Name: the evoked potential operant conditioning system (epocs): a research tool and an emerging therapy for chronic neuromuscular disorders
Uploaded: 2022-08-25T13:00:00
Duration: 10 min 8 s
Description: Watch this Scientific Journal Video about The Evoked Potential Operant Conditioning System EPOCS: A Research Tool and an Emerging Therapy for Chronic Neuromuscular Disorders at JoVE.com

This work was supported by NIH (NIBIB) P41EB018783 (JRW), NIH (NINDS) R01NS114279 (AKT), NIH (NINDS) U44NS114420 (I. Clements, AKT, JRW), NYS SCIRB C33279GG &#38; C32236GG (JRW), NIH (NICHD) P2C HD086844 (S. Kautz), The Doscher Neurorehabilitation Research Program (AKT), and Stratton Albany VA Medical Center.

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M., Velez, J., Falivena, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operant+up-conditioning+of+the+tibialis+anterior+motor-evoked+potential+in+multiple+sclerosis:+feasibility+case+studies.">Operant up-conditioning of the tibialis anterior motor-evoked potential in multiple sclerosis: feasibility case studies.</a> Neural Plasticity. , 4725393 (2018).</li><li>Thompson, A. K., Chen, X. Y., Wolpaw, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+a+simple+motor+skill:+Task-dependent+adaptation+plus+long-term+change+in+the+human+soleus+H-reflex.">Acquisition of a simple motor skill: Task-dependent adaptation plus long-term change in the human soleus H-reflex.</a> Journal of Neuroscience. 29 (18), 5784-5792 (2009).</li><li>Makihara, Y., Segal, R. L., Wolpaw, J. R., Thompson, A. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+H-reflex+as+a+tool+in+neurophysiology:+Its+limitations+and+uses+in+understanding+nervous+system+function.">The H-reflex as a tool in neurophysiology: Its limitations and uses in understanding nervous system function.</a> Muscle &amp; Nerve. 28 (2), 144-160 (2003).</li><li>Pierrot Deseilligny, E., Burke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Circuitry of the Human Spinal Cord: Its Role in Motor Control and Movement Disorders. , (2012).</li><li>Skinner, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Behavior of Organisms: An Experimental Analysis. , (1938).</li><li>Eftekhar, A., Norton, J. J. S., McDonough, C. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplitude+of+the+maximum+motor+response+(Mmax)+in+human+muscles+typically+decreases+during+the+course+of+an+experiment.">Amplitude of the maximum motor response (Mmax) in human muscles typically decreases during the course of an experiment.</a> Experimental Brain Research. 124 (2), 265-270 (1999).</li><li>Devetzoglou-Toliou, S., 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=Recursive+PID+controller+for+automatically+adjusting+M-wave+size+during+H-reflex+operant+conditioning.">Recursive PID controller for automatically adjusting M-wave size during H-reflex operant conditioning.</a> International IEEE/EMBS Conference on Neural Engineering. 10, 1079-1082 (2021).</li><li>Wolpaw, J. R., Seegal, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diurnal+rhythm+in+the+spinal+stretch+reflex.">Diurnal rhythm in the spinal stretch reflex.</a> Brain Research. 244 (2), 365-369 (1982).</li><li>Chen, X. Y., Wolpaw, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythm+in+rat+H-reflex.">Circadian rhythm in rat H-reflex.</a> Brain Research. 648 (1), 167-170 (1994).</li><li>Carp, J. S., Tennissen, A. M., Chen, X. Y., Wolpaw, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diurnal+H-reflex+variation+in+mice.">Diurnal H-reflex variation in mice.</a> Experimental Brain Research. 168 (4), 517-528 (2006).</li><li>Lagerquist, O., Zehr, E. P., Baldwin, E. R., Klakowicz, P. M., Collins, D. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operant+conditioning+of+spinal+stretch+reflexes+in+patients+with+spinal+cord+injuries.">Operant conditioning of spinal stretch reflexes in patients with spinal cord injuries.</a> Experimental Neurology. 130 (2), 202-213 (1994).</li><li>Wolf, S. L., Segal, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+human+biceps+brachii+spinal+stretch+reflex+magnitude.">Reducing human biceps brachii spinal stretch reflex magnitude.</a> Journal of Neurophysiology. 75 (4), 1637-1646 (1996).</li><li>Mrachacz-Kersting, N., 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=Acquisition+of+a+simple+motor+skill:+Task-dependent+adaptation+and+long-term+changes+in+the+human+soleus+stretch+reflex.">Acquisition of a simple motor skill: Task-dependent adaptation and long-term changes in the human soleus stretch reflex.</a> Journal of Neurophysiology. 122 (1), 435-446 (2019).</li><li>Thompson, A. 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JRW and AKT are inventors on three patents related to H-reflex conditioning. The other authors have no competing financial interests or conflicts of interest to report.

