Name: direct-current stimulation and multi-electrode array recording of seizure-like activity in mice brain slice preparation
Uploaded: 2016-06-07T14:15:00
Description: Watch this Scientific Journal Video about Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation at JoVE.com

We are grateful for the technical support from the Neural Circuit Electrophysiology Core at Academia Sinica. This work was supported by the National Science Council (102-2320-B-001-026-MY3 and 100-2311-B-001-003-MY3) and Neuroscience Program of Academia Sinica.

Procedures that involve animal subjects were approved by the Institutional Animal Care and Utilization Committee, Academia Sinica, Taipei, Taiwan.
1. Preparing Experimental Solution and Equipment for Multielectrode Array Recording
<ol>
	<li>Prepare artificial cerebral spinal fluid (aCSF; 124 mM NaCl, 4.4 mM KCl, 1 mM NaH2PO3, 2 mM MgSO4, 2 mM CaCl2, 25 mM NaHCO3, and 10 mM glucose, bubbled with 95% O2 and 5% CO2).</...

<ol>
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In the present study, the effects of the duration and orientation of DCS on ACC seizure-like activity were tested. To obtain stable data in mouse brain slices, how to keep the integrity of the MT-ACC pathway and to avoid damage it is key, especially the steps in which two angled ventral cuts and a dorsal cut of the cortex are made. Moreover, the time to prepare the brain slice can also affect the activity of the brain slice, which should be the shortest time possible to keep the brain fresh and strong. A previous study s...

Epilepsy is a common neurological disorder. Thirty percent of patients with epilepsy suffer from drug-resistant seizures1. Transcranial direct-current stimulation (tDCS) provides a noninvasive approach to control or alter network activities across large brain areas, such as seizures. Clinical studies have shown that tDCS effectively treats intractable seizures2 and can produce both short- and long-term suppressive effects on seizures3-5. However, the therapeutic mechanism of tDCS actions is still unclear. The brain slice model presented is an in vitro method to investigate how the therapeutic mechanism of tDCS actions alters the symptoms of seizure-like brain activities. Accordingly, to achieve its optimal effects, specific stimulation parameters including orientation, field strength, and stimulation duration need to be tested in an experimental model. Previous studies have shown that the orientation of the electric field is important to obtain therapeutic effects6. Thus, testing and arranging the orientation of electrodes relative to the position of the tested brain slice are feasible.
Frontal lobe epilepsy and anterior cingulate cortex (ACC) seizures are often drug-resistant7,8. Some studies have reported the application of tDCS in the cingulate cortex9-11. tDCS is shown to affect vigilance, decision making and emotion through alteration of ACC activities, and can modulate neuronal excitability and seizure activity in this brain region12. Therefore, suppressive effects of tDCS on ACC seizures might be helpful for clinical treatment and the evaluation of alternative treatments.
The present protocol describes the preparation of an electrode in the recording chamber for DCS of a brain slice and its effect on seizure-like activity recording with a multielectrode array (MEA).

