Using Optogenetics to Reverse Neuroplasticity and Inhibit Cocaine Seeking in Rats

This protocol is significant because it uses novel advances in optogenetic techniques and a preclinical model of substance abuse to reverse plasticity in neural circuits that are responsible for promoting relapse. The main advantage of this technique is that it permits circuit specific manipulation of synaptic activity in an awake animal, which has long lasting inhibitory effects on future cue motivated cocaine seeking behavior. This method could be utilized across many other systems and circuits to specifically manipulate behaviorally relevant neuroplasticity.

Start preparing for the surgery by connecting a 26 gauge stainless steel injection cannula to a Hamilton syringe filled with one microliter of concentrated AAV solution. After anesthetizing the animal, shave the surgical area and lubricate the eyes to prevent drying as described in the text manuscript. Next, inject into the rat a body weight volume of the analgesic carprofen subcutaneously through the skin of the upper back, and inject five milliliters of lactated Ringer's solution subcutaneously through the skin of the lower back.

Then perform the intravenous catheter implantation. Immediately following catheter implantation, secure rat in a stereotaxic frame to perform adeno-associated viral injections. Inject a bolus of lidocaine subcutaneously through the skin above the skull in order to locally anesthetize the area, and then make a 0.5 millimeter incision from the front to the rear of the skull.

Remove overlying tissue to expose the surface of the skull. Level the rat's head along the anterior posterior axis and zero stereotaxic coordinates to bregma, then use a dremel tool equipped with a small drill bit to drill three small holes through the skull, and mount stainless steel screws in place with a screwdriver. For the injection of AAV, drill bilateral holes based on the coordinates from the Rat Brain Atlas"for the medial geniculate nucleus or MGN.

The coordinates used relative to bregma are minus 5.4 millimeters on the anterior posterior axis, plus three millimeters on the medial lateral axis, and minus 6.6 millimeters on the dorsal ventral axis. Slowly lower the injection cannula at approximately four millimeters per minute, until cannula are properly positioned in the MGN, and inject concentrated AAV solution at a rate of 0.1 microliters per minute. After infusions are complete, leave injection cannula in place for five minutes to allow for diffusion of the virus away from the cannula and then slowly withdraw cannula from the brain.

Next, proceed to the implantation of optic fibers targeting medial geniculate nucleus lateral amygdala terminals. Use a dremel tool to drill bilateral holes above the lateral amygdala, at coordinates as explained in the manuscript. Use forceps to grasp the ferrule of the optic fiber implant and secure the ferrule to the stereotaxic adapters in place.

Slowly lower fibers at a rate of two millimeters per minute until the tip of the fiber sits in the dorsal portion of the LA.Secure ferrules to the skin, first using a thin layer of Loctite instant adhesive, followed by dental cement. Once dental cement has sufficiently dried, cover ferrules with ferrule sleeves and dust covers. Following surgical procedures, house the rat individually and flush catheters daily with saline containing five milligrams per milliliter of gentamicin and 30 USP units per milliliter of heparin.

24 hours prior to the start of behavioral experiments, food restrict rats to 90%of their free feeding weight. Place the rat in an operant chamber to undergo daily one hour training sessions for self-administration of two milligrams per milliliter of cocaine, under a fixed ratio one schedule of reinforcement. In the chamber, allow the rat to lever press.

A press on the designated active lever results in a cocaine infusion at one milligram per kilogram dose, and a ten second presentation of a compound light and tone cue. A press on the designated inactive lever has no programmed effects. Continue the self-administration experiments for at least 10 days, until the rat successfully earns at least eight infusions per day across three consecutive days.

Failure to reach acquisition criteria by day 20, results in exclusion from the study. After acquisition criteria are successfully met, subject the rat to one hour instrumental extinction sessions for six to 10 days by allowing rats to freely lever press in operant chambers. Continue instrumental extinction daily until an average of maximum 25 lever presses over two consecutive days occurs.

For optogenetic induction of long-term depression or LTD, connect patch cables to a 473 nanometer blue laser diode via a rotary joint suspended above a clean, standard rodent housing cage. Turn on the laser according to operating instructions and connect the laser to a pulse generator. Adjust settings to give the rat the 900 pulses of light at two microsecond, at one hertz.

Measure the light output through the patch cord using a light sensor, and adjust the laser intensity so that the light output through the patch cable is approximately five to seven milliwatts. Place the rat in the clean housing cage and remove dust covers and ferrule sleeves exposing the ferrules. Then connect the patch cords bilaterally to each ferrule.

If set up properly, rodents will be able to move freely around the cage during optogenetic stimulation. After allowing the rat to explore the environment for three minutes, turn on the pulse generator to initiate optogenetic stimulation. Following long-term depression induction, allow rat to remain in the cage for three minutes, before placing it back in the home cage.

24 hours after in vivo optogenetic stimulation, place the rat back into the operant conditioning chambers to undergo a one hour standard cue induced reinstatement session to assess cocaine seeking behavior. A press on the active lever results in a ten second presentation of the light and tone cue. At least one week after the first test, give a second reinstatement test to determine if optogenetic LTD induction results in a long-term suppression of cocaine seeking.

In the representative analysis, acquisition and extinction of cocaine self-administration are shown. Cocaine infusions and active lever responses increased across acquisition, with few inactive lever responses. During cocaine self-administration, the number of active responses gradually increased across each acquisition day before stabilizing during the second week.

On day one of the extinction, and initial boost in lever pressing was observed, followed by a decrease in responding on both levers. Following instrumental extinction, reintroduction of cues reinstated cocaine seeking represented by an increase in the number of active lever responses. Optical LTD caused a significant reduction in active lever presses relative to controls.

Seven days later, rats were tested again. Animals that previously underwent MGN-LA LTD had significantly reduced active lever pressing compared to controls, revealing that optical LTD produced a long-term decrease in cocaine seeking. Ex vivo electrophysiological recordings showed a decrease in the amplitude of optically evoked excitatory postsynaptic current in LA neurons, following optical LTD.

Additionally, the rise slope of excitatory postsynaptic potentials was reduced by ex vivo optical stimulation in the neurons, from animals that had sham stimulation, but not in neurons from animals that had in vivo optical LTD. The placement of optic fiber implants and viral expression were verified histologically in the lateral amygdala and MGN. To successfully reduce cocaine seeking behavior, it is critical to achieve proper light output through the optic fiber implants, and to use sustained, low-frequency stimulation to promote long-term depression.

Following this procedure, neural recording techniques could be used to determine changes in neural activity, and other behavioral tests of cocaine seeking or other learned behaviors could be conducted.