Using Optogenetics to Reverse Neuroplasticity and Inhibit Cocaine Seeking in Rats
Summary
The methods described here outline a procedure used to optogenetically reverse cocaine-induced plasticity in a behaviorally-relevant circuit in rats. Sustained low-frequency optical stimulation of thalamo-amygdala synapses induces long-term depression (LTD). In vivo optogenetically-induced LTD in cocaine-experienced rats resulted in the subsequent attenuation of cue-motivated drug seeking.
Abstract
This protocol demonstrates the steps needed to use optogenetic tools to reverse cocaine-induced plasticity at thalamo-amygdala circuits to reduce subsequent cocaine seeking behaviors in the rat. In our research, we had found that when rats self-administer intravenous cocaine paired with an audiovisual cue, synapses formed at inputs from the medial geniculate nucleus of the thalamus (MGN) onto principal neurons of the lateral amygdala (LA) become stronger as the cue-cocaine association is learned. We hypothesized that reversal of the cocaine-induced plasticity at these synapses would reduce cue-motivated cocaine seeking behavior. In order to accomplish this type of neuromodulation in vivo, we wanted to induce synaptic long-term depression (LTD), which decreases the strength of MGN-LA synapses. To this end, we used optogenetics, which allows neuromodulation of brain circuits using light. The excitatory opsin oChiEF was expressed on presynaptic MGN terminals in the LA by infusing an AAV containing oChiEF into the MGN. Optical fibers were then implanted in the LA and 473 nm laser light was pulsed at a frequency of 1 Hz for 15 minutes to induce LTD and reverse cocaine induced plasticity. This manipulation produces a long-lasting reduction in the ability of cues associated with cocaine to induce drug seeking actions.
Introduction
Substance abuse is a very serious public health issue in the U.S. and worldwide. Despite decades of intense research, there are very few effective therapeutic options1,2. A major setback to treatment is the fact that chronic drug use generates long-term associative memories between environmental cues and the drug itself. Re-exposure to drug-related cues drives physiological and behavioral responses that motivate continued drug use and relapse3. A novel therapeutic strategy is to enact memory-based treatments that aim to manipulate the circuits involved in regulating drug-cue associations. Recently, it was observed that synapses in the lateral amygdala (LA), specifically those arising from the medial geniculate nucleus (MGN) of the thalamus, are strengthened by repeated cue-associated cocaine self-administration, and that this potentiation can support cocaine seeking behavior4,5. Therefore, it was proposed that cue-induced reinstatement could be attenuated by reversing plasticity at MGN-LA synapses.
The ability to precisely target the synaptic plasticity of a specific brain circuit has been a major challenge to the field. Traditional pharmacological tools have had some success in decreasing relapse behaviors, but are limited by the inability to manipulate individual synapses. However, the recent development of in vivo optogenetics has provided the tools needed to overcome these limitations and control neural pathways with temporal and spatial precision6,7,8. By expressing light-sensitive opsins in a specific brain circuit, laser light can then be used to activate or inhibit the circuit. Frequency-dependent optical stimulation can be utilized to specifically manipulate the synaptic plasticity of the circuit in a behaving animal.
This manuscript outlines the procedure taken to manipulate the behaviorally-relevant MGN-LA circuit using in vivo optogenetics. First, the excitatory opsin oChIEF was expressed in the MGN and optical fibers were bilaterally implanted in the LA. Animals were then trained to self-administer cocaine in a cue-dependent fashion, which potentiates the MGN-LA pathway. Next, sustained, low frequency stimulation with 473 nm laser light was used to produce circuit-specific LTD. Reversing the plasticity induced by cocaine use generated a long-lasting reduction in the capacity of cues to trigger actions that are associated with drug seeking behavior.
Protocol
Representative Results
Discussion
As described above, there are several critical steps that are important for achieving the proper experimental results. The protocol will likely only be effective in animals that properly acquire cocaine self-administration, and to date, it has only been tested using the parameters outlined above. It is possible that cocaine dose, schedule of reinforcement, and cue parameters can be modified with likely little effect on behavioral outcomes, with the exception that a second-order schedule of reinforcement may lead to amygd…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors wish to acknowledge support from USPHS grants K01DA031745 (MMT), R01DA042029 (MMT), DA035805 (YHH), F31DA039646 (MTR), T32031111 (MTR), and the Pennsylvania Department of Health.
Materials
| 0.9% Saline | Fisher Scientific | NC0291799 | |
| A.M.P.I. Stimulus Isolator | Iso-Flex | ||
| AAV5.hSyn.oChIEF.tdTomato | Duke Viral Vector Core (via Roger Tsien) | #268 | See Lin et al., 2009; Nabavi et al., 2014 |
| AAV5.hSyn.tdTomato (Control) | Duke Viral Vector Core Control | See Lin et al., 2009; Nabavi et al., 2014 | |
| Artificial Tears (Opthalmic Ointment) | Covetrus | 70349 | |
| ATP Magnesium Salt | Fisher Scientific | A9187 | |
| Betadine | Butler Schein | 38250 | |
| Calcium chloride | Fisher Scientific | C1016 | |
| Cesium chloride | Fisher Scientific | 289329 | |
| Cesium hydroxide | Fisher Scientific | 516988 | |
| Cesium methanesulfonate | Fisher Scientific | C1426 | |
| Cocaine HCl | NIDA Drug Supply Center | 9041-001 | |
| Cryostat | Leica | CM1950 | |
| D-Glucose | Sigma-Aldrich | G8270 | |
| DMSO | Fisher Scientific | BP231-1 | |
| Dual-Channel Temperature Controller | Warner Instruments | TC-344C | |
| EGTA | Fisher Scientific | E3889 | |
| Ethanol | University of Pittsburgh Chemistry Stockroom | 200C5000 | |
| Ferrule Dust Caps | Thor Labs | CAPL | White plastic dust caps for 1.25 mm Ferrules |
| Ferrule Mating Sleeves | Doric Lenses | F210-3011 | Sleeve_BR_1.25, Bronze, 1.25 mm ID |
| Ferrules | Precision Fiber Products | MM-FER2007C-2300 | Ø1.25 mm Multimode LC/PC Ceramic ferrule, Ø230 μm hole size |
| Fiber Optic | Thor Labs | FP200URT | 200 μm core multimode fiber (0.5 NA) |
| Fiber Optic Rotary Joint | Prizmatix | (Ordered from Amazon) | 18 mm diameter, FC-FC connector for fiber |
| Fiber Stripping Tool | Thor Labs | T12S21 | |
| Fluoroshield with DAPI | Sigma-Aldrich | F6057 | |
| Gentamicin | Henry Schein | 6913 | |
| GTP Sodium Salt | Fisher Scientific | G8877 | |
| Hamilton syringe | Hamilton | 80085 | 10 μL volume, 26 gauge, 2 inch, point style 3 |
| Heat Gun | Allied Electronics | 972-6966 | 250 V, 750-800 °F |
| Heat-Curable Epoxy | Precision Fiber Products | PFP-353ND-8OZ | |
| Heparin | Henry Schein | 55737 | |
| HEPES | Sigma-Aldrich | H3375 | |
| Hydrochloric Acid | Fisher Scientific | 219405490 | |
| Isoflurane | Henry Schein | 29405 | |
| Ketamine HCl | Henry Schein | 55853 | Ketamine is a controlled substance and should be handled according to institutional guidelines |
| Lactated Ringer’s | Henry Schein | 9846 | |
| Laser, driver, and laser-to-fiber coupler | OEM Laser Systems | BL-473-00100-CWM-SD-xx-LED-0 | 100 mW, 473-nm, diode-pumped solid-state laser (One option) |
| L-glutathione | Fisher Scientific | G4251 | |
| Lidocaine | Butler Schein | 14583 | |
| Light Sensor | Thor Labs | PM100D | Compact energy meter console with digital display |
| Loctite instant adhesive | Grainger | 5E207 | |
| Magnesium sulfate | Sigma-Aldrich | 203726 | |
| Microelectrode Amplifier/Data Acquisition | Molecular Devices | MULTICLAMP700B / Digidata 1440A | |
| Microinjector pump | Harvard Apparatus | 70-4501 | Dual syringe |
| Micromanipulator | Sutter Instruments | MPC-200/ROE-200 | |
| Microscope | Olympus | BX51WI | Upright microscope for electrophysiology |
| Microscope | Olympus | BX61VS | Epifluorescent slide-scanning microscope |
| N-methyl-D-glucamine | Sigma-Aldrich | M2004 | |
| Orthojet dental cement, liquid | Lang Dental | 1504BLK | black |
| Orthojet dental cement, powder | Lang Dental | 1530BLK | Contemporary powder, black |
| Paraformaldehyde | Sigma-Aldrich | P6148 | |
| Patch Cables | Thor Labs | FP200ERT | Multimode, FT030 Tubing |
| Picrotoxin | Fisher Scientific | AC131210010 | |
| Polishing Disc | Thor Labs | D50FC | |
| Polishing Pad | Thor Labs | NRS913 | 9" x 13" |
| Polishing Paper | Thor Labs | LFG5P | 5 μm grit |
| Polishing Paper | Thor Labs | LFG3P | 3 μm grit |
| Polishing Paper | Thor Labs | LFG1P | 1 μm grit |
| Polishing Paper | Thor Labs | LFG03P | 0.3 μm grit |
| Potassium chloride | Sigma-Aldrich | P9333 | |
| Potassium hydroxide | Fisher Scientific | P5958 | |
| Potassium methanesulfonate | Fisher Scientific | 83000 | |
| QX-314-Cl | Alomone Labs | Q-150 | |
| Rimadyl (Carprofen) | Henry Schein | 24751 | |
| Self-Administration Chambers/Software | Med Associates | MED-NP5L-D1 | |
| Sodium bicarbonate | Sigma-Aldrich | S5761 | |
| Sodium chloride | Sigma-Aldrich | S7653 | |
| Sodium Hydroxide | Sigma-Aldrich | 1064980500 | |
| Sodium L-Ascorbate | Sigma-Aldrich | A7631 | |
| Sodium Pentobarbital | Henry Schein | 24352 | |
| Sodium phosphate | Sigma-Aldrich | S9638 | |
| Sodium phosphocreatine | Fisher Scientific | P7936 | |
| Sodium pyruvate | Sigma-Aldrich | P2256 | |
| Stainless steel machine screws | WW Grainger | 6GB25 | M2-0.40mm Machine Screw, Pan, Phillips, A2 Stainless Steel, Plain, 3 mm Length |
| Stereotaxic adapter for ferrules | Thor Labs | XCL | |
| Stereotaxic Frame | Stoelting | 51603 | |
| Sucrose | Sigma-Aldrich | S8501 | |
| Suture Thread | Fine Science Tools | 18020-50 | Silk thread; Size: 5/0, Diameter: 0.12 mm |
| TEA-Chloride | Fisher Scientific | T2265 | |
| Thiourea | Sigma-Aldrich | T8656 | |
| Vetbond Tissue Adhesive | Covetrus | 001505 | |
| Vibratome | Leica | VT1200S | |
| Xylazine | Butler Schein | 33198 |
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