禁欲ラットで探しているヘロインに長期の食事制限の影響を研究するための手順

The overall goal of this procedure is to study the effects of environmental manipulations, such as chronic food restriction on relapse to heroin seeking in laboratory wraps under withdrawal conditions. This is achieved by first surgically inserting an intravenous catheter into the rat's jugular vein to allow for heroin self-administration following a recovery period. Rats are trained to self-administer heroin in operant conditioning chambers for a period of 10 days.

The next step of the procedure is the abstinence phase where rats are transported to the animal facility and subjected to a period of restricted or unrestricted access to food. Finally, the rats are returned to the operant conditioning chambers to undergo a heroin seeking test where responses on the heroin impaired lever result in activation of a cue, light and tone in absence of heroin delivery. Ultimately, chronically food restricted rats With a history of heroin, self-administration will exhibit a robust increase in heroin seeking under withdrawal conditions, resulting from re-exposure to the drug taking environment and drug associated cues.

The main advantage of this technique over existing methods, such as the reinstatement procedure, is the absence of explicit operant extinction, which is not observed in the human condition. Furthermore, the use of chronic versus acute manipulations allows for greater construct validity with respect to the human condition. This method can help answer key questions in the drug addiction field, such as the common neurological mechanisms underlying drug and food seeking behaviors.

Catheters are prepared in bulk and autoclave sterilized for use over a long period of time. To construct a catheter, first cuts elastic tubing into 12 centimeter long pieces. Use a toothpick or an 18 gauge needle to place a small drop of silicon around the catheter three centimeters from the tip.

Let the silicon dry for 24 hours before autoclaving bend the long tube of the five up cannula, which will serve as a connector in a right angle to the plastic pedestal before autoclaving. To assemble the catheters on the day of the surgery, push the long side of the scholastic tube over the bent metal tube of the five up cannula about halfway to the plastic pedestal. Connect the five up tube via tigon tubing and a blended 22 gauge needle to a one milliliter syringe filled with sterile saline.

Flush saline through the catheter to ensure unobstructed flow. To begin anesthetize the animal before shaving and cleaning the area on the right shoulder and the head with alcohol and a surgical scrub. After applying gel to the eyes to prevent drying, penicillin and saline are injected to protect against infection and dehydration.

Use standard techniques to expose the external jugular vein once isolated from the surrounding tissue. Insert curved forceps under the vein to keep it lifted and slightly stretched. Make a small V-shaped cut on the vein and hold the cut open.

Using a vessel dilator advance the short three centimeter tip of the catheter into the vein towards the heart until the silicone drop is at the incision point. Tie the catheter to the vein with three sutures, one as far coddle as possible. One just rostral to the silicone drop, and the third one as far rostral as possible.

Pass saline through the catheter to ensure that it remains obstructed and that the sutures are not too tight. Next place the rat ventral side down. Incise the head and insert a hemostat to separate the skin from the underlying tissue and create a subcutaneous pocket.

Guide the hemostat under the skin towards the opening on the shoulders where the catheter was inserted. Break through the connective tissue and tightly clamp the catheter at the most rostral point below the base of the five up. Detach the five up and thread the long end of the catheter under the skin and out the incision located at the top of the head.

Use scissors to cut the catheter below where the hemostat was clamped and reattach the long end of the five up. Pull the catheter along the metal shaft of the five up all the way to the plastic pedestal pass saline through to ensure free flow. After closing the wounds with sutures, place the rat in a stereotaxic apparatus.

Once the exposed skull is clean and dry, use a manual drill bit to drill four to five holes into the surface. Next, insert machine screws into the holes to act as scaffolding for the head cap. Place the five up connector between the screws and tuck the excess length of the catheter into the subcutaneous pocket made earlier.

Lastly, use dental cement to affix the five up to the top of the skull. Remove the rat from the stereotaxic apparatus after the cement is fully dried and monitor until fully recovered. Rats are housed individually in operant conditioning chambers.

Enclosed in sound attenuating compartments equipped with a fan. Two retractable levers are mounted nine centimeters above the floor of the right sidewall with a cue light above each lever. A tone module is placed above the active lever and a red house light is positioned at the top center of the left sidewall.

Food and water is available at libitum. The drug pump is attached to the catheter via a liquid swivel and tigon tubing protected with a metal spring. Heroin self-administration is initiated following recovery from surgery and a 24 hour habituation period.

To begin, flush the catheter with a heparin gentamycin solution. Next, connect the five up connector to the Tigon tube and attach the metal spring. Begin each session at the onset of the dark phase with the extension of the active and inactive levers, as well as illumination of the house light and activation of the Q light tone complex.

For 30 seconds, the rats are allowed to self-administer heroin for 10 days during three three hour sessions separated by three hour intervals. Responses on the active lever result in activation of the drug pump and initiation of a 22nd timeout period during which the house light is turned off, and the Q light tone complex above the active lever is activated. Inactive lever responses are recorded, but have no programmable consequences.

After self-administration training, remove the rats from the operant conditioning chambers and individually house them in the animal facility with unrestricted access to food and water. For one drug washout day the next day, reduce the amount of food for the food restricted group to begin the abstinence period while maintaining unrestricted access to food In the sated group. Perform the drug seeking test the morning of abstinence.

Day 14, return the rats to the operant conditioning chambers and attach them to the metal spring. Allow a one or three hour session during which active and inactive lever responses have the same consequences as in self-administration training, excluding the availability of the drug. These results demonstrate that body weights remain stable throughout the heroin.

Self-administration training During abstinence, rats with unrestricted access to chow increase their body weight while those that are food restricted decrease their body weight by approximately 10 to 15%As seen here, infusions and active lever responses increased over the training days, whereas inactive lever responses did not. Following the abstinence period, active lever responding for the food restricted rats significantly increased compared to the sated rats. Whereas inactive lever responding is minimal and comparable between groups Following this procedure.

Other methods such as intracranial injections, microdialysis, western blotting and immunohistochemical techniques can be used to target specific neurochemical and pharmacological systems. After watching this video, you should have a clear understanding of how to surgically implant an intravenous catheter into the rat's jugular vein, as well as how to conduct a heroin self-administration procedure in standard operant conditioning chambers.