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This study is supported by the German Research Foundation (DFG) within the Collaborative Research Center (SFB 1270/1,2 ELAINE 299150580).

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</ol>

The authors have nothing to disclose.

We described a methodological approach to investigate the mechanisms of deep brain stimulation (DBS) in the pathophysiological dystonic neuronal network in the dtsz hamster. Critical steps include precise electrode implantation, rapid cerebellar slice preparation, and stable HD-MEA recording conditions to ensure reliable data acquisition. We demonstrate the surgery procedure, preparation of cerebellar slices, recording, and analysis techniques to explore the ex vivo&#160;cerebellar activity and its modulation by DBS. The electrophysiological measurements using HD-MEA provided real-time recordings of cerebellar spontaneous activity with high temporal and spatial resolution36, which we consider necessary for investigating the mechanisms of pallidal deep brain stimulation in the treatment of dystonia.
Dystonia is currently known as a network disorder characterized by abnormal motor output arising from dysfunctional interactions among basal ganglia, cerebellar, and cortical circuits37,38. Cerebellar abnormalities have been shown to play a significant role in dystonia, as supported by both clinical and experimental studies15,22,39. Furthermore, an anatomical study involving the injection of retrograde rabies virus (RV) into the cerebellar cortex of Cebus monkeys shows that the subthalamic nucleus of the basal ganglia has a substantial projection to the cerebellar cortex40. These findings underscore the importance of exploring cerebellar circuits alongside the basal ganglia networks when studying the therapeutic mechanisms of DBS40,41,42. This method is particularly suitable for studies investigating neuronal interactions and neuromodulation effects in movement disorders.
Regarding the network-wide mechanisms of pallidal DBS, studies have demonstrated that DBS of the internal segment of the globus pallidus (GPi) induces widespread changes in brain activity, not only within the basal ganglia but also across interconnected regions such as the cortico-striatal network43, the thalamus44, and the cerebellum15. These observations support the hypothesis that pallidal DBS can modulate activity throughout the basal ganglia-cerebellar network, contributing to its therapeutic efficacy in dystonia. This demonstrates the need for preclinical studies to investigate DBS mechanisms and the advantage of HD-MEA recordings for capturing network-wide electrophysiological changes. Furthermore, the study by Kotyra et al. (2025) supports the presence of long-lasting neuroplastic remodeling induced by longer-term GPi-DBS within the dystonic motor network45. In vivo&#160;findings showed that pallidal stimulation progressively reduced dystonia severity in dtsz hamsters and that the beneficial effect persisted after stopping stimulation, suggesting that DBS-induced effects are not limited to acute stimulation alone45. In addition, recent electrophysiological studies demonstrated network-wide modulation of synaptic plasticity and spike patterns after longer-term DBS, including increased cerebellar activity15, enhanced excitatory input to motor thalamic neurons, and more clustered excitatory input to cortical M146.
Representative results indicate a network-wide impact of deep brain stimulation (DBS), as evidenced by the normalization of spike activity in the molecular, Purkinje cell, and granular cell layers of the cerebellar cortex in dystonic hamsters (Figure 6). The increased spike activity observed in the dtsz DBS group reached levels comparable to the wild-type controls. This normalization of spike activity supports the therapeutic potential of DBS for treating dystonia. Notably, the dtsz-Sham group showed intermediate spike values compared to the WT group across all cerebellar layers, suggesting that electrode implantation alone may influence cerebellar network activity. This interpretation is consistent with the clinically described microlesion effect in dystonia, where electrode implantation itself may transiently improve symptoms before stimulation onset47. Although the dtsz hamster reproduces key features of paroxysmal generalized dystonia, it does not fully reflect the clinical heterogeneity observed across human dystonia syndromes. Therefore, caution should be taken when translating these findings to human dystonia.
This work addresses technical challenges related to stimulator encapsulation, DBS surgery, slice preparation, and large-scale electrophysiological data analysis. The combination of CMOS-based HD-MEA technology provides a robust platform for in vitro investigation of highly complex neuronal interactions48. Our results contribute to a more nuanced understanding of the pathophysiological dynamics underlying dystonia, offering valuable insights into the network-level mechanisms of DBS therapy. Facilitating a detailed functional analysis of specific brain regions and their interactions contributes to a more differentiated understanding of the pathophysiological dynamics in dystonic neuronal networks, and, on the other hand, provides insight into the therapeutic mechanism of action of DBS.
