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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 association....

Protocol

The experiments described in this protocol were consistent with the guidelines set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of Pittsburgh's Institutional Animal Care and Use Committee. All procedures were performed using adult, naïve Sprague-Dawley rats that weighed 275-325 g upon arrival.

1. Construction of Optic Fiber Implants and Patch Cables

  1. Prepare optic fi.......

Representative Results

A timeline outlining the order of experiments is shown in Figure 1. Throughout behavioral experiments, the number of cocaine infusions as well as the number of responses made on the active lever serves as a measure of the intensity of cocaine-seeking behavior. During the initial days of cocaine self-administration, the number of active responses should gradually increase across each acquisition day, before stabilizing during the second week. Conversely, inactive lever responses should remain.......

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.......

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

NameCompanyCatalog NumberComments
0.9% SalineFisher ScientificNC0291799
A.M.P.I. Stimulus IsolatorIso-Flex
AAV5.hSyn.oChIEF.tdTomatoDuke Viral Vector Core (via Roger Tsien)#268See Lin et al., 2009; Nabavi et al., 2014
AAV5.hSyn.tdTomato (Control)Duke Viral Vector Core ControlSee Lin et al., 2009; Nabavi et al., 2014
Artificial Tears (Opthalmic Ointment)Covetrus70349
ATP Magnesium SaltFisher ScientificA9187
BetadineButler Schein38250
Calcium chlorideFisher ScientificC1016
Cesium chlorideFisher Scientific289329
Cesium hydroxideFisher Scientific516988
Cesium methanesulfonateFisher ScientificC1426
Cocaine HClNIDA Drug Supply Center9041-001
CryostatLeicaCM1950
D-GlucoseSigma-AldrichG8270
DMSOFisher ScientificBP231-1
Dual-Channel Temperature ControllerWarner InstrumentsTC-344C
EGTAFisher ScientificE3889
EthanolUniversity of Pittsburgh Chemistry Stockroom200C5000
Ferrule Dust CapsThor LabsCAPLWhite plastic dust caps for 1.25 mm Ferrules
Ferrule Mating SleevesDoric LensesF210-3011Sleeve_BR_1.25, Bronze, 1.25 mm ID
FerrulesPrecision Fiber ProductsMM-FER2007C-2300Ø1.25 mm Multimode LC/PC Ceramic ferrule, Ø230 μm hole size
Fiber OpticThor LabsFP200URT200 μm core multimode fiber (0.5 NA)
Fiber Optic Rotary JointPrizmatix(Ordered from Amazon)18 mm diameter, FC-FC connector for fiber
Fiber Stripping ToolThor LabsT12S21
Fluoroshield with DAPISigma-AldrichF6057
GentamicinHenry Schein6913
GTP Sodium SaltFisher ScientificG8877
Hamilton syringeHamilton8008510 μL volume, 26 gauge, 2 inch, point style 3
Heat GunAllied Electronics972-6966250 V, 750-800 °F
Heat-Curable EpoxyPrecision Fiber ProductsPFP-353ND-8OZ
HeparinHenry Schein55737
HEPESSigma-AldrichH3375
Hydrochloric AcidFisher Scientific219405490
IsofluraneHenry Schein29405
Ketamine HClHenry Schein55853Ketamine is a controlled substance and should be handled according to institutional guidelines
Lactated Ringer’sHenry Schein9846
Laser, driver, and laser-to-fiber couplerOEM Laser SystemsBL-473-00100-CWM-SD-xx-LED-0100 mW, 473-nm, diode-pumped solid-state laser (One option)
L-glutathioneFisher ScientificG4251
LidocaineButler Schein14583
Light SensorThor LabsPM100DCompact energy meter console with digital display
Loctite instant adhesiveGrainger5E207
Magnesium sulfateSigma-Aldrich203726
Microelectrode Amplifier/Data AcquisitionMolecular DevicesMULTICLAMP700B / Digidata 1440A
Microinjector pumpHarvard Apparatus70-4501Dual syringe
MicromanipulatorSutter InstrumentsMPC-200/ROE-200
MicroscopeOlympusBX51WIUpright microscope for electrophysiology
MicroscopeOlympusBX61VSEpifluorescent slide-scanning microscope
N-methyl-D-glucamineSigma-AldrichM2004
Orthojet dental cement, liquidLang Dental1504BLKblack
Orthojet dental cement, powderLang Dental1530BLKContemporary powder, black
ParaformaldehydeSigma-AldrichP6148
Patch CablesThor LabsFP200ERTMultimode, FT030 Tubing
PicrotoxinFisher ScientificAC131210010
Polishing DiscThor LabsD50FC
Polishing PadThor LabsNRS9139" x 13"
Polishing PaperThor LabsLFG5P5 μm grit
Polishing PaperThor LabsLFG3P3 μm grit
Polishing PaperThor LabsLFG1P1 μm grit
Polishing PaperThor LabsLFG03P0.3 μm grit
Potassium chlorideSigma-AldrichP9333
Potassium hydroxideFisher ScientificP5958
Potassium methanesulfonateFisher Scientific83000
QX-314-ClAlomone LabsQ-150
Rimadyl (Carprofen)Henry Schein24751
Self-Administration Chambers/SoftwareMed AssociatesMED-NP5L-D1
Sodium bicarbonateSigma-AldrichS5761
Sodium chlorideSigma-AldrichS7653
Sodium HydroxideSigma-Aldrich1064980500
Sodium L-AscorbateSigma-AldrichA7631
Sodium PentobarbitalHenry Schein24352
Sodium phosphateSigma-AldrichS9638
Sodium phosphocreatineFisher ScientificP7936
Sodium pyruvateSigma-AldrichP2256
Stainless steel machine screwsWW Grainger 6GB25M2-0.40mm Machine Screw, Pan, Phillips, A2 Stainless Steel, Plain, 3 mm Length
Stereotaxic adapter for ferrulesThor LabsXCL
Stereotaxic FrameStoelting51603
SucroseSigma-AldrichS8501
Suture ThreadFine Science Tools18020-50Silk thread; Size: 5/0, Diameter: 0.12 mm
TEA-ChlorideFisher ScientificT2265
ThioureaSigma-AldrichT8656
Vetbond Tissue AdhesiveCovetrus001505
VibratomeLeicaVT1200S
XylazineButler Schein33198

References

  1. Connors, N. J., Hoffman, R. S. Experimental treatments for cocaine toxicity: A difficult transition to the bedside. Journal of Pharmacology and Experimental Therapeutics. 347 (2), 251-257 (2013).
  2. Makani, R., Pradhan, B., Shah, U., Parikh, T.

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OptogeneticsNeuroplasticityCocaine SeekingRatsAAVMedial Geniculate NucleusIntravenous CatheterStereotaxic SurgeryNeuroscienceAddiction Research

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