Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Canadian Council of Animal Care and the McGill University Animal Care Committee. This research was supported by funds awarded to KG from the Canadian Institutes of Health Research (CIHR). No conflicts of interest declared.

1. Animal Housing and Acclimatization Procedures
<ol>
 <li>Male and female CD-1 mice are housed with same-sex littermates, up to 5 animals per cage, in standard plastic cages containing beta chip bedding and nestlet pads, with wire grid lids to accommodate water bottles and food. Standard mouse chow and water are available ad libitum in the home cages throughout the experiment. </li>
 <li>Mice are maintained on a reverse light-dark schedule (lights on 22:00 to 10:00). </li>
 <li>Mice are allowed to acclimatize to the IVSA testing rooms for one week prior to the start of experimental procedures. </li>
</ol>
2. Operant Training
<ol>
 <li>All behavioural procedures including sweetened milk training and intravenous drug self-administration sessions are carried out using operant chambers measuring 15.9 x 14 x 12.7 cm, equipped with 2 ultra-sensitive mouse levers, dipper cups, stimulus lights and microliter syringe pumps (Med Associates Inc., St. Albans, VT, USA). Chambers are interfaced to a computer using Med Associates Smart CR interface and Med-PC software to program the schedule of reinforcement and collect data. </li>
 <li>Na&iuml;ve mice are habituated to the operant chambers and trained to lever press using a sweetened milk reward (0.1 ml) presented in a dipper cup. Operant milk training is conducted in 1-hr sessions for 5-7 days. Completion of a fixed ratio (FR) schedule on the active lever resulted in the presentation of the dipper cup and the illumination of a stimulus light. The sweetened milk solution consisted of sucrose (10 mg/ml, ACS Reagent, Sigma-Aldrich Inc. St-Louis MO, USA) added to whole milk (3.25% fat content). Operant training with a sweetened milk solution or a liquid food reinforcer is a common technique used to facilitate operant responding in mice 1,2. </li>
</ol>
3. Preparation of Equipment to be Used during Surgery 
Required equipment - 20, 23 and 26 gauge needles, grinder, 1 cc syringes, tygon tubing, soldering iron, heparin, antibiotics and analgesic.
<ol>
 <li>Prepare a needle to guide the insertion of the catheter into the jugular vein by shaving down a 20 gauge needle. The shaft of the needle is shaved down using a grinder to form a channel within the needle to guide the catheter tubing into the vein. The needle channel should be carefully checked for any metal debris that may have been deposited. Any obstructions to the channel should be scraped out using fine tipped forceps. </li>
 <li>Adapt two 1-cc syringes to be used for flushing and checking the catheter. Prepare two syringes by taking 12 cm pieces of Tygon tubing and attach them at one end to 26 gauge needles affixed to 1 cc syringes, and stretching the other ends of the tubing over 23 gauge needles. One syringe should be filled with 0.9% sterile saline and the other with a heparinized ticarcillin solution (33 mg of ticarcillin and 0.3 mg of heparin per 10 ml of sterile saline). </li>
 <li>Prepare catheter cannula caps. Tygon tubing is stretched over a 23 gauge needle, and cut 1 cm from the bevel. Melt the open end of the tubing to create a thick seal. The tubing should be melted so that the entire cannula cap is long enough to fit over the catheter cannula, with the sealed end fitting snugly over the end of the cannula. Note that if the cap is too long, there is a risk that it will be bent and subsequently pierced during the process of attaching the screw-on catheter covers. Thus careful preparation of the catheter caps is recommended, in order to provide a good fit to the cannula.</li>
 <li>Prepare required solutions. All reagents are purchased from Sigma-Aldrich Inc. (St-Louis MO, USA).
 <ol class="lalpha">
 <li> Heparinized Ticarcillin solution for catheter flushing - Dissolve 0.33 g Ticarcillin (disodium salt) and 0.003 g of heparin in 10 ml of sterile saline. 0.03 ml of solution is flushed through the catheter daily. The antibiotic solution is administered to prevent blood clots and infections from developing. </li>
 <li>Amikicin (antibiotic) for subcutaneous injection - A single subcutaneous injection is given following surgery at a dose of 10 mg/kg to prevent post-operative infections. </li>
 <li>Ketoprofen (analgesic) for subcutaneous injection - A single subcutaneous injection is given following surgery at a dose of 5 mg/kg to manage any post-operative pain. </li>
 </ol>
 </li>
</ol>
4. Catheter Implantation Surgery 
Required equipment and reagents: Isoflurane, sterile saline, alcohol (70%), 1 and 3 cc syringes filled with sterile saline, 1-cc adapted syringes, antibiotic and analgesic solutions prepared in section 3 above, mouse catheters (CamCaths, Cambridgeshire, UK), catheter covers (crystal caps from HRS Scientific, Montreal, Quebec), eye lubricant, 4 cm plastic bar to elevate vein (this can be constructed from a plastic Q-tip), polysporin, sterile swabs and gauze, curved and straight forceps, artery clamps, fine scissors. 
<img src="/files/ftp_upload/3739/3739step1.jpg" alt="Step 1" fo:content-width="3in" fo:src="/files/ftp_upload/3739/3739step1highres.jpg"/>
<ol>
 <li>Using standard aseptic techniques the surgical bench, the surgical instruments, and the catheters are sterilized prior to surgery. Proper sterilization techniques include steam autoclaving for the surgical instruments and catheters purchased from CamCaths. Glass bead sterilization can also be used on the tips of the surgical instruments. Ethylene oxide sterilization can be employed on more delicate catheters or materials that risk melting. A more detailed description of rodent surgical aseptic techniques can be found in the attached references 1,2. Set up of the bench, instruments and nose cone for maintenance anaesthesia is illustrated in the photo. </li>
 <li>Mice are anaesthetized with isoflurane gas, and maintained under anaesthesia using a breathing tube under a scavenging system. Eye lubricant (Tears Naturale P.M) is applied to both eyes to prevent them from drying out during the procedure. An appropriate surgical margin is shaved on the animal's back and around the neck. Before any incisions are made, the surgical fields are restricted using sterile drapes. </li>
 <li>In order to prepare the catheter for insertion into the right atrium of the heart, the excess catheter tubing is cut off 1.2 cm from the bulb of the catheter. This is the optimal length established for adult CD-1 mice, approximately 8 weeks of age, 20-25 grams body weight. The length of catheter tubing may need to be adjusted slightly (by trial and error), based on the strain, size and age of the mice. Prior to insertion, the syringe containing sterile saline (section 3.2) is attached to the catheter cannula, and the catheter is flushed and checked for leaks. Keep this syringe attached to the catheter throughout the surgical procedure. It will be used to flush the catheter, and draw back blood in step 4.7. </li>
 <li>Following sterilization with 70% alcohol, a 2 cm long midscapular incision is made starting midway on the back and ending just below the neck in order to accommodate the base of the catheter. Connective tissue should be forced apart with forceps to make space for the catheter base below the skin. </li>
 <li>Placing the animal on its back, a second shallow 1 - 2 cm diagonal incision is made from the right clavicle going upwards to the animals jaw, after the area has been swabbed with 70% alcohol. The jugular vein will be found superficially under the skin of the neck. In preparation for insertion of the catheter, tubing from the base of the catheter is pulled through the incision on the back and brought close to the jugular vein by passing the tubing under the skin just over the right shoulder. The end of the catheter tubing is then attached to an artery clamp and placed at the animal's side to keep it in place. </li>
 <li>The right jugular vein is located by gently moving away superficial connective and adipose tissue from the incision around the animal's neck. Connective tissue around the vein is broken apart using curved forceps and the vein is then elevated using a sterile plastic bar. Loose open suture knots are made around each end of the vein and the catheter tubing is threaded through the top knot and looped over the suture thread to rest unclamped over the right shoulder.</li>
 <li>Prior to insertion, wet both the 20 gauge insertion needle and vein with sterile saline to reduce friction. The needle is held parallel to the vein, and inserted gently near the bottom of the elevated vein (Note: approximately 0.5 cm of the needle tip needs to enter the vein). Using forceps, slide the catheter tubing down the shaft of the needle into the vein. Resistance would indicate that the tubing is within connective tissue and not within the vein. Push 0.03 cc of saline through the vein to make sure that there are no leaks. Leaks would indicate that the vein may be pierced or that the catheter tubing placement needs adjusting. 
 Note: To check if the tubing is within the vein, attempt to draw up some blood using the attached saline syringe. If blood cannot be immediately drawn up, the vein or heart wall may be occluding the catheter tip, or the vein has not been pierced; adjust the tubing and try again. Needle reinsertion may be necessary if blood still cannot be drawn up. </li>
 <li>In order to secure the catheter in place, push the catheter bulb to the insertion point and remove the needle. Tie the bottom knot and then pull the catheter flush against the bar before tying the second knot right above the bulb. Test again to see if blood can be drawn up and loosen the knots slightly if needed. Tuck the catheter tubing under the skin and suture the ventral incision around the animal's neck. Apply Polysporin Heal Fast using a sterile cotton tip applicator, or any other antibiotic ointment preferably containing some analgesic to the closed incision. </li>
 <li>With the animal on its abdomen, place the catheter base under the skin of the back within the prepared incision. Make sure that the excess tubing is minimally looped and well hidden under the catheter base to minimize the chances of the animal chewing and piercing it. Suture the incision on both sides of the catheter base, and apply Polysporin Heal Fast using a sterile cotton tip applicator.</li>
 <li>Flush the catheter with 0.03 cc of the heparinized ticarcillin solution using the syringe with the tubing affixed to it (section 3.2). Cap the cannula with the plastic cannula cap and screw on the white catheter cover. In some mice that are fresh from surgery, blood may leak out of the catheter before the cannula is capped. It is important to re-flush the animal and rapidly replace the cannula cap before blood has the chance to flow out. Animals must be flushed on a daily basis to maintain catheter patency. </li>
 <li>After wiping down the injection area between the animals back legs with 70% ethanol, subcutaneously inject the analgesic ketoprofen at a dose of 5 mg/kg on one side, and the antibiotic amikicin at a dose of 10 mg/kg on the other side.</li>
 <li>After anaesthesia is discontinued, animals are allowed to recover in a clean cage with easy access to food and water for 5 to 7 days. Mice should be placed in a heated cabinet overnight to prevent post-operative hypothermia. </li>
</ol>
<img src="/files/ftp_upload/3739/3739step2.jpg" alt="Step 2" fo:content-width="4.75in" fo:src="/files/ftp_upload/3739/3739step2highres.jpg"/>
5. Behavioural testing &#x2013; Intravenous Self-Administration
<ol>
 <li>Prior to behavioural testing catheters are flushed with 0.9% sterile saline. Mice are then placed in the operant chambers and connected to the infusion lines and infusion pumps. Active lever presses result in a 3.2 sec 18 &mu;l drug infusion coupled with the illumination of a stimulus light. Each lever press is followed by an 8 sec time out period during which the stimulus light stays on. </li>
 <li>Following the operant session, mice catheters are flushed with the heparinized ticarcillin solution before being returned to their home cage. </li>
 <li>Mice are allowed to self-administer for 3 consecutive 2-hr sessions at each dose. Doses were presented in a random order for each mouse, as shown in the following section. </li>
 <li>Catheter patency is assessed daily by ensuring that both the saline and antibiotic solution can be flushed through the catheter. In addition, a ketamine/midazolam test can be conducted as described in the attached reference 5. In brief, signs of anesthesia such as immobility within 5 sec of an infusion of 0.02-0.03 ml ketamine (15 mg/ml), or midazolam (0.75 mg/ml) midazolam is evidence of a patent catheter 5. </li>
</ol>

<ol>
	<li>Rocha, B. A., Ator, R., Emmett-Oglesby, M. W., Hen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Intravenous Cocaine Self-Administration in Mice Lacking 5-HT1B Receptors. Pharmacology Biochemistry and Behavior. 57 (3), 407-412 (1997).</li><li>Zapata, A., Chefer, V. I., Ator, R., Shippenberg, T. S., Rocha, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+sensitization+and+enhanced+dopamine+response+in+the+nucleus+accumbens+after+intravenous+cocaine+self-administration+in+mice.">Behavioural sensitization and enhanced dopamine response in the nucleus accumbens after intravenous cocaine self-administration in mice.</a> European Journal of Neuroscience. 17 (3), 590-596 (2003).</li><li>Pritchett-Corning, K. R., Mulder, G. B., Luo, Y., White, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+Rodent+Surgery+for+the+New+Surgeon.">Principles of Rodent Surgery for the New Surgeon.</a> J. Vis. Exp. (47), e2586 (2011).</li><li>Hoogstraten-Miller, S. L., Brown, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Techniques in Aseptic Rodent Surgery Current Protocols in Immunology. , 1.12-1.14 (2001).</li><li>Thomsen, M., Caine, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+Intravenous+Drug+Self-Administration+in+Rats+and+Mice.">Chronic Intravenous Drug Self-Administration in Rats and Mice.</a> Current Protocols in Neuroscience. . 9, 20.1-20.40 (2005).</li><li>Gill, K. J., Boyle, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confirmation+of+quantitative+trait+loci+for+cocaine+induced+activation+in+the+AcB/BcA+series+of+recombinant+congenic+strains.">Confirmation of quantitative trait loci for cocaine induced activation in the AcB/BcA series of recombinant congenic strains.</a> Pharmacogenetics. 13, 329-338 (2003).</li><li>Gill, K. J., Boyle, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+influences+on+drug-induced+psychomotor+activation+in+mice.">Genetic influences on drug-induced psychomotor activation in mice.</a> Genes, Brain &#38; Behavior. 7, 859-868 (2008).</li><li>Boyle, A. E., Gill, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+AXB/BXA+recombinant+inbred+lines+of+mice+to+the+locomotor+activating+effects+of+cocaine:+A+QTL+analysis.">Sensitivity of AXB/BXA recombinant inbred lines of mice to the locomotor activating effects of cocaine: A QTL analysis.</a> Pharmacogenetics. 11 (3), 255-264 (2001).</li><li>Boyle, A. E., Gill, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+verification+of+previously+identified+QTLs+for+cocaine-induced+activation+using+a+panel+of+B6.A+chromosome+substitution+strains+(CSS)+and+A/JxC57Bl/6J+F2+mice.">A verification of previously identified QTLs for cocaine-induced activation using a panel of B6.A chromosome substitution strains (CSS) and A/JxC57Bl/6J F2 mice.</a> Psychopharmacology. 207 (2), 325-3234 (2009).</li><li>Thomsen, M., Caine, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychomotor+stimulant+effects+of+cocaine+in+rats+and+15+mouse+strains.">Psychomotor stimulant effects of cocaine in rats and 15 mouse strains.</a> Experimental and Clinical Psychopharmacology. 19 (5), 321-341 (2011).</li><li>Thomsen, M., Caine, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+drug+self-administration+in+mice:+practical+considerations.">Intravenous drug self-administration in mice: practical considerations.</a> Behavior genetics. 37 (1), 101-118 (2007).</li></ol>

No conflicts of interest declared.

Animal models of drug abuse are particularly useful in understanding the genetic basis of drug-related behaviours. For instance, mice with different genetic profiles show heritable differences in their sensitivity to cocaine 6-8 and help identify potential gene candidates mediating the phenotypic variability observed 9. The intravenous catheterization procedures described in this paper have been used with considerable success to examine drug IVSA in various strains of mice as well as mice of different genetic backgrounds 10. 
The procedures shown in this video highlight important factors to focus on during and following the catherization surgery in order to obtain reliable intravenous self-administration data. First, the placement of the catheter tubing within the right atrium is vital, in order to prevent catheter failure from blot clots. During surgery it is important to make sure that the catheter end is unimpeded, and not occluded by either the heart or vein tissue. Second, daily catheter flushing is required both before and after operant sessions in order to prevent blockages. Finally, the catheter cannula must be covered constantly with both the cannula caps and crystal covers when the animals are in their home cages, to prevent the entry of debris. Minor blockage of the catheter cannula may be dislodged using a fine 26 gauge needle, however daily flushing with the heparinized antibiotic solution is required, particularly on days when the animals are not tested for IVSA. 
To perform animal survival surgery, a good knowledge of aseptic techniques, analgesia and anaesthesia is necessary. While this video does not replace proper surgical training, it may be used as a guide for researchers wishing to acquire the techniques necessary for this paradigm.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Ticarcillin Disodium 			Salt</td>
 <td>Sigma-Aldrich</td>
 <td>T5639-1G</td>
 <td>1 gram bottle</td>
 </tr>
 <tr>
 <td>Tears 			Naturale P.M (Eye lubricant)</td>
 <td>Alcon</td>
 <td>N/A</td>
 <td>3.5 			gram tube 
This 			eye lubricant can be found in most pharmacies. Any ophthalmic eye 			lubricant can be used.</td>
 </tr>
 <tr>
 <td>Cocaine </td>
 <td>Medisca 			Pharmaceutique</td>
 <td>0723-06</td>
 <td>1 			gram bottle</td>
 </tr>
 <tr>
 <td>Ketamine</td>
 <td>Medisca 			Pharmaceutique</td>
 <td>1754-04</td>
 <td>25 			gram bottle</td>
 </tr>
 <tr>
 <td>Midazolam</td>
 <td>Medisca 			Pharmaceutique</td>
 <td>2519-06</td>
 <td>1 			gram bottle</td>
 </tr>
 <tr>
 <td>Heparin sodium salt</td>
 <td>Sigma-Aldrich</td>
 <td>H4784-250MG</td>
 <td>250 gram bottle</td>
 </tr>
 <tr>
 <td>Ketoprofen</td>
 <td>Sigma-Aldrich</td>
 <td>K1751-1G</td>
 <td>1 gram bottle</td>
 </tr>
 <tr>
 <td>Amikicin Sulphate salt</td>
 <td>Sigma-Aldrich</td>
 <td>A2324-5G</td>
 <td>5 gram bottle</td>
 </tr>
 <tr>
 <td>Sucrose ACS Reagent</td>
 <td>Sigma-Aldrich</td>
 <td>S5016-500G</td>
 <td>500 gram bottle</td>
 </tr>
 <tr>
 <td>Standard mouse catheters 			with soft mesh</td>
 <td>CamCaths</td>
 <td>MIVSA/20/26/BC-1S</td>
 <td>Tubing specifications: 			26SWG;0.451;0.254</td>
 </tr>
 <tr>
 <td>Crystal cap applicator</td>
 <td>HRS Scientific</td>
 <td>313CAC</td>
 <td>This is the white 			catheter cap.</td>
 </tr>
 <tr>
 <td>Tygon Micro Bore Tubing</td>
 <td>Thomas Scientific</td>
 <td>9561S41</td>
 <td>Tubing specifications 			(bore x o.d. x wall thickness in inches): .01 x .03 x .01 </td>
 </tr>
 </tbody>
</table>

methods for intravenous self administration in a mouse model

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

<img src="/files/ftp_upload/3739/3739fig1.jpg" alt="Figure 1" /> 
Figure 1. The pattern of responding for Intravenous drug self-administration will vary by drug, dose range and mouse strain employed. The figure presented shows cocaine self-administration data following successful catherization surgeries using the procedure described in the video. The figure shows the mean (&plusmn;SEM) cocaine infusions earned and mean (&plusmn;SEM) cocaine consumption (mg/kg body weight) across a range of 4 cocaine doses presented in a random order on a FR1 schedule of reinforcement. Abscissa: dose of self-administered drug per infusion. Left Ordinate: total number of infusions earned during the 2-hr operant session. Right Ordinate: total cocaine intake in mg/kg during the 2-hr testing session. All 13 catheters remained patent for the duration of the study (4 weeks). A one-way ANOVA conducted on dose revealed that mice were administering cocaine in a dose-dependent fashion [F (1,12) = 42.8 , p&lt;0.05]. There is an increase in cocaine consumption over the dose-response curve [F (3,36) = 29.6, p&lt;0.05] despite a decrease in lever pressing at the higher doses. Each data point represents the average of 3 testing sessions at each cocaine dose (&plusmn;SEM) collected in CD-1 mice (n=13/ dose, males and females combined). Comparisons of active (drug-reinforced) vs inactive lever responding across the dose response curve were made using two-way ANOVA to ensure that mice were discriminating between the two levers. For the CD1 mice, the analysis revealed a preference for the active lever [F (1,12) = 10.255 , p&lt;0.05] over the entire dose-response curve (data not shown here).

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Methods for Intravenous Self Administration in a Mouse Model

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

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

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53.0K Views.  McGill University Health Centre. The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

Video: Methods for Intravenous Self Administration in a Mouse Model

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

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

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

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

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

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...