Name: non-invasive strategies for chronic manipulation of dreadd-controlled neuronal activity
Uploaded: 2019-08-25T15:00:00
Description: Watch this Scientific Journal Video about Non-invasive Strategies for Chronic Manipulation of DREADD-controlled Neuronal Activity at JoVE.com

This work was supported by the intramural research program at the National Institute of Mental Health (ZIA MH002964-02). We would like to thank the support of the NIMH IRP Rodent Behavioral Core (ZIC MH002952).

All animals were handled in accordance with guidelines of the Animal Care and Use Committees of the National Institute of Mental Health (NIMH). All efforts were made to minimize the pain and the number of animals used.
1. Adeno-associated virus injections in the hippocampus
NOTE: Wild type male mice of mixed background (B6/129 F1 hybrid, 3 months old) were for stereotaxically injected with&#160;an AAV encoding the M3 muscarinic receptor (hM3Dq) into t...

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A., 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=Clozapine+N-Oxide+Administration+Produces+Behavioral+Effects+in+Long-Evans+Rats:+Implications+for+Designing+DREADD+Experiments.">Clozapine N-Oxide Administration Produces Behavioral Effects in Long-Evans Rats: Implications for Designing DREADD Experiments.</a> eNeuro. 3 (5), (2016).</li><li>Gomez, J. L., 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=Chemogenetics+revealed:+DREADD+occupancy+and+activation+via+converted+clozapine.">Chemogenetics revealed: DREADD occupancy and activation via converted clozapine.</a> Science. 357 (6350), 503-507 (2017).</li></ol>

DREADDs have emerged as a popular and effective approach to remotely manipulate neuronal activity17. The design of alternative strategies for CNO delivery will broadly increase the spectrum of options available for specific experimental settings. In addition, non-invasive strategies for the delivery of CNO minimize any potential misinterpretation of results by reducing adverse side effects that can directly impact the animal&#8217;s health. Here, we described two non-invasive strategies for CNO de...

Technical advances in the field of neuroscience have allowed scientists to precisely identify and control the activity of particular neuronal populations1. This has contributed to better understand the basis of neuronal circuits and their impact on animal behavior, as well as, revising established dogmas2,3. Among these novel tools, optogenetic and chemogenetic strategies have had a profound impact not only on the quality of discoveries but also on the way experiments are conceived and designed4. In the present manuscript, we focus on chemogenetic strategies for controlling the activation of neurons via engineered receptor-ligand strategies. Designer receptors exclusively activated by designer drugs (DREADDs) represent one of the most popular chemogenetic tools for the remote control of neuronal activity, as reviewed by Roth 20165. DREADDs utilize modified muscarinic acetylcholine receptors that are specifically activated by an inert ligand, clozapine-N-oxide (CNO)6.
Most studies use CNO administered by intraperitoneal (i.p.) injections, which effectively controls the dosage and timing of engineered receptors activation in an acute fashion. However, when repetitive or chronic DREADD activation is required, the use of multiple i.p. injections become unfeasible. To address this issue, different strategies for the chronic CNO delivery have been reported, including implanted minipumps7 and intracranial cannulas8,9. To different extents, all these strategies cause the animals stress and pain10, and require a surgical intervention that could also have a direct impact on the behavioral responses to be tested11. Here, we describe three non-invasive strategies for the chronic CNO delivery.
For this purpose, mice were stereotaxically injected in the hippocampus with an adeno-associated virus (AAV) encoding an engineered version of the excitatory M3 muscarinic receptor (hM3Dq) that when activated by the ligand CNO leads to the burst-like firing of neurons6. It was previously shown that a single eye-drop containing CNO can effectively elicit a robust activation of DREADD-expressing neurons12. Here we describe a modified method for the repetitive delivery of eye drops. To achieve chronic and sustained control of the designer receptors, we next describe a non-invasive strategy to deliver CNO to mice through the drinking water. Finally, we describe an alternative paradigm for delivering CNO in drinking water during a restricted time window. Mice locomotor activity, as well as drinking behavior and the consumption of sweet caloric solutions, are mostly restricted to the dark portion of the light/dark cycle13,14. Therefore, we adopted a protocol based on the mouse&#8217;s preference for sucrose. By measuring the induction of the immediate-early gene c-Fos in AAV-infected cells, as a readout for neuronal activation12,15, we found that these CNO delivery strategies robustly activate DREADD-controlled neurons over extended durations.

<table>
	<tbody>
		<tr>
			<td>BSA</td>
			<td>Sigma life science</td>
			<td>#A2153-100G</td>
			<td>Lyophilized powder &#8805;96% (agarose gel electrophoresis)</td>
		</tr>
		<tr>
			<td>C57BL/6J mice</td>
			<td>The Jackson laboratory</td>
			<td>#000664</td>
			<td>male mice, 3 months old</td>
		</tr>
		<tr>
			<td>Capillaries</td>
			<td>Drummond Scientific Company</td>
			<td>#3-000-203-G/X</td>
			<td>Outer diameter: 1.14 in.</td>
		</tr>
		<tr>
			<td>Clozapine-N-oxide</td>
			<td>Sigma</td>
			<td>#C0832</td>
			<td>5mg</td>
		</tr>
		<tr>
			<td>Forane</td>
			<td>Baxter</td>
			<td>#NDC 10019-360-60</td>
			<td>Isoflurane, USP</td>
		</tr>
		<tr>
			<td>Microinjector III</td>
			<td>Drummond Scientific Company</td>
			<td>#3-000-207</td>
			<td>Nanoject III - Programmable Nanoliter Injector</td>
		</tr>
		<tr>
			<td>Mounting media</td>
			<td>Invitrogen</td>
			<td>#P36930</td>
			<td>Prolong Gold antifade reagent</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>#15710</td>
			<td>16% aqueous solution (methanol free), 10 ml</td>
		</tr>
		<tr>
			<td>Primary c-Fos Antibody</td>
			<td>Cell signaling technology</td>
			<td>#2250S</td>
			<td>c-Fos (9F6) Rabbit mAb (100&#181;l)</td>
		</tr>
		<tr>
			<td>rAAV5/hSyn-hm3D-mCherry</td>
			<td>UNC Vector Core</td>
			<td></td>
			<td>Titer: ~3x10e12 vg/mL</td>
		</tr>
		<tr>
			<td>rAAV5/hSyn-mCherry</td>
			<td>UNC Vector Core</td>
			<td></td>
			<td>Titer: ~3x10e12 vg/mL</td>
		</tr>
		<tr>
			<td>Secondary Antibody</td>
			<td>Invitrogen</td>
			<td>#A21206</td>
			<td>Alexa Fluor TM 488 Donkey anti-rabbit IgG(H+L), 2mg/ml</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>americanbio.com</td>
			<td>#AB02025-00100</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

We observed that repetitive CNO delivery using eye-drops elicited a robust induction of c-Fos expression in most infected neurons (Figure 1C), showing that the effectiveness of CNO delivery is sustained during the repetitive exposure. Furthermore, a significant induction of c-Fos was observed in samples collected 2 h after CNO treatment, compared to samples obtained 6 h after CNO exposure (Figures 1D-E), demonstrating that changes induced by CNO are time-dependent.
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Watch this Scientific Journal Video about Non-invasive Strategies for Chronic Manipulation of DREADD-controlled Neuronal Activity at JoVE.com

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

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 ...

DREADDs are the most popular chemogenetic approach for remote control of neuronal activity. Here we offer new alternatives for repetitive or chronic DREADD control, focusing on less invasive CNO delivery methods. We describe two strategies for the prolonged CNO delivery in mice by repetitive eyedrops, or chronically through the animal's drinking water.The protocols described here are examples of chronic and noninvasive options for CNO delivery that can be adapted to a variety of experimental designs including different DREADD variants, tissues, or even species. A key issue for a CNO delivery is a animal's stress and pain. These protocols minimize this problem and are easy to perform and adapt to different experimental settings.Before performing the surgery, clean and sterilize the stereotaxic frame and all needed instruments. When the frame is ready, confirm a lack of response to toe pinch in an anesthetized mixed background wild type male mouse, shave the top of the head, and fix the head of the mouse to the frame. Then apply ocular protective lubricant on the eyes and finally, clean the exposed skin with sequential povidone iodine and 70%ethanol scrubs.Use a sterile scalpel to expose the skull and calibrate the frame to the bregma point. Drill at a medialateral coordinate of 2.9 millimeters, and an anterior posterior coordinate of minus 2.7 millimeters to target the hippocampus. When the brain is exposed, use a micro injector and pulled microcapillary pipettes to unilaterally inject 90 nanoliters of the adenoassociated virus into the hippocampus at a dorsal ventral depth of minus three millimeters.Then close the incision with nylon sutures, and remove the animal from the frame for analgesia administration. For repetitive CNO delivery, beginning four weeks post injection, acclimate the animals to handling by scruffing each mouse three minutes daily for three to four days prior to the administration of the eyedrops. When the mice have become acclimated to handling, weigh each mouse to determine the appropriate amount of CNO to be delivered to achieve a one milligram of CNO per kilogram of body weight concentration.Two hours before lights off during the inactive phase, load one to three microliters of the prepared CNO solution into a P10 micropipette and immobilize the mouse via scruffing. Slowly expel the solution until a stable droplet forms on the pipette tip and carefully bring the droplet close to the cornea until the solution is delivered without touching the pipette tip to the mouse's eye. Then return the mouse to its home cage before applying the CNO eyedrop to the eye of the next animal.In cases where CNO needs to be delivered during the mouse active phase, ensure the presence of a dim red light for proper animal handling and CNO delivery. For chronic CNO treatment, beginning four weeks post injection and three days before starting the treatment, replace the regular water bottles with small bottles stopped with rubber spouts and covered with aluminum foil containing 10 milliliters of regular water. Secure to the cages with tape to allow mice to acclimate to the bottles.Measure the daily water consumption for each mouse, and weigh each mouse. Test a range of CNO concentrations to determine the dose that displays the maximum effectiveness with the minimal CNO concentration. Then fill each small bottle with eight milliliters of regular water plus the required amount of CNO.For restricted CNO treatment, beginning four weeks post injection and three days before starting the treatment, place a small bottle containing 10 milliliters of water plus 1%sucrose on the cage away from the original water bottle during the last portion of the animal's active phase. At the end of the exposure, remove the water plus sucrose solutions from each cage and measure the sucrose water consumption for each animal. For CNO delivery, fill the bottles with five milliliters of water plus 1%sucrose and one milligram per kilogram of CNO.Place the bottle in the same position as during the sugar water acclimation period during the last portion of the active phase, removing the bottles and measuring the amount of water, sucrose and CNO consumed as demonstrated. At the end of the experiment, harvest the treated animal brain into fresh fixative. After 9 to 12 hours, cryoprotect the brain tissue in a 30%sucrose solution.When the brain sinks, section the sample on a cryostat. When all of the brain sections have been obtained and the nonspecific binding blocked, incubate the tissue samples with an anti-c-Fos antibody solution at four degrees Celsius overnight with constant agitation. The next morning, wash the samples with three five-minute washes in fresh PBS per wash before incubating the samples with an appropriate fluorescence conjugated secondary antibody.After one hour at room temperature and constant agitation protected from light, obtain digital images of the labeled tissue sections by fluorescence confocal microcroscopy. After loading the images into ImageJ, outline at measure the areas of Adeno-associated infected mCherry positive cells and quantify the number of C false positive cells within this region to obtain the number of activated cells per area. Repetitive CNO delivery using eyedrops elicits a robust induction of cFos expression in most infected neurons demonstrating that the effectiveness of CNO delivery is sustained during the repetitive exposure.Furthermore, a significant induction of cFos is observed in samples collected two hours after CNO treatment compared to samples obtained six hours after CNO exposure, indicating that changes induced by CNO are time dependent. The daily consumption of water plus CNO is not significantly different compared to the total volume of regular water consumed. Similarly, the amount of water plus 1%sucrose consumed during the night is not affected by the addition of CNO, nor are differences in the daily consumption of both water plus CNO, and water plus sucrose plus CNO observed over a five-day experimental period.Similar to the application of CNO eyedrops, a robust induction of cFos is observed after two, but not six hours of CNO drinking water access. In addition, there is a clear threshold of effectiveness for CNO as a low CNO dose does not elicit cFos activation compared to a saline control, whereas higher doses induce a robust and similar cFos induction. We recommend performing a dose response analysis to define the lowest CNO dose that does not significantly reduce neuronal activation, and considering the use clozapine as a ligand.Here we demonstrated using CNO-mediated cFos induction as a result of neuronal activation. However, these protocol can be easily adopted to electrophysiological analysis or behavioral tests. DREADD technology is a powerful tool for remote control of neuronal activity, and designing alternative strategies for CNO delivery.We'll increase the spectrum of options for experimental settings and potential clinical interventions.

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Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

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 ...

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.

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 ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

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 ...