This research was made possible in part by the generous funding of the Knight Campus Undergraduate Scholars program and the Phil and Penny Knight Foundation

All methods detailed here were performed in compliance with IRB protocol and approval (University of Oregon IRB protocol number 10182017.017). Informed consent was obtained from all subjects.
1. Reaching apparatus
<ol>
	<li>Place a large graphics tablet flat on a desktop.</li>
	<li>Use an adjustable 80-20 aluminum frame to position the task monitor 6-8 in above the tablet in parallel, with the screen facing upward (for a blueprint, check here: https://github.com/greenhouselab/Reach_TMS and&#160;Supplementary Figure 1). 
	NOTE: This setup allows for participants to reach across the tablet and acquire targets presented on the task monitor while occluding vision of their reaching arm.</li>
	<li>Use the setup described in Kim et al.3 as a reference.</li>
</ol>
2. Machine interfaces
<ol>
	<li>Connect the tablet to the computer via a USB port. Connect the task monitor to the computer via the HDMI port. Connect the rear TMS port to the computer via a DB-9 cable.</li>
	<li>Connect the EMG system to the computer via a PCI-6220 card DAQ. Connect the photodiode to the EMG system via a BNC cable.</li>
</ol>
3. Photodiode sensor
<ol>
	<li>Attach a photodiode sensor to the BNC cable. Secure the photodiode sensor with tape to the top-right corner of the task monitor, with the sensor facing the screen, &#8804; 1 cm away. 
	NOTE: This will record the timing of stimuli presented on the task monitor as analog data in an independent input channel.</li>
</ol>
4. Software
<ol>
	<li>Download VETA Toolbox26 (https://github.com/greenhouselab/Veta) for MATLAB 2018 to interface with the hardware for data collection.</li>
	<li>Download the reaching task code (https://github.com/greenhouselab/Reach_TMS) developed for the control of experimental parameters and interfacing with the tablet.</li>
</ol>
5. Participant screening and informed consent
<ol>
	<li>Screen the subject for contraindications to TMS. Exclusion criteria include a personal or family history of seizure, headache, brain trauma, fainting, chronic stress or anxiety, problems with sleep, and any neuroactive medication. Additional exclusion criteria include any metal implants in the brain or skull and any recreational drug or alcohol use in the 24 h prior to testing. Inclusion criteria included right-handedness and age between 18 and 35 years.</li>
	<li>Provide a written explanation of the procedure and associated risks, clarifying any further questions the participant may have.</li>
	<li>Obtain informed consent from participants.</li>
</ol>
6. Subject setup
<ol>
	<li>Position the subject in a comfortable chair facing the tablet. Ensure that the knees are flexed to 90&#176; with the legs under the desk.</li>
	<li>Prepare the skin and place EMG electrodes.
	<ol>
		<li>Use fine-grain sandpaper to gently abrade the skin at the site of the right first dorsal interossei (FDI), extensor carpi radialis, and anterior deltoid muscles, as well as the C4 prominence at the base of the neck, to detect electrical artifacts produced by the TMS pulse. 
		NOTE: Muscle recording sites can be adapted based on user needs.</li>
		<li>Swab each abraded area with an alcohol prep pad once per electrode site to clean.</li>
		<li>Place one EMG electrode on each site. Ensure that the electrodes run perpendicular to the muscle fibers. Place the ground electrode on the bony prominence of the right elbow.</li>
		<li>Secure each electrode with medical tape.</li>
	</ol>
	</li>
	<li>Check the quality of the EMG recording. Use the VETA toolbox to visualize all EMG traces and ensure they are free of artifacts. If EMG traces are noisy, ensure the ground is properly placed and that all electrodes make proper contact with the skin.</li>
</ol>
7. Transcranial magnetic stimulation
<ol>
	<li>Turn on the TMS machine.</li>
	<li>Find the TMS hot spot of the right FDI muscle via stimulation of the left M1.
	<ol>
		<li>Place the coil ~5 cm lateral and 2 cm anterior to the vertex of the head, oriented ~45&#176; off the midline.</li>
		<li>Administer TMS pulses once every 4 s while repositioning the coil in increments of approximately 5 mm in the anterior-posterior and medial-lateral plane.</li>