The protocol described above is suitable for demonstrating soleus H-reflex down-conditioning in a typical adult without neurological impairment. The precise parameter values may vary from person to person and particularly as a function of impairment. Whereas the participant&#39;s recruitment curve reached Mmax at a stimulating current of around 25 mA in the video, another person might require 50 mA or more, so the current would be increased in larger steps during recruitment curve measurement. They might also require a longer pulse duration. A third person might be more sensitive and require smaller current settings. The protocol also needs to be adapted according to the muscle that is being conditioned. For example, when targeting the flexor carpi radialis muscle24,25, a lower current setting is generally used; the Voluntary Contraction mode should be used to establish a scale for the background-EMG limits; and greater care must be taken both during the optimization of electrode placement and during the optimization of posture, which must then be kept constant across trials.
The protocol is sensitive to variations in the relationship between stimulator current setting and the amount of current actually delivered to the nerve&#8212;this may be affected by small variations in posture, hydration of the participant, and drying out of the adhesive electrode gel. In H-reflex conditioning, this problem can be mitigated by using M-wave size as an indicator of effective stimulation intensity. It reflects the number of soleus motoneuron efferent axons excited by the stimulus. Thus, if M-wave size is kept constant, it implies that the number of primary afferent axons excited by the stimulus, i.e., the axons that elicit the H-reflex, is also kept constant (see also Crone et al.26). Hence, this M-wave is referred to as the reference response in the software. For this reason, step 4.5.12. mentions that the target M-wave size should be recorded. It is actually more important to keep this response size roughly constant than to keep the nominal current strictly constant. The Sequence tab of the analysis window allows retrospective verification of M-wave constancy over each run; for soleus H-reflex conditioning, this is often sufficient to correct any problems. For greater control, a second monitor may be attached to the computer to display real-time M-wave analytics that guide trial-by-trial manual adjustment. Automation of this control task is an ongoing project27.
Diurnal variation may also affect a person&#39;s electrophysiological responses28,29,30,31. For this reason, it is recommended that all sessions be performed at the same time of day, i.e., within the same 3 h time window.
The success of operant conditioning may be sensitive to the accuracy of the time interval chosen by the operator to define the H-reflex; in particular, the interval should not be too wide. Detailed guidelines for correct interval definition are beyond the scope of the current article. This is also a function that will be automated in future versions of the software.
A critical step in the protocol is step 4.5.6., in which the operator manually increases the stimulator current repeatedly after each fixed number of trials. Mis-counting the trials here or mis-adjusting the current dial can lead to distortion of the resulting recruitment curve. This possibility of user error can be mitigated by enabling the Digitimer Link option, which allows automation of the current adjustment for one particular stimulator model.
This article has focused on H-reflex conditioning, as it is the most fully developed of the potential clinical applications of EPOCS. The existing software helps researchers in the ongoing efforts to hone this protocol toward wide clinical dissemination32. Beyond H-reflex conditioning, EPOCS may also be applied in its current form to a wider variety of stimulation methods and evoked responses. For example, it can equally well trigger a mechanical device that elicits a stretch reflex, which may also be conditioned33,34,35. The approach is adaptable to an individual&#39;s impairments; in one person, down-conditioning the soleus H-reflex improves locomotion by reducing spastic hyperreflexia14; in another, up-conditioning the tibialis anterior MEP improves locomotion by alleviating foot drop36.
While efforts are ongoing to produce a commercial implementation of the protocol, the original software will be maintained in parallel as a research tool to provide the necessary flexibility to expand the field of targeted neuroplasticity. This flexibility is enabled by the modularity and extensibility of the widespread and well-established BCI2000 software platform, on which EPOCS is based. This means that, with minimal intervention by a software engineer, the system is re-configurable for an even wider variety of research purposes. For example, it can be configured to record additional biosignal channels or additional sensors for later analysis (e.g., foot switches and motion tracking sensors) for conditioning during locomotion. It can also be programmed to consider additional triggering criteria for stimulation (e.g., triggering stimulation only at a particular part of the gait cycle) or to trigger additional reinforcement stimuli on successful or unsuccessful trials. Example customization files are provided.
Targeted neuroplasticity is still in its infancy. Its as-yet unexplored avenues are expected to provide great benefits both for developing novel therapeutic approaches (as discussed above) and for elucidating the natural history of disease and the mechanisms of central nervous system function in both health and disease2,32,37. We are, therefore, committed to maintaining and supporting EPOCS as a key tool for realizing this therapeutic and scientific potential.

Over the past decade, targeted neuroplasticity strategies have emerged as a new approach for the rehabilitation of neurological impairments1,2. One such strategy is operant conditioning of an evoked potential. This entails repeatedly eliciting electrophysiological responses that can be measured non-invasively - for example, by electroencephalography (EEG) or surface electromyography (EMG) - and giving the person immediate feedback on the size of each response relative to a criterion level set by the therapist or investigator. Over time, this protocol trains the person to increase or decrease their response and can, consequently, target beneficial change to a central nervous system site that is important in a behavior such as locomotion or reach-and-grasp. The targeted change benefits performance and, in addition, enables better practice that leads to widespread beneficial change that improves the entire behavior. For example, in people with incomplete spinal cord injury (iSCI) in whom clonus impairs locomotion, operant conditioning that reduces the Hoffmann reflex in the soleus muscle of one leg improves locomotor muscle activity in both legs, thereby increasing walking speed and restoring right/left step symmetry1,3,4,5. Another example is that of paired-pulse stimulation, which can lastingly increase the size of the motor-evoked potential (MEP) to transcranial magnetic stimulation, thereby improving reach-and-grasp function in people with chronic hand and arm impairment following iSCI6.
Implementing such protocols demands special-purpose software that must perform multiple functions. Specifically, it must continuously acquire, process, and save electrophysiological signals; it must continuously monitor the state of the nervous system and trigger stimulation appropriately under tight real-time constraints; it must provide continuous moment-by-moment feedback, trial-by-trial feedback, and session-by-session feedback; it must provide a user interface to guide setup and tuning by the investigator or therapist; and, finally, it must store and organize signal data and meta-information in a standardized format.
The evoked potential operant conditioning system (EPOCS) is our answer to this outstanding need. Under the hood, the software is based on BCI2000, an open-source neurotechnology platform that is used in hundreds of laboratories around the world7,8. In EPOCS, BCI2000&#39;s usual user interface is hidden and replaced by a streamlined interface that is optimized for evoked potential operant conditioning protocols.
The current article and its accompanying video illustrate the use of EPOCS in one particular protocol: operant conditioning to reduce the size of the Hoffmann (H-) reflex in the soleus muscle. This response is the electrically elicited analog of the knee-jerk stretch reflex. H-reflex down-conditioning has been shown to reduce the impact of clonus on, and to thereby improve, locomotion in animals with iSCI9,10,11,12,13 and in humans with iSCI, multiple sclerosis, or stroke5,14,15. It can be applied without adverse side effects in animals and people with or without neurological injury16,17.
The operant conditioning protocol functions by performing multiple trials, each lasting several seconds. The sequence of events of one trial is shown schematically in Figure 1, with numbers denoting the following functions:
1. Continuous background EMG is recorded from bipolar surface electrodes over the target muscle (soleus) and its antagonist (tibialis anterior). The background level is evaluated as the mean rectified value of the high-pass-filtered signal in a sliding window.
2. Background EMG level in the target muscle is shown as the height of a bar, continuously updated on the participant&#39;s screen. This helps the participant to keep the activity within a specified range (hatched region).
3. The software judges the appropriate moment for electrical stimulation and triggers the stimulator accordingly. The principal criteria are that at least 5 s must have elapsed since the previous stimulation and that the background EMG level must have remained in the specified range continuously for 2 s.
4. A constant-current stimulator delivers an electrical pulse transcutaneously to the tibial nerve (typically monophasic, with 1 ms duration).
5. The resulting stimulus-locked response is recorded. The software computes the sizes of two components of particular interest: the earlier M-wave, which reflects muscle activation resulting from direct stimulation of the motor axon; and the later H-reflex, which reflects the signal relayed through a reflex arc in the spinal cord18,19,20,21,22. EPOCS refers to these as the reference response and target response, respectively.
6. H-reflex size for the current trial is displayed as the height of a second bar, relative to a desired criterion level that defines a successful or unsuccessful trial. For down-conditioning, the bar is dark green if the H-reflex size fell below the criterion, or bright red if it did not (vice versa for up-conditioning). Simultaneously, the numeric display of the cumulative success rate is updated accordingly. Together, these graphical display elements provide the immediate positive or negative reinforcement on which operant conditioning relies23.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63736/63736fig01.jpg" /> 
Figure 1: Schematic illustration of EPOCS&#39; core functionality during down-conditioning of the soleus H-reflex. The participant views a large monitor screen that shows the background EMG level, the most recent H-reflex size, the number of trials completed so far in the current run of 75, and the running proportion of successful trials for the run. The sequence of events in one trial is denoted by the numbers 1-6, as described in the Introduction. <a href="https://www.jove.com/files/ftp_upload/63736/63736fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A human H-reflex conditioning protocol typically consists of 6 baseline sessions, followed by 24-30 conditioning sessions spread over 10 weeks at a rate of 3 sessions/week, and several follow-up sessions over the subsequent 3-6 months14,16. Each session lasts 60-90 min.
To support this protocol as well as other related protocols, EPOCS has five distinct modes of operation, each served by one of the tabs of its main window, entitled Stimulus Test, Voluntary Contraction, Recruitment Curve, Control Trials, and Training Trials.
In Stimulus Test mode, the software triggers a stimulus every few seconds, not necessarily contingent on the state of the target muscle. The response signals are shown on the screen after each stimulus. This allows the operator to verify the quality of the electrode connections and the EMG signal; to optimize the position of the stimulating and recording electrodes; and to establish the individual&#39;s response morphology.
In Voluntary Contraction mode, the software measures and shows the background EMG level while the participant is encouraged to contract the muscle as much as possible, in the absence of electrical stimulation. In some protocols, the EMG level at maximum voluntary contraction (MVC) is a useful reference for setting the background EMG criteria. In the protocol demonstrated here, this is not necessary, as a stable standing posture standardizes the activity of the soleus muscle sufficiently.
In Recruitment Curve mode, stimulation is contingent on the background EMG level (shown continuously on the screen) remaining in the correct range; response signals are shown on the screen after each stimulus; and the sequence of responses may be analyzed at the end of a run. This allows the operator to determine the start and end of the time intervals in which the responses of interest appear; to determine the relationship between stimulation intensity and response size, both before and after the conditioning runs; and to determine the stimulation intensity to be used for conditioning.
In Control Trials mode, stimulation is contingent on the background EMG level (shown continuously on screen), but no feedback is given about the target response size. The sequence and distribution of response sizes may be analyzed. This mode may be used to gather baseline measurements of response size, or as a control condition for comparison against operant conditioning in a crossover or between-subjects experimental design. It can serve as a basis for setting the performance criterion for operant conditioning at the beginning of each session.
Finally, in Training Trials mode, stimulation is contingent on the background EMG level (shown continuously on screen), and trial-by-trial reinforcement is also provided by showing the target response size, as described above and shown in Figure 1. This is the mode in which operant conditioning is performed.
The next section will guide the reader through the five modes by demonstrating the protocol for down-conditioning the soleus H-reflex in an adult participant without neurological injury.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alcohol swabs</td>
			<td>any</td>
			<td></td>
			<td>For application to skin</td>
		</tr>
		<tr>
			<td>BNC cable (long) x 1</td>
			<td>any</td>
			<td></td>
			<td>Male BNC to male BNC, long enough to reach from digitizer to stimulator</td>
		</tr>
		<tr>
			<td>BNC cable (medium) x 2</td>
			<td>any</td>
			<td></td>
			<td>Male BNC to male BNC, long enough to reach from amplifier to digitizer</td>
		</tr>
		<tr>
			<td>BNC cable (short) x 1</td>
			<td>any</td>
			<td></td>
			<td>Male BNC to male BNC, short (to patch between two digitizer ports)</td>
		</tr>
		<tr>
			<td>BNC tee connector</td>
			<td>any</td>
			<td></td>
			<td>Female-male-female BNC splitter</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Lenovo</td>
			<td>ThinkStation P340</td>
			<td>A wide range of computing hardware is suitable, especially if using a USB digitizer (no PCI slots needed).&#160; Must run Windows 7+. Include standard keyboard &#38; mouse.</td>
		</tr>
		<tr>
			<td>Constant-current stimulator</td>
			<td>Digitimer Ltd.</td>
			<td>DS8R</td>
			<td>The DS8R enjoys EPOCS automation support. If controlled manually,&#160; other constant-current stimulators may be used provided they have an external TTL trigger and can achieve a pulse duration of 1&#160;ms or more.</td>
		</tr>
		<tr>
			<td>Digitizer (option A)</td>
			<td>National Instruments</td>
			<td>USB-6212</td>
			<td>USB digitizer with integrated BNC connectors.</td>
		</tr>
		<tr>
			<td>Digitizer (option B)</td>
			<td>National Instruments</td>
			<td>PCIe-6321</td>
			<td>PCIe digitizer&#8212;requires desktop computer with a free PCI slot, also cable and BNC terminal block (below)</td>
		</tr>
		<tr>
			<td>Digitizer cable (for option B only)</td>
			<td>National Instruments</td>
			<td>SHC68-68-EPM</td>
			<td>Connects PCIe digitizer to BNC terminal block</td>
		</tr>
		<tr>
			<td>Digitizer terminal block (for option B only)</td>
			<td>National Instruments</td>
			<td>BNC-2090A</td>
			<td>19-inch-rack-mountable BNC terminal block</td>
		</tr>
		<tr>
			<td>EMG amplifier system</td>
			<td>Bortec Biomedical Ltd.</td>
			<td>AMT-8</td>
			<td>Analog amplifier + portable unit + long transmission cable + battery pack + two 500-gain active electrode leads (1 bipolar, 1 bipolar with ground)</td>
		</tr>
		<tr>
			<td>Monitor</td>
			<td>any</td>
			<td></td>
			<td>Large enough for the participant to see clearly from the intended viewing distance.</td>
		</tr>
		<tr>
			<td>NeuroPlus electrodes (22&#160;x&#160;22&#160;mm) x 6</td>
			<td>Vermont Medical Inc.</td>
			<td>A10040-60</td>
			<td>Disposable self-adhesive silver/silver-chloride 22&#160;x&#160;22&#160;mm&#160;surface-EMG electrodes. 6 needed per session (11 on participant&#39;s first session)</td>
		</tr>
		<tr>
			<td>NeuroPlus electrode (22&#160;x&#160;35&#160;mm) x 1</td>
			<td>Vermont Medical Inc.</td>
			<td>A10041-60</td>
			<td>Disposable self-adhesive silver/silver-chloride 22 x 35 mm surface-EMG electrode. 1 needed per session.</td>
		</tr>
		<tr>
			<td>Snap lead x 2</td>
			<td>any</td>
			<td>EDR1220</td>
			<td>Leads for stimulating electrodes: 1.5mm DIN to button snap</td>
		</tr>
		<tr>
			<td>Wire</td>
			<td>any</td>
			<td></td>
			<td>8&#8211;10 cm length of single-core insulated wire</td>
		</tr>
	</tbody>
</table>

the evoked potential operant conditioning system (epocs): a research tool and an emerging therapy for chronic neuromuscular disorders

The Evoked Potential Operant Conditioning System (EPOCS) is a software tool that implements protocols for operantly conditioning stimulus-triggered muscle responses in people with neuromuscular disorders, which in turn can improve sensorimotor function when applied appropriately. EPOCS monitors the state of specific target muscles&#8212;e.g., from surface electromyography (EMG) while standing, or from gait cycle measurements while walking on a treadmill&#8212;and automatically triggers calibrated stimulation when pre-defined conditions are met. It provides two forms of feedback that enable a person to learn to modulate the targeted pathway&#39;s excitability. First, it continuously monitors ongoing EMG activity in the target muscle, guiding the person to produce a consistent level of activity suitable for conditioning. Second, it provides immediate feedback of the response size following each stimulation and indicates whether it has reached the target value.
To illustrate its use, this article describes a protocol through which a person can learn to decrease the size of the Hoffmann reflex&#8212;the electrically-elicited analog of the spinal stretch reflex&#8212;in the soleus muscle. Down-conditioning this pathway&#39;s excitability can improve walking in people with spastic gait due to incomplete spinal cord injury. The article demonstrates how to set up the equipment; how to place stimulating and recording electrodes; and how to use the free software to optimize electrode placement, measure the recruitment curve of direct motor and reflex responses, measure the response without operant conditioning, condition the reflex, and analyze the resulting data. It illustrates how the reflex changes over multiple sessions and how walking improves. It also discusses how the system can be applied to other kinds of evoked responses and to other kinds of stimulation, e.g., motor evoked potentials to transcranial magnetic stimulation; how it can address various clinical problems; and how it can support research studies of sensorimotor function in health and disease.

