Research Article

Diazepam Withdrawal Expression is related to Hippocampal NOS-1 Upregulation

Emilce Artur de la Villarmois, María Florencia Constantin, Claudia Bregonzio and Mariela Fernanda Pérez*

Published: 08 January, 2018 | Volume 2 - Issue 1 | Pages: 001-009

Background: Benzodiazepines are usually prescribed for anxiety and sleep disorders in a long-term fashion that may cause drug dependence. Discontinuation after prolonged administration may lead to withdrawal expression, being anxiety the most predominant sign. It has been described that a context-dependent associative learning process underlies diazepam dependence. Nitric oxide is a crucial player in learning and memory processes, hippocampal transmission, as well as in benzodiazepines withdrawal. Considering that previous results from our laboratory showed an increase in hippocampal functional plasticity only in diazepam dependent rats, the aim of the present investigation is to determine whether diazepam dependence could alter neuronal nitric oxide synthase enzyme (NOS-1) expression within the hippocampus, by using western blot.

Results: chronic diazepam-treated animals that developed dependence showed increase in NOS-1 expression in dorsal, but not in ventral hippocampus, while no-dependent or control animals presented similar NOS-1 protein levels.

Conclusion: withdrawal from long-term diazepam exposure could be associated to increased nitric oxide neurotransmission within dorsal hippocampus induced by NOS-1 over-expression. This mechanism could underlie the improved hippocampal synaptic transmission previously observed in diazepam withdrawn animals. Confirmatory experiments need to be addressed to determine the mechanisms by which nitric oxide participates in benzodiazepines withdrawal in order find new molecular targets to develop pharmacological tools to prevent the withdrawal syndrome

Read Full Article HTML DOI: 10.29328/journal.hps.1001006 Cite this Article Read Full Article PDF


Diazepam; Withdrawal; NOS-1, Nitric oxide, Hippocampal plasticity


  1. Greenblatt DJ, Shader RI. Dependence, tolerance, and addiction to benzodiazepines: clinical and pharmacokinetic considerations. Drug Metab Rev. 1978; 8: 13-28. Ref.: https://goo.gl/E5Z8uo
  2. Dickinson B, Rush PA, Radcliffe AB. Alprazolam use and dependence. A retrospective analysis of 30 cases of withdrawal. West J Med. 1990; 152: 604-608. Ref.: https://goo.gl/udDKN5
  3. Quaglio G, Pattaro C, Gerra G, Mathewson S, Verbanck P, et.al. High dose benzodiazepine dependence: description of 29 patients treated with flumazenil infusion and stabilised with clonazepam. Psychiatry Res. 2012; 198: 457-462. Ref.: https://goo.gl/ketbKE
  4. Pérez MF, Maglio LE, Marchesini GR, Molina JC, Ramírez OA. Environmental changes modify the expression of Diazepam withdrawal. Behav Brain Res. 2002; 136: 75-81. Ref.: https://goo.gl/PWLV8f
  5. Wolf ME. Addiction: making the connection between behavioral changes and neuronal plasticity in specific pathways. Mol Interv. 2002; 2: 146-157. Ref.: https://goo.gl/yBEPLQ
  6. Monti MC, Almirón RS, Bignante EA, Ramírez OA. Changes in hippocampal arc protein expression and synaptic plasticity by the presentation of contextual cues linked to drug experience. Synapse. 2010; 64: 39-46. Ref.: https://goo.gl/yf352i
  7. Taubenfeld SM, Muravieva EV, Garcia-Osta A, Alberini CM. Disrupting the memory of places induced by drugs of abuse weakens motivational withdrawal in a context-dependent manner. Proc Natl Acad Sci U.S.A. 2010; 107: 12345-12350. Ref.: https://goo.gl/3Jbs5m
  8. Nestler EJ. Psychogenomics: opportunities for understanding addiction. J Neurosci. 2001; 21: 8324-8327. Ref.: https://goo.gl/XEw8H4
  9. Ciccocioppo R, Sanna PP, Weiss F. Cocaine-predictive stimulus induces drug-seeking behavior and neural activation in limbic brain regions after multiple months of abstinence: reversal by D(1) antagonists. Proc Natl Acad Sci U.S.A. 2001; 98: 1976-1981. Ref.: https://goo.gl/Vv6RK7
  10. Kelley AE. Memory and addiction: shared neural circuitry and molecular mechanisms. Neuron. 2004; 44: 161-179. Ref.: https://goo.gl/tkC1ZZ
  11. Eisch AJ, Mandyam CD. Drug dependence and addiction, II: adult neurogenesis and drug abuse. Am J Psychiatry. 2004; 161: 426. Ref.: https://goo.gl/euWy9n
  12. Del Olmo N, Miguens M, Higuera-Matas A, Torres I, Garcia-Lecumberri C, et al. Enhancement of hippocampal long-term potentiation induced by cocaine self-administration is maintained during the extinction of this behavior. Brain Res. 2006; 1116: 120-126. Ref.: https://goo.gl/w3eTTg
  13. Kim JJ, Fanselow MS. Modality-specific retrograde amnesia of fear. Science. 1992; 256 : 675-677. Ref.: https://goo.gl/oicVog
  14. Phillips RG, LeDoux JE. Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning. Behavioral neuroscience. 1992; 106: 274-285. Ref.: https://goo.gl/jr44xM
  15. Martin SJ, Grimwood PD, Morris RG. Synaptic plasticity and memory: an evaluation of the hypothesis. Annual review of neuroscience. 2000; 23: 649-711. Ref.: https://goo.gl/WBf6vy
  16. Perez MF, Nasif FJ, Marchesini GR, Maglio LE, Ramirez OA. Hippocampus and locus coeruleus activity on rats chronically treated with diazepam. Pharmacology Biochemistry and Behavior. 2001; 69: 431-438. Ref.: https://goo.gl/8Nnq9a
  17. Prast H, Philippu A. Nitric oxide as modulator of neuronal function. Prog Neurobiol. 2001; 64: 51-68. Ref.: https://goo.gl/NBsCkh
  18. Garthwaite J, Boulton CL. Nitric oxide signaling in the central nervous system. Annu Rev Physiol. 1995; 57: 683-706. Ref.: https://goo.gl/8D8VjX
  19. Sousa AM, Prado WA. The dual effect of a nitric oxide donor in nociception. Brain Res. 2001; 897: 9-19. Ref.: https://goo.gl/2G8D97
  20. Cardenas A, Moro MA, Hurtado O, Leza JC, Lizasoain I. Dual role of nitric oxide in adult neurogenesis. Brain Res Brain Res Rev. 2005; 50: 1-6. Ref.: https://goo.gl/MBJzpF
  21. Yamada K, Noda Y, Nakayama S, Komori Y, Sugihara H, et al. Role of nitric oxide in learning and memory and in monoamine metabolism in the rat brain. Br J Pharmacol. 1995; 115: 852-858. Ref.: https://goo.gl/JJxrDs
  22. Quock RM, Nguyen E. Possible involvement of nitric oxide in chlordiazepoxide-induced anxiolysis in mice. Life Sci. 1992; 51: 255-260. Ref.: https://goo.gl/eSmFQc
  23. Del-Bel EA, Oliveira PR, Oliveira JA, Mishra PK, Jobe PC, et al. Anticonvulsant and proconvulsant roles of nitric oxide in experimental epilepsy models. Braz J Med Biol Res. 1997; 30: 971-979. Ref.: https://goo.gl/3GmpVV
  24. Uzbay IT, Oglesby MW. Nitric oxide and substance dependence. Neurosci Biobehav Rev. 2001; 25: 43-52. Ref.: https://goo.gl/br81aH
  25. Nasif FJ, Hu XT, Ramirez OA, Perez MF. Inhibition of neuronal nitric oxide synthase prevents alterations in medial prefrontal cortex excitability induced by repeated cocaine administration. Psychopharmacology (Berl). 2011; 218: 323-330. Ref.: https://goo.gl/Y2oJXw
  26. Gabach LA, Carlini VP, Monti MC, Maglio LE, De Barioglio SR, et al. Involvement of nNOS/NO/sGC/cGMP signaling pathway in cocaine sensitization and in the associated hippocampal alterations: does phosphodiesterase 5 inhibition help to drug vulnerability? Psychopharmacology (Berl). 2013; 229: 41-50. Ref.: https://goo.gl/BPJLzy
  27. Talarek S, Listos J, Fidecka S. Effect of nitric oxide synthase inhibitors on benzodiazepine withdrawal in mice and rats. Pharmacol Rep, 2011, vol: 63 pp. 680-689.
  28. Rawls SM, Rodriguez T, Baron DA, Raffa RB. A nitric oxide synthase inhibitor (L-NAME) attenuates abstinence-induced withdrawal from both cocaine and a cannabinoid agonist (WIN 55212-2) in Planaria. Brain Res. 2006; 1099: 82-87. Ref.: https://goo.gl/6CYxx7
  29. Jain R, Mukherjee K, Mohan D. Effects of nitric oxide synthase inhibitors in attenuating nicotine withdrawal in rats. Pharmacology biochemistry and behavior. 2008; 88: 473-480. Ref.: https://goo.gl/P3ug5B
  30. Vaupel DB, Kimes AS, London ED. Nitric oxide synthase inhibitors. Preclinical studies of potential use for treatment of opioid withdrawal. Neuropsychopharmacology. 1995; 13: 315-322. Ref.: https://goo.gl/sdnuRt
  31. Gabra BH, Afify EA, Daabees TT, Abou Zeit-Har MS. The role of the NO/NMDA pathways in the development of morphine withdrawal induced by naloxone in vitro. Pharmacol Res. 2005; 51: 319-327. Ref.: https://goo.gl/KJJXWy
  32. Uzbay IT, Erden BF, Tapanyigit EE, Kayaalp SO. Nitric oxide synthase inhibition attenuates signs of ethanol withdrawal in rats. Life Sci. 1997; 61: 2197-2209. Ref.: https://goo.gl/VMLjRU
  33. Robbins TW, Everitt BJ. Neurobehavioural mechanisms of reward and motivation. Curr Opin Neurobiol. 1996; 6: 228-236. Ref.: https://goo.gl/UdZYbF
  34. Griffiths RR, Weerts EM. Benzodiazepine self-administration in humans and laboratory animals--implications for problems of long-term use and abuse. Psychopharmacology (Berl). 1997; 134: 1-37. Ref.: https://goo.gl/qhdZQF
  35. Maren S. Pavlovian fear conditioning as a behavioral assay for hippocampus and amygdala function: cautions and caveats. Eur J Neurosci. 2008; 28: 1661-1666. Ref.: https://goo.gl/RZmj2j
  36. Fontanesi LB, Ferreira R, Cabral A, Castilho VM, Brandao ML, et al. Brainstem areas activated by diazepam withdrawal as measured by Fos-protein immunoreactivity in rats. Brain Res. 2007; 1166: 35-46. Ref.: https://goo.gl/Q7PNKZ
  37. Almiron RS, Ramirez OA. Participation of dorsal raphe nucleus in the behavioral alteration observed after discontinuation of chronic diazepam administration: possible neural circuitry involved. Synapse. 2005; 56: 61-68. Ref.: https://goo.gl/W7FcYP
  38. Shen G, Mohamed MS, Das P, Tietz EI. Positive allosteric activation of GABAA receptors bi-directionally modulates hippocampal glutamate plasticity and behaviour. Biochem Soc Trans. 2009; 37: 1394-1398. Ref.: https://goo.gl/28QCid
  39. Wang WS, Chen ZG, Liu WT, Chi ZQ, He L, et al. Dorsal hippocampal NMDA receptor blockade impairs extinction of naloxone-precipitated conditioned place aversion in acute morphine-treated rats by suppressing ERK and CREB phosphorylation in the basolateral amygdala. Br J Pharmacol. 2015; 172: 482-491. Ref.: https://goo.gl/DgVxu2
  40. García-Pardo MP, Escobar-Valero C, Rodríguez-Arias M, Miñarro J, Aguilar MA. Involvement of NMDA glutamate receptors in the acquisition and reinstatement of the conditioned place preference induced by MDMA. Behav Pharmacol. 2015; 26: 411-417. Ref.: https://goo.gl/f4dRLL
  41. Perez MF, Salmiron R, Ramirez OA. NMDA-NR1 and -NR2B subunits mRNA expression in the hippocampus of rats tolerantto Diazepam. Behav. Brain Res. 2003; 144; 119-124. Ref.: https://goo.gl/3fP9vB
  42. File SE, Fernandes C. Dizocilpine prevents the development of tolerance to the sedative effects of diazepam in rats. Pharmacol Biochem Behav. 1994; 47: 823-826. Ref.: https://goo.gl/QoyznY
  43. Khanna JM, Shah G, Weiner J, Wu PH, Kalant H. Effect of NMDA receptor antagonists on rapid tolerance to ethanol. Eur J Pharmacol. 1993; 230: 23-31. Ref.: https://goo.gl/Ch1GAn
  44. Trujillo KA, Akil H. Inhibition of morphine tolerance and dependence by the NMDA receptor antagonist MK-801. Science. 1991; 251: 85-87. Ref.: https://goo.gl/HQ3iiH
  45. Almiron RS, Perez MF, Ramirez OA. MK-801 prevents the increased NMDA-NR1 and NR2B subunits mRNA expression observed in the hippocampus of rats tolerant to diazepam. Brain Res. 2004; 1008: 54-60. Ref.: https://goo.gl/b8JymE
  46. Garthwaite J, Garthwaite G, Palmer RM, Moncada S. NMDA receptor activation induces nitric oxide synthesis from arginine in rat brain slices. Eur J Pharmacol. 1989; 172: 413-416. Ref.: https://goo.gl/gk36Lf
  47. Park JH, Straub VA, O'Shea M. Anterograde signaling by nitric oxide: characterization and in vitro reconstitution of an identified nitrergic synapse. J Neurosci. 1998; 18: 5463-5476. Ref.: https://goo.gl/qeeM8U
  48. Bon CL, Garthwaite J. On the role of nitric oxide in hippocampal long-term potentiation. J Neurosci. 2003; 23: 1941-1948. Ref.: https://goo.gl/uCx7fS
  49. Smith ACW, Scofield MD, Heinsbroek JA, Gipson CD, Neuhofer D, et al. Accumbens nNOS Interneurons Regulate Cocaine Relapse. J Neurosci. 2017; 37: 742-756. Ref.: https://goo.gl/ZrGRa9
  50. Rahmati B, Beik A. Prevention of morphine dependence and tolerance by Nepeta menthoides was accompanied by attenuation of Nitric oxide overproduction in male mice. J Ethnopharmacol. 2017; 199: 39-51. Ref.: https://goo.gl/Arw5ax
  51. Okere CO, Waterhouse BD. Nicotine withdrawal upregulates nitrergic and galaninergic activity in the rat dorsal raphe nucleus and locus coeruleus. Neurosci Lett. 2013; 536 : 29-34. Ref.: https://goo.gl/DaiZf8
  52. Faria MS, Muscara MN, Moreno Junior H, Teixeira SA, Dias HB, et al. Acute inhibition of nitric oxide synthesis induces anxiolysis in the plus maze test. Eur J Pharmacol. 1997; 323: 37-43. Ref.: https://goo.gl/Kj8JLE
  53. Volke V, Wegener G, Vasar E. Augmentation of the NO-cGMP cascade induces anxiogenic-like effect in mice. J Physiol Pharmacol. 2003; 54: 653-660. Ref.: https://goo.gl/oqqvT3
  54. Fanselow MS, Dong HW. Are the dorsal and ventral hippocampus functionally distinct structures? Neuron. 2010; 65: 7-19. Ref.: https://goo.gl/92kbGx


Figure 1

Figure 1

Figure 1

Figure 2

Similar Articles

Recently Viewed

Read More

Most Viewed

Read More