		<li>Beginning at 30% maximum stimulator output, gradually increase the TMS intensity by 2% increments until MEPs are observed.</li>
		<li>Once the optimum location is identified, at which MEPs can be reliably elicited on the majority (~75%) of pulses at the lowest possible stimulator intensity, determine the resting motor threshold (RMT) by finding the intensity level that produces MEPs with a peak-to-peak amplitude of &#62;50 &#181;V on five out of 10 pulses.</li>
		<li>Mark the position by gently placing thin strips of reflective tape on the participant&#39;s head along the perimeter of the coil. Maintain coil positioning either by manually holding the coil or using a stand to support it.</li>
	</ol>
	</li>
</ol>
8. Reaching task setup
<ol>
	<li>Put a Velcro glove on the right hand of the participant to facilitate a relaxed power grip posture.</li>
	<li>Attach the stylus to the glove and advise the subject to keep the hand relaxed between reaching movements.</li>
	<li>Communicate the task instructions, which are as follows: Guide the cursor to the home position on the bottom of the screen. You will see a cue at one of two target locations. When the target fills in with color, reach through the target as fast and as accurately as possible. Then return to the home position. Indicate locations of home positions, cues, and targets (Figure 1A).</li>
	<li>Coach the participant to slice through targets with the stylus as quickly and as accurately as possible. Turn off the lights in the task room to obscure the participant&#39;s vision of arm movements and improve the visibility of the task monitor.</li>
</ol>
9. Task design
<ol>
	<li>Control visual stimulus presentation with Psychtoolbox 3.0 in Matlab 2018 (Supplementary Coding File 1).</li>
	<li>Use the following parameters to match the current data: 20 practice trials; 270 test trials; TMS on 4/5 of test trials; TMS either coincides with the preparatory cue onset (baseline TMS) or 100 ms before the imperative cue (delay TMS) with equal frequency; 1/10 of total trials are catch trials, in which the imperative cue does not appear; the home position is a circle with a 2 cm radius positioned in the bottom center of the workspace; two circular targets with 1 cm radius are positioned 15 cm from the home position at +45&#176; and -45&#176; away from the midline.</li>
	<li>Set the event order and durations as follows: preparatory cue at 900 ms and imperative cue at 900 ms.</li>
</ol>
10. TMS administration
<ol>
	<li>The VETA toolbox concurrently administers TMS and records EMG&#160;https://github.com/greenhouselab/Veta.</li>
	<li>Control the timing of the TMS pulses with the VETA toolbox to coincide with the chosen behavioral events (i.e., the onset of the preparatory cue or 100 ms preceding target onset).</li>
	<li>Deliver TMS with sufficient frequency to ensure a sufficient number of MEPs for analysis. 
	NOTE: As written, the task code will deliver a TMS pulse on 4/5 of total trials either at the onset of the preparatory cue-to elicit baseline MEPs-or 100 ms before the imperative cue-to elicit delayed MEPs. Parameters can be adjusted in the code according to user needs. Trials without TMS can be used to evaluate behavioral performance in the absence of TMS. This is useful for determining any possible influence of TMS on performance.</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimum+number+of+trials+required+for+within-+and+between-session+reliability+of+TMS+measures+of+corticospinal+excitability.">Minimum number of trials required for within- and between-session reliability of TMS measures of corticospinal excitability.</a> Neuroscience. 320, 205-209 (2016).</li><li>Koch, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interplay+between+posterior+parietal+and+ipsilateral+motor+cortex+revealed+by+twin-coil+transcranial+magnetic+stimulation+during+reach+planning+toward+contralateral+space.">Functional interplay between posterior parietal and ipsilateral motor cortex revealed by twin-coil transcranial magnetic stimulation during reach planning toward contralateral space.</a> The Journal of Neuroscience. 28 (23), 5944-5953 (2008).</li><li>Goldenkoff, E. 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All authors declare that there are no conflicts of interest

The methods outlined above offer a novel approach to studying motor preparation in the context of reaching behaviors. Although reaching represents a popular model task in the study of motor control and learning, there is a need for precisely evaluating the CS dynamics associated with reaching behavior. TMS offers a noninvasive, temporally precise method of capturing CS activity at discrete time points during reaching. The approach described here combines two independent subfields-TMS and reaching-into a ...

Goal-directed reaching is a fundamental motor behavior that allows humans to interact with and manipulate the external environment. The study of reaching in the fields of motor physiology, psychology, and neuroscience has produced rich and extensive literature that includes a variety of methodologies. Early studies of reaching used direct neural recordings in non-human primates to investigate neural activity at the level of single neurons1,2. More recent studies have investigated reaching using behavioral paradigms that employ sensorimotor adaptation to explore the nature of motor learning and control3,4,5. Such behavioral tasks combined with functional magnetic resonance imaging and electroencephalography can measure whole brain activity during reaching in humans6,7. Other studies have applied online TMS to investigate various features of reach preparation and execution8,9,10,11,12,13,14. However, there remains a need for an open-source and flexible approach that combines the behavioral assessment of reaching with TMS. While the utility of combining TMS with behavioral protocols is very well established15, here, we specifically examine the application of TMS within the context of reaching using an open-source approach. This is novel in that other groups who have published using this combination of methods have not made their tools readily available, prohibiting direct replication. This open-source approach facilitates replication, data sharing, and the possibility of multi-site studies. Additionally, should others wish to pursue novel research questions with similar tools, the open-source code can act as a launch pad for innovation, as it is readily adaptable.
TMS offers a noninvasive means of probing the motor system at precisely controlled time points16. When applied over the primary motor cortex (M1), TMS can elicit a measurable deflection in the electromyogram of a targeted muscle. The amplitude of this voltage wave, known as the motor evoked potential (MEP), provides an index of the momentary excitability state of the corticospinal (CS) pathway-a resultant analog of all excitatory and inhibitory influences on the CS pathway17. In addition to providing a reliable within-subject measurement of intrinsic CS excitability, TMS can be combined with other behavioral or kinematic metrics to investigate the relationships between CS activity and behavior in a temporally precise manner. Many studies have utilized a combination of TMS and electromyography (EMG) to address a variety of questions about the motor system, particularly since this combination of methods makes it possible to investigate MEPs under a vast array of behavioral conditions15. One area where this has proven particularly useful is in the study of action preparation, most often through the study of single-joint movements18. However, there are comparatively fewer TMS studies of naturalistic multi-joint movements such as reaching.
The current goal was to design a delayed-response reaching task that includes behavioral kinematics, online single-pulse TMS administration, and simultaneous EMG recording from multiple muscles. The task includes a two-dimensional point-to-point reaching paradigm with online visual feedback using a horizontally oriented monitor such that visual feedback matches reach trajectories (i.e., a 1:1 relationship during veridical feedback and no transformation between visual feedback and motion). The current design also includes a set of trials with a visuomotor perturbation. In the provided example, this is a 20&#176; rotational shift in the cursor feedback. Previous studies have used a similar reaching paradigm to address questions about the mechanisms and computations associated with sensorimotor adaptation19,20,21,22,23,24,25. Furthermore, this approach makes it possible to assess motor system excitability dynamics at precise time points during online motor learning.