Figure 2 shows the effectiveness of the above protocol in measuring M-wave and H-reflex recruitment curves and in measuring the distribution of H-reflex sizes at constant stimulation intensity. It also illustrates the overall effectiveness of the multi-session protocol in changing the H-reflex size in neurologically unimpaired participants and in improving locomotor function in participants with incomplete spinal cord injury.
Figure 2A shows a screenshot of the analysis window following a run performed in Recruitment Curve mode during H-reflex operant conditioning (see protocol step 4.5.). In the lower half of the window (the Sequence pane), the horizontal axis shows trial number&#8212;hence, stimulus intensity increases from left to right. H-reflex size (green circles) rises then falls as a function of stimulus intensity, whereas M-wave size (brown triangles) rises then saturates. Figure 2B shows a screenshot of the analysis window following a run performed in Control Trials or Training Trials mode during H-reflex operant conditioning (see protocol step 4.6. and step 4.7.). In the lower panel (the &#34;Distribution&#34; pane), the histogram of H-reflex sizes facilitates the selection of an appropriate criterion level for subsequent up- or down-conditioning. In Figure 2C, H-reflex size in neurologically unimpaired participants is plotted as a function of session number across 6 baseline sessions, 24 conditioning sessions, and 4 follow-up sessions. Data were collected from 15 participants (8 male, 7 female) of whom 2 participated in both up- and down-conditioning arms. Participants were aged 21-55 years. All the participants gave informed consent. The protocol was approved by the institutional review board (IRB) of the New York State Department of Health (approval number 05-058). Thompson et al.16 provides further details. Figure 2D shows the beneficial effect of soleus H-reflex down-conditioning in participants with chronic lower limb impairment following incomplete spinal cord injury. Successful conditioning was associated with an improvement in gait symmetry and in walking speed relative to baseline. Data were collected from 13 participants (9 male, 4 female) aged 28-68 years, who gave informed consent. The protocol was approved by the IRB of Helen Hayes Hospital (approval number 07-07). Thompson et al.14 provides further details.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63736/63736fig02.jpg" /> 
Figure 2: Representative results. (A) Screenshot of the recruitment curve analysis window. (B) Screenshot of the Control Trials or Training Trials analysis window. (C) Contrasting effects of up- and down-conditioning of the soleus H-reflex in uninjured participants. Red upward triangles show mean H-reflex size from N = 6 successfully up-conditioned participants (out of 8); blue downward triangles show mean responses from N = 8 successfully down-conditioned participants (out of 9). Error bars denote standard error. This image has been modified from Thompson et al.16. (D) Therapeutic effect of soleus H-reflex down-conditioning on walking speed and gait symmetry in people with chronic impairment following incomplete spinal cord injury. The bars contrast results for N = 6 participants whose H-reflexes were successfully down-conditioned against N = 4 participants from the control condition (no operant conditioning) and N = 3 participants in whom the down-conditioning protocol failed to reduce reflex size. Error bars denote standard error. Each asterisk indicates a p-value below 0.05 on a paired t-test comparing pre- against post-conditioning measurements. This image has been modified from Thompson et al.14. <a href="https://www.jove.com/files/ftp_upload/63736/63736fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about The Evoked Potential Operant Conditioning System EPOCS: A Research Tool and an Emerging Therapy for Chronic Neuromuscular Disorders at JoVE.com

