Supported by the 2014-2015 Neurosurgery Research and Education Foundation (NREF) award and a 2014 Grant-In-Aid Award from Louisiana State University Shreveport School of Medicine (J.A.W.). We thank S. Harold and C.M. Keller for their invaluable technical assistance and teaching.

<ol>
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The authors have no competing financial interests with regards to this methodology.

Although the exact mechanisms of deep brain stimulation are not fully characterized, DBS efficacy for both motor and psychiatric disorders may result from a dynamic interaction between the electrical therapy and the functioning of various subcortical and cortical brain regions over time6,22-26. While non-contingent methods of methamphetamine delivery to rodents are well-described27,28, these methods are most appropriate for discrete investigations of drug pharmacokinetics and neurochemical effects27-29. Operant IV drug self-administration, by incorporating an element of motivation for drug, is ideally suited for the study of how electrical therapies like DBS interact with pathological behaviors over time. The procedures we describe examine the effects of DBS in one environment on contingent methamphetamine use in a different environment.
There are three key steps in our IV methamphetamine self-administration paradigm: 1) Induction of rapid acquisition and escalation of drug intake during long access sessions, 2) Maintenance of a stable, high rate of drug intake during subsequent short access sessions and 3) Development of a front loading pattern of drug taking. This paradigm can be accomplished in a 2 to 3 week timeframe with 10 - 12 rats per experiment, which is both cost-effective and ideally suited to test the effects of DBS given the potentially limited lifespan of head caps in rodents using psycho-stimulants. This procedure, like other paradigms that incorporate a period of long access19,30,31 reasonably simulates some aspects of substance use disorders; it demonstrates both escalation of use and high motivation to obtain drug with early session &#8220;drug-loading,&#8221; which are important aspects of human dependence versus recreational use19,30. Rodents who have long access exposure to IV methamphetamine also demonstrate cognitive deficits32, distinct responses to pharmacological treatment33, pharmacokinetics34 and neurochemical changes35 that are more similar to humans suffering from chronic methamphetamine use disorder than rodents with only short access exposure.
Likewise there are three key steps in our deep brain stimulation procedure: 1) Habituation to the DBS environment, including the head tether connection, for one or two &#8220;mock&#8221; sessions, 2) Daily, intermittent delivery of active stimulation using a commercial system, and 3) DBS disconnection and subsequent transport to the drug setting. This paradigm is designed to mimic the process of non-invasive therapies like TMS rather than that of traditional continuous DBS. Fully implanted, programmable DBS systems like those used for common movement disorders3 will be marginally feasible in patients suffering from psycho-stimulant dependence for several aforementioned reasons. Intermittent electrical treatment strategies that do not involve high-risk surgery and aftercare, like TMS, may be better adapted to this patient population. The methods we have described will allow investigators to develop and refine treatment strategies that can modify drug-related behavior while being delivered outside of the drug environment in a restricted timeframe. There is accumulating evidence that transient intracranial electrical stimulation that is patterned after specific neurophysiological deficits23 or combined with systemic pharmacotherapy36 exert long-lasting positive effects on psychiatric and drug-related behaviors for several weeks after the treatment has ceased.
The needs for initial excellent surgical technique and for ongoing care of multiple surgical sites during intense drug-use are the main limitations of this methodology. If either the IV catheter or the DBS electrodes become non-operational and/or infected, the rat must exit the study. Jugular catheter and intracranial electrode placements under strict sterile technique are best learned from experienced investigators prior to initiating these procedures independently.
This procedure is amenable to several modifications and future investigations, including examination of: 1) alternate stimulation parameters (e.g.,-stimulation waveform, pulse width, frequency, amplitude), 2) other potential therapeutic brain targets (e.g.,-nucleus accumbens core, medial prefrontal cortex, midbrain, habenula), 3) different DBS delivery patterns (e.g.,- daily DBS delivery, weekly DBS delivery, DBS at various intervals prior to operant sessions, DBS before acquisition), and, perhaps most exciting, 4) combinations of short-duration DBS and pharmaceutical agents that imitate optogenetic stimulation of selective pathways and exert enduring behavioral modifications36.

Methamphetamine is a psychostimulant that produces an intense and prolonged euphoria due to an acute increase in synaptic monoamines, particularly dopamine. Methamphetamine dependence is an epidemic health problem with an estimated 25 to 34 million users globally and no proven treatment1,2. There is a substantial need to develop novel therapeutic strategies for methamphetamine dependence. Deep brain stimulation (DBS) is a neurosurgical procedure that uses a brain &#8220;pacemaker&#8221; to normalize disruptive neuronal firing patterns that occur in certain diseases, including Parkinson&#8217;s disease, dystonia, and essential tremor3. Recent human case reports suggest that DBS may also be an effective treatment for alcohol and drug dependence, but preclinical evidence regarding psychostimulants (e.g., cocaine, methamphetamine) is limited4-8.
Continuous deep brain stimulation, as it is currently practiced, requires exceptional cooperation from the patient and his/her family. Meticulous wound care and personal hygiene are required to protect the underlying pacemaker hardware, which is susceptible to infection even in patients who are not using intravenous drugs with resultant bacteremia. Regular follow-up of the DBS device is also necessary given the open loop design of the system; experienced physicians alter the settings of modern DBS to decrease target symptoms during routine clinic appointments3. This treatment paradigm will be limited in cocaine and methamphetamine users due to their challenging psychosocial situations. Several rodent studies have imitated this impractical paradigm by examining DBS effects when the therapy is delivered continuously during cocaine self-administration procedures in the drug-use environment9-11.
Non-invasive discontinuous techniques that do not require indwelling hardware, like transcranial magnetic stimulation (TMS), may be a better option for treatment of substance use disorders12. TMS is delivered non-invasively using an external headcoil to generate electrical fields in a particular brain target during daily, intermittent treatments. The recent advent of H coil or &#8220;deep&#8221; TMS allows deeper brain structures to be stimulated, in addition to cortical sites, expanding its potential use13,14. Both therapies are delivered discontinuously during a series of sessions in a different environment than that of primary drug use and have shown promise in both human and rodent trials for drug dependence13,15-17. The window to treat methamphetamine dependent patients will likely be during periods of sobriety such as court-mandated rehabilitation, not during street binges when they can experience violent or erratic behavior18. As such, the aim of this article is to describe the delivery of electrical stimulation that is temporally and spatially separate from the drug-use environment, which more closely approximates what is possible in humans, for the treatment of IV methamphetamine dependence.

<table><tbody><tr><td>Rodent operant chambers</td><td>Med Associates, Inc</td><td>ENV-008CT</td><td>Med Associates Inc. PO Box 319 St. Albans, Vermont 05478 USA Phone: (802) 527-2343</td></tr><tr><td>Kopf Small Animal Stereotaxic Instrument with Digital Display Console</td><td>Kopf Instruments</td><td>Model 940</td><td>Kopf Phone: 1-877-352-3275 Fax: 1-818-352-3275 Email: sales@kopfinstruments.net</td></tr><tr><td>Z-Series 3-DSP Bioamp Processor</td><td>Tucker Davis Technologies</td><td>RZ5D</td><td>Tucker-Davis Technologies&amp;nbsp;11930 Research Circle Alachua, FL 32615&amp;nbsp;USA Ph: 386-462-9622&amp;nbsp;www.tdt.com</td></tr><tr><td>Z-Series 32-Channel Stimulator</td><td>Tucker Davis Technologies</td><td>IZ2-32</td><td>Software is accompanied by a manual that discusses how to program experiments using the OpenEx platform, which can be accessed here: http://www.tdt.com/files/manuals/OpenEx_User_Guide.pdf</td></tr><tr><td>48 Volt LI-ION Battery Pack for IZ2 Stimulator</td><td>Tucker Davis Technologies</td><td>LZ48-200</td><td></td></tr><tr><td>32-Channel Splitter Box for PZ5</td><td>Tucker Davis Technologies</td><td>S-BOX_PZ5</td><td></td></tr><tr><td>OpenEx Ext Software Package for Multi-Channel Neural Recording</td><td>Tucker Davis Technologies</td><td>OpenEx</td><td></td></tr><tr><td>Platinum-iridium stimulating electrodes</td><td>Plastics One Inc</td><td>MS303/8-B/SPC ELECT PT 2C TW .005&quot;</td><td>Plastics One Inc P.O.Box 21465, S.W. Roanoke, VA 24018, PH 540-772-7950</td></tr><tr><td>2-channel cables between stimulator and commutator</td><td>Plastics One Inc</td><td>305-441/2 W/ Spring</td><td></td></tr><tr><td>2-channel cables between commutator and electrode pedestal</td><td>Plastics One Inc</td><td>305-305 W/ Spring</td><td></td></tr><tr><td>4-channel commutators</td><td>Plastics One Inc</td><td>SL2+2C and SL2+SC/SB</td><td></td></tr></tbody></table>

a general method for evaluating deep brain stimulation effects on intravenous methamphetamine self-administration

Substance use disorders, particularly to methamphetamine, are devastating, relapsing diseases that disproportionally affect young people. There is a need for novel, effective and practical treatment strategies that are validated in animal models. Neuromodulation, including deep brain stimulation (DBS) therapy, refers to the use of electricity to influence pathological neuronal activity and has shown promise for psychiatric disorders, including drug dependence. DBS in clinical practice involves the continuous delivery of stimulation into brain structures using an implantable pacemaker-like system that is programmed externally by a physician to alleviate symptoms. This treatment will be limited in methamphetamine users due to challenging psychosocial situations. Electrical treatments that can be delivered intermittently, non-invasively and remotely from the drug-use setting will be more realistic. This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence. Methamphetamine dependence is rapidly developed in rodents using an operant paradigm of intravenous (IV) self-administration that incorporates a period of extended access to drug and demonstrates both escalation of use and high motivation to obtain drug.

Following placement of an IV jugular catheter and intracranial DBS electrodes, rodents can be successfully trained to self-administer IV methamphetamine after a brief recovery period. Figure 3 shows that rats will acquire and escalate methamphetamine self-administration after 2 days of extended-access to drug with an average of 168 &#177;&#160;12 infusions per session by day 4.
Rats are then moved to a daily 2-hr schedule of operant training for two reasons: 1) to prevent methamphetamine toxicity and severe behavioral alterations with persistent, prolonged access and 2) to establish a relatively stable rate of responding that can be manipulated by various therapeutic interventions. Figure 4 shows that the average number of total infusions per short access session over the second week is 75 &#177; 8 and generally varies by less than 10% day-to-day. Figure 5 demonstrates that rats develop an increased motivation to take drug as shown by the emergence of a &#8220;front-loading&#8221; pattern of intake by day 6 of training as compared to day 1. Once this develops, the effect is largely sustained over subsequent sessions (data not shown).
Figure 6 shows that bilateral DBS delivered in the non-drug environment resulted in a marked decrease of operant IV methamphetamine self-administration on three of five days compared to the sham stimulated group. The nucleus accumbens shell was targeted given its known involvement in drug-consummatory behavior8 using the following stereotactic coordinates relative to bregma (AP + 1.6, DV - 8.5, ML&#160;&#177;&#160;2.4). Stimulation parameters and duration were loosely based on previous published experience with DBS for the treatment of psychiatric disease8,20,21 but can be adjusted depending on the experimenter&#8217;s needs. Error bars are moderate and not all days reach significance indicating the range of responses that can be seen in behavioral assessments despite a clear treatment effect. Increasing the number of rats per experiment can help compensate for this natural variability. 11 animals were initially used for this experiment. One animal was euthanized for poor feeding post-operatively, one animal was excluded due to seizures, and one animal was excluded due to DBS electrode malfunction leaving us with a total of 8 animals (N = 4 Sham; N = 4 active DBS). In general starting with about 10 - 12 rats for each experiment will allow for its successful completion.
<img alt="Figure 1" src="/files/ftp_upload/53266/53266fig1.jpg" />
Figure 1. Visual Programming Language. The investigator uses a visual programming language, like the example shown here, to design a program that can deliver brain stimulation to multiple animals simultaneously at user-entered parameters. <a href="https://www.jove.com/files/ftp_upload/53266/53266fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53266/53266fig2.jpg" />
Figure 2. Visual Control Panel. Prior to the start of the experiment, the investigator specifies the desired frequency, pulse width, and amplitude on the left side of a visual control panel. Here stimulation parameters are: current intensity 200 &#956;A; pulse width 61 msec; pulse frequency 130 Hz. Once stimulation is initiated, the waveform for the active current delivery is displayed on the right. <a href="https://www.jove.com/files/ftp_upload/53266/53266fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53266/53266fig3.jpg" />
Figure 3. Acquisition of IV Methamphetamine Self-Administration. Total operant responding (360&#160;min) data were analyzed using a repeated-measures ANOVA with the daily session defined as the repeated measure. All analyses that were&#160;p&#160;&#60;0.05 were considered significant. Data is mean &#177; standard error. Total methamphetamine infusions during the daily 6-hr operant sessions over the first four days of operant training. + P &#60;0.05 compared to sessions 1 and 2. <a href="https://www.jove.com/files/ftp_upload/53266/53266fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53266/53266fig4.jpg" />
Figure 4. Maintenance of IV Methamphetamine Self-Administration. Total operant responding (120&#160;min) data were analyzed using a repeated-measures ANOVA with the daily session defined as the repeated measure. All analyses that were&#160;p&#160;&#60;0.05 were considered significant. Data is mean &#177; standard error. Total methamphetamine infusions during the daily 2-hr operant sessions over the second week of operant training, demonstrating stable but intense drug-taking behavior. <a href="https://www.jove.com/files/ftp_upload/53266/53266fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53266/53266fig5.jpg" />
Figure 5. Development of Motivation to Take Drug. Operant responding was totaled every 15 min for the first hour and data were analyzed using a repeated-measures ANOVA with each 15-min quadrant defined as the repeated measure. All analyses that were&#160;p&#160;&#60;0.05 were considered significant. Data is mean &#177;&#160;standard error. A &#8220;front-loading&#8221; pattern is not present on day 1 of operant training but develops by the second week, indicating a strong motivation to take drug. + P &#60;0.05 compared to 30, 45, and 60 min, ++ P &#60;0.05 compared to 45 and 60 min, +++ P &#60;0.05 compared to 60 min. <a href="https://www.jove.com/files/ftp_upload/53266/53266fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53266/53266fig6.jpg" />
Figure 6. DBS Effects on IV Methamphetamine Self-Administration. Total operant responding in the first hour (60&#160;min) data were analyzed using a mixed ANOVA with a between subject variable of treatment (DBS vs Sham) and a repeated measure of daily session. All analyses that were&#160;p &#60;0.05 were considered significant. Data is mean &#177; standard error. Bilateral pre-operant deep brain stimulation significantly reduced the number of methamphetamine infusions over the first 60 min of operant responding on treatment days 3, 4, and 7. + P &#60;0.05 compared to sham group and baseline responding. Responding returned to baseline levels after daily treatment ended. <a href="https://www.jove.com/files/ftp_upload/53266/53266fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A General Method for Evaluating Deep Brain Stimulation Effects on Intravenous Methamphetamine Self-Administration at JoVE.com

A General Method for Evaluating Deep Brain Stimulation Effects on Intravenous Methamphetamine Self-Administration

This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence.

Substance use disorders, particularly to methamphetamine, are devastating, relapsing diseases that disproportionally affect young people.  There is a need ...

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Behavior

15.1K Views.  Louisiana State University. This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence.

Video: A General Method for Evaluating Deep Brain Stimulation Effects on Intravenous Methamphetamine Self-Administration

Novel Apparatus and Method for Drug Reinforcement

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction. Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in animal research to model various components of drug reinforcement, consumption, and addiction in humans. For this study, we used a novel approach to studying drug reinforcement in rats by combining traditional CPP and self-administration methodologies. We assembled an apparatus using two Med Associate operant chambers, sensory stimuli, and a Plexiglas-constructed neutral zone. These modifications allowed our experiments to encompass motivational aspects of drug intake through self-administration and drug-free assessment of drug/cue conditioning strength with the CPP test. In our experiments, rats self-administered cocaine (0.75 mg/kg/inj, i.v.) during either four (e.g., the "short-term") or eight (e.g., the "long-term") alternating-day sessions in an operant environment containing distinctive sensory cues (e.g., olfactory and visual). On the alternate days, in the other (differently-cued) operant environment, saline was available for self-infusion (0.1 ml, i.v.). Twenty-four hours after the last self-administration/cue-pairing session, a CPP test was conducted. Consistent with typical CPP findings, there was a significant preference for the chamber associated with cocaine self-administration. In addition, in animals undergoing the long-term experiment, a significant positive correlation between CPP magnitude and the number of cocaine-reinforced lever responses. In conclusion, this apparatus and approach is time and cost effective, can be used to examine a wide array of topics pertaining to drug abuse, and provides more flexibility in experimental design than CPP or self-administration methods alone.

Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans. In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats.

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction.  Operant drug ...

Neuroscience

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to screen different neurostimulation patterns in hemiparkinsonian (unilateral lesioned) rats. It consists of creating a unilateral Parkinson&#39;s lesion by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle, implanting chronic stimulation electrodes into the subthalamic nucleus and evaluating motor outcomes at the end of 24 hr periods of cable-bound external neurostimulation. The stimulation was conducted with constant current stimulation. The amplitude was set 20% below the individual threshold for side effects. The motor outcome evaluation was done by the assessment of spontaneous paw use in the cylinder test according to Shallert and by the assessment of skilled reaching in the staircase test according to Montoya. This protocol describes in detail the training in the staircase box, the cylinder test, as well as the use of both in hemiparkinsonian rats. The use of both tests is necessary, because the staircase test seems to be more sensitive for fine motor skill impairment and exhibits greater sensitivity to change during neurostimulation. The combination of the unilateral Parkinson model and the two behavioral tests allows the assessment of different stimulation parameters in a standardized way.

Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to ...

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

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

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

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

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

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue reactivity, the sensitivity to cues previously linked with the drug-taking experience, plays a prominent role in human craving during abstinence. Cue reactivity can be assessed as the attentional orientation toward drug-associated cues that is measurable as appetitive approach behavior in both preclinical and human studies. Herein describes an assessment of cue reactivity in rats trained to self-administer cocaine. Cocaine self-administration is paired with the presentation of discrete cues that act as conditioned reinforcers (i.e., house light, stimulus light, infusion pump sounds). Following a period of abstinence, lever presses in the cocaine self-administration context accompanied by the discrete cues previously paired with cocaine infusion are measured as cue reactivity. This model is useful to explore neurobiological mechanisms underlying cue reactivity processes as well as to assess pharmacotherapies to suppress cue reactivity and therefore, modify relapse vulnerability. Advantages of the model include its translational relevance, and its face and predictive validities.&#160;The primary limitation of the model is that the cue reactivity task can only be performed infrequently and must only be used in short duration (e.g., 1 hour), otherwise rats will begin to extinguish the pairing of the discrete cues with the cocaine stimulus. The model is extendable to any positively reinforcing stimulus paired with discrete cues; though particularly applicable to drugs of abuse, this model may hold future applications in fields such as obesity, where palatable food rewards can act as positively reinforcing stimuli.

Cue reactivity is conceptualized as sensitivity to cues linked with drug-taking experiences that contribute to craving and relapse in abstinent humans. Cue reactivity is modeled in rats by measuring attentional orientation toward drug-associated cues that results in appetitive approach behavior in a cue reactivity test following self-administration and forced abstinence.

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue ...

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 ...

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement learning. Understanding how the diverse neuronal inputs into the VTA control phasic DA release can provide a better picture of the circuitry that controls reward processing and reinforcement learning. Here, we describe a method that combines intra-VTA cannula infusions of pharmacological agonists and antagonists with stimulation-evoked phasic DA release (combined infusion and stimulation, or CIS) as measured by in vivo fast-scan cyclic voltammetry (FSCV). Using CIS-FSCV in anesthetized rats, a phasic DA response can be evoked by electrically stimulating the VTA with a bipolar electrode fitted with a cannula while recording in the nucleus accumbens core. Pharmacological agonists or antagonists can be infused directly at the stimulation site to investigate specific VTA receptors&#39; roles in driving phasic DA release. A major benefit of CIS-FSCV is that VTA receptor function can be studied in vivo, building on in vitro studies.

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry CIS-FSCV to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

The goal of this protocol is to directly manipulate ventral tegmental area receptors to study their contribution to subsecond dopamine release.

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement ...