biosensors

Article Dopamine and Striatal Firing Respond to Frequency-Dependent DBS Detected by Microelectrode Arrays in the Rat Model of Parkinson’s Disease

Guihua Xiao 1,2 , Yilin Song 1,2, Yu Zhang 1,2, Yu Xing 1,2, Shengwei Xu 1,2, Mixia Wang 1,2, Junbo Wang 1,2, Deyong Chen 1,2, Jian Chen 1,2 and Xinxia Cai 1,2,*

1 State Key Laboratory of Transducer Technology, Aerospace Information Research Institute, Chinese Academy of Sciences, Beijing 100190, China; [email protected] (G.X.); [email protected] (Y.S.); [email protected] (Y.Z.); [email protected] (Y.X.); [email protected] (S.X.); [email protected] (M.W.); [email protected] (J.W.); [email protected] (D.C.); [email protected] (J.C.) 2 University of Chinese Academy of Sciences, Beijing 100049, China * Correspondence: [email protected]; Tel.: +86-010-5888-7193

 Received: 24 August 2020; Accepted: 21 September 2020; Published: 28 September 2020 

Abstract: (1) Background: Deep stimulation (DBS) is considered as an efficient treatment method for alleviating motor symptoms in Parkinson’s disease (PD), while different stimulation frequency effects on the specific neuron patterns at the cellular level remain unknown. (2) Methods: In this work, nanocomposites-modified implantable microelectrode arrays (MEAs) were fabricated to synchronously record changes of dopamine (DA) concentration and striatal neuron firing in the striatum during subthalamic nucleus DBS, and different responses of medium spiny projecting (MSNs) and fast spiking interneurons (FSIs) to DBS were analyzed. (3) Results: DA concentration and striatal neuron spike firing rate showed a similar change as DBS frequency changed from 10 to 350 Hz. Note that the increases in DA concentration (3.11 0.67 µM) and neural spike firing rate (15.24 2.71 Hz) were maximal ± ± after the stimulation at 100 Hz. The MSNs firing response to DBS was significant, especially at 100 Hz, while the FSIs remained stable after various stimulations. (4) Conclusions: DBS shows the greatest regulatory effect on DA concentration and MSNs firing rate at 100 Hz stimulation. This implantable MEA in the recording of the neurotransmitter and neural spike pattern response to DBS provides a new insight to understand the mechanism of PD at the cellular level.

Keywords: MEA; DBS; frequency-dependent; dopamine; MSNs

1. Introduction Parkinson’s disease (PD) is one of the most common neurodegenerative diseases faced by the old that is related to the basal ganglia circuit [1,2]. It consists of the subthalamic nucleus (STN), globus pallidus internus (GPi), cortex, striatum, substantia nigra (SN) and so on [3]. (DBS) in the STN or GPi has been considered as one of the most effective treatment methods to alleviate the symptoms of movement disorders based on actual therapeutic effects [4,5]. It has been applied in the clinical treatment of patients who were diagnosed with PD. However, the DBS parameters would influence the treatment effects significantly, and some researchers reported that different stimulation frequencies would induce distinct results [6–8]. Many studies demonstrated that high-frequency DBS on STN plays an effective role in therapeutic outcome. Most studies just focused on behavioral improvement by adjusting the parameters [9,10]. The majority of patients

Biosensors 2020, 10, 136; doi:10.3390/bios10100136 www.mdpi.com/journal/biosensors Biosensors 2020, 10, 136 2 of 12 showed a significant improvement during DBS at a high frequency of around 100 Hz [11,12]. Dystonia was improved when the DBS frequency was above 60 Hz [13–15]. In contrast, symptoms did not improve during DBS at the low frequency of 10 Hz [16]. This research is helpful for us to confirm the treatment effects, however the DBS mechanism in neuron modulation remains unknown. The cellular mechanism is varied and complex. Meanwhile, different stimulation frequencies induced different therapeutic effects which makes it necessary to reveal the real modulation mechanism at the cellular level underlying the DBS frequency dependence. DBS is hypothesized to alleviate the symptoms of neurological disorders by modulating the abnormal neural activities. Dopamine (DA) concentration degeneration in the substantia nigra (SN) is thought to be the predominant and origin reason for PD [17]. It plays an important role in movement regulation and high DA concentrations are found in the striatum [18]. DA is inherently electrochemically active and can be converted to a reporter molecule easily. Except for the DA communication between neurons, it will transfer the neural information by cell , forming the neural spike. Medium spiny projecting neurons (MSNs) and fast spiking interneurons (FSIs) are the two typical patterns that exist in striatal neurons. These neurons play an important role in movement modulation which has been reported in previous research [19,20]. It is crucial to explore which kind of neural spike pattern would respond to DBS. Obtaining the DA variation and neural spike changes during STN-DBS will provide us more detail on neuron variations to understand the reason. Presently, the neural activities detection technique was based on the traditional implantable micro devices. The researchers focused on single-mode electrode recording such as electrical detection or DA concentration release. A metal wire microelectrode was widely used to record electrical signals due to its low cost and easy fabrication [21]. However, it is impossible to reproduce with certain space and distribution. For neurochemical recording, a microdialysis probe, which is an indirect technique, could be used to monitor various chemicals [22]. This work aims to fabricate an implantable microelectrode array with integrated multi-function involving DA concentration detection and neural spike pattern monitoring. Analyzing the manipulation of DA and the neural spike pattern in the striatum during STN-DBS could help us to understand the treatment mechanism, supplying chances to meliorate the therapy efficacy on different stimulation frequencies. In this work, a multichannel microelectrode array (MEA) was fabricated to record dual-mode signals including DA and neural spike simultaneously. Surface Pt black and reduced graphene oxidase (Pt/rGO) nanoparticles modification could decrease the impedance. It is also sensitive to the DA concentration, and an exclusion layer of the Nafion coating can alter the recording properties of the MEA to exclude common interferences in the brain. The modified MEA was implanted into the striatum to examine the variations of DA and neural spike pattern during STN stimulation at different frequencies. Electrical stimulation was applied at various frequencies (10, 60, 100, 210 and 350 Hz) in the anesthetized rats. The DA concentration and neural spike firing rate were analyzed at the same time dimension, and the different responses of different neuronal patterns to DBS were compared in the paper. Furthermore, integrating the results of electrochemical, neural spike firing, stimulation parameters and clinical treatment effects would further improve the understanding of the DBS treatment mechanism.

2. Materials and Methods

2.1. Reagents Leadacetate and chloroplatinic acid were purchased from Sinopharm Chemical Reagent company (Beijing, China). Graphene oxide nanocomposites solution (2 mg/mL) was purchased from Xianfeng Corporation (Nanjing, China). Nafion solution (20%), ascorbic acid (AA), uric acid (UA), dopamine (DA), 5-hydroxytryptamine (5-HT), dihydroxyphenylacetic acid (Dopac) and glutamate (Glu) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Saline (0.9% NaCl) was purchased from the ShuangHe Corporation (Beijing, China). Biosensors 2020,, 10,, 136x FOR PEER REVIEW 33 of of 12

2.2. Apparatus 2.2. Apparatus The DA concentration was recorded with the amperometry method performed on a four‐ channelThe electrochemical DA concentration workstation was recorded (BioLogic with the VMP3, amperometry Grenoble, method France) performed driven by on EC a‐lab four-channel software. electrochemicalThe neural spike workstation signals were (BioLogic recorded VMP3, on a Grenoble, 128‐channel France) neuron driven data by recording EC-lab software. system (Blackrock The neural spikeMicrosystems, signals were Salt recorded Lake, UT, on USA). a 128-channel The MEA neuron is connected data recording to the dual system‐mode (Blackrock recording Microsystems, system and Saltattached Lake, to UT, the USA). handle The of MEA a micropositioner is connected to (model the dual-mode 2662, David recording KOPF systeminstrument, and attached Tujunga, to CA, the handleUSA). The of a “zero” micropositioner depth could (model be set 2662, when David the MEA KOPF tips instrument, touched the Tujunga, dura mater CA, USA).of the Thebrain. “zero” The depthimplanting could bedepth set when of the the MEA MEA in tips the touched brain the was dura recorded mater of and the brain.displayed The implanting on the screen depth of of the MEAmicropositioner. in the brain wasThe recorded stimulation and displayed was carried on the screenout on of thea micropositioner.commercial stimulation The stimulation electrode was carried(Microprobes, out on aGaithersburg, commercial stimulation MD, USA) electrodeconnected (Microprobes, to a stimulus Gaithersburg, generator (USB MD,‐ME16 USA)‐FAI connected‐Sytem, toMultiChannel a stimulus generator Systems, (USB-ME16-FAI-Sytem,Reutlingen, Germany). MultiChannel Systems, Reutlingen, Germany).

2.3. MEA Fabrication Procedures The MEA was fabricated using photolithographic methods as previous steps based on the on wafer (Supplementary Materials Figure S1) [23], [23], and the completed MEA is shown in Figure1 1a.a. MoreMore thanthan 200200 MEAsMEAs couldcould bebe designeddesigned onon aa singlesingle 4-inch 4‐inch wafer. wafer. TheThe MEAMEA was was designed designed with twotwo shanks. shanks. Two Two rectangle rectangle DA DA recording recording channels channels and six and round six neuralround spikeneural recording spike recording channels werechannels distributed were distributed on the tips on to the record tips to the record in situ the neural in situ activities. neural activities. The individual The individual MEA was MEA released was fromreleased the from 4-inch the wafer 4‐inch and wafer absolutely and absolutely cleaned by cleaned acetone by solution acetone andsolution oxygen and plasma. oxygen Theplasma. bonding The padsbonding of MEA pads wereof MEA connected were connected to the printed to the circuit printed board. circuit The board. 8 bare The electrodes 8 bare electrodes were coated were with coated Pt blackwith Pt nanoparticles black nanoparticles and reduced and graphenereduced graphene oxide (rGO) oxide nanocomposites (rGO) nanocomposites to decrease to the decrease impedance. the Theimpedance. to noiseThe signal ratio could to noise be decreased ratio could significantly be decreased by nanocomposites significantly modification. by nanocomposites Then, 0.5% Nafionmodification. in ethanol Then, was 0.5% specifically Nafion in drop-coated ethanol was onto specifically the DA recording drop‐coated sites onto with the a microsyringe DA recording (10 sites uL) underwith a themicrosyringe . (10 The uL) Nafion under the coating microscope. film was The dried Nafion for 20 coating min at film 100 ◦wasC. Electrode dried for S120 andmin S5at were100 °C. prepared Electrode for S1 DA and recording S5 were sites, prepared and thefor otherDA recording electrodes sites, were and used the to other record electrodes the striatal were neuron used firingto record rate. the DA striatal recording neuron sites firing were calibratedrate. DA recording using the amperometrysites were calibrated method using to obtain the aamperometry proportional variationmethod to in obtain DA content a proportional from the variation oxidation in of DA DA content [23,24 ].from A three-electrode the oxidation of setup DA [23,24]. was used A three in the‐ electrochemicalelectrode setup was signals used detection in the electrochemical which is performed signals ondetection the BioLogic which VMP3is performed potentiostat. on the BioLogic The two DAVMP3 channels potentiostat. were set The as two the workingDA channels electrode. were Anset Agas the/AgCl working reference electrode. electrode An and Ag/AgCl an integrated reference Pt counterelectrode electrode and an integrated were included Pt counter in the electrode three-electrode were included configuration. in the three‐electrode configuration.

Figure 1.1. CharacterizationCharacterization of theof the microelectrode microelectrode array array (MEA) (MEA) structure. structure. (a) The electrodes(a) The electrodes are distributed are ondistributed the tips withon the dopamine tips with (DA)-sensitivedopamine (DA) electrodes‐sensitive (S1,electrodes S5) and (S1, electrical S5) and electrodes electrical electrodes (S2–S4, S6–S8). (S2– (S4,b) TheS6–S8). SEM (b image) The SEM of the image Pt/rGO of nanoparticles-modified the Pt/rGO nanoparticles electrode.‐modified electrode.

2.4. In Vivo Testing of MEAs Sprague-DawleySprague‐Dawley ratsrats (270 (270 g) g) were were used used in the in followingthe followingin vivo in experiments.vivo experiments. Protocols Protocols for animal for useanimal were use approved were approved by the institutionalby the institutional animal animal care and care use and committee. use committee. The animal The animal surgeries surgeries were carriedwere carried out with out permissionwith permission from thefrom Ethical the Ethical Committee Committee of Peking of Peking University University using standardusing standard sterile Biosensors 2020, 10, 136 4 of 12 procedures. The rat model of PD induced by 6-hydroxydopamine (6-OHDA) was supplied by Peking University. Firstly, the rat was anesthetized with 20% urethane and fixed on the stereotaxic frame properly. The skull was exposed by craniotomy. The lambdoidal suture of the bregma was marked as the reference location. Then, the recording site and stimulating site were located according to the distance from the bregma. The recording site was the striatum (AP: 0.36 mm, ML: 2.20 mm, DV: 3.50 mm) and the stimulating site was the STN (AP: 3.60 mm, ML: 2.50 mm, DV: 8.10 mm). − − − A skull nail was placed on the skull of the cerebellum. An Ag/AgCl reference electrode was implanted (AP: 0.36 mm, ML: 2.20 mm, DV: 0.30 mm) to form a three-electrode setup. − − 2.5. Data Acquisition and Analysis A three-electrode configuration was used in the DA recording including an MEA working electrode, a Ag/AgCl reference electrode and an integrated Pt auxiliary electrode. The BioLogic VMP3 potentiostat was used to record the DA response. Six-channel neurophysiological and two-channel electrochemical signals were recorded simultaneously. The neurophysiological signal was recorded by Blackrock Microsystems and sampled at the rate of 30 kHz. A high-pass filter (200 Hz) was applied to obtain the spike firing, while a low-pass filter (200 Hz) was applied to obtain the LFPs. A commercial bipolar electrode with a biphasic pulse train was used as the stimulation electrode. The stimulation was delivered with a multichannel base station (MC Technologies) and stimulus isolation units giving a rectangular pulse. The stimulation and recording system are shown in Figure S2. The neural spike data were sorted in the Offline Sorter software with the valley seeking algorithm. The K-means cluster analysis method was used to clarify the neuron discharges. Two major patterns of neural spike were identified involving MSNs and FSIs. Neuroexplorer software was used to analyze the firing mode and firing rate of the neural spike. The stimulation electrode was implanted into the STN to stimulate the neurons electrically. The modified MEA was implanted into the striatum to record the neuron activities. The effects of the dual-mode signals during STN stimulation at various frequencies (10, 60, 100, 210 and 350 Hz) were examined.

3. Results

3.1. Neural Spike Firing Variations during DBS This MEA contains two shanks that are 7 mm in length. Each shank contains four sensitive sites geometrically distributed on the tips. The completed MEA structure is shown in Figure2. The tips of the MEA were inserted into the striatum to record the dual-mode signals involving DA concentration and neural spike firing. The DA electrodes (S1, S5) and electrical electrodes (S2–S4, S6–S8) were designed as shown in Figure2. The rectangle-shape electrodes are specific for DA recording, while the other round-shape electrodes are specific for neural spike firing recording. Figure2b shows the surface morphology of the electrode modified with Pt black and rGO nanoparticles. Note that the Pt nanoparticles are distributed and covered on the wrinkled graphene to improve the impedance performance. The Pt/rGO-modified electrical electrodes were tested in NaCl solution to obtain the background noise. As shown in Figure S3, the background noise of the bare electrode is much bigger than the Pt/rGO electrode, which makes it impossible to monitor the neural spike with the bare electrode. On the contrary, the signal to noise ratio (SNR) could achieve 5.6 by using the Pt/rGO-modified MEA. We obtained the neural spike when the SNR was bigger than 3. The impedance of the Pt/rGO electrode was tested, as shown in Figure S4, which decreased significantly by 22.6-fold at 1 kHz. The typical calibration of the DA electrode was carried out as shown in Figure2a. The sensitivity during detection was 9.7 pA/µM (Figure2b), while the selectivity is shown in Figure2c. The results demonstrated that the DA electrode could specifically recognize the DA molecule and reject the other common molecules. Biosensors 2020, 10, 136 5 of 12 Biosensors 2020, 10, x FOR PEER REVIEW 5 of 12

FigureFigure 2. ElectrochemistryElectrochemistry measurement measurement of of the the DA DA electrode. electrode. ( (aa)) A A typical typical calibration calibration curve curve of of one one representativerepresentative DA DA electrode electrode in in NaCl NaCl solution solution by by adding adding 10 10 μLµ DAL DA to tomake make the the final final concentration concentration 10 μ10M.µ M.(b) (bFitting) Fitting plot plot of ofthe the current current and and concentration concentration of of DA. DA. (c ()c )The The selectivity selectivity testing testing of of the the DA DA electrode.electrode. Amounts ofof 5 5µ μMM AA, AA, UA, UA, DA, DA, DA, DA, 5-HT, 5‐HT, Dopac Dopac and Gluand were Glu addedwere added into the into NaCl the solution NaCl solutionone by one. one by one.

TheThe commercial commercial bipolar bipolar stimulation stimulation electrode was implanted into the STN. STN. Then, Then, the the stimulation stimulation electrodeelectrode was was fixed fixed on the skull by dental cement to avoidavoid movementmovement duringduring testing.testing. The The MEA MEA connectorconnector was was connected connected to to the the dual dual-mode‐mode recording recording system, system, and and joint joint to to the the depth depth micropositioner. micropositioner. Then, the the Pt/rGO Pt/rGO-modified‐modified MEA MEA was was implanted implanted into into the the striatum striatum to to record record the the dual dual-mode‐mode signals. signals. All recording equipment was prepared properly. The stimulation waveform was was characterized by by severalseveral standard standard parameters parameters including including amplitude, amplitude, pulse pulse width, width, stimulation stimulation frequency, frequency, pulse pulse number number andand stimulation stimulation duration. duration. In In this this work, work, the the frequency frequency parameter parameter training training was wasmainly mainly performed. performed. The pulseThe pulse width width (60 μ (60s), µstimulations), stimulation duration duration (10 (10 s) and s) and current current intensity intensity (300 (300 μµA)A) were were stable stable and consistentconsistent with thosethose routinelyroutinely used used in in Parkinson Parkinson disease disease (PD) (PD) models models [25 [25].]. Frequencies Frequencies of 10, of 60,10, 100,60, 100,210 and210 and 350 Hz350 wereHz were selected selected as the as frequenciesthe frequencies training training parameter parameter along along with with 300 300µA μ intensity,A intensity, 10 s 10duration s duration and and 60 µ 60s pulse μs pulse width. width. WhenWhen the the MEA MEA was was implanted implanted into into the the striatum striatum at at the the speed speed of of 1 1 um/s, um/s, stable stable neural neural spike spike firing firing was recognized and recorded. The The typical typical neural neural spike spike firing firing response response of of six six channels channels is is shown shown in in FigureFigure 33.. The staticstatic neuralneural firingfiring waswas recordedrecorded forfor 55 minmin beforebefore stimulation. stimulation. Then,Then, fivefive frequenciesfrequencies were applied in the STN, relatively. The interval between the the two two stimuli stimuli was was five five minutes, minutes, which which is is longlong enough enough for for the the effects effects of the previous stimulus to to disappear. disappear. As As can can be be seen, seen, the the low low-frequency‐frequency stimulationstimulation such as 10 10 Hz Hz has has no no significant significant effects effects on neural spike firing firing (Figure 3 3a).a). AtAt 60 Hz, neural spikespike firing firing is is increased in two channels after stimulation, while while the the other channels channels show show a a small small increase.increase. At At 100 100 Hz, Hz, note note that that neural neural spike spike firing firing is is increased increased significantly. significantly. The The neural neural spike spike fires fires intensivelyintensively after stimulation.stimulation. At At 210 210 and and 350 350 Hz, Hz, the the increase increase in neuralin neural spike spike firing firing goes goes down. down. The highThe high frequency of around 100 Hz of DBS plays a different role in modulating neural spike firing. Too high or too low frequency stimulation would induce less modulation on neural spike firing. Biosensors 2020, 10, 136 6 of 12 frequency of around 100 Hz of DBS plays a different role in modulating neural spike firing. Too high or tooBiosensors low frequency 2020, 10, x FOR stimulation PEER REVIEW would induce less modulation on neural spike firing. 6 of 12

FigureFigure 3. The3. The neural neural spike spike firingfiring variations during during deep deep brain brain stimulation stimulation (DBS) (DBS) with withdifferent diff erent frequenciesfrequencies of 10of 10 (a ),(a 60), 60 (b (),b), 100 100 ( c(c),), 210210 (d)) and and 350 350 Hz Hz (e ().e ).The The stimulation stimulation (300 μ (300A intensity,µA intensity, 10 s 10 s duration and 60 μs pulse width) is presented with the gray box. duration and 60 µs pulse width) is presented with the gray box.

3.2.3.2. Dual-Mode Dual‐Mode Signals Signals Variations Variations during during DBS The neurotransmitter is released from the vesical to the synaptic cleft during stimulation. The The neurotransmitter is released from the vesical to the synaptic cleft during stimulation. neurotransmitter and neural spike discharge are the two main methods to communicate with each The neurotransmitter and neural spike discharge are the two main methods to communicate with neuron. The DA variation (S1, S5) and the corresponding firing rate responses of two channels (S2, eachS6) neuron. were extracted The DA as variation shown in (S1, Figure S5) 4. and The the dual corresponding‐mode signals firingof a 110 rate s time responses span including of two channelsthe (S2, S6)before were (50 extracted s), during as (10 shown s) and in Figure after 4(50. The s) dual-modestimulation signalstimes were of a 110extracted s time to span analyze including the the beforesynchronous (50 s), during relevance (10 s) of and DA after and (50 the s) firing stimulation rate. The times gray wererectangle extracted in each to curve analyze represents the synchronous the relevanceduration of of DA stimulation and the which firing lasted rate. for The 10 gray s. The rectangle relationship in eachunderlying curve DA represents concentration, the duration firing of stimulationrate and whichstimulation lasted frequency for 10 s.was The analyzed. relationship Figure 4a underlying shows the DAextracellular concentration, DA concentration firing rate and stimulationvariation frequency (S1, S5) evoked was by analyzed. STN stimulation. Figure4 aFigure shows 4b theshows extracellular the neural spike DA concentration firing rate variation variation (S1,before S5) evoked and after by stimulation. STN stimulation. Note that Figure different4b stimulation shows the frequencies neural spike evoke firing different rate responses variation of before DA and the neural spike firing rate in the striatum. They show a similar change trend as the frequency and after stimulation. Note that different stimulation frequencies evoke different responses of DA increases. The DA concentration and neural spike firing rate respond to the stimulation when the and the neural spike firing rate in the striatum. They show a similar change trend as the frequency frequency is increased from 10 to 350 Hz. At 100 Hz, the release of the DA content and the firing rate increases. The DA concentration and neural spike firing rate respond to the stimulation when the Biosensors 2020, 10, 136 7 of 12

Biosensors 2020, 10, x FOR PEER REVIEW 7 of 12 frequency is increased from 10 to 350 Hz. At 100 Hz, the release of the DA content and the firing rate are maximum. The The low low frequency frequency (10 (10 Hz) Hz) has has no nosignificant significant effects eff ectson the on DA the DAconcentration concentration and and neuralneural spike spike firing. firing. As the As frequency the frequency of the of stimulation the stimulation increases, increases, the dual-mode the dual‐mode signals signals are modulated are modulated significantly. While the stimulation is too high, for example, at 210 and 350 Hz, the DA significantly. While the stimulation is too high, for example, at 210 and 350 Hz, the DA concentration concentration and neural spike firing still increase, but they are much less than their states during and neural100 HzBiosensors spikestimulation. 2020 firing, 10, x still FOR increase,PEER REVIEW but they are much less than their states during 100 Hz7 of stimulation.12

are maximum. The low frequency (10 Hz) has no significant effects on the DA concentration and neural spike firing. As the frequency of the stimulation increases, the dual‐mode signals are modulated significantly. While the stimulation is too high, for example, at 210 and 350 Hz, the DA concentration and neural spike firing still increase, but they are much less than their states during 100 Hz stimulation.

FigureFigure 4. Dual-mode 4. Dual‐mode signals signals variations variations duringduring different different frequencies frequencies applied. applied. (a) The (a) Thevariation variation of DA of DA concentration,concentration, and and (b )(b neural) neural spike spike firing firing raterate after subthalamic subthalamic nucleus nucleus (STN) (STN)-DBS‐DBS with withdifferent diff erent stimulationstimulation frequencies. frequencies. The The gray gray box box means means the duration duration of of the the stimulation. stimulation. Figure 4. Dual‐mode signals variations during different frequencies applied. (a) The variation of DA FigureFigure5 showsconcentration, 5 shows the the mean and mean (b) variations neural variations spike firing of of the the rate DADA after concentration subthalamic nucleus and and (STN)neural neural‐DBS spike with spike firing different firing rate rateunder under the dithefferent different stimulationstimulation stimulation frequencies. frequencies. frequencies. The gray Note boxNote means that that thethe the duration DA DA concentration ofconcentration the stimulation. and and neural neural spike spike firingfiring raterate show show a similar change as the stimulation frequency changes. When the stimulation frequency a similar changeFigure as the 5 shows stimulation the mean frequencyvariations of changes. the DA concentration When the and stimulation neural spike frequency firing rate under increased from increased from 10 to 100 Hz, the DA concentration and neural spike firing rate increased and reached 10 to 100 Hz,the different the DA stimulation concentration frequencies. and neural Note that spike the DA firing concentration rate increased and neural and spike reached firing the rate maximum the maximum value at 100 Hz. However, both signals decreased when the stimulation frequency value at 100show Hz. a However,similar change both as signals the stimulation decreased frequency when changes. the stimulation When the frequency stimulation increasedfrequency from 100 increasedincreased from from100 10to to350 100 Hz. Hz, When the DA the concentration stimulation and frequency neural spike is firing too low rate increased(10 Hz) or and too reached high (350 to 350Hz), Hz. thethe When variation maximum the of stimulation value the dual at 100‐mode frequencyHz. However,signals isis toobothless lowthansignals (10the decreased Hz)frequency or too when at high 100 the Hz. (350stimulation There Hz), may the frequency variationbe a subtle of the dual-modefrequencyincreased signals around from is less 100 100 thanHz to that350 the Hz.could frequency When modulate the atstimulation 100 the Hz. dual frequency There‐mode may signals is too be lowat a subtle a (10higher Hz) frequency orvalue too highthan around (350 those at 100 Hz that100 could Hz.Hz), modulate In the combination variation the of dual-mode thewith dual the‐mode clinical signals signals therapeutic is at less a higher than theeffects valuefrequency achieved than at 100 those byHz. 100There at 100Hz may Hz.stimulation, be a In subtle combination the withmodulation the clinicalfrequency of therapeutic dopamine around 100 and eHzffects thatneural could achieved spike modulate firing by 100 the may dual Hz be‐ stimulation,mode one way signals in atwhich the a higher modulation the valuestimulation than of those dopamine works. at and 100 Hz. In combination with the clinical therapeutic effects achieved by 100 Hz stimulation, the neural spikemodulation firing may of dopamine be one and way neural in which spike firing the stimulation may be one way works. in which the stimulation works.

Figure 5. Mean increase variation of DA concentration and neural spike firing rate at the different frequency stimulations. Biosensors 2020, 10, x FOR PEER REVIEW 8 of 12

BiosensorsFigure2020 5., 10Mean, 136 increase variation of DA concentration and neural spike firing rate at the different 8 of 12 frequency stimulations.

3.3.3.3. MSNs MSNs and and FSIs FSIs Respond Respond Different Different to to DBS DBS InIn the the neural neural spike spike recording, recording, MSNs MSNs and and FSIs FSIs were were extracted extracted from from the the neuron neuron discharge discharge data. data. TheseThese two two kinds kinds of of neurons neurons show show a different a different response response to the to stimulation. the stimulation. As shown As shown in Figure in Figure 6, three6, typicalthree typical channels channels of neural of neural spike spikefiring firing were wereobtained obtained from fromthe recording the recording data. data. Figure Figure 6a–e6 showa–e show the spikethe spike firing firing mode mode of MSNs of MSNs under under different diff erentfrequency frequency stimulations, stimulations, while whileFigure Figure 6f–j show6f–j showthe spike the firingspike firingmode modeof FSIs of FSIsunder under the corresponding the corresponding frequency frequency stimulations. stimulations. The The stimulation stimulation duration duration is is markedmarked byby thethe gray gray box. box. Note Note that that the MSNsthe MSNs are modulated are modulated by the stimulationby the stimulation significantly, significantly, especially especiallyin the high-frequency in the high‐frequency stimulation. stimulation. At 100 Hz, At the 100 neural Hz, the spike neural discharge spike ofdischarge MSNs fires of MSNs intensively fires intensivelyafter stimulation. after stimulation. However, However, the low-frequency the low‐frequency stimulation stimulation has no significant has no significant effects on effects the MSNs on thedischarge. MSNs Asdischarge. shown in As Figure shown6f–j, thein FSIsFigure show 6f–j, no significantthe FSIs show response no tosignificant the stimulation. response The to neural the stimulation.spike firing ofThe FSIs neural remains spike stable firing after of FSIs stimulation remains stable regardless after ofstimulation the different regardless frequency of the stimulations. different frequencyStimulation stimulations. shows a diff Stimulationerent modulation shows function a different on modulation the specific neurons.function on the specific neurons.

FigureFigure 6. 6. MediumMedium spiny projectingprojecting neurons neurons (MSNs) (MSNs) (a –(ea)– ande) and fast fast spiking spiking interneurons interneurons (FSIs) (FSIs) (f–j) firing(f–j) firingduring during different different frequencies frequencies applied applied in the in STN-DBS. the STN‐DBS.

TheThe neural neural spike spike waveform waveform of of MSNs MSNs and and FSIs FSIs is is shown shown in in Figure Figure 77a.a. The mean MSNs pattern withwith a a higher higher amplitude amplitude and and duration duration was was extracted extracted from from 3926 3926 spikes, spikes, while while the the FSIs FSIs with with a a lower lower amplitudeamplitude and and duration duration were were extracted extracted from from 1493 1493 spikes. spikes. The The mean mean firing firing rate rate of of MSNs MSNs and and FSIs FSIs underunder the didifferentfferent stimulations stimulations is shownis shown in Figure in Figure7b. Di 7b.fferent Different frequency frequency stimulations stimulations show di ffshowerent differentmodulations modulations on the MSNs on the and MSNs FSIs. As and can FSIs. be seen, As can the MSNsbe seen, firing the rateMSNs is a fffiringected rate by stimulation is affected andby stimulationthe different and increases the different are presented increases under are presented the different under frequency the different stimulations. frequency At 100stimulations. Hz, the mean At 100firing Hz, rate the of mean MSNs firing increased rate of fromMSNs 4.30 increased0.58 to from 19.43 4.30 3.20± 0.58 Hz, to while19.43 ± the 3.20 FSIs Hz, did while not the significantly FSIs did ± ± notincrease, significantly as shown increase, by the as small shown increase by the from small 4.08 increase0.86 from to 5.24 4.080.64 ± 0.86 Hz to after 5.24 STN-DBS. ± 0.64 Hz after The results STN‐ ± ± DBS.give usThe a results new perspective give us a new on the perspective mechanism on of the DBS. mechanism MSNs intensively of DBS. MSNs fired intensively to maintain fired normal to maintainactivities. normal DBS could activities. change DBS the could firing change of di fftheerent firing neurons of different to control neurons the behavior.to control the Note behavior. that the NoteMSNs that increased the MSNs by 3.48-fold increased at by the 3.48 100‐ Hzfold stimulation, at the 100 Hz while stimulation, the other stimulationwhile the other parameter stimulation shows parametera lesser eff showsect on a the lesser neural effect discharge. on the neural Moreover, discharge. the FSIs Moreover, were not the a ffFSIsected were under not theaffected stimulation under thewhich stimulation demonstrated which the demonstrated same level ofthe the same firing level rate of before the firing and rate after before stimulation and after from stimulation low frequency from lowto high frequency frequency. to high frequency. Biosensors 2020, 10, 136 9 of 12 Biosensors 2020, 10, x FOR PEER REVIEW 9 of 12

FigureFigure 7. 7.Response Response of of MSNs MSNs and and FSIs FSIs during during DBS. DBS. (a) The (a) meanThe mean waveform waveform of MSNs of MSNs and FSIs. and (b FSIs.) The ( meanb) The firingmean rate firing variations rate variations of MSNs of and MSNs FSIs and before FSIs and before after and DBS. after DBS. 4. Discussion 4. Discussion The increase in the DA concentration was obtained at the termination of the stimulus after The increase in the DA concentration was obtained at the termination of the stimulus after applying a frequency of 60 Hz or higher. In contrast, the concentration would reach a new steady-state applying a frequency of 60 Hz or higher. In contrast, the concentration would reach a new steady‐ level after applying a low frequency at 10 Hz. As shown in Wightman’s results, the frequency is the state level after applying a low frequency at 10 Hz. As shown in Wightman’s results, the frequency main parameter to influence the DA concentration [26]. They developed a kinetic model to understand is the main parameter to influence the DA concentration [26]. They developed a kinetic model to the DA release and uptake process. At low frequency, the DA release is less than the uptake, and the understand the DA release and uptake process. At low frequency, the DA release is less than the uptake occurs to balance the concentration of released DA. However, at high frequency, the uptake uptake, and the uptake occurs to balance the concentration of released DA. However, at high occurs at a saturated status. The released DA cumulate until the pause of the stimulation. From the frequency, the uptake occurs at a saturated status. The released DA cumulate until the pause of the frequency parameter training, the DA release and firing rate change most at 100 Hz stimulation. stimulation. From the frequency parameter training, the DA release and firing rate change most at The aim of stimulation training was to obtain a balance between the optimal parameter settings 100 Hz stimulation. The aim of stimulation training was to obtain a balance between the optimal to provide maximal symptom suppression and minimal side effects. DBS at high frequency was parameter settings to provide maximal symptom suppression and minimal side effects. DBS at high considered as an effective treatment for various movement disorders. Based on the previous behavior frequency was considered as an effective treatment for various movement disorders. Based on the research under different frequency stimulations, the DA release and MSNs firing rate increases may previous behavior research under different frequency stimulations, the DA release and MSNs firing play a significant role in improving symptoms. rate increases may play a significant role in improving symptoms. MSNs and FSIs respond differently to the DBS at different frequencies. As can be seen, different MSNs and FSIs respond differently to the DBS at different frequencies. As can be seen, different kinds of neurons show a different function in the neuron disease. It will be effective to modulate certain kinds of neurons show a different function in the neuron disease. It will be effective to modulate neurons to improve the treatment of PD disease. As shown in Li’s research, they showed different certain neurons to improve the treatment of PD disease. As shown in Li’s research, they showed responses of projection neurons and interneurons, and corticofugal projection neurons in layer V of the different responses of projection neurons and interneurons, and corticofugal projection neurons in motor cortex were activated during STN-DBS [27]. Many researchers found that different kinds of layer V of the motor cortex were activated during STN‐DBS [27]. Many researchers found that neurons respond differently to stimulation such as light and they show a specific connection [28,29]. different kinds of neurons respond differently to stimulation such as light and they show a specific A disorder of the basal ganglia circuit is the main pathological basis of Parkinson’s disease involving connection [28,29]. A disorder of the basal ganglia circuit is the main pathological basis of Parkinson’s the cortex, striatum, STN and so on [30]. The balance between direct and indirect pathways in the disease involving the cortex, striatum, STN and so on [30]. The balance between direct and indirect basal ganglia circuits is the key to maintain normal motor function [31]. The direct pathway makes the pathways in the basal ganglia circuits is the key to maintain normal motor function [31]. The direct movement occur easily, while the indirect pathway hinders the movement. In the normal state, the main pathway makes the movement occur easily, while the indirect pathway hinders the movement. In functions of DA are to activate the direct pathway and inhibit the indirect pathway which is conducive the normal state, the main functions of DA are to activate the direct pathway and inhibit the indirect to the occurrence of movement. However, the DA concentration is significantly decreased in PD disease pathway which is conducive to the occurrence of movement. However, the DA concentration is which makes the direct pathway activities decreased while making the indirect pathway activities significantly decreased in PD disease which makes the direct pathway activities decreased while increased [32,33]. The balance between the direct and indirect pathways is broken, which leads to making the indirect pathway activities increased [32,33]. The balance between the direct and indirect dyskinesia. DBS is considered as an effective method to increase DA concentration to improve PD pathways is broken, which leads to dyskinesia. DBS is considered as an effective method to increase symptoms [34]. The activation of direct pathway MSNs by stimulation could also improve movement DA concentration to improve PD symptoms [34]. The activation of direct pathway MSNs by behavior such as freezing, locomotor initiation and bradykinesia [19,35]. The implanted microelectrode stimulation could also improve movement behavior such as freezing, locomotor initiation and arrays provide us a new method to obtain the neural activities at a cellular scale. bradykinesia [19,35]. The implanted microelectrode arrays provide us a new method to obtain the neural activities at a cellular scale.

Biosensors 2020, 10, 136 10 of 12

5. Conclusions A dual-mode microelectrode array was fabricated to record the DA concentration and specific neuron spike firing during STN-DBS. The different stimulation frequencies were trained and applied into the deep brain. The recorded DA neurotransmitter and neural spike presented a similar change as the frequency varied from 10 to 350 Hz. Moreover, the stimulation frequency around 100 Hz evoked the highest DA concentration and neural spike firing rate. The MSNs pattern was significantly modulated by stimulation, while the FSIs were not modulated. It can be inferred that the different neural spike patterns play a different role in PD animals. The specific dual-mode activities were crucial to enhance our knowledge in a microscopic view. The silicon-based microelectrode is widely used in deep brain detection. Neural activities involving a neurotransmitter and neuron spike firing would provide more information between neurons. In future work, the pathogenesis and therapeutic effect of neurologic diseases will be mainly studied by using MEAs.

Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6374/10/10/136/s1, Figure S1: The fabrication process of the MEA, Figure S2: The detection system including DA recording and electrical recording under stimulation, Figure S3: Background noise comparison before and after modification of Pt/rGO, Figure S4: Impedance comparison before and after modification of Pt/rGO. Author Contributions: Conceptualization, G.X. and X.C.; methodology, Y.S. and Y.Z.; software, Y.X.; validation, M.W. and S.X.; formal analysis, G.X. and Y.S.; investigation, J.W.; resources, J.C. and D.C.; data curation, G.X.; writing—original draft preparation, G.X.; writing—review and editing, G.X. and Y.X.; visualization, G.X.; supervision, X.C.; project administration, X.C.; funding acquisition, X.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by NSFC (61960206012, 61527815, 61971400, 61775216, 61771452), National Key R & D Program of Nano Science and Technology of China (2017YFA0205902) and the Key Research Programs (QYZDJ-SSW-SYS015) of Frontier Sciences, CAS. Acknowledgments: The authors thank Hongyan Zhao and Guogang Xing from Peking University for establishing the rat model of Parkinson’s disease. Conflicts of Interest: The authors declare no conflict of interest.

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