NMDA Receptors and NO:cGMP Signaling Pathway Mediate the Diazepam-Induced Sensitization to Withdrawal Signs in Mice

Abstract

The goal of the present study was to examine the effects of N-methyl-aspartate (NMDA) receptor antagonists-memantine and ketamine and the drugs modifying the NO:cGMP pathway-NG-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI), the endogenous precursor of NO-L-arginine, and the guanylyl cyclase inhibitor-methylene blue (MB) on the development of sensitization to withdrawal signs precipitated after chronic, interrupted treatment with diazepam, a benzodiazepine receptor agonist, in mice. To develop the sensitization, the mice were divided into groups: continuously and sporadically (with two diazepam-free periods) treated with diazepam (15 mg/kg, sc). To precipitate the withdrawal syndrome (clonic and tonic seizures, and death), pentylenetetrazole (55 mg/kg, sc) with the benzodiazepine receptor antagonist, flumazenil (5.0 mg/kg, ip), were administered after the last injection of diazepam or saline. Memantine (2.5, 5.0 mg/kg), and ketamine (2.5, 5.0 mg/kg), L-NAME (100, 200 mg/kg) and 7-NI (20 and 40 mg/kg), L-arginine (250, 500 mg/kg) and MB (5 and 10 mg/kg) were administered ip in sporadically diazepam-treated mice during the diazepam-free periods. Our results indicated that both NMDA receptor antagonists and drugs that inhibit the NO:cGMP pathway, except L-arginine (the endogenous donor of NO), attenuated the diazepam-induced sensitization to withdrawal signs in mice. Thus, NMDA receptors and the NO:cGMP pathway are involved in the mechanisms of sensitization to benzodiazepine withdrawal.

Keywords: Diazepam; NMDA receptor; NO:cGMP pathway; Sensitization; Withdrawal.

Fig. 1

Effects of memantine (MEM; 2.5 and 5.0 mg/kg, ip) and ketamine (KET; 2.5; 5.0 mg/kg ip) on the development of sensitization to diazepam withdrawal signs. NMDA antagonists were injected in mice once a day for 3 days during diazepam-free periods. The withdrawal signs were induced 48 h after the cessation of diazepam treatment by simultaneous injection of subthreshold dose of pentylenetetrazole (PTZ; 55 mg/kg, sc) with flumazenil (FLU; 5.0 mg/kg, ip). Data represent the number of clonic, tonic, and death incidents in %. The values of the number of withdrawal signs in mice treated with diazepam (DZ; 21 days) + Saline + FLU + PTZ were assumed to be 100%. The number of clonic and tonic episodes in 10 mice and the mortality rate are presented below X axis. One-way ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001; N = 10

Fig. 2

Effects of L-NAME (100 and 200 mg/kg, ip) on the development of sensitization to diazepam withdrawal signs. L-NAME was injected in mice once a day for 3 days during diazepam-free periods. The withdrawal signs were induced 48 h after the cessation of diazepam treatment by simultaneous injection of subthreshold dose of pentylenetetrazole (PTZ; 55 mg/kg, sc) with flumazenil (FLU; 5.0 mg/kg, ip). Data represent the number of clonic, tonic, and death incidents in %. The values of the number of withdrawal signs in mice treated with diazepam (DZ; 21 days) + Saline + FLU + PTZ were assumed to be 100%. The number of clonic and tonic episodes in 10 mice and the mortality rate are presented below X axis. N = 10, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Effects of 7-nitroindazol (7-NI; 20 and 40 mg/kg, ip) on the development of sensitization to diazepam withdrawal signs. 7-NI was injected in mice once a day for 3 days during diazepam-free periods. The withdrawal signs were induced 48 h after the cessation of diazepam treatment by simultaneous injection of subthreshold dose of pentylenetetrazole (PTZ; 55 mg/kg, sc) with flumazenil (FLU; 5.0 mg/kg, ip). Data represent the number of clonic, tonic, and death incidents in %. The values of the number of withdrawal signs in mice treated with diazepam (DZ; 21 days) + Saline + FLU + PTZ were assumed to be 100%. The number of clonic and tonic episodes in 12 mice and the mortality rate are presented below X axis. N = 12, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Effects of methylene blue (MB; 5.0 and 10 mg/kg, ip) on the development of sensitization to diazepam withdrawal signs. MB was injected in mice once a day for 3 days during diazepam-free periods. The withdrawal signs were induced 48 h after the cessation of diazepam treatment by simultaneous injection of subthreshold dose of pentylenetetrazole (PTZ; 55 mg/kg, sc) with flumazenil (FLU; 5.0 mg/kg, ip). Data represent the number of clonic, tonic and death incidents in %. The values of the number of withdrawal signs in mice treated with diazepam (DZ; 21 days) + Saline + FLU + PTZ were assumed to be 100%. The number of clonic and tonic episodes in 12 mice and the mortality rate are presented below X axis. N = 12, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Effects of L-arginine (L-ARG; 250 and 500 mg/kg, ip) on the development of sensitization to diazepam withdrawal signs. L-NAME was injected in mice once a day for 3 days during diazepam-free periods. The withdrawal signs were induced 48 h after the cessation of diazepam treatment by simultaneous injection of subthreshold dose of pentylenetetrazole (PTZ; 55 mg/kg, sc) with flumazenil (FLU; 5.0 mg/kg, ip). Data represent the number of clonic, tonic, and death incidents in %. The values of the number of withdrawal signs in mice treated with diazepam (DZ; 21 days) + Saline + FLU + PTZ were assumed to be 100%. The number of clonic and tonic episodes in 10 mice and the mortality rate are presented below X axis. N=10. *P<0.05, ***P<0.001