The Evoked Potential Operant Conditioning System EPOCS: A Research Tool and an Emerging Therapy for Chronic Neuromuscular Disorders

The Evoked Potential Operant Conditioning System aids scientific investigation of sensorimotor function and can administer targeted neurobehavioral training that can impact sensorimotor rehabilitation in neuromuscular disorders. This article describes its capabilities and illustrates its application in modifying a simple spinal reflex to achieve lasting improvement in motor function.

The Evoked Potential Operant Conditioning System (EPOCS): A Research Tool and an Emerging Therapy for Chronic Neuromuscular Disorders

The Evoked Potential Operant Conditioning System (EPOCS) is a software tool that implements protocols for operantly conditioning stimulus-triggered muscle ...

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SCIENTISTS IN ACTION

Operant Sensation Seeking in the Mouse

Operant methods are powerful behavioral tools for the study of motivated behavior. These 'self-administration' methods have been used extensively in drug addiction research due to their high construct validity. Operant studies provide researchers a tool for preclinical investigation of several aspects of the addiction process. For example, mechanisms of acute reinforcement (both drug and non-drug) can be tested using pharmacological or genetic tools to determine the ability of a molecular target to influence self-administration behavior1-6. Additionally, drug or food seeking behaviors can be studied in the absence of the primary reinforcer, and the ability of pharmacological compounds to disrupt this process is a preclinical model for discovery of molecular targets and compounds that may be useful for the treatment of addiction3,7-9. One problem with performing intravenous drug self-administration studies in the mouse is the technical difficulty of maintaining catheter patency. Attrition rates in these experiments are high and can reach 40% or higher10-15. Another general problem with drug self-administration is discerning which pharmacologically-induced effects of the reinforcer produce specific behaviors. For example, measurement of the reinforcing and neurological effects of psychostimulants can be confounded by their psychomotor effects. Operant methods using food reinforcement can avoid these pitfalls, although their utility in studying drug addiction is limited by the fact that some manipulations that alter drug self-administration have a minimal impact on food self-administration. For example, mesolimbic dopamine lesion or knockout of the D1 dopamine receptor reduce cocaine self-administration without having a significant impact on food self-administration 12,16. 
Sensory stimuli have been described for their ability to support operant responding as primary reinforcers (i.e. not conditioned reinforcers)17-22. Auditory and visual stimuli are self-administered by several species18,21,23, although surprisingly little is known about the neural mechanisms underlying this reinforcement. The operant sensation seeking (OSS) model is a robust model for obtaining sensory self-administration in the mouse, allowing the study of neural mechanisms important in sensory reinforcement24. An additional advantage of OSS is the ability to screen mutant mice for differences in operant behavior that may be relevant to addiction. We have reported that dopamine D1 receptor knockout mice, previously shown to be deficient in psychostimulant self-administration, also fail to acquire OSS24. This is a unique finding in that these mice are capable of learning an operant task when food is used as a reinforcer. While operant studies using food reinforcement can be useful in the study of general motivated behavior and the mechanisms underlying food reinforcement, as mentioned above, these studies are limited in their application to studying molecular mechanisms of drug addiction. Thus, there may be similar neural substrates mediating sensory and psychostimulant reinforcement that are distinct from food reinforcement, which would make OSS a particularly attractive model for the study of drug addiction processes. The degree of overlap between other molecular targets of OSS and drug reinforcers is unclear, but is a topic that we are currently pursuing. While some aspects of addiction such as resistance to extinction may be observed with OSS, we have found that escalation 25 is not observed in this model24. Interestingly, escalation of intake and some other aspects of addiction are observed with self-administration of sucrose26. Thus, when non-drug operant procedures are desired to study addiction-related processes, food or sensory reinforcers can be chosen to best fit the particular question being asked. 
In conclusion, both food self-administration and OSS in the mouse have the advantage of not requiring an intravenous catheter, which allows a higher throughput means to study the effects of pharmacological or genetic manipulation of neural targets involved in motivation. While operant testing using food as a reinforcer is particularly useful in the study of the regulation of food intake, OSS is particularly apt for studying reinforcement mechanisms of sensory stimuli and may have broad applicability to novelty seeking and addiction.

In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.

Operant methods are powerful behavioral tools for the study of motivated behavior.  These 'self-administration' methods have been used extensively in drug ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

A Human-machine-interface Integrating Low-cost Sensors with a Neuromuscular Electrical Stimulation System for Post-stroke Balance Rehabilitation

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing delivery of oxygen and nutrients. About half of the stroke survivors are left with some degree of disability. Innovative methodologies for restorative neurorehabilitation are urgently required to reduce long-term disability. The ability of the nervous system to reorganize its structure, function and connections as a response to intrinsic or extrinsic stimuli is called neuroplasticity. Neuroplasticity is involved in post-stroke functional disturbances, but also in rehabilitation. Beneficial neuroplastic changes may be facilitated with non-invasive electrotherapy, such as neuromuscular electrical stimulation (NMES) and sensory electrical stimulation (SES). NMES involves coordinated electrical stimulation of motor nerves and muscles to activate them with continuous short pulses of electrical current while SES involves stimulation of sensory nerves with electrical current resulting in sensations that vary from barely perceivable to highly unpleasant. Here, active cortical participation in rehabilitation procedures may be facilitated by driving the non-invasive electrotherapy with biosignals (electromyogram (EMG), electroencephalogram (EEG), electrooculogram (EOG)) that represent simultaneous active perception and volitional effort. To achieve this in a resource-poor setting, e.g., in low- and middle-income countries, we present a low-cost human-machine-interface (HMI) by leveraging recent advances in off-the-shelf video game sensor technology. In this paper, we discuss the open-source software interface that integrates low-cost off-the-shelf sensors for visual-auditory biofeedback with non-invasive electrotherapy to assist postural control during balance rehabilitation. We demonstrate the proof-of-concept on healthy volunteers.

A novel low-cost human-machine interface for interactive post-stroke balance rehabilitation system is presented in this article. The system integrates off-the-shelf low-cost sensors towards volitionally driven electrotherapy paradigm. The proof-of-concept software interface is demonstrated on healthy volunteers.

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...

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 ...

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (&#8805;8) as needed for transcriptional profiling experiments.

This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing.

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or ...

A Protocol for Transcranial Photobiomodulation Therapy in Mice

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide range of neurological and psychiatric disorders, and memory enhancement in age-related cognitive decline and neurodegenerative diseases. We describe a laboratory protocol for transcranial photobiomodulation therapy (PBMT) in mice. Aged BALB/c mice (18 months old) are treated with a 660 nm laser transcranially, once daily for 2 weeks. Laser transmittance data shows that approximately 1% of the incident red light on the scalp reaches a 1 mm depth from the cortical surface, penetrating the dorsal hippocampus. Treatment outcomes are assessed by two methods: a Barnes maze test, which is a hippocampus-dependent spatial learning and memory task evaluation, and measuring hippocampal ATP levels, which is used as a bioenergetics index. The results from the Barnes task show an enhancement of the spatial memory in laser-treated aged mice when compared with age-matched controls. Biochemical analysis after laser treatment indicates increased hippocampal ATP levels. We postulate that the enhancement of memory performance is potentially due to an improvement in hippocampal energy metabolism induced by the red laser treatment. The observations in mice could be extended to other animal models since this protocol could potentially be adapted to other species frequently used in translational neuroscience, such as rabbit, cat, dog, or monkey. Transcranial photobiomodulation is a safe and cost-effective modality which may be a promising therapeutic approach in age-related cognitive impairment.

Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide ...