, Volume 233, Issue 4, pp 715–725 | Cite as

Negative allosteric modulation of GABAA receptors inhibits facilitation of brain stimulation reward by drugs of abuse in C57BL6/J mice

  • Matthew E. Tracy
  • Matthew L. Banks
  • Keith L. SheltonEmail author
Original Investigation



There is an emerging body of evidence that implicates a crucial role of γ-aminobutyric acid subtype A (GABAA) receptors in modulating the rewarding effects of a number of abused drugs. Modulation of GABAA receptors may therefore represent a novel drug-class independent mechanism for the development of abuse treatment pharmacotherapeutics.


We tested the hypothesis that the GABAA receptor benzodiazepine-site (BDZ) negative modulator Ro15-4513 would reduce the reward-related effects of three pharmacologically dissimilar drugs; toluene vapor, d-methamphetamine, and diazepam using intracranial self-stimulation (ICSS) in mice. We also examined whether Ro15-4513 attenuated dopamine release produced by d-methamphetamine in an in vivo microdialysis procedure.


Ro15-4513 abolished ICSS reward facilitation produced by all three abused drugs at Ro15-4513 doses which had no effect on ICSS when administered alone. In contrast, the BDZ antagonist flumazenil only attenuated the ICSS-facilitating effects of diazepam. Administration of the same dose of Ro15-4513 which abolished drug-facilitated ICSS produced a 58 % decrease in d-methamphetamine-stimulated dopamine in the nucleus accumbens of mice relative to d-methamphetamine alone.


These results demonstrate that negative modulation of GABAA receptors can produce profound reductions in reward-related effects of a diverse group of drugs that activate the mesolimbic reward pathway through different mechanisms. These data suggest that pharmacological modulation of GABAA receptors may represent a viable pathway for the development of drug abuse pharmacotherapies.


Benzodiazepine D-methamphetamine Ro15-4513 Intracranial self-stimulation (ICSS) Toluene Inhalant abuse GABA Brain stimulation reward (BSR) Flumazenil Negative allosteric modulation 



Dr. Shelton and Matt Tracy declare that the NIH has funded their research under R01DA-020553 and F31DA-034469, respectively. Dr. Banks declares that NIH has funded his research. During the past 3 years, he has received compensation as a collaborator with Perdue Pharmaceuticals for projects related to opioid pharmacology and drug development.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethics approval

All procedures were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University and were in accordance with NIH guidelines (National Research Council 2011).


  1. Aickin M, Gensler H (1996) Adjusting for multiple testing when reporting research results: the Bonferroni vs Holm methods. Am J Public Health 86:726–728PubMedCentralCrossRefPubMedGoogle Scholar
  2. Apawu AK, Mathews TA, Bowen SE (2015) Striatal dopamine dynamics in mice following acute and repeated toluene exposure. Psychopharmacology (Berl) 232:173–184. doi: 10.1007/s00213-014-3651-x CrossRefGoogle Scholar
  3. Bauer CT, Banks ML, Blough BE, Negus SS (2013) Use of intracranial self-stimulation to evaluate abuse-related and abuse-limiting effects of monoamine releasers in rats. Br J Pharmacol 168:850–862. doi: 10.1111/j.1476-5381.2012.02214.x PubMedCentralCrossRefPubMedGoogle Scholar
  4. Beckley JT, Woodward JJ (2013) Volatile solvents as drugs of abuse: focus on the cortico-mesolimbic circuitry. Neuropsychopharmacology 38:2555–2567. doi: 10.1038/npp.2013.206 PubMedCentralCrossRefPubMedGoogle Scholar
  5. Beckstead MJ, Weiner JL, Eger EI 2nd et al (2000) Glycine and gamma-aminobutyric acid(A) receptor function is enhanced by inhaled drugs of abuse. Mol Pharmacol 57:1199–1205PubMedGoogle Scholar
  6. Bishop BE, Laverty R (1989) Dose-dependent reduction by Ro 15–4513 in mice of the effects of ethanol and some other general depressant drugs. Eur J Pharmacol 162:265–271CrossRefPubMedGoogle Scholar
  7. Bossert JM, Franklin KBJ (2003) Reinforcing versus anticonvulsant drugs: effects on intracranial self-stimulation rate-frequency M50 indices. Behav Brain Res 144:243–247CrossRefPubMedGoogle Scholar
  8. Brauer LH, Ambre J, De Wit H (1996) Acute tolerance to subjective but not cardiovascular effects of d-amphetamine in normal, healthy men. J Clin Psychopharmacol 16:72–76CrossRefPubMedGoogle Scholar
  9. Carlezon WA, Chartoff EH (2007) Intracranial self-stimulation (ICSS) in rodents to study the neurobiology of motivation. Nat Protoc 2:2987–2995. doi: 10.1038/nprot.2007.441 CrossRefPubMedGoogle Scholar
  10. Chan M-H, Chung S-S, Stoker AK et al (2012) Sarcosine attenuates toluene-induced motor incoordination, memory impairment, and hypothermia but not brain stimulation reward enhancement in mice. Toxicol Appl Pharmacol 265:158–165. doi: 10.1016/j.taap.2012.10.004 PubMedCentralCrossRefPubMedGoogle Scholar
  11. Chan M-H, Tsai Y-L, Lee M-Y, et al. (2015) The group II metabotropic glutamate receptor agonist LY379268 reduces toluene-induced enhancement of brain-stimulation reward and behavioral disturbances. Psychopharmacology (Berl). doi: 10.1007/s00213-015-3973-3Google Scholar
  12. Creed MC, Ntamati NR, Tan KR (2014) VTA GABA neurons modulate specific learning behaviors through the control of dopamine and cholinergic systems. Front Behav Neurosci 8:8. doi: 10.3389/fnbeh.2014.00008 PubMedCentralCrossRefPubMedGoogle Scholar
  13. D’Souza D (2015) Ability of partial inverse agonist, iomazenil, to block ethanol effects in humans. NCT01590277.Google Scholar
  14. De Vry J, Slangen JL (1985) The Ro 15–1788 cue: evidence for benzodiazepine agonist and inverse agonist properties. Eur J Pharmacol 119:193–197CrossRefPubMedGoogle Scholar
  15. Di Scala G, Oberling P, Rocha B, Sandner G (1992) Conditioned place preference induced by Ro 16–6028, a benzodiazepine receptor partial agonist. Pharmacol, Biochem Behav 41:859–862CrossRefGoogle Scholar
  16. Dixon CI, Morris HV, Breen G et al (2010) Cocaine effects on mouse incentive-learning and human addiction are linked to alpha2 subunit-containing GABAA receptors. Proc Natl Acad Sci U S A 107:2289–2294. doi: 10.1073/pnas.0910117107 PubMedCentralCrossRefPubMedGoogle Scholar
  17. Engin E, Bakhurin KI, Smith KS et al (2014) Neural basis of benzodiazepine reward: requirement for α2 containing GABAA receptors in the nucleus accumbens. Neuropsychopharmacology 39:1805–1815. doi: 10.1038/npp.2014.41 PubMedCentralCrossRefPubMedGoogle Scholar
  18. File SE, Dingemanse J, Friedman HL, Greenblatt DJ (1986) Chronic treatment with Ro 15–1788 distinguishes between its benzodiazepine antagonist, agonist and inverse agonist properties. Psychopharmacology (Berl) 89:113–117CrossRefGoogle Scholar
  19. Fish EW, Riday TT, McGuigan MM et al (2010) Alcohol, cocaine, and brain stimulation-reward in C57Bl6/J and DBA2/J mice. Alcohol Clin Exp Res 34:81–89. doi: 10.1111/j.1530-0277.2009.01069.x CrossRefPubMedGoogle Scholar
  20. Hondebrink L, Meulenbelt J, van Kleef RGDM et al (2011) Modulation of human GABAA receptor function: a novel mode of action of drugs of abuse. Neurotoxicology 32:823–827. doi: 10.1016/j.neuro.2011.05.016 CrossRefPubMedGoogle Scholar
  21. Hsu JC (1996) Multiple comparisons: theory and methods, first edit. Chapman & Hall, LondonCrossRefGoogle Scholar
  22. Ikemoto S, Bonci A (2014) Neurocircuitry of drug reward. Neuropharmacology 76(Pt B):329–341. doi: 10.1016/j.neuropharm.2013.04.031 CrossRefPubMedGoogle Scholar
  23. Invernizzi R, Pozzi L, Samanin R (1991) Release of dopamine is reduced by diazepam more in the nucleus accumbens than in the caudate nucleus of conscious rats. Neuropharmacology 30:575–578CrossRefPubMedGoogle Scholar
  24. Jhou TC, Fields HL, Baxter MG et al (2009) The rostromedial tegmental nucleus (RMTg), a GABAergic afferent to midbrain dopamine neurons, encodes aversive stimuli and inhibits motor responses. Neuron 61:786–800. doi: 10.1016/j.neuron.2009.02.001 PubMedCentralCrossRefPubMedGoogle Scholar
  25. Kida T, Noguchi J, Zhang M-R et al (2003) Metabolite analysis of [11C] Ro15-4513 in mice, rats, monkeys and humans. Nucl Med Biol 30:779–784CrossRefPubMedGoogle Scholar
  26. Koob GF (1992) Drugs of abuse: anatomy, pharmacology and function of reward pathways. Trends Pharmacol Sci 13:177–184. doi: 10.1016/0165-6147(92)90060-J CrossRefPubMedGoogle Scholar
  27. Leitl MD, Onvani S, Bowers MS et al (2013) Pain-related depression of the mesolimbic dopamine system in rats: expression, blockade by analgesics, and role of endogenous κ-opioids. Neuropsychopharmacology 39:614–624. doi: 10.1038/npp.2013.236 PubMedCentralCrossRefPubMedGoogle Scholar
  28. Lukas SE, Mendelson JH, Benedikt RA (1986a) Instrumental analysis of ethanol-induced intoxication in human males. Psychopharmacology (Berl) 89:8–13CrossRefGoogle Scholar
  29. Lukas SE, Mendelson JH, Benedikt RA, Jones B (1986b) EEG alpha activity increases during transient episodes of ethanol-induced euphoria. Pharmacol, Biochem Behav 25:889–895CrossRefGoogle Scholar
  30. McBride WJ, Murphy JM, Lumeng L, Li TK (1988) Effects of Ro 15–4513, fluoxetine and desipramine on the intake of ethanol, water and food by the alcohol-preferring (P) and -nonpreferring (NP) lines of rats. Pharmacol, Biochem Behav 30:1045–1050CrossRefGoogle Scholar
  31. Melón LC, Boehm SL (2011) GABAA receptors in the posterior, but not anterior, ventral tegmental area mediate Ro15-4513-induced attenuation of binge-like ethanol consumption in C57BL/6J female mice. Behav Brain Res 220:230–237. doi: 10.1016/j.bbr.2011.02.014 PubMedCentralCrossRefPubMedGoogle Scholar
  32. Miczek KA, Weerts EM (1987) Seizures in drug-treated animals. Science 235:1127a. doi: 10.1126/science.235.4793.1127a CrossRefPubMedGoogle Scholar
  33. Miller DW, Yourick DL, Tessel RE (1989) Antagonism of methoxyflurane-induced anesthesia in rats by benzodiazepine inverse agonists. Eur J Pharmacol 173:1–10CrossRefPubMedGoogle Scholar
  34. Miller LL, Leitl MD, Banks ML et al (2015) Effects of the triple monoamine uptake inhibitor amitifadine on pain-related depression of behavior and mesolimbic dopamine release in rats. Pain 156:175–184. doi: 10.1016/j.pain.0000000000000018 PubMedCentralCrossRefPubMedGoogle Scholar
  35. Moody EJ, Skolnick P (1988) The imidazobenzodiazepine Ro 15–4513 antagonizes methoxyflurane anesthesia. Life Sci 43:1269–1276CrossRefPubMedGoogle Scholar
  36. National Drug Intelligence Center (2011) The economic impact of illicit drug use on American society. Washington D.C, United States Department of JusticeGoogle Scholar
  37. National Research Council (2011) Guide for the care and use of laboratory animals, 8th edn. National Academies Press, Washington, D.CGoogle Scholar
  38. Negus SS, Miller LL (2014) Intracranial self-stimulation to evaluate abuse potential of drugs. Pharmacol Rev 66:869–917. doi: 10.1124/pr.112.007419 PubMedCentralCrossRefPubMedGoogle Scholar
  39. Nieh EH, Kim S-Y, Namburi P, Tye KM (2013) Optogenetic dissection of neural circuits underlying emotional valence and motivated behaviors. Brain Res 1511:73–92. doi: 10.1016/j.brainres.2012.11.001 PubMedCentralCrossRefPubMedGoogle Scholar
  40. Oades RD, Halliday GM (1987) Ventral tegmental (A10) system: neurobiology. 1. Anatomy and connectivity. Brain Res 434:117–165CrossRefPubMedGoogle Scholar
  41. Páez-Martínez N, Ambrosio E, García-Lecumberri C et al (2008) Toluene and TCE decrease binding to mu-opioid receptors, but not to benzodiazepine and NMDA receptors in mouse brain. Ann N Y Acad Sci 1139:390–401. doi: 10.1196/annals.1432.031 CrossRefPubMedGoogle Scholar
  42. Paxinos G, Franklin KBJ (2007) The mouse brain in stereotaxic coordinates, 3rd edn. Academic, San DiegoGoogle Scholar
  43. Pokk P, Zharkovsky A (1997) The effects of flumazenil, Ro 15–4513 and beta-CCM on the behaviour of control and stressed mice in the plus-maze test. J Physiol Pharmacol 48:253–261PubMedGoogle Scholar
  44. Potier MC, Prado de Carvalho L, Dodd RH et al (1988) In vivo binding of (3H)Ro15-1788 in mice: comparison with the in vivo binding of (3H)flunitrazepam. Life Sci 43:1287–1296CrossRefPubMedGoogle Scholar
  45. Riegel AC, Zapata A, Shippenberg TS, French ED (2007) The abused inhalant toluene increases dopamine release in the nucleus accumbens by directly stimulating ventral tegmental area neurons. Neuropsychopharmacol Off Publ Am Coll Neuropsychopharmacol 32:1558–1569. doi: 10.1038/sj.npp.1301273 CrossRefGoogle Scholar
  46. Robinson JE, Agoglia AE, Fish EW et al (2012) Mephedrone (4-methylmethcathinone) and intracranial self-stimulation in C57BL/6J mice: comparison to cocaine. Behav Brain Res 234:76–81. doi: 10.1016/j.bbr.2012.06.012 PubMedCentralCrossRefPubMedGoogle Scholar
  47. Rowlett JK, Lelas S (2007) Comparison of zolpidem and midazolam self-administration under progressive-ratio schedules: consumer demand and labor supply analyses. Exp Clin Psychopharmacol 15:328–337. doi: 10.1037/1064-1297.15.4.328 CrossRefPubMedGoogle Scholar
  48. Rudolph U, Knoflach F (2011) Beyond classical benzodiazepines: novel therapeutic potential of GABAA receptor subtypes. Nat Rev Drug Discov 10:685–697. doi: 10.1038/nrd3502 PubMedCentralCrossRefPubMedGoogle Scholar
  49. Russo SJ, Nestler EJ (2013) The brain reward circuitry in mood disorders. Nat Rev Neurosci 14:609–625. doi: 10.1038/nrn3381 CrossRefPubMedGoogle Scholar
  50. Schaefer GJ, Michael RP (1989) Interactions between RO 15–4513 and ethanol on brain self-stimulation and locomotor activity in rats. Pharmacol, Biochem Behav 34:785–790CrossRefGoogle Scholar
  51. Shelton KL, Grant KA (2001) Effects of naltrexone and Ro 15–4513 on a multiple schedule of ethanol and Tang self-administration. Alcohol Clin Exp Res 25:1576–1585CrossRefPubMedGoogle Scholar
  52. Shelton KL, Nicholson KL (2013) Benzodiazepine-like discriminative stimulus effects of toluene vapor. Eur J Pharmacol 720:131–137PubMedCentralCrossRefPubMedGoogle Scholar
  53. Smith AJ, Alder L, Silk J et al (2001) Effect of alpha subunit on allosteric modulation of ion channel function in stably expressed human recombinant gamma-aminobutyric acid(A) receptors determined using (36)Cl ion flux. Mol Pharmacol 59:1108–1118PubMedGoogle Scholar
  54. Stanton A. Glantz (2006) Primer of Biostatistics, 6th Edition. McGraw-Hill Education/MedicalGoogle Scholar
  55. Steffensen SC, Lee RS, Stobbs SH, Henriksen SJ (2001) Responses of ventral tegmental area GABA neurons to brain stimulation reward. Brain Res 906:190–197CrossRefPubMedGoogle Scholar
  56. Stinchcomb A, Bowers BJ, Wehner JM (1989) The effects of ethanol and Ro 15–4513 on elevated plus-maze and rotarod performance in long-sleep and short-sleep mice. Alcohol 6:369–376CrossRefPubMedGoogle Scholar
  57. Straub CJ, Carlezon WAJ, Rudolph U (2010) Diazepam and cocaine potentiate brain stimulation reward in C57BL/6J mice. Behav Brain Res 206:17–20. doi: 10.1016/j.bbr.2009.08.025 CrossRefPubMedGoogle Scholar
  58. Tan KR, Brown M, Labouèbe G et al (2010) Neural bases for addictive properties of benzodiazepines. Nature 463:769–774. doi: 10.1038/nature08758 PubMedCentralCrossRefPubMedGoogle Scholar
  59. Tan KR, Yvon C, Turiault M et al (2012) GABA neurons of the VTA drive conditioned place aversion. Neuron 73:1173–1183. doi: 10.1016/j.neuron.2012.02.015 CrossRefPubMedGoogle Scholar
  60. Taylor SR, Badurek S, Dileone RJ, et al. (2014) GABAergic and Glutamatergic Efferents of the Mouse Ventral Tegmental Area. J Comp Neurol. doi: 10.1002/cne.23603Google Scholar
  61. Tracy ME, Slavova-Hernandez GG, Shelton KL (2014) Assessment of reinforcement enhancing effects of toluene vapor and nitrous oxide in intracranial self-stimulation. Psychopharmacology (Berl) 231:1339–1350. doi: 10.1007/s00213-013-3327-y CrossRefGoogle Scholar
  62. van Zessen R, Phillips JL, Budygin EA, Stuber GD (2012) Activation of VTA GABA neurons disrupts reward consumption. Neuron 73:1184–1194. doi: 10.1016/j.neuron.2012.02.016 PubMedCentralCrossRefPubMedGoogle Scholar
  63. Venault P, Chapouthier G (2007) From the behavioral pharmacology of beta-carbolines to seizures, anxiety, and memory. Sci World J 7:204–223. doi: 10.1100/tsw.2007.48 CrossRefGoogle Scholar
  64. Votey SR, Bosse GM, Bayer MJ, Hoffman JR (1991) Flumazenil: a new benzodiazepine antagonist. Ann Emerg Med 20:181–188CrossRefPubMedGoogle Scholar
  65. Williams JM, Stafford D, Steketee JD (2005) Effects of repeated inhalation of toluene on ionotropic GABAA and glutamate receptor subunit levels in rat brain. Neurochem Int 46:1–10. doi: 10.1016/j.neuint.2004.07.006 CrossRefPubMedGoogle Scholar
  66. Xia Y, Driscoll JR, Wilbrecht L et al (2011) Nucleus accumbens medium spiny neurons target non-dopaminergic neurons in the ventral tegmental area. J Neurosci 31:7811–7816. doi: 10.1523/JNEUROSCI.1504-11.2011 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Matthew E. Tracy
    • 1
  • Matthew L. Banks
    • 1
  • Keith L. Shelton
    • 1
    Email author
  1. 1.Department of Pharmacology and ToxicologyVirginia Commonwealth UniversityRichmondUSA

Personalised recommendations