X.H.T.W. is a W.M. Keck Foundation Distinguished Young Scholar in Medical Research, and is also supported by NIH/NHLBI grants R01-HL089598 and R01HL091947, and Muscular Dystrophy Association grant #69238. This work is also supported in part by the Fondation Leducq Alliance for CaMKII Signalling in Heart. N.L. is the recipient of the 2009-2010 Michel Mirowski International Fellowship in Cardiac Pacing and Electrophysiology from Heart Rhythm Society and 2009-2012 American Heart Association Postdoc Fellowship.

Part 1. Surgical preparation
<ol>
	<li>In case programmed electrical stimulation experiments are performed as survival surgeries in mice sterile conditions will be necessary. However, the most common type of experiment is terminal in nature, for which regular clear surgical techniques suffice.</li>
 <li>The mouse is anesthetized using 2% isoflurane in 0.5 L/min 100% O2. </li>
 <li>Hair clippers are used to shave the fur from the neckline to mid chest level.</li>
 <li>The anesthetized mouse is placed in a supine position, with its limbs taped onto ECG electrodes incorporated in a heating board (Indus Instruments, Houston, TX). And the surgical area is disinfected with 10% Povidone Iodine. A recommended system includes a rectal temperature probe connected to a heating pad controlled by a thermoanalyzer system, such that the body temperature is maintained at 37.0 °C ± 1.0 °C.</li>
</ol>
Part 2. Cannulation and insertion of EP catheter into right atrium and ventricle
<ol>
	<li>After confirming by a toe-pinch that the mouse is fully anesthetized, an 1/2 inch incision is made to the right of the midline with the caudal terminus at the level of the clavicle. </li>
 <li>Subcutaneous tissue, salivary glands, and lymphatic tissues are separated by means of blunt dissection to visualize the right jugular vein. </li>
 <li>The proximal end of the vein is tied with a 6-0 suture. Pulling gently on this suture will keep the internal jugular vein straight while inserting the catheter. Another suture is placed under the vein at the distal end of the visualized segment. This suture will be tied around the catheter once the catheter is placed optimally within the heart, to ensure hemostasis and maintain the catheter in the desired position (Figure 1). </li>
 <li>The computer-based data acquisition is now started to record surface ECG and 4 leads of intracardiac electrograms simultaneously (i.e., IOX-2 acquisition software, Emka Technologies, VA, USA). For intracardiac electrogram recording, electrodes on the catheter are connected to an external stimulator in "recording" mode (i.e., model STG3008, MultiChannel Systems, Reutlingen, Germany). </li>
 <li>Using micro-scissors, a small incision is made in the longitudinal direction of the vein, and the 1.1F octapolar catheter (EPR-800, Millar Instruments, Houston, TX) is advanced through the vein into the right atrium. Gently pulling on the proximal suture will help keep the internal jugular vein straight and will allow easier passage of the catheter into the right atrium and ventricle. Proper catheter position is verified by visualization of the waveforms of the 4 intracardiac electrograms at the level of the apex of the right ventricle, the base of the right ventricle, the atrioventricular node, and the right atrium, respectively (Figure 2). </li>
 <li>The distal suture is now tied off to secure the catheter position and prevent possible bleeding.</li>
</ol>
Part 3. Programmed Electrical Stimulation
<ol>
	<li>In the case of selective atrial pacing, the electrode pair located inside the atrium is switched from "recoding" mode to "stimulation" mode, while the other electrode pairs remain in "recording" mode. </li>
 <li>To determine whether a mouse has an increased vulnerability to atrial arrhythmias, programmed electrical stimulation of the right atrium is performed. First, the atrial pacing threshold is determined by applying 2-ms current pulses (at least 50) at different basic cycle lengths (BCL) to test for consistency of stimulus capture. The BCL of the initial pulse train is slightly lower than the intrinsic BCL, and is decreased by 10 ms (for example, 100 ms, 90 ms, 80 ms, and 70 ms). The typical current amplitude required for stimulus capture is 100-200 μA.</li>
 <li>The sinus node recovery time (SNRT) is measured after applying a 15-s atrial pacing train at a BCL of 100 ms. SNRT is defined as the interval between the last stimulus in the pacing train and the onset of first spontaneous sinus beat. </li>
 <li>The atrial effective refractory period (AERP) is determined by applying series of atrial pacing trains at a fixed BCLs (i.e., 100 ms) with a coupled shorter S2 premature stimulus. The S1-S2 interval is progressively reduced by 2-ms in each pacing train from 70ms to 20ms. The AERP is defined as the longest S1-S2 coupling interval for atria that failed to generate a propagated beat with S2 (S1 being the regular train pulse, and S2 the premature stimulus). Between each stimulation protocol, there was a recovery period of at least 30 seconds.</li>
 <li>The effective refractory period of the atrioventricular (AV) node (AVNERP) is determined by applying series of atrial pacing trains at BCL of 100 ms with a coupled S2 premature stimulus. The S1-S2 interval is progressively reduced by 2 ms each pacing train from 70 ms to 20 ms. The AVNERP is defined as the longest S1-S2 coupling interval at which the premature stimulation delivered to the atrium is followed by a His potential but not by a QRS complex. Between each stimulation protocol, there was a recovery period of at least 30 seconds. </li>
 <li>Inducibility of atrial arrhythmias, including atrial fibrillation (AF), may be tested using the burst pacing protocol described by Verheule et al.2 A series of 2 second bursts is applied to determine inducibility of atrial arrhythmias. The first 2 second burst has a cycle length (CL) of 40 ms, and each successive 2 second burst has a CL of 2-ms shorter than the previous burst, until the final CL of 20 ms. In the absence of an arrhythmogenic substrate, the heart will resume sinus rhythm immediately following the pacing protocol. In the case of atrial flutter, there will be a regular pattern of fast A waves seen on the atrial electrogram, whereas the frequency of the ventricular response is typically slower, as seen on the ventricular electrogram. In case of atrial fibrillation, the surface ECG will show irregular RR intervals in the absence of P waves. Additionally, the atrial electrogram will reveal rapid and irregular A waves, whereas the ventricular electrogram will reveal irregular and slower ventricular waves (Figure 3). Arrhythmia induction protocols are typically performed in triplicate, and an arrhythmia is considered present if it can be evoked in at least 2 out of 3 trials.3,4.</li>
</ol>
Part 4. Removal of Catheter
<ol>
	<li>After all pacing protocols are finished, data acquisition is stopped. The suture at the distal end of the catheter is gently cut off, in order to release the catheter. </li>
 <li>In case of terminal EP studies, the knot is gently loosened to release the catheter.</li>
 <li>At the conclusion of the study, the mouse will be humanely euthanized while under isoflurane using cervical dislocation.</li>
</ol>
Representative Results
Surface ECG and intracardiac electrograms are recorded simultaneously throughout the in vivo electrophysiology study, and are reviewed in detail following the completion of all protocols. The baseline electrophysiological parameters include PR interval, PQ interval, QRS duration, QT intervals, and QRS morphology. These parameters can be measured manually, or automatically using the data acquisition software IOX-2 or ECG-AUTO (Emka Technologies, VA, USA). 
The SNRT, AERP, and AVNERP provide information regarding the sinus node "pacemaker" function, atrial, and AV nodal conduction properties, respectively. Examples of pacing-induced episodes of atrial fibrillation can be found in the paper by Chelu et al.4
<img src="/files/ftp_upload/1730/1730fig1.jpg" alt="Rat cardiovascular surgery diagram; jugular vein cannulation and heart diagram; scale comparison." data-altupdatedat="1747911167000" data-alt="Figure 1"> Figure 1. Illustration of intracardiac catheterization for electrophysiology studies in mice. A. Right internal jugular vein is isolated and cannulated. The distal end of vein is tied off once proper catheter position is achieved. B. The mouse is placed in a supine position for this procedure. C. Cartoon depicting the intracardiac position of catheter. Pair of electrodes are positioned at the level of the apex of the right ventricle, the base of the right ventricle, the atrioventricular node, and the right atrium, respectively. D. Close-up of the 1.1F octapolar catheter. Figure modified from Mathur et al. with permission of Circ Arrhtyhm Electrophys 5.
<img src="/files/ftp_upload/1730/1730fig2.jpg" alt="Electrocardiogram (ECG) tracing; diagram of heart electrical activity; waveform and interval analysis." data-altupdatedat="1747911167000" data-alt="Figure 2"> Figure 2. Representative Surface ECG and Intracardiac Electrograms in a Mouse. (A) Surface ECG in lead II configuration showing regular sinus rhythm with a frequency of 540 beats per minute. (B-E) Bipolar intracardiac electrogram recordings at the level of the right atrium (B), atrioventricular node (C), and base of the right ventricle (D), and the apex of the right ventricle (E), respectively. Note that the intracardiac A-wave in panel B corresponds to the P-wave on the surface ECG. The V-wave on the ventricular electrogram corresponds to the QRS wave on the surface ECG.
<img src="/files/ftp_upload/1730/1730fig3.jpg" alt="Electrophysiology diagram with Lead II-ECG, atrial, and ventricular electrograms for cardiac analysis." data-altupdatedat="1747911167000" data-alt="Figure 3"> Figure 3. Representative surface ECG and intracardiac electrograms in a mouse that developed atrial fibrillation after atrial burst pacing.

<ol>
	<li>Nattel, S., Shiroshita-Takeshita, A., Brundel, B. J., Rivard, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+atrial+fibrillation:+lessons+from+animal+models.">Mechanisms of atrial fibrillation: lessons from animal models.</a> Prog Cardiovasc Dis. 48, 9-9 (2005).</li><li>Verheule, S., Sato, T., Everett, T. t., Engle, S. K., Otten, D., Rubart-von der Lohe, M., Nakajima, H. O., Nakajima, H., Field, L. J., Olgin, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+vulnerability+to+atrial+fibrillation+in+transgenic+mice+with+selective+atrial+fibrosis+caused+by+overexpression+of+TGF-beta1.">Increased vulnerability to atrial fibrillation in transgenic mice with selective atrial fibrosis caused by overexpression of TGF-beta1.</a> Circ Res. 94, 1458-1458 (2004).</li><li>Sood, S., Chelu, M. G., Oort, R. J. v. a. n., Skapura, D., Santonastasi, M., Dobrev, D., Wehrens, X. H., H, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+calcium+leak+due+to+FKBP12.6+deficiency+in+mice+facilitates+the+inducibility+of+atrial+fibrillation.">Intracellular calcium leak due to FKBP12.6 deficiency in mice facilitates the inducibility of atrial fibrillation.</a> Heart Rhythm. 5, 1047-1047 (2008).</li><li>Chelu, M. G., Sarma, S., Sood, S., Wang, S., van Oort, R. J., Skapura, D. G., Li, N., Santonastasi, M., Muller, F. U., Schmitz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calmodulin+kinase+II-mediated+sarcoplasmic+reticulum+Ca2++leak+promotes+atrial+fibrillation+in+mice.">Calmodulin kinase II-mediated sarcoplasmic reticulum Ca2+ leak promotes atrial fibrillation in mice.</a> J Clin Invest. 119, 1940-1940 (2009).</li><li>Mathur, N., Subeena, S., Wang, S., van Oort, R. J., Sarma, S., Li, N., Skapura, D., Bayle, J. H., Valderrabano, M., Wehrens, X. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sudden+Infant+Death+Syndrome+in+Mice+With+an+Inherited+Mutation+in+RyR.">Sudden Infant Death Syndrome in Mice With an Inherited Mutation in RyR.</a> Circ Arrhythmia Electrophysiol. , (2009).</li></ol>

During cardiac catheterization, extensive bleeding during catheterization could increase the heart rate due to hypovolemia. In this case, an intraperitoneal injection of sterile saline (0.3 1.0 mL) could normalize filling pressure and reduce hemodynamic stress in the mouse.
Exposure to isoflurane longer than 2 hours, or higher concentrations of isoflurane (&gt;2 %) could suppress cardiac and respiratory functions in the mouse. Therefore, it is recommended that all studies be completed in le...

programmed electrical stimulation in mice

Genetically-modified mice have emerged as a preferable animal model to study the molecular mechanisms underlying conduction abnormalities, atrial and ventricular arrhythmias, and sudden cardiac death.1 Intracardiac pacing studies can be performed in mice using a 1.1F octapolar catheter inserted into the jugular vein, and advanced into the right atrium and ventricle. Here, we illustrate the steps involved in performing programmed electrical stimulation in mice. Surface ECG and intracardiac electrograms are recorded simultaneously in the atria, atrioventricular junction, and ventricular myocardium, whereas intracardiac pacing of the atrium is performed using an external stimulator. Thus, programmed electrical stimulation in mice provides unique opportunities to explore molecular mechanisms underlying conduction defects and cardiac arrhythmias.

Watch this Scientific Journal Video about Programmed Electrical Stimulation in Mice at JoVE.com

Programmed Electrical Stimulation in Mice

Programmed electrical stimulation provides the ability to determine conduction properties of the heart, and the possibility to induce and terminate cardiac arrhythmias using various pacing protocols. Using a transvenous catheter, intracardiac electrogram recordings can be obtained in mice following programmed electrical stimulation protocols to identify arrhythmogenic substrates.

Genetically-modified mice have emerged as a preferable animal model to study the molecular mechanisms underlying conduction abnormalities, atrial and ...

programmed-electrical-stimulation-in-mice

Research

JoVE Journal

Medicine

20.4K Views.  Baylor College of Medicine (BCM). Geprogrammeerde elektrische stimulatie biedt de mogelijkheid tot geleiding eigenschappen van het hart te bepalen, en de mogelijkheid om te induceren en te beëindigen hartritmestoornissen met behulp van verschillende pacing protocollen. Met behulp van een katheter transveneuze, kan intracardiale elektrogram opnames worden verkregen bij muizen na geprogrammeerde elektrische stimulatie protocollen aritmogene substraten te identificeren.

Video: Programmed Electrical Stimulation in Mice

Long-term Potentiation of Perforant Pathway-dentate Gyrus Synapse in Freely Behaving Mice

Studies of long-term potentiation of synaptic efficacy, an activity-dependent synaptic phenomenon having properties that make it attractive as a potential cellular mechanism underlying learning and information storage, have long been used to elucidate the physiology of various neuronal circuits in the hippocampus, amygdala, and other limbic and cortical structures. With this in mind, transgenic mouse models of neurological diseases represent useful platforms to conduct long-term&#160;potentiation (LTP) studies to develop a greater understanding of the role of genes in normal and abnormal synaptic communication in neuronal networks involved in learning, emotion and information processing. This article describes methodologies for reliably inducing LTP in the freely behaving mouse. These methodologies can be used in studies of transgenic and knockout freely&#160;behaving mouse models of neurodegenerative diseases.

Transgenic and knockout mouse models of neurological diseases are useful for studying the role of genes in normal and abnormal neurophysiology. This article describes methodologies which can be used to study long-term potentiation, a cellular mechanism which may underlie learning and memory, in transgenic and knockout freely&#160;behaving mouse models of neuropathology.

Studies of long-term potentiation of synaptic efficacy, an activity-dependent synaptic phenomenon having properties that make it attractive as a potential ...

Behavior

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

Two Different Real-Time Place Preference Paradigms Using Optogenetics within the Ventral Tegmental Area of the Mouse

Understanding how neuronal activation leads to specific behavioral output is fundamental for modern neuroscience. Combining optogenetics in rodents with behavioral testing in validated paradigms allows the measurement of behavioral consequences upon stimulation of distinct neurons in real-time with high spatial and temporal selectivity, and thus the establishment of causal relationships between neuronal activation and behavior. Here, we describe a step-by-step protocol for a real-time place preference (RT-PP) paradigm, a modified version of the classical conditioned place preference (CPP) test. The RT-PP is performed in a three-compartment apparatus and can be utilized to answer if optogenetic stimulation of a specific neuronal population is rewarding or aversive. We also describe an alternative version of the RT-PP protocol, the so-called neutral compartment preference (NCP) protocol, which can be used to confirm aversion. The two approaches are based on extensions of classical methodology originating from behavioral pharmacology and recent implementation of optogenetics within the neuroscience field. Apart from measuring place preference in real time, these setups can also give information regarding conditioned behavior. We provide easy-to-follow step-by-step protocols alongside examples of our own data and discuss important aspects to consider when applying these types of experiments.

Here we present two easy-to-follow step-by-step protocols for place preference paradigms using optogenetics in mice. Using these two different setups, preference and avoidance behaviors can be solidly assessed within the same apparatus with high spatial and temporal selectivity, and in a straightforward manner.

Understanding how neuronal activation leads to specific behavioral output is fundamental for modern neuroscience. Combining optogenetics in rodents with ...

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of single neurons and region-specific transmitter release, up to whole neuronal circuitries in the central nervous system, and allows the direct measurement of behavioral outcomes. Neurons express optogenetic tools via an injection of viral vectors carrying the DNA of choice, such as Channelrhodopsin2 (ChR2). Light is brought into specific brain regions via chronic optical implants that terminate directly above the target region. After two weeks of recovery and proper tool-expression, mice can be repeatedly used for behavioral tests with optogenetic stimulation of the neurons of interest.
Optogenetic modulation has a high temporal and spatial resolution that can be accomplished with high cell specificity, compared to the commonly used methods such as chemical or electrical stimulation. The light does not harm neuronal tissue and can therefore be used for long-term experiments as well as for multiple behavioral experiments in one mouse. The possibilities of optogenetic tools are nearly unlimited and enable the activation or silencing of whole neurons, or even the manipulation of a specific receptor type by light.
The results of such behavioral experiments with integrated optogenetic stimulation directly visualizes changes in behavior caused by the manipulation. The behavior of the same animal without light stimulation as a baseline is a good control for induced changes. This allows a detailed overview of neuronal types or neurotransmitter systems involved in specific behaviors, such as anxiety. The plasticity of neuronal networks can also be investigated in great detail through long-term stimulation or behavioral observations after optical stimulation. Optogenetics will help to enlighten neuronal signaling in several kinds of neurological diseases.

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Neuroscience

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for various neurological and psychiatric disorders. HFS in the deep region of the brain, also named deep-brain stimulation (DBS), is becoming increasingly important in clinical trials. Recent progress in the field of high-frequency DBS (HF-DBS) surgery has begun to spread the possibility of utilizing this invasive technique to other situations, such as treatment for major depression disorder (MDD), obsessive-compulsive disorder (OCD), and so on. 
Despite these expanding indications, the underlying mechanisms of the beneficial effects of HF-DBS remain enigmatic. To address this question, one approach is to use implanted electrodes that sparsely activate distributed subpopulations of neurons by HFS. It has been reported that HFS in the anterior nucleus of the thalamus could be used for the treatment of refractory epilepsy in the clinic. The underlying mechanisms might be related to the increased neurogenesis and altered neuronal activity. Therefore, we are interested in exploring the physiological alterations by the detection of neuronal activity as well as neurogenesis in the mouse dentate gyrus (DG) before and after HFS treatment. 
In this manuscript, we describe methodologies for HFS to target the activation of the DG in mice, directly or indirectly and in an acute or chronic manner. In addition, we describe a detailed protocol for the preparation of brain slices for c-fos and Notch1 immunofluorescent staining to monitor the neuronal activity and signaling activation and for bromodeoxyuridine (BrdU) labeling to determine the neurogenesis after the HF-DBS induction. The activation of the neuronal activity and neurogenesis after the HF-DBS treatment provides direct neurobiological evidence and potential therapeutic benefits. Particularly, this methodology can be modified and applied to target other interested brain regions such as the basal ganglia and subthalamic regions for specific brain disorders in the clinic.

This protocol shows how to set up a reliable HFS method in mice. Neurons throughout the hippocampal dentate gyrus are electrically stimulated by HFS directly and indirectly in vivo. Neuronal activity and molecular signaling are examined by c-fos and Notch1 immunofluorescent staining, respectively; neurogenesis is quantified by bromodeoxyuridine labeling assay.

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for ...

Transcranial Direct Current Stimulation (tDCS) in Mice

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for several neuropsychiatric diseases. The biological effects of tDCS are not fully understood, which is in part explained due to the difficulty in obtaining human brain tissue. This protocol describes a tDCS mouse model that uses a chronically implanted electrode allowing the study of the long-lasting biological effects of tDCS. In this experimental model, tDCS changes the cortical gene expression and offers a prominent contribution to the understanding of the rationale for its therapeutic use.

Transcranial Direct Current Stimulation tDCS in Mice

Transcranial direct current stimulation (tDCS) is a therapeutic technique proposed to treat psychiatric diseases. An animal model is essential for understanding the specific biological alterations evoked by tDCS. This protocol describes a tDCS mouse model that uses a chronically implanted electrode.

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for ...

Mapping Cerebral Blood Flow and Electrical Fields: Electrode Placement in Mice

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and variations in the substrate supply to the brain. This paper describes a protocol for measuring CBF responses to transcranial alternating current stimulation (tACS). Dose-response curves are estimated both from the CBF change occurring with tACS (mA) and from the intracranial electric field (mV/mm). We estimate the intracranial electrical field based on the different amplitudes measured by glass microelectrodes within each side of the brain. In this paper, we describe the experimental setup, which involves using either bilateral laser Doppler (LD) probes or laser speckle imaging (LSI) to measure the CBF; as a result, this setup requires anesthesia for the electrode placement and stability. We present a correlation between the CBF response and the current as a function of age, showing a significantly larger response at higher currents (1.5 mA and 2.0 mA) in young control animals (12-14 weeks) compared to older animals (28-32 weeks) (p &lt; 0.005 difference). We also demonstrate a significant CBF response at electrical field strengths &lt;5 mV/mm, which is an important consideration for eventual human studies. These CBF responses are also strongly influenced by the use of anesthesia compared to awake animals, the respiration control (i.e., intubated vs. spontaneous breathing), systemic factors (i.e., CO2), and local conduction within the blood vessels, which is mediated by pericytes and endothelial cells. Likewise, more detailed imaging/recording techniques may limit the field size from the entire brain to only a small region. We describe the use of extracranial electrodes for applying tACS stimulation, including both homemade and commercial electrode designs for rodents, the concurrent measurement of the CBF and intracranial electrical field using bilateral glass DC recording electrodes, and the imaging approaches. We are currently applying these techniques to implement a closed-loop format for augmenting the CBF in animal models of Alzheimer's disease and stroke.

Author Spotlight: Stimulation-Based Approach to Improve Cerebral Blood Flow in Alzheimer's Model 

We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations.

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and ...

Selective Manipulation of Theta Oscillations in a Mouse Model

Source: Bender, F. et al., <a href="https://app.jove.com/v/57349">Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice.</a> J. Vis. Exp (2018)
This video demonstrates a protocol to modulate theta oscillations in a genetically engineered mouse model with light-sensitive ion channels in its GABAergic medial septum neurons. By delivering light pulses to the hippocampus, the protocol aims to synchronize neuronal activity and generate theta oscillations, which are recorded as local field potentials.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
PV-Cre ...

Programmed Electrical Stimulation in Mice

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