<table border="0" cellpadding="0" cellspacing="0" width="1018">
	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1017px;">Anesthetic:</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isoflurane</td>
			<td>Halocarbon Products Corporation&#160;</td>
			<td>NDC 12164-002-25</td>
			<td style="width:187px;">4%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">aCSF (total:1L):</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D(+)-Glucose</td>
			<td>MERCK</td>
			<td>1.08337.1000</td>
			<td style="width:187px;">10 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydrogen carbonate</td>
			<td>MERCK</td>
			<td>1.06329.0500</td>
			<td style="width:187px;">25 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride</td>
			<td>MERCK</td>
			<td>1.06404.1000</td>
			<td style="width:187px;">124 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">(+)-Sodium L-ascorbate, &#62;=98%</td>
			<td>SIGMA</td>
			<td>A4034-100G</td>
			<td style="width:187px;">0.15 g / 2 c.c</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium sulfate, anhydrous,ReagentPlus</td>
			<td>SIGMA</td>
			<td>M7506-500G</td>
			<td style="width:187px;">2 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride dihydrate</td>
			<td>MERCK</td>
			<td>1.02382.1000</td>
			<td style="width:187px;">2 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dihydrogen phosphate monohydrate</td>
			<td>MERCK</td>
			<td>1.06346.1000</td>
			<td style="width:187px;">1 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium chloride</td>
			<td>May &#38; Baker LTD Dagenham England</td>
			<td>MS 7616</td>
			<td style="width:187px;">4.4 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Drugs:</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(+)-Bicuculline</td>
			<td>TOCRIS</td>
			<td>0130</td>
			<td style="width:187px;">5 &#181;M in aCSF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-Aminopyridine</td>
			<td>TOCRIS</td>
			<td>0940</td>
			<td style="width:187px;">250 &#181;M in aCSF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Brain slice Preparation:</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Vibratome</td>
			<td>Vibratome</td>
			<td>Series 1000</td>
			<td style="width:187px;">Block slicing into 500 &#181;m thick slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">MEA system:</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:355px;">Multielectrode array (MEA) probes: 6 x 10 planar MEA</td>
			<td>Multi Channel Systems</td>
			<td style="width:184px;">60MEA500/30iR-Ti-pr MEAS 6x10</td>
			<td style="width:187px;">electrode diameter, 30 &#181;m; electrode spacing, 500 &#181;m; impedance, 50 k&#937; at 200 Hz</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:355px;">Multielectrode array (MEA) probes: 8 x 8 MEA&#160;</td>
			<td>Ayanda Biosystems</td>
			<td style="width:184px;">60MEA200/10iR-Ti-pr MEAS 8x8</td>
			<td style="width:187px;">pyramidal-shaped electrode; diameter, 40 &#181;m; tip height, 50 &#181;m; electrode spacing, 200 &#181;m; impedance, 1000 k&#937; at 200 Hz</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:355px;">A 60-channel amplifier was used with a band-pass filter set between 0.1 Hz and 3 KHz at 1200X amplification</td>
			<td>Multi-Channel Systems</td>
			<td>MEA-1060-BC</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MC Rack software at a 10 KHz sampling rate</td>
			<td>Multi-Channel Systems</td>
			<td></td>
			<td style="width:187px;">Software for data collect and recordings</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">control of a pulse generator</td>
			<td>Multi-Channel Systems</td>
			<td>STG 1002</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">slice anchor kits and hold-downs</td>
			<td>Warner Instruments</td>
			<td style="width:184px;">SHD-26H/10; WI64-0250</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peristaltic Pump-minipuls3</td>
			<td>Gilsom</td>
			<td style="width:184px;">MINIPULS3</td>
			<td>perfusion rate : 8 ml/min</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Stimulation system:</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Isolated stimulator</td>
			<td>A-M Systems</td>
			<td>Model 2100</td>
			<td style="width:187px;">intensity of &#177;350 &#956;A , duration of 200 &#956;s</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tungsten electrode</td>
			<td>A-M Systems</td>
			<td>575300</td>
			<td style="width:187px;">placed in thalamus</td>
		</tr>
	</tbody>
</table>

direct-current stimulation and multi-electrode array recording of seizure-like activity in mice brain slice preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Preparation of the Thalamocingulate Slice and MEA Recording System Setup
The MT-ACC slice from mice is a special slice preparation that allows exploration of the electrophysiological properties of the thalamocingulate pathway. Figure 1A shows the way in which the MT-ACC slice was prepared. The brain of the mouse was quickly removed and kept in cool oxygenated aCSF (Figure 1A, a, b). To reveal su...

Watch this Scientific Journal Video about Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation at JoVE.com

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

The overall goal of this experimental procedure, is to access the arrangement of electrodes in the recording chamber for direct current stimulation of a brain slice and it's effect on seizure-like activity recorded with a multi-electrode array. This better can help answer key questions in direct current stimulation, and a multi-electrode array recorded seizure-like activity in mouse brain slice. An advantage of this technique is the effects of direct current stimulation can be evaluated in the specific pathway of the mouse brain slice.Demonstrating the procedure will be Hsiang Chin, and Wei-Jen Chang, technicians from my laboratory. In this procedure, sterilize all of the surgical instruments with 75%ethanol solution before the experiment. Then, expose the skull of the animal and trim off the remaining muscle.Next, peel away the dorsal surface of the skull using rongeurs, and trim away the sides. Subsequently, using a spatula, cut the olfactory bulbs and nerve connections along the ventral surface of the brain before removing it. Quickly transfer the brain to a beaker filled with ice cold, oxygenated, ACSF.To prepare slices containing the MT-ACC pathway, make a sagittal cut, 2 mm lateral to the midline in each hemisphere, to display the subcortical anatomy. Then, make an angled cross cut parallel to the visible fiber tract in the striatum. Make the second angled cross cut from the connection between the cerebellum and visual cortex, to the midpoint between the anterior commissure and the optic tract, that are ventral and parallel to the thalamus cingulate pathway.After that, attach the brain block to an angular plate with a cyanoacrylate adhesive. Make a cut just above the turning point of the pathway. After that, unfold the plate, flatten it, and glue it on to the chamber stage of a vibratome.Subsequently section the brain slices at 500 micrometers thickness, and keep them in ice cold oxygenated ACSF. Transfer a slice to the recording chamber at 32 degrees Celsius with continuous profusion of oxygenated ACSF for one hour. Confirm the placement of a MEA probe on the multichannel system.Use one tube to guide the ACSF into the MEA chamber and the other tube to guide the ACSF out of the chamber. Continuously perfuse the preparation with warm, oxygenated ACSF at 30 degrees Celsius. Using a wet cotton swab, transfer a brain slice to the MEA.Carefully move the brain slice to orient the ACC above the electrodes, then, stabilize the brain slice with a slice anchor to ensure a good electrical connection between the slice and the electrodes. Afterward, place the anode electrode proximal to the ACC and the cathode electrode distal to the ACC. Record the field strength by the two field orientations of MEA.Then deliver the electric currents using a stimulator. Adjust the distance between the two silver chloride electrodes to about 1.5 to 2 cm and adjust the stimulators current strength to make the DCS between 0.5 and 2 milliamps. In this procedure, place a tungsten electrode in MT, and deliver pulses from the stimulator to the thalamic region of the slice.Next, use various current intensities, to determine the threshold that elicits an ACC response. Move the tungsten electrode along the thalamus cingulate pathway to obtain the optimal response profile. Record 1-20 ACC responses and use the software to automatically average all of the ACC responses evoked my MT stimulation.To induce seizure-like activity, add 250 micromolar 48P and 5 micromolar bicuculline to the perfusion solution, and continue to profuse the slice for two to three hours. Maintain the pump at a relatively fast perfusion rate, which can help prevent the build up of a pH gradient. Next, place the tungsten electrode at MT and deliver electrical stimulation to obtain an ACC response profile.Record 10-20 sweeps and average the responses. Afterward, replace the prefusion solution with fresh ACSF to wash out the drugs. In this procedure, ensure the generation of uniform electric fields, by passing currents between two parallel silver chloride coated silver wires that are placed inside the MEA chamber.If there are no issues, the DCS should stay between 0.5 and 2 milliamps. Next, turn off the DCS and stimulate the thalamus with a tungsten electrode. To obtain maximal synaptic responses in ACC record 10-20 responses and then average them.Then, turn on the DCS and stimulate the thalamus simultaneously. Evaluate the amplitude changes of the thalamic stimulation evoked ACC response during DCS. Now, turn off the DCS, add 250 micromolar 48P, and 5 micromolar bicuculline to the perfusion solution, and wait 2-3 hours.If the drugs effect the brain slice, the slice should produce cortical seizure responses. Subsequently, collect 10-20 anterior cingulate cortical responses, and measure the amplitude and duration of the electrically evoked cortical seizure responses. Nest, turn on the DCS and stimulate the thalamus simultaneously.Evaluate the changes in the amplitude and duration of the evoked cortical seizure responses during DCS application. After that, replace the perfusion solution with fresh ACSF to wash out the drugs. This figure shows different evoked responses, which include the thalamic stimulation evoked responses in ACC, drug-induced, seizure-like activity, and both the thalamic stimulation and drug induced seizure-like activity.This figure shows the affect of different orientations in DCS, such as different orientations in the electric field, thalamic stimulation evoked responses with DCS, and the effect of cathodal DCS on seizure-like activity. And in this figure, 15 minutes of cathodal DCS is shown the have effectively induced long term depression and depressed evoked responses. The seizure duration was significantly decresed after 15 minutes of cathodal DCS compared with no DCS application.faster, this technically can be done in four hours if it is performed properly. Following this procedure, other matters like transcranial magnetic stimulation can be performed, in order to answer additional questions like providing noninvasive approaches for controlling resistance seizures. After this development, this technique paved the way for researchers in the field of noninvasive therapy to explore seizure treatments in of animal model.After watching this video, you should have a good understanding of how to use the current stimulation to assess seizure-like activity in brain slices.

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Neuroscience

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

Electrode Positioning and Montage in Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a non-invasive and safe alternative to change cortical excitability2. The effects of one session of tDCS can last for several minutes, and its effects depend on polarity of stimulation, such as that cathodal stimulation induces a decrease in cortical excitability, and anodal stimulation induces an increase in cortical excitability that may last beyond the duration of stimulation6. These effects have been explored in cognitive neuroscience and also clinically in a variety of neuropsychiatric disorders &#x2013; especially when applied over several consecutive sessions4. One area that has been attracting attention of neuroscientists and clinicians is the use of tDCS for modulation of pain-related neural networks3,5. Modulation of two main cortical areas in pain research has been explored: primary motor cortex and dorsolateral prefrontal cortex7. Due to the critical role of electrode montage, in this article, we show different alternatives for electrode placement for tDCS clinical trials on pain; discussing advantages and disadvantages of each method of stimulation.

Transcranial direct current stimulation (tDCS) is an established technique to modulate cortical excitability1,2. It has been used as an investigative tool in neuroscience due to its effects on cortical plasticity, easy operation, and safe profile. One area that tDCS has been showing encouraging results is pain alleviation 3-5.

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While these systems can provide a wealth of behavioral information, the cost and complexity of these systems can be prohibitive for many labs. We have developed a low-cost assay for measuring locomotive behavior and seizure movement in D. melanogaster. The system uses a web-cam to capture images that can be processed using a combination of inexpensive and free software to track the distance moved, the average velocity of movement and the duration of movement during a specified time-span. To demonstrate the utility of this system, we examined a group of D. melanogaster mutants, the Bang-sensitive (BS) paralytics, which are 3-10 times more susceptible to seizure-like activity (SLA) than wild type flies. Using this novel system, we were able to detect that the BS mutant bang senseless (bss) exhibits lower levels of exploratory locomotion in a novel environment than wild type flies. In addition, the system was used to identify that the drug metformin, which is commonly used to treat type II diabetes, reduces the intensity of SLA in the BS mutants.

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Using a webcam and a combination of free and inexpensive software programs, locomotive patterns in Drosophila melanogaster can be analyzed to detect differences in the speed, distance, and time of various motor activities.

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

Transcranial Direct Current Stimulation (tDCS) in Mice

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for several neuropsychiatric diseases. The biological effects of tDCS are not fully understood, which is in part explained due to the difficulty in obtaining human brain tissue. This protocol describes a tDCS mouse model that uses a chronically implanted electrode allowing the study of the long-lasting biological effects of tDCS. In this experimental model, tDCS changes the cortical gene expression and offers a prominent contribution to the understanding of the rationale for its therapeutic use.

Transcranial Direct Current Stimulation tDCS in Mice

Transcranial direct current stimulation (tDCS) is a therapeutic technique proposed to treat psychiatric diseases. An animal model is essential for understanding the specific biological alterations evoked by tDCS. This protocol describes a tDCS mouse model that uses a chronically implanted electrode.

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for ...

Stimulation Location Determination using a 3D Digitizer with High-Definition Transcranial Direct Current Stimulation

The abundance of neuroimaging data and rapid development of machine learning has made it possible to investigate brain activation patterns. However, causal evidence of brain area activation leading to a behavior is often left missing. Transcranial direct current stimulation (tDCS), which can temporarily alter brain cortical excitability and activity, is a noninvasive neurophysiological tool used to study causal relationships in the human brain. High-definition transcranial direct current stimulation (HD-tDCS) is a noninvasive brain stimulation (NIBS) technique that produces a more focal current compared to conventional tDCS. Traditionally, the stimulation location has been roughly determined through the 10-20 EEG system, because determining precise stimulation points can be difficult. This protocol uses a 3D digitizer with HD-tDCS to increase accuracy in determination of stimulation points. The method is demonstrated using a 3D digitizer for more accurate localization of stimulation points in the right temporo-parietal junction (rTPJ).

Presented here is a protocol to achieve higher accuracy in determination of stimulation location combining a 3D digitizer with high-definition transcranial direct current stimulation.

The abundance of neuroimaging data and rapid development of machine learning has made it possible to investigate brain activation patterns. However, ...

In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological characteristics in the intact spinal network and holds significant advantages relative to classical in vitro or extracellular recording techniques. An advantage of in vivo intracellular recordings is that this method can be performed on adult animals with a fully mature nervous system, and therefore many observed physiological mechanisms can be translated to practical applications. In this methodological paper, we describe this procedure combined with externally applied constant current stimulation, which mimics polarization processes occurring within spinal neuronal networks. Trans-spinal direct current stimulation (tsDCS) is an innovative method increasingly used as a neuromodulatory intervention in rehabilitation after various neurological injuries as well as in sports. The influence of tsDCS on the nervous system remains poorly understood and the physiological mechanisms behind its actions are largely unknown. The application of the tsDCS simultaneously with intracellular recordings enables us to directly observe changes of motoneuron membrane properties and characteristics of rhythmic firing in response to the polarization of the spinal neuronal network, which is crucial for the understanding of tsDCS actions. Moreover, when the presented protocol includes the identification of the motoneuron with respect to an innervated muscle and its function (flexor versus extensor) as well as the physiological type (fast versus slow) it provides an opportunity to selectively investigate the influence of tsDCS on identified components of spinal circuitry, which seem to be differently affected by polarization. The presented procedure focuses on surgical preparation for intracellular recordings and stimulation with an emphasis on the steps which are necessary to achieve preparation stability and reproducibility of results. The details of the methodology of the anodal or cathodal tsDCS application are discussed while paying attention to practical and safety issues.

This protocol describes in vivo intracellular recording of rat lumbar motoneurons with simultaneous trans-spinal direct current stimulation. The method enables us to measure membrane properties and to record rhythmic firing of motoneurons before, during and after anodal or cathodal polarization of the spinal cord.

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological ...

Transcranial Direct Current Stimulation (tDCS) for Memory Enhancement

Memory enhancement is one of the great challenges in cognitive neuroscience and neurorehabilitation. Among various techniques used for memory enhancement, transcranial direct current stimulation (tDCS) is emerging as an especially promising tool for improvement of memory functions in a non-invasive manner. Here, we present a tDCS protocol that can be applied for memory enhancement in healthy-participant studies as well as in aging and dementia research. The protocol uses weak constant anodal current to stimulate cortical targets within cortico-hippocampal functional network engaged in memory processes. The target electrode is placed either on the posterior parietal cortex (PPC) or the dorsolateral prefrontal cortex (DLPFC), while the return electrode is placed extracranially (i.e., on the contralateral cheek). In addition, we outline a more advanced method of oscillatory tDCS, mimicking a natural brain rhythm to promote hippocampus-dependent memory functions, which can be applied in a personalized and non-personalized manner. We present illustrative results of associative and working memory improvement following single tDCS sessions (20 minutes) in which the described electrode montages were used with current intensities between 1.5 mA and 1.8 mA. Finally, we discuss crucial steps in the protocol and methodological decisions that must be made when designing a tDCS study on memory.

Transcranial Direct Current Stimulation tDCS for Memory Enhancement

A protocol for memory enhancement by using transcranial direct current stimulation (tDCS) targeting dorsolateral prefrontal and posterior parietal cortices, as core cortical nodes within hippocampo-cortical network, is presented. The protocol has been well evaluated in healthy-participant studies and is applicable to aging and dementia research as well.

Memory enhancement is one of the great challenges in cognitive neuroscience and neurorehabilitation. Among various techniques used for memory enhancement, ...

Understanding Motor Neuron Responses to Spinal Cord Stimulation

The Ex vivo Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons During Spinal Cord Stimulation

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to execute sensorimotor behaviors, directly studying the electrical responses of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation. To simultaneously record diverse stimulus characteristics and cellular responses, a patch-clamp is a good method to study the electrophysiological characteristics at a single-cell scale. However, there are still some complex difficulties in achieving this goal, including maintaining cell viability, quickly separating the spinal cord from the bony structure, and using the SCS to successfully induce action potentials. Here, we present a detailed protocol using patch-clamp to study the electrical responses of motor neurons to SCS with high spatiotemporal resolution, which can help researcher improve their skills in separating the spinal cord and maintaining the cell viability at the same time to smoothly study the electrical mechanism of SCS on motor neuron and avoid unnecessary trial and mistake.

Author Spotlight: Advancing Spinal Cord Stimulation - Exploring the Cellular Responses of Motor Neurons Through Patch-Clamp Electrophysiology 

This protocol describes a method using a patch-clamp to study the electrical responses of motor neurons to spinal cord stimulation (SCS) with high spatiotemporal resolution, which can help researchers improve their skills in separating the spinal cord and maintaining cell viability simultaneously.

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to ...