Because reaching has proven to be a fruitful behavior for investigating learning/adaptation, assessing CS excitability in the context of this behavior has enormous potential to shed light on the neural substrates involved in these behaviors. These may include local inhibitory influences, changes in tuning properties, the timing of neural events, etc., as have been established in non-human primate research. However, these features have been more difficult to quantify in humans and clinical populations. Neural dynamics can also be investigated in the absence of overt movement in humans using the combined TMS and EMG approach (i.e., during the preparation of movement or at rest).
The tools presented are open-source, and the code is easily adaptable. This novel paradigm will produce important insights into the mechanisms involved in the preparation, execution, termination, and adaptation of reaching movements. Moreover, this combination of methods has the potential to uncover relationships between electrophysiology and reaching behavior in humans.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Port Native PCI Express&#160;</td>
			<td>StarTech.com</td>
			<td>RS232 Card with 16950 UART&#160;</td>
			<td>Must be compatible with desktop computer</td>
		</tr>
		<tr>
			<td>Adjustable 80-20 aluminum frame</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol prep pads</td>
			<td>any</td>
			<td></td>
			<td>EMG preparation</td>
		</tr>
		<tr>
			<td>Bagnoli Bipolar Electrodes</td>
			<td>Delsys</td>
			<td>DE 2.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Bagnoli Reference Electrode</td>
			<td>Delsys</td>
			<td>USX2000</td>
			<td>2&#8221; (5cm) Round</td>
		</tr>
		<tr>
			<td>Bagnoli-8 EMG System</td>
			<td>Delsys</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chair</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer monitor for EMG/TMS</td>
			<td>n/a</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Desk</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop Computer</td>
			<td>Dell</td>
			<td>xps 8930</td>
			<td>RAM: 16 GB, Storage: 1TB, Graphics: 1060 6GB&#160;</td>
		</tr>
		<tr>
			<td>EMG electrodes</td>
			<td>Delsys</td>
			<td>Sensor Adhesive Interface</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine grain sandpaper</td>
			<td>any</td>
			<td></td>
			<td>EMG preparation</td>
		</tr>
		<tr>
			<td>Graphics tablet</td>
			<td>Wacom</td>
			<td>Intuos-4 XL</td>
			<td></td>
		</tr>
		<tr>
			<td>Handle of paint roller</td>
			<td>any</td>
			<td></td>
			<td>to be used as stylus handle, hollowed out center must be large enough for stylus to sit securely inside&#160;</td>
		</tr>
		<tr>
			<td>Medical tape</td>
			<td>any</td>
			<td></td>
			<td>To secure EMG electrodes</td>
		</tr>
		<tr>
			<td>PCI-6220 card DAQ</td>
			<td>National Instruments</td>
			<td></td>
			<td>To interface EMG system</td>
		</tr>
		<tr>
			<td>Photodiode Sensor</td>
			<td>Vishay</td>
			<td>BPW21R</td>
			<td>To record timing of task events into EMG trace.</td>
		</tr>
		<tr>
			<td>Rear TMS port</td>
			<td>Magstim</td>
			<td></td>
			<td>Included with TMS machine</td>
		</tr>
		<tr>
			<td>Right-handed polyethylene glove</td>
			<td>any</td>
			<td></td>
			<td>Cut out thumb and index finger of glove to expose FDI muscle</td>
		</tr>
		<tr>
			<td>Sensory Adhesive Interface, 2-slot</td>
			<td>Delsys</td>
			<td>SC-F01</td>
			<td></td>
		</tr>
		<tr>
			<td>Stylus</td>
			<td>Wacom</td>
			<td>Intuos-4 grip pen</td>
			<td></td>
		</tr>
		<tr>
			<td>Tablet-to-Computer USB cable&#160;</td>
			<td>any</td>
			<td></td>
			<td>Included in Tablet purchase</td>
		</tr>
		<tr>
			<td>Task Monitor</td>
			<td>Asus</td>
			<td>VG248</td>
			<td></td>
		</tr>
		<tr>
			<td>TMS coil</td>
			<td>Magstim</td>
			<td>D70 Remote Coil</td>
			<td>7cm diameter, figure-of-eight coil</td>
		</tr>
		<tr>
			<td>TMS machine</td>
			<td>Magstim</td>
			<td>200-2</td>
			<td></td>
		</tr>
		<tr>
			<td>TMS-to-Computer DB9 cable</td>
			<td>any</td>
			<td></td>
			<td>Connects to PCIe Serial Card</td>
		</tr>
		<tr>
			<td>Velcro</td>
			<td>any</td>
			<td></td>
			<td>To be placed on glove and stylus handle</td>
		</tr>
	</tbody>
</table>

assessing corticospinal excitability during goal-directed reaching behavior

Reaching is a widely studied behavior in motor physiology and neuroscience research. While reaching has been examined using a variety of behavioral manipulations, there remain significant gaps in the understanding of the neural processes involved in reach planning, execution, and control. The novel approach described here combines a two-dimensional reaching task with transcranial magnetic stimulation (TMS) and concurrent electromyography (EMG) recording from multiple muscles. This method allows for the noninvasive detection of corticospinal activity at precise time points during the unfolding of reaching movements. The example task code includes a delayed response reaching task with two possible targets displayed &#177; 45&#176; off the midline. Single pulse TMS is delivered on the majority of task trials, either at the onset of the preparatory cue (baseline) or 100 ms prior to the imperative cue (delay). This sample design is suitable for investigating changes in corticospinal excitability during reach preparation. The sample code also includes a visuomotor perturbation (i.e., cursor rotation of &#177; 20&#176;) to investigate the effects of adaptation on corticospinal excitability during reach preparation. The task parameters and TMS delivery can be adjusted to address specific hypotheses about the state of the motor system during reaching behavior. In the initial implementation, motor evoked potentials (MEPs) were successfully elicited on 83% of TMS trials, and reach trajectories were recorded on all trials.

Successful execution of the described methods includes the recording of tablet data, EMG traces, and reliable elicitation of MEPs. An experiment was completed that included 270 test trials with TMS delivered on 4/5 of the trials (216 trials).
Data were collected from 16 participants (eight females; eight males) aged 25 &#177; 10 years, all of whom self-reported as right-handed. We assessed the effectiveness of the visual perturbation on behavioral performance by deriving a learning function fo...

Watch this Scientific Journal Video about Assessing Corticospinal Excitability During Goal-Directed Reaching Behavior at JoVE.com

Assessing Corticospinal Excitability During Goal-Directed Reaching Behavior

Reaching is a fundamental skill that allows humans to interact with the environment. Several studies have aimed to characterize reaching behavior using a variety of methodologies. This paper offers an open-source application of transcranial magnetic stimulation to assess the state of corticospinal excitability in humans during reaching task performance.

Reaching is a widely studied behavior in motor physiology and neuroscience research. While reaching has been examined using a variety of behavioral ...

This method combines a flexible behavioral reaching paradigm with transcranial magnetic stimulation electromyography all from a single computer.This is significant because it streamlines existing approaches.The main advantages of this technique are that it is openly available and offers flexibility in terms of behavioral task and TMS parameters.This setup can easily be adapted for use with clinical populations, as it is versatile and mobile.The method offers unique insights into corticospinal excitability and muscle activity during behavioral reaching and motor adaptation.Place a large graphics tablet flat on a desktop.Use an adjustable 80-20 aluminum frame to position the task monitor six to eight inches above the tablet in parallel with the screen facing upward.Next, position the subject in a comfortable chair facing the tablet and ensure that the knees are flexed to 90

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Neuroscience

1.6K Views.  University of Oregon. This method combines a flexible behavioral reaching paradigm with transcranial magnetic stimulation electromyography all from a single computer.This is significant because it streamlines existing approaches.The main advantages of this technique are that it is openly available and offers flexibility in terms of behavioral task and TMS parameters.This setup can easily be adapted for use with clinical populations, as it is versatile and mobile.The method offers unique insights into corticospinal...

Video: Assessing Corticospinal Excitability During Goal-Directed Reaching Behavior

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. Chemotherapy-induced peripheral neuropathy produces symptoms of numbness and paraesthesia in the limbs and may progress to difficulties with fine motor skills and walking, leading to functional impairment. In addition to producing troubling symptoms, chemotherapy-induced neuropathy may limit treatment success leading to dose reduction or early cessation of treatment. Neuropathic symptoms may persist long-term, leaving permanent nerve damage in patients with an otherwise good prognosis3. As chemotherapy is utilised more often as a preventative measure, and survival rates increase, the importance of long-lasting and significant neurotoxicity will increase.
There are no established neuroprotective or treatment options and a lack of sensitive assessment methods. Appropriate assessment of neurotoxicity will be critical as a prognostic factor and as suitable endpoints for future trials of neuroprotective agents. Current methods to assess the severity of chemotherapy-induced neuropathy utilise clinician-based grading scales which have been demonstrated to lack sensitivity to change and inter-observer objectivity4. Conventional nerve conduction studies provide information about compound action potential amplitude and conduction velocity, which are relatively non-specific measures and do not provide insight into ion channel function or resting membrane potential. Accordingly, prior studies have demonstrated that conventional nerve conduction studies are not sensitive to early change in chemotherapy-induced neurotoxicity4-6. In comparison, nerve excitability studies utilize threshold tracking techniques which have been developed to enable assessment of ion channels, pumps and exchangers in vivo in large myelinated human axons7-9.
Nerve excitability techniques have been established as a tool to examine the development and severity of chemotherapy-induced neurotoxicity10-13. Comprising a number of excitability parameters, nerve excitability studies can be used to assess acute neurotoxicity arising immediately following infusion and the development of chronic, cumulative neurotoxicity. Nerve excitability techniques are feasible in the clinical setting, with each test requiring only 5 -10 minutes to complete. Nerve excitability equipment is readily commercially available, and a portable system has been devised so that patients can be tested in situ in the infusion centre setting. In addition, these techniques can be adapted for use in multiple chemotherapies.
In patients treated with the chemotherapy oxaliplatin, primarily utilised for colorectal cancer, nerve excitability techniques provide a method to identify patients at-risk for neurotoxicity prior to the onset of chronic neuropathy. Nerve excitability studies have revealed the development of an acute Na+ channelopathy in motor and sensory axons10-13. Importantly, patients who demonstrated changes in excitability in early treatment were subsequently more likely to develop moderate to severe neurotoxicity11. However, across treatment, striking longitudinal changes were identified only in sensory axons which were able to predict clinical neurological outcome in 80% of patients10. These changes demonstrated a different pattern to those seen acutely following oxaliplatin infusion, and most likely reflect the development of significant axonal damage and membrane potential change in sensory nerves which develops longitudinally during oxaliplatin treatment10. Significant abnormalities developed during early treatment, prior to any reduction in conventional measures of nerve function, suggesting that excitability parameters may provide a sensitive biomarker.

This abstract describes a novel method to assess the development of neurotoxicity in patients receiving chemotherapy treatment. While conventional assessment methods are limited in their ability to detect early changes in nerve function, nerve excitability techniques provide early identification of patients at risk of severe neurotoxicity and insight into pathophysiology.

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their worlds when trying to understand their behavior and ecology. Sensitivity to the ultraviolet spectrum (UV; 300 - 400 nm) is found across multiple genera of birds, fish, reptiles, amphibians, and even mammals. This protocol describes a technique for examining organisms for the presence of UV-reflecting structures and a method for testing whether these cues are used as social signals in the context of mate choice. A spectrophotometer is used to detect the presence of UV reflectance and variation in reflective intensity between individuals and sexes. An example of this technique is presented in which a dichotomous mate choice test exposes sexually receptive individuals to opposite sex individuals whose visual appearance can be manipulated by filters that either transmit full spectrum or block UV wavelengths. This system allowed for the determination that female, but not male, sailfin mollies (Poecilia latipinna) were using UV markings as part of their mating decisions. These types of studies serve to expand our knowledge of the range of organisms that utilize UV and provide insight into how UV plays a role in their lives.

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

This protocol outlines the use of spectrophotometry to detect ultraviolet-reflecting structures on organisms (in this example, the sailfin molly Poecilia latipinna) and describes dichotomous choice tests for fish that allow inferences to be made on the role of ultraviolet cues during mate selection.

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Operant Protocols for Assessing the Cost-benefit Analysis During Reinforced Decision Making by Rodents

Reinforcement-guided decision making is the ability to choose between competing courses of action based on the relative value of the benefits and their consequences. This process is integral to the normal human behavior and has been shown to be disrupted by neurological and psychiatric disorders such as addiction, schizophrenia, and depression. Rodents have long been used to uncover the neurobiology of human cognition. To this end, several behavioral tasks have been developed; however, most are non-automated and are labor-intensive. The recent development of the open-source microcontroller has enabled researchers to automate operant-based tasks for assessing a variety of cognitive tasks, standardizing the stimulus presentation, improving the data recording and consequently, improving the research output. Here, we describe an automated delay-based reinforcement-guided decision-making task, using an operant T-maze controlled by custom-written software programs. Using these decision-making tasks, we show the changes in the local field potential activities in the anterior cingulate cortex of a rat whilst it performs a delay-based cost-and-benefit decision-making task.

A cost-benefit analysis is a weighing-scale approach that the brain performs during the course of decision making. Here, we propose a protocol to train rats on an operant-based decision-making paradigm where rats choose higher rewards at the expense of waiting for 15 s to receive them.

Reinforcement-guided decision making is the ability to choose between competing courses of action based on the relative value of the benefits and their ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Measuring Neural Mechanisms Underlying Sleep-Dependent Memory Consolidation During Naps in Early Childhood

Sleep is critical for daily functioning. One important function of sleep is the consolidation of memories, a process that makes them stronger and less vulnerable to interference. The neural mechanisms underlying the benefit of sleep for memory can be investigated using polysomnography (PSG). PSG is a combination of physiological recordings including signals from the brain (EEG), eyes (EOG), and muscles (EMG) that are used to classify sleep stages. In this protocol, we describe how PSG can be used in conjunction with behavioral memory assessments, actigraphy, and parent-report to examine sleep-dependent memory consolidation. The focus of this protocol is on early childhood, a period of significance as children transition from biphasic sleep (consisting of a nap and overnight sleep) to monophasic sleep (overnight sleep only). The effects of sleep on memory performance are measured using a visuospatial memory assessment across periods of sleep and wakeful-rest. A combination of actigraphy and parent report is used to assess sleep rhythms (i.e., characterizing children as habitual or non-habitual nappers). Finally, PSG is used to characterize sleep stages and qualities of those stages (such as frequencies and the presence of spindles) during naps. The advantage of using PSG is that it is the only tool currently available to assess sleep quality and sleep architecture, pointing to the relevant brain state that supports memory consolidation. The main limitations of PSG are the length of time it takes to prepare the recording montage and that recordings are typically taken over one sleep bought. These limitations can be overcome by engaging young participants in distracting tasks during application and combining PSG with actigraphy and self/parent-report measures to characterize sleep cycles. Together, this unique combination of methods allows for investigations into how naps support learning in preschool children.

This protocol describes methods used to examine neural mechanisms underlying sleep-dependent memory consolidation during naps in early childhood. It includes procedures for examining the effect of sleep on behavioral memory performance, as well as the application and recording of both polysomnography and actigraphy.

Sleep is critical for daily functioning. One important function of sleep is the consolidation of memories, a process that makes them stronger and less ...

Simultaneous Application of Transcranial Direct Current Stimulation during Virtual Reality Exposure

Transcranial direct current stimulation (tDCS) is a form of non-invasive brain stimulation that changes the likelihood of neuronal firing through modulation of neural resting membranes. Compared to other techniques, tDCS is relatively safe, cost-effective, and can be administered while individuals are engaged in controlled, specific cognitive processes. This latter point is important as tDCS may predominantly affect intrinsically active neural regions. In an effort to test tDCS as a potential treatment for psychiatric illness, the protocol described here outlines a novel procedure that allows the simultaneous application of tDCS during exposure to trauma-related cues using virtual reality (tDCS+VR) for veterans with posttraumatic stress disorder (NCT03372460). In this double-blind protocol, participants are assigned to either receive 2 mA tDCS, or sham stimulation, for 25 minutes while passively watching three 8-minute standardized virtual reality drives through Iraq or Afghanistan, with virtual reality events increasing in intensity during each drive. Participants undergo six sessions of tDCS+VR over the course of 2-3 weeks, and psychophysiology (skin conductance reactivity) is measured throughout each session. This allows testing for within and between session changes in hyperarousal to virtual reality events and adjunctive effects of tDCS. Stimulation is delivered through a built-in rechargeable battery-driven tDCS device using a 1 (anode) x 1 (cathode) unilateral electrode set-up. Each electrode is placed in a 3 x 3 cm (current density 2.22 A/m2) reusable sponge pocket saturated with 0.9% normal saline. Sponges with electrodes are attached to the participant&#8217;s skull using a rubber headband with the electrodes placed such that they target regions within the ventromedial prefrontal cortex. The virtual reality headset is placed over the tDCS montage in such a way as to avoid electrode interference.

This manuscript outlines a novel protocol to allow the simultaneous application of transcranial direct current stimulation during exposure to warzone trauma-related cues using virtual reality for veterans with posttraumatic stress disorder.