Name: non-invasive assessment of changes in corticomotoneuronal transmission in humans
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Description: Watch this Scientific Journal Video about Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans at JoVE.com

This study was supported by a grant from the Swiss National Science Foundation (316030_128826).

This protocol was approved by the local ethics committee and the experiments are in accordance with the Declaration of Helsinki (1964).
1. Subject Preparation
NOTE: Subject instructions - Before starting with the experiment, instruct each subject about the purpose of the study and potential risk factors. For transcranial magnetic stimulation (TMS), medical risks include any history of epileptic seizure, mental implants in eyes and/or ...

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	<li>Lemon, R. N., Kirkwood, P. A., Maier, M. A., Nakajima, K., Nathan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+and+indirect+pathways+for+corticospinal+control+of+upper+limb+motoneurons+in+the+primate.">Direct and indirect pathways for corticospinal control of upper limb motoneurons in the primate.</a> Prog.Brain Res. , 263-279 (2004).</li><li>Jankowska, E., Padel, Y., Tanaka, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+of+pyramidal+tract+cells+to+alpha-motoneurones+innervating+hind-limb+muscles+in+the+monkey.">Projections of pyramidal tract cells to alpha-motoneurones innervating hind-limb muscles in the monkey.</a> J. 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The H-reflex conditioning procedure described here has been specifically addressed to assess acute changes in transmission over the corticomotoneuronal synapse following repetitive activation of the corticospinal pathway28. In this respect, H-reflex conditioning has highlighted that rTMS does not only affect excitability of cortical structures but also has an effect on the corticomotoneural transmission at the corticomotoneural synapse. However, this method may indeed have broader application as c...

In primates, the corticospinal tract constitutes the major descending pathway controlling voluntary actions1. The corticospinal pathway connects motor cortical areas to spinal &#945;-motoneurons via direct monosynaptic corticomotoneuronal connections and via indirect oligo- and polysynaptic connections2,3. Although the motor cortex can easily be excited non-invasively by Transcranial Magnetic Stimulation (TMS), the evoked electromyographic response to this stimulation is often difficult to interpret. The reason for this is that the compound Motor Evoked Potential (MEP) can be influenced by changes in the excitability of intracortical and corticospinal neurons, spinal interneurons and spinal &#945;-motoneurons4,5,6,7. Several noninvasive electrophysiological techniques and stimulation protocols aim at determining whether changes in corticospinal excitability and transmission are caused by changes at the cortical or spinal level. Commonly, changes in the amplitude of the electrically evoked H-reflex are used as &#39;indicative&#39; of alterations of excitability at the motoneuron pool. However, it was previously shown that the H-reflex depends not only on the excitability of the motoneuron pool but is also modulated by other factors such as presynaptic inhibition8,9 or homosynaptic post-activation depression5,10. Another limitation when comparing MEPs and H-reflexes is the disability to detect excitability changes at the interneuronal level11,12. In addition to these drawbacks, the motoneurons might be differently activated by peripheral nerve stimulation than with TMS so that changes in the motoneuronal excitability would affect these responses in a different kind of way compared to responses mediated via the corticospinal pathway13,14,15.
Another method used to separate spinal from cortical effects represents Transcranial Electrical Stimulation (TES) of the motor cortex16. Applied at low stimulation intensities, TES was argued to be unaffected by changes in cortical excitability. As both TES and TMS activate the &#945;-motoneurons via the corticospinal pathway, the comparison of magnetically and electrically evoked MEPs provides a more attractive method to draw conclusions on the cortical nature of changes in the size of the MEPs than the comparison between H-reflexes and MEPs. However, when stimulation intensity is increased, TES-evoked MEPs are also influenced by changes in cortical excitability17,18. This problem can be circumvented when electrical stimulation is not applied to the motor cortex but at the cervicomedullary junction. However, although electrical stimulation can evoke cervicomedullary motor evoked potentials (cMEPs) in upper limb and lower limb muscles, most subjects perceive electrical stimulation at the brainstem (and cortex) as extremely unpleasant and painful. A less painful alternative is to activate the corticospinal pathway at the cervicomedullary junction by use of magnetic stimulation at the inion19. It is generally accepted that Cervicomedullary Magnetic Stimulation (CMS) activates many of the same descending fibers as motor cortical TMS and that changes in cortical excitability can be detected by comparing MEPs with cMEPs19. Increases in the excitability of intracortical cells and corticomotoneuronal cells are thought to facilitate the cortically evoked MEP without a concurrent change in the cervicomedullary evoked MEP.
However, in most subjects it is impossible to obtain magnetically evoked cMEPs in the lower extremity at rest20,21. One approach to overcome this problem is to elevate the excitability of spinal motoneurons by voluntary precontracting the target muscle. However, it is well known that slight changes in contraction strength influence the size of the cMEP. Thus, it is difficult to compare different tasks. In addition, changes in the motoneuronal excitability due to pre-contraction will influence MEPs and cMEPs but not necessarily to the same extent. Finally, by comparing compound MEPs with compound cMEPs some information contained in the descending volleys is lost. This has been revealed by studies involving conditioning of the H-reflex of soleus, tibialis anterior, and carpi radialis muscles by magnetic motor cortical stimulation12,22. By combining peripheral nerve stimulation and TMS over the motor cortex with specific interstimulus intervals (ISI), it is possible to study facilitatory and inhibitory effects of the different descending volleys on the H-reflex. This technique is greatly inspired by the spatial facilitation technique used to determine transmission in neural pathways in animal experiments and may be seen as a non-invasive, indirect version of that technique23. While the H-reflex is not only important to differentiate between different fractions of the corticospinal pathway (fast versus slower corticospinal projections) it is also essential to elevate spinal excitability in a controlled and comparable way. Thus, at rest and during activity, this combination of stimulation techniques allows assessment of changes in different fractions of the corticospinal pathway with a high temporal resolution, i.e. in the fastest, presumably monosynaptic corticomotoneuronal connections and in slower oligo- and polysynaptic pathways12,22,24,25. Recently, this technique was extended by not only conditioning the H-reflex with TMS over the motor cortex (M1-conditioning) but also by additional conditioning stimulation at the cervicomedullary junction (CMS-conditioning)26. By comparing effects between M1- and CMS-conditioning, this technique allows pathway specific differentiation with a high temporal resolution and it allows interpretations to be made on cortical versus spinal mechanisms. Furthermore and most importantly with respect to the current study, this technique allows assessment of transmission at the corticomotoneural synapse when considering the early facilitation. The early facilitation of the H-reflex is in all likelihood caused by activation of direct, monosynaptic corticomotoneural projections to the spinal motoneurons12,26. To test the fastest corticospinal pathways and thus, the early facilitation, the H-reflex has to be elicited 2 to 4 ms before the TMS. The reason for this is the slightly shorter latency of the MEP (around 32 ms; see27) compared to the H-reflex (around 34 ms; see25). Eliciting the H-reflex shortly before applying TMS, leads to convergence of the ascending and fastest descending excitations at the level of the spinal motoneurons. When TMS is applied over the cervicomedullary junction, the descending volley will arrive around 3 - 4 ms earlier at the spinal motoneuron pool than after stimulation over M1. For CMS-conditioning, peripheral nerve stimulation should therefore be evoked 6 - 8 ms before the magnetic pulse. A change of the early facilitation after CMS-conditioning indicates differential transmission at the synapse between the corticospinal tract and the &#945;-motoneuron28. In the current study, this recently developed technique was used to differentiate spinal from cortical effects following low frequency repetitive TMS (rTMS). More specifically, we hypothesized that if the early facilitation with M1-conditioning is reduced following the rTMS intervention but the early facilitation following CMS-conditioning is not, the effect should be purely cortical in origin. In contrast, if the early facilitation with CMS-conditioning also changes, this alteration should be related to mechanisms taking place at the spinal level. More specifically, as the early facilitation of the H-reflex is thought to be caused by activation of direct, corticomotoneuronal projections to the spinal motoneurones12,29, a change of the CMS-and M1-conditioned H-reflex at the time of the early facilitation should indicate an altered corticomotoneuronal transmission i.e. synaptic efficacy28.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Self-adhesive EMG electrodes</td>
			<td>Blue sensor N, Ambu, Ballerup, Denmark</td>
			<td></td>
			<td>Used to record EMG signals</td>
		</tr>
		<tr>
			<td>Electrical stimulator</td>
			<td>Digitimer DS7A, Hertfordshire, UK</td>
			<td></td>
			<td>Used to elicit the soleus H-reflex</td>
		</tr>
		<tr>
			<td>Stimulating electrode</td>
			<td>Blue sensor N, Ambu, Ballerup, Denmark</td>
			<td></td>
			<td>Used to elicit the soleus H-reflex</td>
		</tr>
		<tr>
			<td>Magnetic stimulator no1</td>
			<td>Magstim Rapid2 TMS stimulator, Magstim Company Ltd., Whitland, UK</td>
			<td></td>
			<td>Used to elicit contralateral motor evoked potentials in the soleus muscle</td>
		</tr>
		<tr>
			<td>Coil no1: 90 mm figure-of-eight coil&#160;</td>
			<td>Magstim Company Ltd., Whitland, UK</td>
			<td></td>
			<td>Used to elicit contralateral motor evoked potentials in the soleus muscle</td>
		</tr>
		<tr>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>Stimulator no1 and coil no1 were used in the original publication (Taube et al. 2014; Cerebral Cortex)</td>
		</tr>
		<tr>
			<td>Magnetic stimulator no2</td>
			<td>MagPro X100 with MagOption, MagVenture A/S, Farum, Denmark</td>
			<td></td>
			<td>Used to elicit contralateral motor evoked potentials in the soleus muscle</td>
		</tr>
		<tr>
			<td>Coil no2: 95-mm focal &#8220;butterfly-shaped&#8221; coil (D-B80)&#160;</td>
			<td>MagVenture A/S, Farum, Denmark</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Stimulator no2 and coil no2 were used in the video session</td>
		</tr>
		<tr>
			<td>Magnetic stimulator no3</td>
			<td>Magstim Company Ltd., Whitland, UK</td>
			<td></td>
			<td>Used to stimulate at the cervicomedullary junction</td>
		</tr>
		<tr>
			<td>Coil no3: double-cone magnetic coil</td>
			<td>Magstim Company Ltd., Whitland, UK</td>
			<td></td>
			<td>Used to stimulate at the cervicomedullary junction</td>
		</tr>
		<tr>
			<td>Image-guided TMS navigational system no1</td>
			<td>Brainsight 2, Rouge Research, Montreal, Canada</td>
			<td></td>
			<td>Used in the original publication (Taube et al. 2014; Cerebral Cortex) to monitor coil position throughout the experiment</td>
		</tr>
		<tr>
			<td>Image-guided TMS navigational system no2</td>
			<td colspan="2">TMS Navigator SW-Version 2.0, LOCALITE GmbH, Sankt Augustin, Germany</td>
			<td>Used for the video session</td>
		</tr>
		<tr>
			<td>Literature:&#160;</td>
			<td><a name="RANGE!B15"></a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Taube et al. 2014</td>
			<td colspan="3">Taube, W., Leukel, C., Nielsen, J. B. &#38; Lundbye-Jensen, J. Repetitive Activation of the Corticospinal Pathway by Means of rTMS may Reduce the Efficiency of Corticomotoneuronal Synapses. Cerebral cortex, doi:10.1093/cercor/bht359 (2014).</td>
		</tr>
	</tbody>
</table>

non-invasive assessment of changes in corticomotoneuronal transmission in humans

The corticospinal pathway is the major pathway connecting the brain with the muscles and is therefore highly relevant for movement control and motor learning. There exists a number of noninvasive electrophysiological methods investigating the excitability and plasticity of this pathway. However, most methods are based on quantification of compound potentials and neglect that the corticospinal pathway consists of many different connections that are more or less direct. Here, we present a method that allows testing excitability of different fractions of the corticospinal transmission. This so called H-reflex conditioning technique allows one to assess excitability of the fastest (monosynaptic) and also polysynaptic corticospinal pathways. Furthermore, by using two different stimulation sites, the motor cortex and the cervicomedullary junction, it allows not only differentiation between cortical and spinal effects but also assessment of transmission at the corticomotoneural synapse. In this manuscript, we describe how this method can be used to assess corticomotoneural transmission after low-frequency repetitive transcranial magnetic stimulation, a method that was previously shown to reduce excitability of cortical cells. Here we demonstrate that not only cortical cells are affected by this repetitive stimulation but also transmission at the corticomotoneuronal synapse at the spinal level. This finding is important for the understanding of basic mechanisms and sites of neuroplasticity. Besides investigation of basic mechanisms, the H-reflex conditioning technique may be applied to test changes in corticospinal transmission following behavioral (e.g., training) or therapeutic interventions, pathology or aging and therefore allows a better understanding of neural processes that underlie movement control and motor learning.

Occurrence of the early facilitation after M1- and CMS-conditioning
H-reflex conditioning with TMS over M1 resulted in an early facilitation that occurred around ISI -3 &#38; -4 ms. The early facilitation after CMS-conditioning occurred around 3 ms earlier (ISI -6 &#38; -7 ms, respectively). Exemplary ISI-curves of one subject are displayed in Figure 1. In the present study, the early facil...