In this study, technical problems led to increased background noise and disrupted stable signal acquisition during HD-MEA recordings. Common troubleshooting steps include addressing noise sources, ensuring proper grounding, and maintaining stable tissue-electrode contact. In cases of high noise levels or unstable measurements, grounding, cable connections, and nearby electrical devices should be checked first. Air bubbles on the electrode array can also impair tissue-electrode contact and recording stability and must be carefully removed (Figure 4). Furthermore, stable data transmission from the amplifier to the computer via a high-quality USB-C-to-USB-C cable must be ensured. Electrodes with persistent noise should be excluded from further analysis. While recording extracellular signals with HD-MEA allows for high spatial resolution of the network activity and, together with the superimposed microscopic image, assignment to the individual layers of the cerebellar cortex, it cannot be ruled out that the detected extracellular activity was generated by crossing axons from a layer other than the assigned one. Furthermore, statistical analyses were performed at the slice level, as each slice represented an independent electrophysiological recording under standardized experimental conditions, although some slices originated from the same animal.
During DBS, further problems can occur due to electrode loosening or a technical defect in the stimulator. To minimize these issues, ensure proper electrode fixation and maintain sterile handling conditions throughout the procedure. This happened in 5% of all trials in the present study. To minimize electrode loosening, the skull surface must be dried before fixing the electrode with adhesive or dental cement to improve adhesion and prevent slippage. To ensure sterility and avoid mechanical contamination, the stimulator, electrode tip, and monofilament thread must only be handled with forceps or hemostasis forceps. Additionally, gloves should be disinfected with ethanol and dried before handling the animal to minimize the risk of infection. Rapid preparation of cerebellar brain slices is essential for high-quality electrophysiological recordings. However, this meant that the position of the implanted DBS electrodes could not be verified post-hoc. In another study investigating the effect of different stimulation parameters on dystonia severity in the dtsz-Hamster, the same stimulation electrodes and surgical techniques were used. Here, we were able to confirm the correct electrode position in 94 % of trials37.
In conclusion, this protocol provides a powerful platform for studying the electrophysiological mechanisms of DBS and contributes valuable insights into its mechanism of action. The dtsz hamster uniquely enables preclinical studies of the pallidal DBS effects on the dystonic phenotype, together with ex vivo&#160;high-resolution recording of neuronal network activity. Our results support the concept of cerebellar involvement in dystonia and highlight the importance of network-level analysis in understanding and optimizing neuromodulation therapies.

Dystonia is the third most common movement disorder1 characterized by distinct clinical signs and symptoms2,3, although it may sometimes be mistaken for other motor disorders4,5,6. The most prominent clinical features include involuntary muscle contractions, repetitive movements, and abnormal and/ or twisted posture7. These features may be accompanied by symptoms such as pain, impaired motor performance, and fatigue, which may worsen during periods of stress, anxiety, or physical exhaustion8,9,10,11. The pathophysiology of dystonia remains not totally understood12,13. Although studies have traditionally focused on the basal ganglia, increasing evidence supports a network-level concept of the disorder, involving multiple brain regions, including the cerebellum14,15,16,17. This protocol aims to investigate extracellular network activity across distinct layers of the cerebellar cortex to elucidate their role in the pathophysiology of dystonia and to understand the network-wide mechanisms of deep brain stimulation (DBS).
Deep brain stimulation (DBS) targeting the globus pallidus internus (GPi) or the subthalamic nucleus (STN) is often used to treat certain forms of dystonia18,19, particularly those that are drug-resistant. Additionally, the treatment outcome is heterogeneous and unpredictable, as patients with similar clinical manifestations may respond differently to the same therapeutic approach20. A more comprehensive understanding of the neural network underlying dystonia could improve treatment outcomes21. Therefore, large-scale and experimental approaches are needed, which can only be achieved through preclinical studies in animal models. Combining in vivo DBS experiments with ex vivo&#160;high-density microelectrode array (HD-MEA) recordings enables high-resolution monitoring of the (almost) physiological neuronal network activity to better understand the pathophysiology of dystonia and the mechanisms of DBS.
The basal ganglia and cerebellum each influence motor control, at least in part, through their connections with the primary motor cortex (M1)22. An anatomical study with a striatal lesion and surgical removal of the cerebellum supports this idea23. In our study, we use the animal model of the dystonic dtsz hamster24,25, which exhibits cerebellar abnormalities associated with dystonia26. Using high-density microelectrode arrays (HD-MEAs), we investigated cerebellar cortical network activity in dystonic and healthy hamsters. However, conventional electrophysiological approaches lack the spatial resolution to capture layer-specific network dynamics, whereas this method addresses this limitation. On the other hand, we investigated the effect of deep brain stimulation in the globus pallidus internus on the cerebellar cortical networks.
The HD-MEA system offers the advantage of detailed electrophysiological recordings in parallel, enabling analysis across the layers of the cerebellar cortex (granular, Purkinje, and molecular layers), with each layer playing an essential role in the processing of movement sequences27. The granular layer, densely packed with granule cells, receives and relays sensory and motor information from mossy fibers, allowing initial signal integration before further processing27. The Purkinje layer, which contains Purkinje cells, transmits inhibitory signals to the deep cerebellar nuclei, where the granular and molecular layers modulate the outgoing signal. The molecular layer, rich in dendrites and interneurons, supports complex modulation through synaptic interactions with Purkinje cells, fine-tuning motor output28. HD-MEA, unlike other recording techniques, is not limited by spatial resolution or sampling area, allowing the unique contributions of each layer to be captured in the altered neural dynamics seen in dystonia15. This approach provides higher spatial resolution and parallel recording capabilities compared to single-electrode or low-density recording methods. There are HD-MEA systems based on the complementary metal-oxide-semiconductor (CMOS)29,30 technology, combined with the Active Pixel Sensor (APS) concept, that provide functional imaging for visualizing the electrical activity of large populations of neurons over time31,32. With this technique, the dynamics of each layer of the cerebellar cortex can be monitored, and specific synaptic transmission processes can be investigated by washing with various substances. This method is particularly suitable for studies investigating complex neuronal network activity and layer-specific alterations in neurological disease models.
This work addresses technical challenges regarding encapsulation of the stimulation system as well as&#160;tissue preparation, data acquisition, and the analysis of a large electrophysiological dataset. With this work, we present an option for in vitro&#160;investigation of highly complex neuronal processing, which, in our case, on the one hand, contributes to a more differentiated understanding of the pathophysiological dynamics in dystonic neuronal networks and, on the other hand, provides knowledge about the therapeutic mechanism of action of DBS.

<table><tbody><tr><td>3% H2O2</td><td>Caelo</td><td>15301730</td><td></td></tr><tr><td>50 ml conical tube</td><td>Greiner Bio-One</td><td></td><td>Falcon-type conical tube</td></tr><tr><td>Agar Petri dish</td><td>Handmade&amp;nbsp;</td><td></td><td></td></tr><tr><td>Anaesthesia Unit</td><td>BioMedical Instruments</td><td>Univentor 1200</td><td></td></tr><tr><td>BioCam DupleX</td><td>3Brain</td><td>BioCAM DupleX</td><td>CMOS-HD-MEA platform</td></tr><tr><td>Blade (5*)</td><td>Apollo Herkenrath</td><td>09-025-056</td><td></td></tr><tr><td>Brainwave Software 5</td><td>3Brain</td><td>Version 5</td><td>CMOS-HD-MEA platform</td></tr><tr><td>Breathing mask</td><td>Narishige</td><td>GM-4</td><td></td></tr><tr><td>Bulk Fill Flowable: Compula Refill</td><td>Dentsply Sirona</td><td>267-0195</td><td>For cement</td></tr><tr><td>Capsule Dispenser Gun&amp;nbsp;</td><td>Dentsply Sirona</td><td>296-0003</td><td>For cement</td></tr><tr><td>Carbogen</td><td>Air Liquide</td><td>Air Liqiuide (House system)</td><td></td></tr><tr><td>Cavity Block Glass</td><td>AIM</td><td>AIM</td><td>40 mm x 40 mm</td></tr><tr><td>Clamp (3*)</td><td>DIEFFENBACH</td><td>12-1003-04</td><td>stainless steel 4cm</td></tr><tr><td>Computer</td><td>Dell</td><td>Precission 3431</td><td></td></tr><tr><td>CorePlate&lt;sup&gt;TM&lt;/sup&gt; 1W 38/60</td><td>3Brain</td><td>Accura HD-MEA</td><td>CMOS-HD-MEA chip</td></tr><tr><td>Drill accessory (13)</td><td>FST</td><td>19007-07</td><td>Tip: 7mm</td></tr><tr><td>Drill accessory (13)</td><td>FST</td><td>19007-09</td><td>Tip: 9mm</td></tr><tr><td>Eco wipes - wipe dispenser</td><td>Dr Schumacher</td><td>00-915SE001 (1x1)&amp;nbsp;</td><td></td></tr><tr><td>Eco wipes - wipe rolls</td><td>Dr Schumacher</td><td>00-915SE001 (6x1)</td><td></td></tr><tr><td>ELASTOSIL</td><td>Wacker</td><td>RT 601 A/B</td><td></td></tr><tr><td>Electrode</td><td>Microprobes</td><td>SNEX-100</td><td></td></tr><tr><td>Ethanol</td><td>Carl Roth GmbH + Co. KG</td><td>Art-Nr. T913.3</td><td></td></tr><tr><td>Excavator (10)</td><td>HLW Dental-Instruments Germany</td><td>S-10.28-6</td><td></td></tr><tr><td>Forceps (5)</td><td>FST</td><td>18025-10</td><td>Stainless Steel 10cm</td></tr><tr><td>Forceps (6)</td><td>Aesculap</td><td>BD311R</td><td>9cm</td></tr><tr><td>Forceps (7)</td><td>FST</td><td>91100-12</td><td>Stainless Steel 12cm</td></tr><tr><td>Forceps (8)</td><td>FST</td><td>11231-20</td><td>Stainless Steel 11cm</td></tr><tr><td>Forceps (8*)&amp;nbsp;</td><td>Karl Hammacher</td><td>9.160 160</td><td>Wironit 10,5cm</td></tr><tr><td>Forceps (9)</td><td>FST</td><td>11200-33</td><td>Dumoxel 13,5cm</td></tr><tr><td>Forceps (9)</td><td>FST</td><td>11200-33</td><td>Dumoxel 13,5cm</td></tr><tr><td>Heated surgical table+integrated gas exhaust</td><td>BioMedical Instruments</td><td>BioMedical Instruments</td><td></td></tr><tr><td>heliobond</td><td>Ivoclar Vivadent</td><td>42040</td><td></td></tr><tr><td>Ice Machine</td><td>Scotsman</td><td>AF 80 AS / WS</td><td></td></tr><tr><td>Inflow and outflow needles</td><td>BD Microlance&amp;nbsp;</td><td>1,25 x70mm</td><td></td></tr><tr><td>Isathal 10mg/g</td><td>Dechra</td><td>Zul.-Nr. 400216.00.00</td><td>Fucidin</td></tr><tr><td>Isoflurane</td><td>Sedana medical</td><td>N001254</td><td></td></tr><tr><td>Mili-Q Water Filter</td><td>Elga</td><td>PURELAB flex 3</td><td></td></tr><tr><td>Needle Holder (4)</td><td>FST</td><td>12501-13</td><td>Tungsten Carbide 13cm</td></tr><tr><td>Octenisept</td><td>Sch&amp;uuml;lke &amp;amp; Mayr</td><td></td><td>Antiseptic solution&amp;nbsp;</td></tr><tr><td>Paper Filter (4*)</td><td>Carl Roth GmbH + Co. KG</td><td>AP75.1</td><td></td></tr><tr><td>Parafilm</td><td>Sigma Aldrich</td><td>HS234526C</td><td></td></tr><tr><td>Platinum anchor</td><td>3Brain</td><td>3Brain</td><td></td></tr><tr><td>Retractor Tip (14)</td><td>FST</td><td>18200-10</td><td>2.5mm</td></tr><tr><td>Reused Electrode (11)</td><td>Microprobes</td><td>SNEX-100</td><td>For Coordinates</td></tr><tr><td>Ringer</td><td>B. Braun</td><td>3570030</td><td></td></tr><tr><td>Scissor (1)</td><td>FST</td><td>14078-10</td><td>Stainless Steel 10cm</td></tr><tr><td>Scissor (2)</td><td>Aesculap</td><td>BC144R</td><td></td></tr><tr><td>Scissor (3)</td><td>FST</td><td>14058-09</td><td>Stainless Steel 9cm</td></tr><tr><td>Scissor (9*)</td><td>FST</td><td>91401-12</td><td>Stainless Steel 12cm</td></tr><tr><td>Screw (12)</td><td>Online-Schrauben.de</td><td>DIN 84 A2 M 1x2</td><td></td></tr><tr><td>Shaver</td><td>Exacta</td><td>GT415</td><td></td></tr><tr><td>STELLA (software defined implantable modular platform) stimulation system</td><td>non-commercial; developement of the University of Rostock</td><td>open source (https://github.com/SFB-ELAINE/STELLA)</td><td>battery-driven DBS stimulator</td></tr><tr><td>Sterelizer 250</td><td>FST</td><td>18000-45</td><td></td></tr><tr><td>Stereotaxic</td><td>Narishige</td><td>Model SR-AR</td><td></td></tr><tr><td>Super glue</td><td>UHU</td><td>64212</td><td></td></tr><tr><td>Tear cream (Panthenol Nose Cream)</td><td>Jenapharm</td><td>5541249</td><td></td></tr><tr><td>Vibratome&amp;nbsp;</td><td>Laica</td><td>VT12005</td><td></td></tr><tr><td>Xylocain Gel 2%</td><td>Aspen Pharma</td><td>1138060</td><td></td></tr><tr><td>Weigh Boat</td><td>Carl Roth GmbH + Co. KG</td><td>HYT9.1</td><td></td></tr><tr><td>Weigher</td><td>Voltcraft</td><td>TS-5000/1</td><td></td></tr><tr><td>Compounds used for solutions preparation</td><td></td><td></td><td></td></tr><tr><td>Calcium chloride (CaCl2)</td><td>Sigma-Aldrich</td><td>10043-52-4</td><td></td></tr><tr><td>D-Glucose(C6H12O6)</td><td>Sigma-Aldrich</td><td>50-99-7</td><td></td></tr><tr><td>Magnesium Chloride (MgCl2)</td><td>Sigma-Aldrich</td><td>7786-30-3</td><td></td></tr><tr><td>Potassium Chloride(KCl)</td><td>Sigma-Aldrich</td><td>7447-40-7</td><td></td></tr><tr><td>Sodium Bicarbonate (NaHCO3)</td><td>Sigma-Aldrich</td><td>144-55-8</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma-Aldrich</td><td>7647-14-5</td><td></td></tr><tr><td>Sodium phosphate monobasic (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>7558-80-7</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>57-50-1</td><td></td></tr></tbody></table>

investigating the network-wide mechanisms of pallidal deep brain stimulation using high-density microelectrode arrays

Investigating The Network-Wide Mechanisms of Pallidal Deep Brain Stimulation Using High-Density Microelectrode Arrays

Dystonia is a neurological movement disorder characterized by abnormal muscle control affecting voluntary movements and sustained postures. Although dystonia is associated with basal ganglia dysfunction, increasing evidence suggests that the cerebellum is involved in its pathophysiology. This protocol presents in vivo pallidal deep brain stimulation (DBS) in a dystonic hamster model (dtsz hamster) combined with ex vivo&#160;high-density microelectrode array (HD-MEA) recordings to investigate cerebellar activity. The protocol includes (1) DBS surgery for electrode and stimulator implantation, (2) acute cerebellar slice preparation, (3) high-resolution electrophysiological recordings, and (4) data analysis. Representative results indicate that 11 days of pallidal DBS restored spike activity toward normal levels in the molecular, Purkinje, and granular layers, consistent with cerebellar network involvement in dystonia. This protocol enables detailed analysis of cerebellar activity and its modulation by DBS, providing a platform to investigate network-level mechanisms underlying neuromodulation therapies.

Pallidal DBS increased the number of spikes to normal levels in cortical layers between groups.
We analyzed the total number of spikes in three layers of the cerebellar cortex&#8212;molecular layer (ML), Purkinje cell layer (PL), and granular cell layer (GL) &#8212; across four experimental groups: wild-type (WT, N = 8), dtsz (N = 8), dtsz-Sham (N = 7), and dtsz-DBS (N = 7), with analyses performed on slices obtained from these animals.
While dystonic tissue (dtsz) generated significantly fewer spikes, pallidal DBS increased the number of spikes to levels comparable to those of healthy controls (Table&#160;3). The Kruskal-Wallis H test further confirmed this, showing a statistically significant increase in the number of spikes in the ML for the dtsz DBS group compared to both the dtsz and dtsz-Sham groups (&#967;2(3) = 20.728, p = 0.001; Figure&#160;6A). The dtsz-DBS group also showed a similar increase in spikes in PL (&#967;2(3) = 16.855, p = 0.001; Figure&#160;6B) and GL (&#967;2(3) = 13.164, p. = 0.004; Figure&#160;6C), reaching values comparable to those of the WT group (Table 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/70507/70507fig1.jpg" /> 
Figure 1. Overview of surgical instruments. The image shows an arranged tray containing a set of surgical and microsurgical instruments designed explicitly for procedures involving the implantation of deep electrodes and stimulators. The instruments are organized in sequential order of use, enhancing procedural efficiency. They are placed on tissue to maintain cleanliness and ensure easy accessibility during the procedure. This arrangement focuses on precision, sterility, and readiness for complex surgical workflows. (1) Scissors blunt/blunt, (2) Suture scissors, (3) Scissors sharp/sharp, (4) Needle holder, (5) Suture tying forceps, (6) Splinter forceps/curved, (7) Forceps serrated, (8) Forceps standard short, (9) Forceps standard, long, (10) Micro spatula, (11) Reused electrode, (12) Screws, (13) Drill accessories, (14) Retractor Tip/clamp, (15) Glass cavity block to be filled with 0.9&#160;% NaCl and xylocaine. For more information, check the material list. <a href="https://www.jove.com/files/ftp_upload/70507/70507fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/70507/70507fig2.jpg" /> 
Figure 2. Experimental setup for anesthetizing and positioning the animal in the stereotaxic apparatus for implantation of DBS electrodes and the stimulator. (A) The animal is gently placed inside a custom-made induction chamber to initiate anesthesia. (B) The induction chamber is connected to a breathing mask to ensure proper administration of anesthesia (induction with isoflurane at 4%) while maintaining safety during handling. (C) Once anesthetized, the animal is transferred to a heating plate with a connected breathing mask (maintenance with isoflurane at 3%) to prevent hypothermia and ensure continuous anesthesia during shaving. (D) The animal is carefully positioned in the stereotaxic frame. A longitudinal incision is made, and clamps are used to retract the skin. Four dots indicate screw placement. Screws are inserted into the marked areas, and two additional dots are marked for electrode placement. (E) The electrode is inserted into the GPi and secured with glue before the stereotaxic holder is removed. For better fixation, cement is applied around the electrodes. Interrupted longitudinal sutures are used to close the surgical site, and (F) the stimulator is placed in the subcutaneous flank area of the animal. The animal remains alive post-surgery, and the arrow indicates the location of the stimulator. <a href="https://www.jove.com/files/ftp_upload/70507/70507fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/70507/70507fig3.jpg" /> 
Figure 3. Illustration of brain sectioning and slicing. (A) surgical instruments for the dissection procedure. (1*) Homemade Agar Petri dish, (2*) Agar Petri dish, (3*) Clamp fixing the carbogen tube, (4*) Paper filter, (5*) Blade, (6*) Super glue, (7*) Vibratome plate/holder, (8*) Curved forceps, (9*) Scissor, Sharp/Blunt. (B) Brain sample preparation: after dissection, the brain is cut, and the dashed lines indicate the orientation of the coronal plane sectioning. (C) Parasagittal sectioning: the brain is cut along the specified parasagittal plane. (D) Tissue fixation: The resulting brain tissue is affixed to a plate/holder with glue to secure it for precise sectioning. (E) Vibratome sectioning: The plate with attached tissue is transferred to a vibratome, where ultra-thin slices are obtained (200 &#181;m). (F) Final slices: Precise parasagittal cerebellar slices are produced, ready for electrophysiological recording35. For more information, check the material list. <a href="https://www.jove.com/files/ftp_upload/70507/70507fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/70507/70507fig4.jpg" /> 
Figure 4. Illustration of the steps to get a good cell activity. (A) Cleaning: This step involves preparing the HD-MEA by cleaning its surface to ensure that no dirt interferes with the attachment (Contact pad, A-1) or recording process (flat area, A-2). (B) Slice position: The tissue slice is carefully positioned on the HD-MEA (B-1), ensuring proper alignment for effective electrode contact (B-2). A thin tip of the pipette (arrow) is used to remove the remaining ACSF, allowing the anchor to be inserted. (C) Attachment of the slice: the slice is securely positioned on the HD-MEA using the anchor. At this step, it is crucial to place a drop of ACSF and then carefully remove the excess liquid using a pipette (C-1; arrow) and a twisted piece of precision wipe paper (C-2). This ensures the slice remains stable and well-connected to the electrodes for accurate recording. (D) Perfusion: Finally, a perfusion system is connected and initiated to maintain the tissue slice&#39;s viability during the experiment, where the inflow has to be on top of the slice and the outflow close to the wall (D-2). It is also essential to remove all bubbles from the ground to minimize noise during recording (indicated by the arrow). Additional grounding is attached to both the inflow and outflow tubing (D-1). The red scale bars are 1.5&#160;mm long. <a href="https://www.jove.com/files/ftp_upload/70507/70507fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/70507/70507fig5.jpg" /> 
Figure 5. Metrics for amplitude and standard deviation in HD-MEA recordings using Brain Wave 5 software (BW5) to evaluate the signal quality. (A) Peak-to-Peak Amplitude metric selected to extract intrinsic electrode amplitude values (scale: 500&#160;&#181;V). (B) Standard Deviation metric selected to quantify baseline noise across electrodes (scale: 30&#160;&#181;V). <a href="https://www.jove.com/files/ftp_upload/70507/70507fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/70507/70507fig6.jpg" /> 
Figure 6: Effects of pallidal DBS on spike activity across cerebellar cortical layers: (A) molecular layer (ML), (B) Purkinje cell layer (PL), and (C) granular layer (GL). The Box-Whisker plots display the mean and median with a cross and horizontal line, respectively, along with the 25th and 75th percentiles. The respective groups are wild type (WT, n&#160;=&#160;22&#160;slices), dtsz without surgery (n&#160;=&#160;20&#160;slices), dtsz DBS inactive (Sham, n&#160;=&#160;20&#160;slices), and dtsz-DBS active (n&#160;=&#160;20&#160;slices). Statistical significance was determined using the Kruskal-Wallis test, followed by Dunn&#39;s pairwise comparisons (*p .&#60; 0.05). <a href="https://www.jove.com/files/ftp_upload/70507/70507fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Molecular weight [g/ mol]</td>
			<td>Final concentration [mol/ l]</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>58.44</td>
			<td>0.124</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.55</td>
			<td>0.003</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>180.16</td>
			<td>0.01</td>
		</tr>
		<tr>
			<td>CaCl2 (water-free)</td>
			<td>110.98</td>
			<td>0.0025</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>84,007</td>
			<td>0.026</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>119.98</td>
			<td>0.00125</td>
		</tr>
		<tr>
			<td>pH&#160;</td>
			<td colspan="2">7.35 to 7.40 (adjustable with HCl)</td>
		</tr>
		<tr>
			<td>Osmolality [mosmol/ kg]</td>
			<td colspan="2">290 to 300</td>
		</tr>
	</tbody>
</table>
Table 1: Artificial cerebrospinal fluid (ACSF) solution: Composition and final molar concentrations of components used to prepare ACSF for cerebellar slice experiments. pH was adjusted to 7.35&#8211;7.40 with HCl, and osmolality was maintained between 290&#8211;300&#160;mOsm&#183;kg-1.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Molecular weight [g/mol]</td>
			<td>Final concentration [mol/L]</td>
		</tr>
		<tr>
			<td>Saccharose</td>
			<td>342.30</td>
			<td>0.075</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.55</td>
			<td>0.0025</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>58.44</td>
			<td>0.087</td>
		</tr>
		<tr>
			<td>CaCl2 (water-free)</td>
			<td>110.98</td>
			<td>0.0005</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>95.21</td>
			<td>0.007</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>180.16</td>
			<td>0.01</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>84.01</td>
			<td>0.025</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>119.98</td>
			<td>0.00125</td>
		</tr>
		<tr>
			<td>pH&#160;</td>
			<td colspan="2">7.35 to 7.40 (adjustable with HCl)</td>
		</tr>
		<tr>
			<td>Osmolality [mosmol/ kg]</td>
			<td colspan="2">320 to 330</td>
		</tr>
	</tbody>
</table>
Table 2: Sucrose solution. Composition and final molar concentrations of the sucrose solution used during cerebellar slice preparation. pH was adjusted to 7.35&#8211;7.40, and osmolality was maintained between 320&#8211;330 mOsm&#183;kg-1.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Groups:</td>
			<td>WT&#160;</td>
			<td>dtsz&#160;</td>
			<td>dtsz-Sham</td>
			<td>dtsz-DBS&#160;</td>
		</tr>
		<tr>
			<td>n</td>
			<td>22</td>
			<td>20</td>
			<td>20</td>
			<td>20</td>
		</tr>
		<tr>
			<td colspan="5">Coefficient of variance</td>
		</tr>
		<tr>
			<td>ML</td>
			<td>0.53</td>
			<td>0.55</td>
			<td>0.6</td>
			<td>0.35</td>
		</tr>
		<tr>
			<td>PL</td>
			<td>0.57</td>
			<td>0.57</td>
			<td>0.52</td>
			<td>0.46</td>
		</tr>
		<tr>
			<td>GL</td>
			<td>0.48</td>
			<td>0.66</td>
			<td>0.61</td>
			<td>0.51</td>
		</tr>
		<tr>
			<td colspan="5">Number of spikes</td>
		</tr>
		<tr>
			<td>ML</td>
			<td>287240&#177;32605</td>
			<td>163434&#177;19998</td>
			<td>234209&#177;31284</td>
			<td>345915&#177;27113</td>
		</tr>
		<tr>
			<td>PL</td>
			<td>413223&#177;50171</td>
			<td>194277&#177;24786</td>
			<td>304568&#177;35692</td>
			<td>412707&#177;42509</td>
		</tr>
		<tr>
			<td>GL</td>
			<td>390817&#177;40139</td>
			<td>211274&#177;31193</td>
			<td>325533&#177;44318</td>
			<td>373208&#177;42220</td>
		</tr>
	</tbody>
</table>
Table 3: Summary of spike metrics across cerebellar layers and experimental groups. WT &#8211; wild type, non-dystonic control hamsters; dtsz - native dtsz hamsters; dtsz-DBS - dtsz hamsters continuously stimulated for 11 days; dtsz-Sham - dtsz hamsters undergoing the surgery, however, with no active DBS; ML &#8211; molecular layer; PL &#8211; Purkinje cell layer; GL. &#8211; granular cell layer.

Here, we describe a protocol for ex vivo high-density microelectrode array recordings from acute cerebellar brain slices of a dystonic hamster model that received in vivo. continuous pallidal deep brain stimulation for 11 days.

Dystonia is a neurological movement disorder characterized by abnormal muscle control affecting voluntary movements and sustained postures. Although ...

We investigate how deep brain stimulation of the globus pallidus alters network activity of the cerebellum, and in this way restores neuronal function in dystonia. The high-density microelectrode arrays enables the simultaneous recording of motor loop circuits to investigate the network-wide effects of deep brain stimulation in movement disorders such as dystonia. To begin, house the electronics of the STELLA Stimulation System in a custom 3D printed polyethylene terephthalate glycol or PETG capsule.Use silicone to seal the area between the lid and the capsule and the housing at the cable exit. After drying, inspect the silicone for visible gaps and repeat the sealing if gaps are present. After full solidification of the silicone, prepare the epoxy resin at a 100:35 mixing ratio by weight.Use the Vevor RS-1.5 to remove air bubbles from the resin under vacuum. Before assembly, apply a thin layer of Vaseline to the mating surface of the custom silicone mold. Place the stimulator capsule into the mold.Slowly fill it with epoxy resin until the stimulator and cable junctions are fully covered, avoiding air bubbles during filling. After resin curing, remove the capsule from the mold. Use a fine file or polishing tool to smooth the epoxy surface and eliminate sharp edges.Immerse the capsule in liquid silicone to apply a final thin silicone coating. Use a plastic pipette to apply additional silicone around the cable near the junction area. Use a stick to remove excess silicone before curing to avoid coating thickness.To perform a post-encapsulation functionality test before surgical implantation or experimental use, place the STELLA Stimulation System in Ringer's solution and activate the magnetic built-in self-test function. Confirm the correct light emitting diode or LED blink pattern of 2 by 2, indicating proper stimulation output and rule out short circuits, open circuits, or low battery conditions. After testing, turn the device off to save battery.Prepare the workplace with dissection tools, sucrose solution, Petri dishes, and the vibratome setup. Add ice cold slushy sucrose solution to a Petri dish containing filter paper at the bottom and maintain continuous carbogenation for brain extraction and slicing. Transfer the brain excised within 15 minutes following the 11-day deep brain stimulation or DBS period into the prepared Petri dish containing ice cold slushy sucrose.Use a blade to remove the cerebellum by making a corneal cut through the pons. Then remove the cerebellar hemisphere through a parasagittal cut. Prepare the vibratome plate with a drop of super glue.Carefully remove the cerebellum from the Petri dish. Dab the parasagittal section plane onto filter paper to dry it before gluing this side. Place the vibratome plate into the tray.Fill the tray with enough half slushy sucrose solution to completely submerge the cerebellum. Gas the tray with carbogen. Cut parasagittal cerebellar slices with a thickness of 200 micrometers at a constant speed of 0.08 millimeters per second.Store the slices in artificial cerebrospinal fluid or ACSF in a beaker constantly gassed with carbogen at room temperature. Allow the slices to recover in carbonated ACSF at room temperature for approximately 40 minutes before transfer to the high-density microelectrode array or HD-MEA for rewarming and stabilization before recording. Clean the gold plated pads with 100%ethanol and the electrode array with 70%ethanol and a soft brush.Then, rinse the electrode array with Milli-Q water. Prior to tissue slice placement, insert the CorePlate single well into the recording setup. Connect the HD-MEA with CorePlate TM single well into the recording system.Pipette approximately 2 milliliters of ACSF into the well without air bubbles. Start live acquisition in BrainWave 5 or BW5 software and set the parameters like Cockpit Mode and Tissue Model. Verify that the background noise falls within the optimal range of 9-20 microvolts.Record for at least 30 seconds. After stopping the recording, open the saved file for analysis. To analyze the noise interface, click on Results and select Playback.Click on Media Export and under the Raw Traces Dropdown menu select Activity Map to analyze the peak to peak amplitude and standard deviation to determine the baseline noise. Next, use a Pasteur pipette to place the tissue slice in the center of the CorePlate single well under a microscope and remove the ACSF. Position the anchor on top of the slice to fix it.Use a 3 milliliter Pasteur pipette to apply and remove a single drop of ACSF to wash the slice, repeating 5-10 times. Use a twisted paper wick to remove the residual ACSF. Apply drops of ACSF to the center of the well to improve contact between the slice and electrodes for optimal recording.Position the inflow line above the slice and the outflow line away from it. Remove any bubbles from the ground electrode before proceeding with data acquisition and analysis. The analysis of the total number of spikes in molecular Purkinje cell and granular cell layers of the cerebellar cortex showed that dystonic tissue slices generated significantly fewer spikes, whereas Pallidal DBS increased the number of spikes to levels comparable to those of healthy controls.The Kruskal-Wallis H test further confirmed a statistically significant increase in the number of spikes in the molecular layer of cerebellar cortex for the dystonic DBS group compared to both the untreated dystonic and dystonic sham group. The dystonic DBS group also showed an increase in spikes in the Purkinje cell layer and the granular cell layer, reaching values comparable to those of the control group. Maintaining stable HD-MEA recording through all measures with proper grounding, good keep electrode contact and low noise experimental conditions is critical.This protocol enables high resolution measurement of cerebellar network activity and the effects of its modulation by deep brain stimulation.

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