The overall goal of this neurophysiological experiment is to assess changes in transmission at the Corticomotoneuronal synapses in humans after repetitive Transcranial Magnetic Stimulation. The H-Reflex conditioning technique can help and psyche questions in the field of neuroscience, such as, better to understand basic mechanisms and sites of neuroplasticity. We were especially interested in the plasticity taking place between the corticospinal tract and the motor neurone, the Corticomotoneuronal synapse.
The main advantage of this technique, compared to measure and compound potentials, is that it is possible to measure synaptic input produced by distinct Corticospinal connections to spinal motor neurons. There are at least two further advantages of conditioned H-reflexes compared to compound potentials. First, it's possible to detect influences of even weak Corticospinal volleys and second, it's possible to assess effects of longer latency Corticospinal volleys.
Prior to the experiment, first screen the subject for TMS medical risks such as, history of epileptic seizures, obtain written and informed consent and inform the subject about the purpose of the experiment. Prepare the skin for surface electrode placement over the soleus muscle by shaving, disinfecting with propanol and applying light skin abrasion. Then, place surface electrodes to measure electrophysiological responses.
Check the EMG signals on the screen, while the subject performs plantar and dorsiflexion. For H-Reflex conditioning, first use tape to fixate a five by five centimeter anode on the anterior aspect of the knee, just underneath the patella. Apply stimulation with square wave pulses lasting one millisecond and move the cathode on the popliteal fossa, until the optimal position for stimulation is located.
Then, place a self-adhesive electrode on the skin and fix in place with tape. Prior to the experiment, first record an HM recruitment curve. Calculate H-Reflexes and M-Waves as peak to peak EMG amplitudes online in the recording software.
Adjust the size of the H-Reflex to 20%of the maximum M-Wave. First, perform the calibration procedure for the neuro-navigational system. Use a figure eight coil to stimulate the subject's motor cortical area, contralateral to the placed electrodes.
To find the optimal stimulation spot, first place the coil one centimeter in front of the vertex. Point that handle of the coil backwards to evoke a posterior to anterior flux of induced current in the center of the coil. Identify the position where motor evoked potentials can be evoked with minimum stimulation intensity.
At this point, place the subject's head on the table and use rigid foam to prevent motion. Use an image guided TMS navigational system for a monitoring coil and head position throughout the experiment. Then, fixate the coil to a stand and the subject's head to the chair and fixate the coil with Velcro strips to the head.
Next, determine the minimum intensity required to evoke motor-evoked potentials with peak to peak EMG amplitudes larger than 50 micro-volts in six out of 10 consecutive trial. This is the subject's resting motor threshold. Also place a double cone magnetic coil at the cervicomedullary junction to excite axons of the corticospinal tract.
Position the coil so that the first derivative of the induced current is cranially directed and that its central position is on or near the enium. To begin, first take care to ensure that the size of the control H-Reflex stays constant throughout the experiment. If a deviation is detected, adjust the stimulation intensity prior to the consecutive trial.
Now, adjust the stimulation intensity of the TMS coil over the motor cortex to between 90%and 100%of the subject's motor threshold. Also set the cervicomedullary stimulation intensity to 100%of the maximum stimulator output. Begin by conditioning the H-Reflex with stimulation over the motor cortex at rest.
Apply TMS and peripheral nerve stimulation by varying the timing between the two stimuli to allow assessment of changes in Corticomotoneuronal transmission. Start with an interstimulus interval of negative five milliseconds, meaning peripheral nerve stimulation is illicited five seconds before TMS, then alter this interval in steps of milliseconds from negative five milli seconds to one millisecond. Depending on the interstimulus interval, the H-Reflex can be shifted forward in relationship to the descending volley where it can be shifted to the right, so that the slower corticospinal pathways can be tested.
Vary the interval randomly from trial to trial, to eliminate bias due to order of stimuli and set the pause between stimulation trials to four seconds. Early facilitation should occur around intervals of negative four to negative two milliseconds. Next, condition the H-Reflex using magnetic stimulation over the cervicomedullary junction.
Use interstimulus intervals between negative nine and negative three milliseconds in steps of one millisecond. Apply these TMS intervals over both locations together in each trial and record the resulting control H-Reflex and motor-evoked potentials. Use the control H-Reflex as a reference for the conditioned H-Reflexes and the control motor-evoked potential to ensure comparable stimulation conditions.
Once pre-measurement is completed, set the stimulation intensity to 1.2 times the subject's motor threshold and apply the slow repetitive TMS intervention. Stimulate over the primary motor cortex at one hertz for 20 minutes to induce long lasting suppression of corticospinal excitability. An H-Reflex conditioning curve after M1-conditioning is displayed here while the H-Reflex conditioning curve after CMS-conditioning is shown here.
Early facilitation with TMS over the primary motor cortex occurs around interstimulus intervals of negative three milliseconds. Note that the early facilitation after CMS-conditioning is occurring approximately three to four milliseconds earlier at an interstimulus interval of around negative seven milliseconds. Here, the averages of 10 traces for one representative subject before and after the rTMS intervention are displayed.
It can be seen that the conditioned H-Reflexes representing the early facilitation, are reduced after both M1 and CMS-conditioning. Whereas, the control H-Reflexes remain unchanged. And here, the mean of two subjects is displayed showing the same pattern reduction in both M1 and CMS-conditioned to H-Reflexes without any change in the control H-Reflex.
Once mastered, this protocol can be done in around 90 to 120 minutes. While performing this procedure, it's important to keep the H-Reflex size constant. After watching this video, you should have a good understanding of how to apply conditioning stimuli over the primary motor cortex and the cervicomedullary junction.
This is used to detect the early facilitation, which represents synaptic input to spinal motor neurons mediated by the fastest corticospinal connections.
