MSB is supported by the Alcohol Beverage Medical Research Foundation, a Center for Translational Research Award (UL1 TR000058), the National Institutes for Alcohol Abuse and Alcoholism (P50 AA022537), and startup funds provided by the Virginia Higher Education Equipment Trust Fund and the VCU School of Medicine.

<ol>
	<li>Koob, G. F., Volkow, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurocircuitry+of+addiction.">Neurocircuitry of addiction.</a> Neuropsychopharmacology. 35 (1), 217-238 (2010).</li><li>Kalivas, P. W., Peters, J., Knackstedt, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+and+brain+circuits+in+drug+addiction.">Animal models and brain circuits in drug addiction.</a> Mol Interv. 6 (6), 339-344 (2006).</li><li>Bossert, J. M., Marchant, N. J., Calu, D. J., Shaham, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reinstatement+model+of+drug+relapse:+recent+neurobiological+findings,+emerging+research+topics,+and+translational+research.">The reinstatement model of drug relapse: recent neurobiological findings, emerging research topics, and translational research.</a> Psychopharmacology (Berl). 229 (3), 453-476 (2013).</li><li>Connor, E. C., Chapman, K., Butler, P., Mead, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+predictive+validity+of+the+rat+self-administration+model+for+abuse+liability).">The predictive validity of the rat self-administration model for abuse liability).</a> Neurosci Biobehav Rev. 35 (3), 912-938 (2011).</li><li>Watson, D. J., 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=Selective+blockade+of+dopamine+D3+receptors+enhances+while+D2+receptor+antagonism+impairs+social+novelty+discrimination+and+novel+object+recognition+in+rats:+a+key+role+for+the+prefrontal+cortex.">Selective blockade of dopamine D3 receptors enhances while D2 receptor antagonism impairs social novelty discrimination and novel object recognition in rats: a key role for the prefrontal cortex.</a> Neuropsychopharmacology. 37 (3), 770-786 (2012).</li><li>Takahashi, A., Shimamoto, A., Boyson, C. O., DeBold, J. F., Miczek, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA(B)+receptor+modulation+of+serotonin+neurons+in+the+dorsal+raphe+nucleus+and+escalation+of+aggression+in+mice.">GABA(B) receptor modulation of serotonin neurons in the dorsal raphe nucleus and escalation of aggression in mice.</a> J Neurosci. 30 (35), 11771-11780 (2010).</li><li>Spencer, R. C., Klein, R. M., Berridge, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychostimulants+act+within+the+prefrontal+cortex+to+improve+cognitive+function.">Psychostimulants act within the prefrontal cortex to improve cognitive function.</a> Biol Psychiatry. 72 (3), 221-227 (2012).</li><li>Bowers, M. S., 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=Activator+of+G+protein+signaling+3:+a+gatekeeper+of+cocaine+sensitization+and+drug+seeking.">Activator of G protein signaling 3: a gatekeeper of cocaine sensitization and drug seeking.</a> Neuron. 42 (2), 269-281 (2004).</li><li>Abudara, V., 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=The+connexin43+mimetic+peptide+Gap19+inhibits+hemichannels+without+altering+gap+junctional+communication+in+astrocytes.">The connexin43 mimetic peptide Gap19 inhibits hemichannels without altering gap junctional communication in astrocytes.</a> Front Cell Neurosci. 8, 306 (2014).</li><li>Cahill, E., 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=D1R/GluN1+complexes+in+the+striatum+integrate+dopamine+and+glutamate+signalling+to+control+synaptic+plasticity+and+cocaine-induced+responses.">D1R/GluN1 complexes in the striatum integrate dopamine and glutamate signalling to control synaptic plasticity and cocaine-induced responses.</a> Mol Psychiatry. 19 (12), 1295-1304 (2014).</li><li>McFarland, K., Kalivas, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circuitry+mediating+cocaine-induced+reinstatement+of+drug-seeking+behavior.">The circuitry mediating cocaine-induced reinstatement of drug-seeking behavior.</a> J Neurosci. 21 (21), 8655-8663 (2001).</li><li>Fuchs, R. A., Branham, R. K., See, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+neural+substrates+mediate+cocaine+seeking+after+abstinence+versus+extinction+training:+a+critical+role+for+the+dorsolateral+caudate-putamen.">Different neural substrates mediate cocaine seeking after abstinence versus extinction training: a critical role for the dorsolateral caudate-putamen.</a> J Neurosci. 26 (13), 3584-3588 (2006).</li><li>Ebersberger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+meningeal+innervation:+aspects+and+consequences+of+chemosensitivity+of+meningeal+nociceptors.">Physiology of meningeal innervation: aspects and consequences of chemosensitivity of meningeal nociceptors.</a> Microsc Res Tech. 53 (2), 138-146 (2001).</li><li>Netter, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Musculoskeletal System Part I: Anatomy; Physiology, and Metabolic Disorders. 8, (1987).</li><li>Ferguson, S. M., Eskenazi, D., Ishikawa, M., Wanat, M. J., Phillips, P. E., Dong, Y., Roth, B. L., Neumaier, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+neuronal+inhibition+reveals+opposing+roles+of+indirect+and+direct+pathways+in+sensitization.">Transient neuronal inhibition reveals opposing roles of indirect and direct pathways in sensitization.</a> Nat Neurosci. 14 (1), 22-24 (2011).</li><li>Gonzalez-Perez, O., Guerrero-Cazares, H., Quinones-Hinojosa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+deep+brain+structures+with+microinjections+for+delivery+of+drugs,+viral+vectors,+or+cell+transplants.">Targeting of deep brain structures with microinjections for delivery of drugs, viral vectors, or cell transplants.</a> J Vis Exp. (46), (2010).</li><li>Inquimbert, P., Moll, M., Kohno, T., Scholz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+injection+of+a+viral+vector+for+conditional+gene+manipulation+in+the+mouse+spinal+cord.">Stereotaxic injection of a viral vector for conditional gene manipulation in the mouse spinal cord.</a> J Vis Exp. (73), (2013).</li><li>Schier, C. J., Mangieri, R. A., Dilly, G. A., Gonzales, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+of+ethanol+during+operant+ethanol+self-administration+and+ethanol+determination+by+gas+chromatography.">Microdialysis of ethanol during operant ethanol self-administration and ethanol determination by gas chromatography.</a> J Vis Exp. (67), (2012).</li><li>Fornari, R. V., 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=Rodent+stereotaxic+surgery+and+animal+welfare+outcome+improvements+for+behavioral+neuroscience.">Rodent stereotaxic surgery and animal welfare outcome improvements for behavioral neuroscience.</a> J Vis Exp. (59), e3528 (2012).</li><li>Geiger, B. M., Frank, L. E., Caldera-Siu, A. D., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survivable+stereotaxic+surgery+in+rodents.">Survivable stereotaxic surgery in rodents.</a> J Vis Exp. (20), e880 (2008).</li><li>Richardson, N. R., Roberts, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+ratio+schedules+in+drug+self-administration+studies+in+rats:+a+method+to+evaluate+reinforcing+efficacy.">Progressive ratio schedules in drug self-administration studies in rats: a method to evaluate reinforcing efficacy.</a> J Neurosci Methods. 66 (1), 1-11 (1996).</li><li>Cheeta, S., Brooks, S., Willner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+reinforcer+sweetness+and+the+D2/D3+antagonist+raclopride+on+progressive+ratio+operant+performance.">Effects of reinforcer sweetness and the D2/D3 antagonist raclopride on progressive ratio operant performance.</a> Behav Pharmacol. 6 (2), 127-132 (1995).</li><li>Roberts, D. C., Morgan, D., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+a+rat+addicted+to+cocaine.">How to make a rat addicted to cocaine.</a> Prog Neuropsychopharmacol Biol Psychiatry. 31 (8), 1614-1624 (2007).</li><li>Arnold, J. M., Roberts, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critique+of+fixed+and+progressive+ratio+schedules+used+to+examine+the+neural+substrates+of+drug+reinforcement.">A critique of fixed and progressive ratio schedules used to examine the neural substrates of drug reinforcement.</a> Pharmacol Biochem Behav. 57, 441-447 (1997).</li><li>Hodos, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+ratio+as+a+measure+of+reward+strength.">Progressive ratio as a measure of reward strength.</a> Science. 134 (3483), 943-944 (1961).</li><li>Depoortere, R. Y., Li, D. H., Lane, J. D., Emmett-Oglesby, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parameters+of+self-administration+of+cocaine+in+rats+under+a+progressive-ratio+schedule.">Parameters of self-administration of cocaine in rats under a progressive-ratio schedule.</a> Pharmacol Biochem Behav. 45 (3), 539-548 (1993).</li><li>Bowers, M. S., 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=Nucleus+accumbens+AGS3+expression+drives+ethanol+seeking+through+G+betagamma.">Nucleus accumbens AGS3 expression drives ethanol seeking through G betagamma.</a> Proc Natl Acad Sci U S A. 105 (34), 12533-12538 (2008).</li><li>Bull, C., 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=Rat+Nucleus+Accumbens+Core+Astrocytes+Modulate+Reward+and+the+Motivation+to+Self-Administer+Ethanol+after+Abstinence.">Rat Nucleus Accumbens Core Astrocytes Modulate Reward and the Motivation to Self-Administer Ethanol after Abstinence.</a> Neuropsychopharmacology. 39 (12), 2835-2845 (2014).</li><li>Hopf, F. W., Chang, S. J., Sparta, D. R., Bowers, M. S., Bonci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motivation+for+alcohol+becomes+resistant+to+quinine+adulteration+after+3+to+4+months+of+intermittent+alcohol+self-administration.">Motivation for alcohol becomes resistant to quinine adulteration after 3 to 4 months of intermittent alcohol self-administration.</a> Alcohol Clin Exp Res. 34 (9), 1565-1573 (2010).</li><li>Hopf, F. W., 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=Reduced+nucleus+accumbens+SK+channel+activity+enhances+alcohol+seeking+during+abstinence.">Reduced nucleus accumbens SK channel activity enhances alcohol seeking during abstinence.</a> Neuron. 65 (5), 682-694 (2010).</li><li>Salerni, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Engineering Adhesives for Repeated Sterilization. , (2015).</li><li>Gundersen, H. J., Jensen, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficiency+of+systematic+sampling+in+stereology+and+its+prediction.">The efficiency of systematic sampling in stereology and its prediction.</a> J Microsc. 147 (Pt 3), 229-263 (1987).</li><li>Mastakov, M. Y., Baer, K., Xu, R., Fitzsimons, H., During, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+injection+of+rAAV+with+mannitol+enhances+gene+expression+in+the+rat+brain.">Combined injection of rAAV with mannitol enhances gene expression in the rat brain.</a> Mol Ther. 3 (2), 225-232 (2001).</li><li>Burger, C., Nguyen, F. N., Deng, J., Mandel, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+mannitol-induced+hyperosmolality+amplifies+rAAV2-mediated+striatal+transduction+to+a+greater+extent+than+local+co-infusion.">Systemic mannitol-induced hyperosmolality amplifies rAAV2-mediated striatal transduction to a greater extent than local co-infusion.</a> Mol Ther. 11 (2), 327-331 (2005).</li><li>Agulhon, C., 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=Modulation+of+the+autonomic+nervous+system+by+acute+glial+cell+Gq-GPCR+activation+in+vivo.">Modulation of the autonomic nervous system by acute glial cell Gq-GPCR activation in vivo.</a> J Physiol. 591 (22), 5599-5609 (2013).</li><li>Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S., Roth, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolving+the+lock+to+fit+the+key+to+create+a+family+of+G+protein-coupled+receptors+potently+activated+by+an+inert+ligand.">Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand.</a> PNAS. 104 (12), 5163-5168 (2007).</li><li>Cruikshank, S. J., 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=Potent+block+of+Cx36+and+Cx50+gap+junction+channels+by+mefloquine.">Potent block of Cx36 and Cx50 gap junction channels by mefloquine.</a> PNAS. 101 (33), 12364-12369 (2004).</li><li>Miguel-Hidalgo, J., Shoyama, Y., Wanzo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infusion+of+gliotoxins+or+a+gap+junction+blocker+in+the+prelimbic+cortex+increases+alcohol+preference+in+Wistar+rats.">Infusion of gliotoxins or a gap junction blocker in the prelimbic cortex increases alcohol preference in Wistar rats.</a> J Psychopharmacol. 23 (5), 550-557 (2008).</li><li>Bowers, M. S., Lake, R. W., Rubinchik, S., Dong, J. Y., Kalivas, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidation+of+Homer+1a+function+in+the+nucleus+accumbens+using+adenovirus+gene+transfer+technology.">Elucidation of Homer 1a function in the nucleus accumbens using adenovirus gene transfer technology.</a> Ann N Y Acad Sci. 1003, 419-421 (2003).</li><li>Mackler, S. 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=NAC-1+is+a+brain+POZ/BTB+protein+that+can+prevent+cocaine-induced+sensitization+in+the+rat.">NAC-1 is a brain POZ/BTB protein that can prevent cocaine-induced sensitization in the rat.</a> J Neurosci. 20 (16), 6210-6217 (2000).</li><li>Geyer, M. A., Swerdlow, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+startle+response,+prepulse+inhibition,+and+habituation.">Measurement of startle response, prepulse inhibition, and habituation.</a> Curr Protoc Neurosci. 8, Unit 8.7 (2001).</li><li>Bedford, J. A., Bailey, L. P., Wilson, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cocaine+reinforced+progressive+ratio+performance+in+the+rhesus+monkey.">Cocaine reinforced progressive ratio performance in the rhesus monkey.</a> Pharmacol Biochem Behav. 9 (5), 631-638 (1978).</li></ol>

None of the authors have competing or conflicting interests.

The procedure presented here is an efficient means to manufacture microinjection cannulae and microinjectors that will aid in elucidating the molecular substrates of motivated behavior. This method offers several advantages. First, by manufacturing one&#8217;s own implants and microinjectors, novel experimental parameters can be rapidly optimized; i.e., one does not need to wait for custom made components to arrive. Second, due to the small diameter of the cannula, more cannulae can be simultaneously implanted. This shortens the required surgical time, which can improve survivability, and also allows multiple implants per animal. Third, the software used to control the operant chambers readily accommodates progressive ratio schedules since a fixed ratio paradigm can be rapidly converted to a progressive ratio paradigm by simply applying an event transition parameter list that contains the desired reinforcement schedule.
To be broadly useful, a generic microinjection procedure was presented that should be broadly applicable for the microinjection of nearly any reagent currently available. Consequently, we anticipate that this technique will continue to be of similar high utility in the future with minor modification. By changing only a few variables, this approach can be applied to a wide number of reagents. Parameters that would most commonly be manipulated include the length that the microinjector protrudes from the cannula, volume of injection, and rate of injection. For example, one may want the injector to protrude further from the cannula tip to avoid the glial scar that typically forms around chronic implants. Additionally, one may wish to inject a larger volume. For striatal virus microinjections, a volume of 1 &#956;L is typically used and this volume is typically injected over a longer period of time (frequently 7 &#8211; 10 min plus 3 &#8211; 10 min additional diffusion time) compared to that used for pharmacological reagents (typically 0.3 &#8211; 0.5 &#956;L over 2 &#8211; 3 min plus 1 &#8211; 3 min additional diffusion time). The user should consult the literature and/ or empirically determine the parameters best suited for their needs. Regardless, the success of this procedure is critically dependent upon 4 variables: 1) cannula length, 2) microinjector length, 3) quality of microinjector spray pattern, and 4) the system integrity prior to injection. Because microinjection location is dependent on the depth that the microinjector protrudes from the cannula, it is imperative that both cannulae (Step 1.2.8) and microinjector length (post-bending, Step 2.2.1) are both precisely known and uniform between all subjects. This can easily be controlled by readily rejecting any implement that is not the required length at the final re-measuring. Moreover, the injection location can only be predicted if it occurs immediately beneath the guide cannula. Thus, any microinjector that does not spray a long, fine stream upon testing (Steps 2.4.6) should be rejected. A quality injection is also related to the integrity of the system prior to injection. If after dispensing all water from the injector (prior to filling with reagent) multiple spots are observed on the lab-wipe, then a leak needs to be remedied (Note on Step 2.4.8). Further, if the bubble (Step 2.4.9) that separates the drug from the water in the PE20 tubing is not one, single bubble (after filling the microinjector with reagent), then the injector is partially clogged. This clog could either prevent or divert the injection. This too can be readily remedied (Note on step 2.4.8).
Should one wish to microinject while the animal is in the stereotaxic frame there are three alternatives. First, one could increase the length of the microinjector collar such that it can be held firmly by the stereotaxic manipulator and also extend far enough to allow connection to PE20 tubing. Second, one could temporally implant a cannula and use the standard microinjector presented here. Third, one could use drawn and polished glass pipettes. 16,17
A significant limitation of the procedure presented here is that it is best conducted in well-handled rats that are familiar with the procedure. Rats used for the data described in the results section required no special handling procedures because the same investigator handled the rats on a daily basis for over 2 months. This included daily observation and manipulation of the surgical implant for at least 2 weeks. However, rats can be rapidly habituated by a number of techniques that are used to prior to the pre-pulse inhibition assay, which can be affected by stress. These special habituation techniques have been nicely detailed previously.43 In addition to these procedures, it is advisable that rats be habituated to the microinjection procedure where shortened microinjectors are used during &#8216;sham&#8217; injections. During these sham injections, it is critical that the microinjector not protrude into the tissue in order to limit tissue damage. In other words, the microinjector should be bent no longer than 14 mm. Thus, the thorough habituation required for optimal implementation of this technique could be viewed as a limitation.
While several behavioral paradigms exist to measure motivation, the progressive ratio is commonly used to quantify the effort that the subject is willing to exert to obtain a reinforcer. The progressive ratio paradigm produces a measure known as breakpoint, which is often defined as the maximal number of lever presses in the last completed ratio; i.e., maximal responding that generated a reinforcer.21 The progressive ratio is sensitive to reinforcer magnitude. For example, higher cocaine (or sucrose) doses produce a higher breakpoint and lower cocaine (or sucrose) doses yield a lower breakpoint.21,22 Accordingly, breakpoint is a routinely used proxy for motivation and/or reinforcing efficacy.21,23-26 Because the intention of the breakpoint is to determine when the animal stops responding, an important parameter of the progressive ratio paradigm is session length. Finite session lengths can put a false cap on breakpoint values and this can be exacerbated by pre-treatments that abnormally decrease the rate of self-administration or that increase post-reinforcement pausing. This confound can be overcome by any number of approaches; e.g., sessions that terminate when the animal has withheld responding by some multiple of the average inter-infusion interval.44 A more commonly applied variant of this approach is to terminate sessions once responding has been withheld by some empirically determined value that is held constant across subjects. We have provided the method to apply this approach in Step 4.4.9.11.

Rodents and humans respond in remarkably similar ways to behaviorally relevant stimuli1-3. This suggests that rodents are appropriate subjects for elucidating the molecular substrates of behavior and complex psychiatric conditions4. Understanding the molecular substrates of complex behavioral processes, such as motivation, frequently requires brain microinjection. Both the brain microinjection technique and a primary motivation assay will be presented here. Rats will be used as subjects, but these procedures can readily be adapted to well-handled mice. Included herein are procedures for the manufacture of the required cannulae, obturators (dummy cannulae or stylets), and microinjectors. The method presented is significantly more flexible and more cost-efficient than prefabricated implements. This flexibility will prove valuable when optimizing conditions. Importantly, because the microinjection procedure can be used to test a myriad of hypotheses; the techniques presented here should be broadly applicable. For example, receptor ligands can be microinjected to understand neurochemistry3,5,6; cell-permeable peptides and small-molecules can be microinjected to understand intracellular signaling pathways7-10; toxins, ion channel blockers, or antagonist cocktails can be microinjected to understand circuitry1,11,12.
While the generic protocol presented here can be readily adapted by the user for their particular needs, the procedure is particularly well suited for behavioral assays since microinjection occurs in awake rodents that are only under mild hand restraint. No anesthesia or special restraints are required. This is possible because the brain itself lacks pain sensation. However, if anesthesia is not used, microinjection must occur through cannulae that were previously stereotaxically implanted. This is because nociceptors are present on the scalp, meninges,13 which are the membranes surrounding the brain, and the periosteum,14 which is the membrane covering the skull. It should be noted that microinjection under anesthesia is sometimes desirable. One example is when the virus is being injected, and one may wish to inject virus directly through either stainless steal needles15 or glass pipettes because this can reduce tissue damage and improve transduction efficiency.16,17 The microinjectors described below can be modified for this purpose and suggestions on how to do this can be found in the Discussion. Because other JoVE articles have demonstrated stereotaxic brain cannula implantation,18-20 these procedures will not be covered here.
We present these microinjection procedures together with an assay that quantifies motivation. Several rodent models of motivated behavior are currently in use, such as the runway box and barrier scaling. Here, we describe how to use an operant progressive ratio schedule of reinforcement to quantify motivation where operant responding is being maintained by a reinforcer. Responding on the progressive ratio is responsive to reinforcer magnitude.21,22 Accordingly, this assay is routinely used as a proxy for motivation and/or reinforcing efficacy. 21,23-30 Because several excellent reviews have covered this topic in detail,21,24 we will focus mainly on practical concerns.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cannula Tubing</td>
			<td>Amazon Supply/ Small Parts</td>
			<td>HTXX-26T-60</td>
			<td>26 gauge, Hypotube S/S 316-TW 26GA</td>
		</tr>
		<tr>
			<td>Obturator</td>
			<td>Amazon Supply/ Small Parts</td>
			<td>GWXX-0080-30-05</td>
			<td>33 gauge, Wire S/S 316LVM 0.008 IN</td>
		</tr>
		<tr>
			<td>Microinjector Wire</td>
			<td>MicroGroup</td>
			<td>33RW 304</td>
			<td>33 gauge</td>
		</tr>
		<tr>
			<td>Super Glue</td>
			<td>Loctite</td>
			<td>3924AC</td>
			<td>Liquid, Non-gel, can be autoclaved</td>
		</tr>
		<tr>
			<td>Microinjector Plastic Tubing</td>
			<td>Becton Dickson</td>
			<td>427406</td>
			<td>PE20</td>
		</tr>
		<tr>
			<td>Medium Weight Hemostats</td>
			<td>World Precision Instruments</td>
			<td>501241-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ruler</td>
			<td>Fisher</td>
			<td>09-016</td>
			<td>150 mm</td>
		</tr>
		<tr>
			<td>#7 Forceps</td>
			<td>Stoelting</td>
			<td>52100-77</td>
			<td>Dumont, Dumostar</td>
		</tr>
		<tr>
			<td>Rotary Tool</td>
			<td>Dremmel</td>
			<td>285</td>
			<td>Two-speeds</td>
		</tr>
		<tr>
			<td>Cut-off Disc</td>
			<td>McMaster Carr</td>
			<td>3602</td>
			<td>15/16&#34; x 0.025&#34;</td>
		</tr>
		<tr>
			<td>Microinjection Pump</td>
			<td>Harvard Apparatus</td>
			<td>PhD 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ul Glass Syringe</td>
			<td>Hamilton</td>
			<td>7001KH</td>
			<td>Needle Style: 25s/2.75&#34;/3</td>
		</tr>
		<tr>
			<td>Cotton Tipped Applicator</td>
			<td>Fisher</td>
			<td>23-400-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Wipes</td>
			<td>Kimwipes</td>
			<td>34133</td>
			<td></td>
		</tr>
		<tr>
			<td>Operant Software</td>
			<td>Coulbourn</td>
			<td>Graphic State</td>
			<td></td>
		</tr>
		<tr>
			<td>Operant Chambers</td>
			<td>Coulborun</td>
			<td>Habitest</td>
			<td></td>
		</tr>
	</tbody>
</table>

rodent brain microinjection to study molecular substrates of motivated behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

Here we illustrate examples of results that can be obtained with the above procedure. Shown first is a typical area that is transfected by viral vectors (Figure 3). In general, a transfected volume of ~1 mm3 is obtained in striatal tissue. The volume of the transfected region can be quantified across serial sections using Cavalieri&#8217;s method.32 Importantly, the transfected volume depends on many factors such as the type of tissue being transfected; viral serotype; promoter; rate and volume injected; number of particles injected; injectate pH; and whether hyperosmolar reagents, such as mannitol, were used.33,34 Typically, we microinject 1012 particles/ml, 1 &#181;l/side over 10 min, and allow 7 additional min prior to replacing obturators. Additionally, the injectate is generally around pH 8. Next, we show that motivation can be manipulated by either transgene expression (Figure 4) or pharmacological reagent microinjection (Figure 5).
In Figure 4, we expressed a Designer Receptor Exclusively Activated by Designer Drugs (DREADDs). The DREADD receptor-coding region was followed by an Internal Ribosome Entry Site and a mCitrine cassette. The mCitrine allows convenient visualization of transfected cells. The DREADD was coupled to the heterotrimeric G-protein G&#945;q. Activation of the G&#945;q-coupled DREADD can stimulate astrocytes,28,35 and the DREADD itself can be activated by systemic administration of clozapine-N-oxide (CNO, 3 mg/kg, i.p).14,22,36 Rats were trained to lever respond for ethanol reinforcement, where 3 lever presses yielded 1 chance to drink during daily 1 hr sessions over 60 contiguous days. Next, rats were forced into abstinence, and G&#945;q-coupled DREADDs were expressed in nucleus accumbens core astrocytes. After 3 week abstinence, the motivation to self-administer ethanol was measured by breakpoint.21,27-30 Activation of nucleus accumbens core astrocytes, via systemic CNO administration, decreased the motivation of rats to resume ethanol self-administration after abstinence compared to vehicle. Importantly, CNO had no effect in an equally trained cohort that was expressing Green Fluorescent Protein instead of the DREADD.
<img alt="Figure 3" src="/files/ftp_upload/53018/53018fig3.jpg" /> 
Figure 3. Representative ventral striatal region transfected by microinjected virus. This image illustrates the region of nucleus accumbens astrocytes that were transfected by following the above method. Data are reprinted from Bull et al.13 with permission of the copyright holder. Details can be found in that publication and the accompanying supplementary material. <a href="https://www.jove.com/files/ftp_upload/53018/53018fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53018/53018fig4.jpg" /> 
Figure 4. Motivation of rats to self-administer ethanol was reduced by activating a transgene that was over-expressed in nucleus accumbens core astrocytes. Virus was microinjected and motivation measured via breakpoint after allowing one week for the virus to express. The transgene expressed was a Designer Receptor Exclusively Activated by Designer Drugs (DREADDs). A one-way ANOVA (F(2,30)=3.29, p=0.04) followed by a Scheff&#233; post-hoc revealed that activation of the DREADD by systemic administration of clozapine-N-oxide (CNO, 3 mg/kg, ip) significantly reduced the motivation of rats to self-administer ethanol after abstinence compared to vehicle CNO had no effect in an equally trained cohort that was expressing Green Fluorescent Protein (GFP) instead of the DREADD. Data are reprinted from Bull et al.13 with permission of the copyright holder. Details can be found in that publication and the accompanying supplementary material.
<img alt="Figure 5" src="/files/ftp_upload/53018/53018fig5.jpg" /> 
Figure 5. Microinjection of gap junction blockers increased the motivation of rats to self-administer ethanol after abstinence. Two gap junction blockers were evaluated for their effect on the motivation (measured via breakpoint) of rats to self-administer ethanol: mefloquine and 18-&#945;-glycyrrhetinic acid (18-&#945;). A two-way ANOVA revealed that microinjection of gap-junction blockers into the nucleus accumbens core increased the motivation of rats to self-administer ethanol after abstinence (F(3,40)=5.56, p=0.003). Data are reprinted from Bull et al.13 with permission of the copyright holder. Details can be found in that publication and the accompanying supplementary material.

Watch this Scientific Journal Video about Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior at JoVE.com

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

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JoVE Journal

Behavior

14.1K Views.  Virginia Commonwealth University School of Medicine. Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented. 

Video: Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

This article describes how to microinject viral vectors into mouse brain and then test in a conditioned place preference paradigm that includes an acquisition, extinction and reinstatement phase.

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here with the goal of uncovering rhythm disorders, such as beat deafness. Beat deafness is characterized by poor performance in perceiving durations in auditory rhythmic patterns or poor synchronization of movement with auditory rhythms (e.g., with musical beats). These tasks include the synchronization of finger tapping to the beat of simple and complex auditory stimuli and the detection of rhythmic irregularities (anisochrony detection task) embedded in the same stimuli. These tests, which are easy to administer, include an assessment of both perceptual and sensorimotor timing abilities under different conditions (e.g., beat rates and types of auditory material) and are based on the same auditory stimuli, ranging from a simple metronome to a complex musical excerpt. The analysis of synchronized tapping data is performed with circular statistics, which provide reliable measures of synchronization accuracy (e.g., the difference between the timing of the taps and the timing of the pacing stimuli) and consistency. Circular statistics on tapping data are particularly well-suited for detecting individual differences in the general population. Synchronized tapping and anisochrony detection are sensitive measures for identifying profiles of rhythm disorders and have been used with success to uncover cases of poor synchronization with spared perceptual timing. This systematic assessment of perceptual and sensorimotor timing can be extended to populations of patients with brain damage, neurodegenerative diseases (e.g., Parkinson&#8217;s disease), and developmental disorders (e.g., Attention Deficit Hyperactivity Disorder).

Behavioral tasks that allow for the assessment of perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) are presented. Synchronization of finger tapping to the beat of an auditory stimuli and detecting rhythmic irregularities provides a means of uncovering rhythm disorders.

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

A Dual Task Procedure Combined with Rapid Serial Visual Presentation to Test Attentional Blink for Nontargets

When viewers search for targets in a rapid serial visual presentation (RSVP) stream, if two targets are presented within about 500 msec of each other, the first target may be easy to spot but the second is likely to be missed. This phenomenon of attentional blink (AB) has been widely studied to probe the temporal capacity of attention for detecting visual targets. However, with the typical procedure of AB experiments, it is not possible to examine how the processing of non-target items in RSVP may be affected by attention. This paper describes a novel dual task procedure combined with RSVP to test effects of AB for nontargets at varied stimulus onset asynchronies (SOAs). In an exemplar experiment, a target category was first displayed, followed by a sequence of 8 nouns. If one of the nouns belonged to the target category, participants would respond &#8216;yes&#8217; at the end of the sequence, otherwise participants would respond &#8216;no&#8217;. Two 2-alternative forced choice memory tasks followed the response to determine if participants remembered the words immediately before or after the target, as well as a random word from another part of the sequence. In a second exemplar experiment, the same design was used, except that 1) the memory task was counterbalanced into two groups with SOAs of either 120 or 240 msec and 2) three memory tasks followed the sequence and tested remembrance for nontarget nouns in the sequence that could be anywhere from 3 items prior the target noun position to 3 items following the target noun position. Representative results from a previously published study demonstrate that our procedure can be used to examine divergent effects of attention that not only enhance targets but also suppress nontargets. Here we show results from a representative participant that replicated the previous finding.&#160;

This paper describes a novel dual task procedure combined with Rapid Serial Visual Presentation to look at attentional blink at varied stimulus onset asynchronies. This experimental procedure differed from others in that it tested attentional blink for nontargets.

When viewers search for targets in a rapid serial visual presentation (RSVP) stream, if two targets are presented within about 500 msec of each other, the ...

Using the Activity-based Anorexia Rodent Model to Study the Neurobiological Basis of Anorexia Nervosa

Anorexia nervosa (AN) is a psychiatric illness characterized by excessively restricted caloric intake and abnormally high levels of physical activity. A challenging illness to treat, due to the lack of understanding of the underlying neurobiology, AN has the highest mortality rate among psychiatric illnesses. To address this need, neuroscientists are using an animal model to study how neural circuits may contribute toward vulnerability to AN and may be affected by AN. Activity-based anorexia (ABA) is a bio-behavioral phenomenon described in rodents that models the key symptoms of anorexia nervosa. When rodents with free access to voluntary exercise on a running wheel experience food restriction, they become hyperactive &#8211; running more than animals with free access to food. Here, we describe the procedures by which ABA is induced in adolescent female C57BL/6 mice. On postnatal day 36 (P36), the animal is housed with access to voluntary exercise on a running wheel. After 4 days of acclimation to the running wheel, on P40, all food is removed from the cage. For the next 3 days, food is returned to the cage (allowing animals free food access) for 2 hr daily. After the fourth day of food restriction, free access to food is returned and the running wheel is removed from the cage to allow the animals to recover. Continuous multi-day analysis of running wheel activity shows that mice become hyperactive within 24 hr following the onset of food restriction. The mice run even during the limited time during which they have access to food. Additionally, the circadian pattern of wheel running becomes disrupted by the experience of food restriction. We have been able to correlate neurobiological changes with various aspects of the animals&#8217; wheel running behavior to implicate particular brain regions and neurochemical changes with resilience and vulnerability to food-restriction induced hyperactivity.

Here we present a protocol to induce activity-based anorexia (ABA) in female adolescent mice. ABA is a condition of hyperactivity evoked by imposing food restriction on rodents with access to a running wheel. This phenomenon is being used as a model to study the underlying neurobiology of anorexia nervosa.

Anorexia nervosa (AN) is a psychiatric illness characterized by excessively restricted caloric intake and abnormally high levels of physical activity. A ...

Combined Shuttle-Box Training with Electrophysiological Cortex Recording and Stimulation as a Tool to Study Perception and Learning

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the necessary equipment is now available by several commercial companies. This protocol provides a detailed description of a two-way shuttle-box avoidance learning paradigm in rodents (here Mongolian gerbils; Meriones unguiculatus) in combination with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical electrophysiological in vivo recordings. The detailed protocol is applicable to study multiple aspects of learning behavior and perception in different rodent species.
Site-specific ICMS of auditory cortical circuits as conditioned stimuli here is used as a tool to test the perceptual relevance of specific afferent, efferent and intracortical connections. Distinct activation patterns can be evoked by using different stimulation electrode arrays for local, layer-dependent ICMS or distant ICMS sites. Utilizing behavioral signal detection analysis it can be determined which stimulation strategy is most effective for eliciting a behaviorally detectable and salient signal. Further, parallel multichannel-recordings using different electrode designs (surface electrodes, depth electrodes, etc.) allow for investigating neuronal observables over the time course of such learning processes. It will be discussed how changes of the behavioral design can increase the cognitive complexity (e.g. detection, discrimination, reversal learning).

Shuttle-box avoidance learning is well-established in behavioral neuroscience. This protocol describes how shuttle-box learning in rodents can be combined with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical in vivo recordings as a tool to study multiple aspects of learning and perception.

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For example, in the cat, visual information about an impending obstacle can modulate stepping for avoidance. Locomotor adaptation can also occur independent of vision, as sudden tactile inputs to the leg by an expected obstacle can modify the stepping of all four legs for avoidance. Such complex locomotor coordination involves supraspinal structures, such as the parietal cortex. This protocol describes the use of reversible, cooling-induced cortical deactivation to assess parietal cortex contributions to memory-guided obstacle locomotion in the cat. Small cooling loops, known as cryoloops, are specially shaped to deactivate discrete regions of interest to assess their contributions to an overt behavior. Such methods have been used to elucidate the role of parietal area 5 in memory-guided obstacle avoidance in the cat.

Complex locomotion in naturalistic environments requiring careful coordination of the limbs involves regions of the parietal cortex. The following protocol describes the use of reversible cooling-induced deactivation to demonstrate the role of parietal area 5 in memory-guided obstacle avoidance in the walking cat.

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For ...

Central and Divided Visual Field Presentation of Emotional Images to Measure Hemispheric Differences in Motivated Attention

Two dominant theories on lateralized processing of emotional information exist in the literature. One theory posits that unpleasant emotions are processed by right frontal regions, while pleasant emotions are processed by left frontal regions. The other theory posits that the right hemisphere is more specialized for the processing of emotional information overall, particularly in posterior regions.
Assessing the different roles of the cerebral hemispheres in processing emotional information can be difficult without the use of neuroimaging methodologies, which are not accessible or affordable to all scientists. Divided visual field presentation of stimuli can allow for the investigation of lateralized processing of information without the use of neuroimaging technology. 
This study compared central versus divided visual field presentations of emotional images to assess differences in motivated attention between the two hemispheres. The late positive potential (LPP) was recorded using electroencephalography (EEG) and event-related potentials (ERPs) methodologies to assess motivated attention. Future work will pair this paradigm with a more active behavioral task to explore the behavioral impacts on the attentional differences found.

This study compared central versus divided visual field presentations of emotional images to assess differences in motivated attention between the two hemispheres. The late positive potential (LPP) was recorded using electroencephalography (EEG) and event-related potentials (ERPs) methodologies to assess motivated attention.

Two dominant theories on lateralized processing of emotional information exist in the literature. One theory posits that unpleasant emotions are processed ...

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, and issues commands to start or stop feeding. Deciphering the processes underlying the decision-making of timely and moderate feeding carries major implications in our understanding of physiological and psychological disorders related to feeding control. Simple, quantitative, and robust methods are required to measure the food ingestion of animals after experimental manipulation, such as forcibly increasing the activities of certain target neurons. Here, we introduced dye-labeling-based feeding assays to facilitate the neurogenetic study of feeding control in adult fruit flies. We review available feeding assays, and then describe our methods step-by-step from setup to analysis, which combine thermogenetic and optogenetic manipulation of neurons controlling feeding motivation with dye-labeled food intake assay. We also discuss the advantages and limitations of our methods, compared with other feeding assays, to help readers choose an appropriate assay.

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, ...

Stress-Enhanced Fear Learning, a Robust Rodent Model of Post-Traumatic Stress Disorder

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders such as post-traumatic stress disorder (PTSD). To understand the biological mechanisms of fear dysregulation in PTSD, it is important to start with a valid animal model of the disorder. This protocol describes the methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of PTSD, in both rats and mice. SEFL was developed to recapitulate critical aspects of PTSD, including long-term sensitization of fear learning caused by an acute stressor. SEFL uses aspects of Pavlovian fear conditioning but produces a distinct and robust sensitized fear response far greater than normal conditional fear responses. The trauma procedure involves placing a rodent in a conditioning chamber and administering 15 unsignaled shocks randomly distributed over 90 minutes (for rat experiments; for mouse experiments, 10 unsignaled shocks randomly distributed over 60 minutes are used). On day 2, rodents are placed in a novel conditioning context where they receive a single shock; then, on day 3 they are placed back in the same context as on day 2 and tested for changes in freezing levels. Rodents that previously received the trauma display enhanced levels of freezing on the test day compared to those that received no shocks on the first day. Thus, with this model, a single highly stressful experience (the trauma) produces extreme fear of the stimuli associated with the traumatic event.

Here we describe the detailed methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of post-traumatic stress disorder, in both rats and mice. The model utilizes aspects of Pavlovian fear conditioning and freezing as an index of enhanced fear in rodents.

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders ...