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/pubmed?term=((Neurocircuitry+of+addiction%5BTitle%5D)+AND+%22Neuropsychopharmacology%22%5BJournal%5D)">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/pubmed?term=((Animal+models+and+brain+circuits+in+drug+addiction%5BTitle%5D)+AND+%22Mol+Interv%22%5BJournal%5D)">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/pubmed?term=((The+reinstatement+model+of+drug+relapse%3A+recent+neurobiological+findings%2C+emerging+research+topics%2C+and+translational+research%5BTitle%5D)+AND+%22Psychopharmacology+%28Berl%29%22%5BJournal%5D)">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/pubmed?term=((The+predictive+validity+of+the+rat+self-administration+model+for+abuse+liability%29%5BTitle%5D)+AND+%22Neurosci+Biobehav+Rev%22%5BJournal%5D)">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/pubmed?term=((Selective+blockade+of+dopamine+D3+receptors+enhances+while+D2+receptor+antagonism+impairs+social+novelty+discrimination+and+novel+object+recognition+in+rats%3A+a+key+role+for+the+prefrontal+cortex%5BTitle%5D)+AND+%22Neuropsychopharmacology%22%5BJournal%5D)">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/pubmed?term=((GABA%28B%29+receptor+modulation+of+serotonin+neurons+in+the+dorsal+raphe+nucleus+and+escalation+of+aggression+in+mice%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Psychostimulants+act+within+the+prefrontal+cortex+to+improve+cognitive+function%5BTitle%5D)+AND+%22Biol+Psychiatry%22%5BJournal%5D)">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/pubmed?term=((Activator+of+G+protein+signaling+3%3A+a+gatekeeper+of+cocaine+sensitization+and+drug+seeking%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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/pubmed?term=((The+connexin43+mimetic+peptide+Gap19+inhibits+hemichannels+without+altering+gap+junctional+communication+in+astrocytes%5BTitle%5D)+AND+%22Front+Cell+Neurosci%22%5BJournal%5D)">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/pubmed?term=((D1R%2FGluN1+complexes+in+the+striatum+integrate+dopamine+and+glutamate+signalling+to+control+synaptic+plasticity+and+cocaine-induced+responses%5BTitle%5D)+AND+%22Mol+Psychiatry%22%5BJournal%5D)">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/pubmed?term=((The+circuitry+mediating+cocaine-induced+reinstatement+of+drug-seeking+behavior%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Different+neural+substrates+mediate+cocaine+seeking+after+abstinence+versus+extinction+training%3A+a+critical+role+for+the+dorsolateral+caudate-putamen%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Physiology+of+meningeal+innervation%3A+aspects+and+consequences+of+chemosensitivity+of+meningeal+nociceptors%5BTitle%5D)+AND+%22Microsc+Res+Tech%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aNetter%2C+FH+%22Musculoskeletal+System+Part+I%3A+Anatomy%3B+Physiology%2C+and+Metabolic+Disorders%22">Musculoskeletal System Part I: Anatomy; Physiology, and Metabolic Disorders</a>. 8, 1st ed, CIBA-Geigy. Summit, New Jersey. (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/pubmed?term=((Transient+neuronal+inhibition+reveals+opposing+roles+of+indirect+and+direct+pathways+in+sensitization%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Targeting+of+deep+brain+structures+with+microinjections+for+delivery+of+drugs%2C+viral+vectors%2C+or+cell+transplants%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Stereotaxic+injection+of+a+viral+vector+for+conditional+gene+manipulation+in+the+mouse+spinal+cord%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Microdialysis+of+ethanol+during+operant+ethanol+self-administration+and+ethanol+determination+by+gas+chromatography%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Rodent+stereotaxic+surgery+and+animal+welfare+outcome+improvements+for+behavioral+neuroscience%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Survivable+stereotaxic+surgery+in+rodents%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Progressive+ratio+schedules+in+drug+self-administration+studies+in+rats%3A+a+method+to+evaluate+reinforcing+efficacy%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">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/pubmed?term=((Effects+of+reinforcer+sweetness+and+the+D2%2FD3+antagonist+raclopride+on+progressive+ratio+operant+performance%5BTitle%5D)+AND+%22Behav+Pharmacol%22%5BJournal%5D)">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/pubmed?term=((How+to+make+a+rat+addicted+to+cocaine%5BTitle%5D)+AND+%22Prog+Neuropsychopharmacol+Biol+Psychiatry%22%5BJournal%5D)">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/pubmed?term=((A+critique+of+fixed+and+progressive+ratio+schedules+used+to+examine+the+neural+substrates+of+drug+reinforcement%5BTitle%5D)+AND+%22Pharmacol+Biochem+Behav%22%5BJournal%5D)">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/pubmed?term=((Progressive+ratio+as+a+measure+of+reward+strength%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Parameters+of+self-administration+of+cocaine+in+rats+under+a+progressive-ratio+schedule%5BTitle%5D)+AND+%22Pharmacol+Biochem+Behav%22%5BJournal%5D)">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/pubmed?term=((Nucleus+accumbens+AGS3+expression+drives+ethanol+seeking+through+G+betagamma%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Rat+Nucleus+Accumbens+Core+Astrocytes+Modulate+Reward+and+the+Motivation+to+Self-Administer+Ethanol+after+Abstinence%5BTitle%5D)+AND+%22Neuropsychopharmacology%22%5BJournal%5D)">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/pubmed?term=((Motivation+for+alcohol+becomes+resistant+to+quinine+adulteration+after+3+to+4+months+of+intermittent+alcohol+self-administration%5BTitle%5D)+AND+%22Alcohol+Clin+Exp+Res%22%5BJournal%5D)">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/pubmed?term=((Reduced+nucleus+accumbens+SK+channel+activity+enhances+alcohol+seeking+during+abstinence%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSalerni%2C+CM+%22Engineering+Adhesives+for+Repeated+Sterilization%22">Engineering Adhesives for Repeated Sterilization</a>. , Henkel. Available from: 
 <a target="_blank" href="http://www.henkelna.com/us/content_data/99736_Engineering_Adhesives_for_Repeated_Sterilization.pdf">http://www.henkelna.com/us/content_data/99736_Engineering_Adhesives_for_Repeated_Sterilization.pdf</a> (2015).</li>
<li>Gundersen, H. J., Jensen, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+efficiency+of+systematic+sampling+in+stereology+and+its+prediction%5BTitle%5D)+AND+%22J+Microsc%22%5BJournal%5D)">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/pubmed?term=((Combined+injection+of+rAAV+with+mannitol+enhances+gene+expression+in+the+rat+brain%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">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/pubmed?term=((Systemic+mannitol-induced+hyperosmolality+amplifies+rAAV2-mediated+striatal+transduction+to+a+greater+extent+than+local+co-infusion%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">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/pubmed?term=((Modulation+of+the+autonomic+nervous+system+by+acute+glial+cell+Gq-GPCR+activation+in+vivo%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">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/pubmed?term=((Evolving+the+lock+to+fit+the+key+to+create+a+family+of+G+protein-coupled+receptors+potently+activated+by+an+inert+ligand%5BTitle%5D)+AND+%22PNAS%22%5BJournal%5D)">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/pubmed?term=((Potent+block+of+Cx36+and+Cx50+gap+junction+channels+by+mefloquine%5BTitle%5D)+AND+%22PNAS%22%5BJournal%5D)">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/pubmed?term=((Infusion+of+gliotoxins+or+a+gap+junction+blocker+in+the+prelimbic+cortex+increases+alcohol+preference+in+Wistar+rats%5BTitle%5D)+AND+%22J+Psychopharmacol%22%5BJournal%5D)">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/pubmed?term=((Elucidation+of+Homer+1a+function+in+the+nucleus+accumbens+using+adenovirus+gene+transfer+technology%5BTitle%5D)+AND+%22Ann+N+Y+Acad+Sci%22%5BJournal%5D)">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/pubmed?term=((NAC-1+is+a+brain+POZ%2FBTB+protein+that+can+prevent+cocaine-induced+sensitization+in+the+rat%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Measurement+of+startle+response%2C+prepulse+inhibition%2C+and+habituation%5BTitle%5D)+AND+%22Curr+Protoc+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Cocaine+reinforced+progressive+ratio+performance+in+the+rhesus+monkey%5BTitle%5D)+AND+%22Pharmacol+Biochem+Behav%22%5BJournal%5D)">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><tbody><tr><td>Cannula Tubing</td><td>Amazon Supply/ Small Parts</td><td>HTXX-26T-60</td><td>26 G, 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&quot; x 0.025&quot;</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&quot;/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.remotexs.ntu.edu.sg/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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Nanyang Technological University

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JoVE Journal

Behavior

14.3K 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

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

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 ...

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied1,2.
In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells.
The protocol is most advantageous for parallel analysis of multiple samples (e.g. single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies.

This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays.

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific ...

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or knockdown gene expression in a relatively focused region without significant damage to the brain tissue. After recovery, the injected mouse can be tested on various behavioral tasks such as the Open Field Test (OFT) and the Forced Swim Test (FST). The OFT is designed to assess locomotion and the anxious phenotype in mice by measuring the amount of time that a mouse spends in the center of a novel open field. A more anxious mouse will spend significantly less time in the center of the novel field compared to controls. The FST assesses the anti-depressive phenotype by quantifying the amount of time that mice spend immobile when placed into a bucket of water. A mouse with an anti-depressive phenotype will spend significantly less time immobile compared to control animals. The goal of this protocol is to use the stereotactic injection of a lentivirus in conjunction with behavioral tests to assess how genetic factors modulate animal behaviors.

Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST).

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

Biology

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Neuroscience

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

Adaptable Angled Stereotactic Approach for Versatile Neuroscience Techniques

Stereotactic surgery is an essential tool in the modern neuroscience lab. However, the ability to precisely and accurately target difficult-to-reach brain regions still presents a challenge, particularly when targeting brain structures along the midline. These challenges include avoiding of the superior sagittal sinus and third ventricle and the ability to consistently target selective and discrete brain nuclei. In addition, more advanced neuroscience techniques (e.g., optogenetics, fiber photometry, and two-photon imaging) rely on targeted implantation of significant hardware to the brain, and spatial limitations are a common hindrance. Presented here is a modifiable protocol for stereotactic targeting of rodent brain structures using an angled coronal approach. It can be adapted to 1) mouse or rat models, 2) various neuroscience techniques, and 3) multiple brain regions. As a representative example, it includes the calculation of stereotactic coordinates for targeting of the mouse hypothalamic ventromedial nucleus (VMN) for an optogenetic inhibition experiment. This procedure begins with the bilateral microinjection of an adeno-associated virus (AAV) encoding a light-sensitive chloride channel (SwiChR++) to a Cre-dependent mouse model, followed by the angled bilateral implantation of fiberoptic cannulae. Using this approach, findings show that activation of a subset of VMN neurons is required for intact glucose counterregulatory responses to insulin-induced hypoglycemia.

Described here is a stereotactic procedure that can target challenging and difficult-to-reach brain regions (due to spatial limitations) using an angled coronal approach. This protocol is adaptable to both mouse and rat models and can be applied to diverse neuroscientific applications, including cannula implantation and microinjections of viral constructs.

Stereotactic surgery is an essential tool in the modern neuroscience lab. However, the ability to precisely and accurately target difficult-to-reach brain ...

Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and deafferentation of neuronal groups, which are both invasive methods that permanently impair the integrity of the target area. These methods are accompanied by collateral effects that can affect the analysis of acute and temporal regulatory mechanisms. The stereotaxic implantation of guide cannulas aimed at specific brain regions, followed by a recovery period, allows researchers to microinject different drugs after the disappearance of the undesired effects of the surgery. Tetrodotoxin has been used to determine the roles of several brain areas in diverse physiological processes because it transiently inhibits the sodium-dependent action potentials, thus blocking all neural activity in the target region. This protocol combines this method with strategies for the assessment of the estrous cycle and ovulation to reveal the role of discrete brain regions in the regulation of ovulation at particular times of any given stage of the estrous cycle. Awake and unrestrained rats (Rattus norvegicus) were used to avoid the blocking effects that anesthetics and stress hormones exert on ovulation. This protocol can be easily adapted to other species, brain targets and pharmacological agents to study different physiological processes. Future improvements to this method include the design of a microinjection system using glass capillaries of small diameter instead of guide cannulas. This will reduce the amount of tissue damaged during the implantation and decrease the spread of the infused drugs outside the target area.

This protocol describes the construction of a low-cost microinjection system, its stereotaxic implantation into deep-brain structures and the procedure for timed microinjections of tetrodotoxin in awake and unrestrained rats. The goal is to reveal the participation of hypothalamic structures in the regulation of ovulation by inhibiting their neural activity.

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and ...

Stereotaxic Intracranial Delivery of Chemicals, Proteins or Viral Vectors to Study Parkinson's Disease

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in the substantia nigra. Affected brain areas display intraneuronal fibrillar inclusions consisting mainly of alpha-synuclein (asyn) proteins. No animal model thus far has recapitulated all characteristics of this disease. Here, we describe the use of stereotaxic injection to deliver chemicals, proteins, or viral vectors intracranially in order to mimic various aspects of PD. These methods are well-established and widely used throughout the PD field. Stereotaxic injections are incredibly flexible; they can be adjusted in concentration, age of animal used for injection, brain area targeted and in animal species used. Combinations of substances allow for rapid variations to assess treatments or alter severity of the pathology or behavioral deficits. By injecting toxins into the brain, we can mimic inflammation and/or a severe loss of dopaminergic neurons resulting in substantial motor phenotypes. Viral vectors can be used to transduce cells to mimic genetic or mechanistic aspects. Preformed fibrillar asyn injections best recapitulate the progressive phenotype over an extended period of time. Once these methods are established, it can be economical to generate a new model compared to creating a new transgenic line. However, this method is labor intensive as it requires 30 minutes to four hours per animal depending on the model used. Each animal will have a slightly different targeting and therefore create a diverse cohort which on one hand can be challenging to interpret results from; on the other hand, help mimic a more realistic diversity found in patients. Mistargeted animals can be identified using behavioral or imaging readouts, or only after being sacrificed leading to smallercohort size after the study has already been concluded. Overall, this method is a rudimentary but effective way to assess a diverse set of PD aspects.

We describe how to successfully inject solutions into specific brain areas of rodents using a stereotaxic frame. This survival surgery is a well-established method used to mimic various aspects of Parkinson's disease.

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in ...

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

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