light/dark cycle starting at 08:00 h) with free access to food and water. All experiments were carried out in accordance with the guidelines of the Academia Sinica Institutional Animal Care and Utilization Committee.

Surgery PV-Chr2 mice were initially anesthetized with 4% isoflurane mixed oxygen air and then placed in a stereotaxic apparatus. Animals were maintained under anesthesia with 1.6% isoflurane in oxygen during the surgery. Mouse body temperatures were maintained at 36.5~37.5°C with a homeothermic blanket system (Model 50–7079, Harvard Apparatus, Holliston, MA).

Recording and light stimulationOne Michigan electrode was inserted into the somatosensory cortex to record the seizure, and one electrode was inserted into the corresponding thalamic regions to stimulate and record nRT activity.

Aims:

1.Use Pv-ChR2 mice as model, and verify the expression of channelrhodopsin.2.Induce cortical seizure-like activity by injecting 4-Aminopyridine (4-AP, potassium channel blocker).3.Use light to specifically activate nRT neuron output to investigate the effect of nRT activation during seizure.

Seizure inductionTo study the effect of nRT activation to cortical seizure, cortical seizure-like activity were induced by corpus callosum (CC) stimulation after applying of potassium channel blocker 4-AP.

Figure 1. (A) An example of PV-ChR2-EYFP be expressed in nRT. (B) Enlarge nRT region.(C) Immunostaining result of PV. (D) Immunostaining result of DAPI. (E) Merge.

The expression of channelrhodopsinA DB

D

Green: intrinsik EYFPRed:Anti-PVBlue: Anti-DAPI

Figure 2. (A) 400μm horizontal brain slice containing the nRT and thalamus. (B) nRT neurons in response to 10Hz, 20Hz, 50Hz, 100Hz, 20ms, 50ms, 100ms and 1s. light stimulation. (C) Spikes from nRT and thalamus neurons in response to light stimulation. Unit activity in nRT are significantly increased during light stimulation. Unit activity in thalamus are slightly enhanced during light stimulation and significantly increased after 500ms of light stimulation. (D) Average of spike counts from nRT and thalamus neurons in response to different pulse duration of light stimulation.

Figure 4. Cortical and thalamic responses with different light duration and frequency stimulation. (A) In the upper panel position of 16 channel electrode. 3-16 Channels are in S1, one optic fiber implanted in nRT region. In the lower panel, another electrode 7-16 channels are in VPM. (B) Field potential and current source density response after difference light stimulation in RT. (C) High pass counts with different light stimulation conditions in S1 and Thalamus.

Light stimulation in nRT changes the firing pattern in S1 and thalamus

Figure 3. (A) Position of 16 channel electrode. Upper 3-7 channel are in the nRT. (B) Summation of unit activity from 20 sweeps, noted that during laser stimulation (blue line) the spiking activity are larger in channels located in nRT. Channel 4 was selected and unit activity from 20 sweeps are shown on the right. Statistical result showed that during laser stimulation, the unit activity are significantly enhanced. (C) Position of 16 channel electrode. Upper 2-9 channel are within the thalamus. (D) Summation of unit activity from 20 sweeps, noted that during laser stimulation (blue line) the spiking activity are decreased in channel located within the thalamus. Channel 3 was selected and unit activity from 20 sweeps are shown on the right. Statistical result showed that during laser stimulation, the unit activity in thalamus are significantly decrease, and significantly enhanced after laser stimulation.

Functional test of PV-ChR2 mice in vivo

ConclusionLight stimulation can activate reticular thalamus and thalamus by activating ChR2-expression neurons.High frequency and long duration of light are more effective in suppressing seizure.GABAB receptor may participate in suppressing seizure-like activity.Location of optic fiber is crucial, those in the nRT and thalamus are more effective in suppressing seizure.

Figure 5. (A) Cortical and thalamic seizure-like activities are induced by CC electric stimulation after applying of potassium channel blocker 4-AP. (Electrode stimulate 4 seconds , 10Hz, 500uA). (B) CC induced seizure-like activities are suppressed by 10s light stimulation after CC stimulate. (C) Seizure-like activities are partial reverse after applying of GABAB antagonist CGP 46381 100uM.

Light stimulation in nRT suppressing corpus callosum induced seizure

Different light stimulation condition in suppressing seizure

Figure 7. (A) Statistical result showed that during laser stimulation, high frequency and long duration of light stimulation conditions lead to seizure induced rate significantly decrease (B) Dots on the representative brain map indicated optical stimulation sites. Those with lower seizure induced rate near nRT region.

C

B D

C

C

A B

D

A BS!BF

nRT

VPM

C

CC stim

S1 response

Th response

CC stim

Light 10s

CC stim

S1 response

CC stimLight 10s

Th response

S1 response

Th response

CC stim

CC stim

Light 10s Light 10s

Apply 4-AP 100mM Apply GABAB antagonist CGP 46381 100uMA B C

Effects of GABA antagonist on cortical seizure suppression

Figure 6. light suppression effects and GABA antagonist partial reverse seizure-like activity. (A) Average discharge of different light stimulation or drugs in S1 (B) Average discharge of different light stimulation or drugs in Th. Significant level was set at *P<0.05, **P<0.01 ***p<0.005. Error bars are s.e.m. values.

Bicuculline: GABAA antagonistCGP 46381: GABAB antagonist

A B

Optical stimulation site

Suppress seizure>80% Suppress seizure<50 Suppress seizure 50~80%

A

B

Modified BAC DNA:Promoter region (Parvalbumin)

ChR2-EYFP BGHpA 1.4 3’UTR…FRTATG

(Zhao et al., Nat. Methods 2011)

Eletrode in RT Eletrode in thalamus

100Hz50Hz

250ms 500ms