Watch this Scientific Journal Video about Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans at JoVE.com

Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans

The aim of the present study was to assess changes in transmission at the corticomotoneuronal synapses in humans after repetitive transcranial magnetic stimulation. For this purpose, an electrophysiological method is introduced that allows assessment of pathway specific corticospinal transmission, i.e. differentiation of fast, direct corticospinal pathways from polysynaptic connections.

The corticospinal pathway is the major pathway connecting the brain with the muscles and is therefore highly relevant for movement control and motor ...

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.

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Research

JoVE Journal

Neuroscience

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

Visual Evoked Potential Recordings in Mice Using a Dry Non-invasive Multi-channel Scalp EEG Sensor

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable because of the plunger-spring-barrel structural facet and mechanical strengths resulting from metal materials. The whole process for acquiring the VEP responses in vivo from a mouse consists of four steps: (1) sensor assembly, (2) animal preparation, (3) VEP measurement, and (4) signal processing. This paper presents representative measurements of VEP responses from multiple mice with a submicro-voltage signal resolution and sub-hundred millisecond temporal resolution. Although the proposed method is safer and more convenient compared to other previously reported animal EEG acquiring methods, there are remaining issues including how to enhance the signal-to-noise ratio and how to apply this technique with freely moving animals. The proposed method utilizes easily available resources and shows a repetitive VEP response with a satisfactory signal quality. Therefore, this method could be utilized for longitudinal experimental studies and reliable translational research exploiting non-invasive paradigms.

We designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable. This paper describes the whole process from manufacturing the proposed EEG electrode to signal processing of visual evoked potential (VEP) signals measured on a mouse scalp using a dry non-invasive multi-channel EEG sensor.

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Non-invasive Strategies for Chronic Manipulation of DREADD-controlled Neuronal Activity

Chemogenetic strategies have emerged as reliable tools for remote control of neuronal activity. Among these, designer receptors exclusively activated by designer drugs (DREADDs) have become the most popular chemogenetic approach used in modern neuroscience. Most studies deliver the ligand clozapine-N-oxide (CNO) using a single intraperitoneal injection, which is suitable for the acute activation/inhibition of the targeted neuronal population. There are, however, only a few examples of strategies for chronic modulation of DREADD-controlled neurons, the majority of which rely on the use of delivery systems that require surgical intervention. Here, we expand on two non-invasive strategies for delivering the ligand CNO to chronically manipulate neural population in mice. CNO was administered either by using repetitive (daily) eye-drops, or chronically through the animal&#39;s drinking water. These non-invasive paradigms result in robust activation of the designer receptors that persisted throughout the CNO treatments. The methods described here offer alternatives for the chronic DREADD-mediated control of neuronal activity and may be useful for experiments designed to evaluate behavior in freely moving animals, focusing on less-invasive CNO delivery methods.

Here we describe two non-invasive methods to chronically control neuronal activity using chemogenetics in mice. Eye-drops were used to deliver clozapine-N-oxide (CNO) daily. We also describe two methods for prolonged administration of CNO in drinking water. These strategies for chronic neuronal control require minimal intervention reducing animals&#8217; stress.

Chemogenetic strategies have emerged as reliable tools for remote control of neuronal activity. Among these, designer receptors exclusively activated by ...

Non-Invasive Modulation and Robotic Mapping of Motor Cortex in the Developing Brain

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries unique challenges in children. Similarly, transcranial direct current stimulation (tDCS) can improve motor learning in adults but has only recently been applied to children. The use of tDCS and emerging techniques like high&#8211;definition tDCS (HD-tDCS) require special methodological considerations in the developing brain. Robotic TMS motor mapping may confer unique advantages for mapping, particularly in the developing brain. Here, we aim to provide a practical, standardized approach for two integrated methods capable of simultaneously exploring motor cortex modulation and motor maps in children. First, we describe a protocol for robotic TMS motor mapping. Individualized, MRI-navigated 12x12 grids centered on the motor cortex guide a robot to administer single-pulse TMS. Mean motor evoked potential (MEP) amplitudes per grid point are used to generate 3D motor maps of individual hand muscles with outcomes including map area, volume, and center of gravity. Tools to measure safety and tolerability of both methods are also included. Second, we describe the application of both tDCS and HD-tDCS to modulate the motor cortex and motor learning. An experimental training paradigm and sample results are described. These methods will advance the application of non-invasive brain stimulation in children.

We demonstrate protocols for the modulation (tDCS, HD-tDCS) and mapping (robotic TMS) of the motor cortex in children.

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries ...

Statistical Modelling of Cortical Connectivity Using Non-invasive Electroencephalograms

Non-invasive electrophysiological recordings are useful for the evaluation of nervous system function. These techniques are inexpensive, fast, replicable, and less resource-intensive than imaging. Further, the functional data produced have excellent temporal resolution, which is not achievable with structural imaging.
Current applications of electroencephalograms (EEG) are limited by data processing methods. Standard analysis techniques using raw time series data at individual channels are very limited methods of interrogating nervous system activity. More detailed information about cortical function can be achieved by examining relationships between channels and deriving statistical models of how areas are interacting, allowing visualization of connectivity between networks.
This manuscript describes a method for deriving statistical models of cortical network activity by recording EEG in a standard manner, then examining the interelectrode coherence measures to assess relationships between the recorded areas. Higher order interactions can be further examined by assessing the covariance between the coherence pairs, producing high-dimensional &#34;maps&#34; of network interactions. These data constructs can be examined to assess cortical network function and its relationship to pathology in ways not achievable with traditional techniques.
This approach offers greater sensitivity to network level interactions than is achievable with raw time series analysis. It is, however, limited by the complexity of drawing specific mechanistic conclusions about the underlying neural populations and the high volumes of data generated, requiring more advanced statistical techniques for evaluation, including dimensionality reduction and classifier-based approaches.

Standard EEG analysis techniques offer limited insight into nervous system function. Deriving statistical models of cortical connectivity offers far greater ability to investigate underlying network dynamics. Improved functional assessment opens new possibilities for diagnosis, prognostication, and outcome prediction in nervous system diseases.

Non-invasive electrophysiological recordings are useful for the evaluation of nervous system function. These techniques are inexpensive, fast, replicable, ...

Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly useful for disorders in which identifiable neuronal circuitry plays a key role, such as epilepsy and movement disorders. Currently available surgical modalities, while effective, generally involve an invasive surgical procedure, which can result in surgical injury to non-target tissues. Consequently, it would be of value to expand the range of surgical approaches to include a technique that is both non-invasive and neurotoxic.
Here, a method is presented for producing focal, neuronal lesions in the brain in a non-invasive manner. This approach utilizes low-intensity focused ultrasound together with intravenous microbubbles to transiently and focally open the Blood Brain Barrier (BBB). The period of transient BBB opening is then exploited to focally deliver a systemically administered neurotoxin to a targeted brain area. The neurotoxin quinolinic acid (QA) is normally BBB-impermeable, and is well-tolerated when administered intraperitoneally or intravenously. However, when QA gains direct access to brain tissue, it is toxic to the neurons. This method has been used in rats and mice to target specific brain regions. Immediately after MRgFUS, successful opening of the BBB is confirmed using contrast enhanced T1-weighted imaging. After the procedure, T2 imaging shows injury restricted to the targeted area of the brain and the loss of neurons in the targeted area can be confirmed post-mortem utilizing histological techniques. Notably, animals injected with saline rather than QA do demonstrate opening of the BBB, but dot not exhibit injury or neuronal loss. This method, termed Precise Intracerebral Non-invasive Guided surgery (PING) could provide a non-invasive approach for treating neurological disorders associated with disturbances in neural circuitry.

The goal of the protocol is to provide a method for producing non-invasive neuronal lesions in the brain. The method utilizes Magnetic Resonance-guided Focused Ultrasound (MRgFUS) to open the Blood Brain Barrier in a transient and focal manner, in order to deliver a circulating neurotoxin to the brain parenchyma.

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly ...

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain functions. Most of our current understanding of microglia is based on experiments performed in the mouse. Human microglia differ from mouse microglia, and thus response and characteristics of mouse microglia may not always represent that of human microglia. Further, due to ethical and technical difficulties, research on human microglia is restricted to in vitro culture system, which does not capitulate in vivo characteristics of microglia. To overcome these issues, a simplified method to non-invasively transplant induced pluripotent stem cell-derived human microglia (iPSMG) into the immunocompetent mice brain via a transnasal route in combination with pharmacological depletion of endogenous microglia using a colony-stimulating factor 1 receptor (CSF1R) antagonist is developed. This protocol provides a way to non-invasively transplant cells into the mouse brain and may therefore be valuable for evaluating the in vivo role of human microglia in physiological and pathological brain functions.

The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain ...