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Effects of chronic flurazepam treatment on firing rate of rat substantia nigra pars reticulata neurons
BRE 22950
Short Communications
Effects of chronic flurazepam treatment on firing rate of rat substantia nigra pars reticulata neurons Jennifer L. Tyma, Howard C. Rosenberg, Elizabeth I. Tietz and T.H. Chiu Department of Pharmacology, Medical College of Ohio, Toledo, OH 43699 (U. S. A. )
(Accepted 15 March 1988) Key words: Benzodiazepine: Tolerance: Substantia nigra pars reticulata; Flurazepam
The effect of a benzodiazepine, flurazepam, on the spontaneous activity of neurons in the pars reticulata of the substantia nigra was studied in chloral hydrate anesthetized rats. Flurazepam produced a dose-related suppression of neuronal activity. In rats that were chronically treated with flurazepam, tolerance to flurazepam was present after 7 and 28 days, but not after only 3 days of treatment. Tolerance persisted at least 2, but not 7 days after 4 weeks of chronic treatment.
Chronic benzodiazepine (BZ) exposure has been shown to produce tolerance to the sedative, muscle relaxant and anticonvulsant actions of BZs 2"3'6"8-11. Changes produced by chronic treatment, such as alterations in the G A B A / B Z receptor complex or its function, may be the basis for tolerance to various BZ actions. For example, with the chronic BZ treatment used in the present study a temporal relationship was found between B Z receptor down-regulation and tolerance to ataxia produced by large doses of flurazepam (FZP) 6 or diazepam i°. This treatment also produced tolerance to diazepam's anticonvulsant activity, though the time course differed from that of receptor down-regulation H. This study investigated the possibility that tolerance to BZs could be measured by the action of BZs to suppress spontaneous activity of neurons in the pars reticulata of substantia nigra (SNpr) 13"18. The SNpr appears to be a useful site for studying the acute and chronic actions of BZs 12-15'18'19. Using the method described by Ross et al. 13, dose-response data were recorded during intravenous infusion of a water-soluble BZ, FZP, in control and chronically treated rats. Once tolerance was found, the time course for its production and reversal was deter-
mined and compared to similar information from studies of tolerance measured behaviorally, and studies of receptor regulation. It was thought that comparison of the rates of development and reversal of these p h e n o m e n a might provide some insight as to possible mechanisms of tolerance. Male S p r a g u e - D a w l e y rats were given FZP according to an established chronic treatment protocol 6, in which a saccharin-flavored F Z P solution was provided as the sole source of drinking water. During the first week, the FZP concentration was adjusted to provide 100 mg/kg/day. During weeks 2 - 4 , 150 mg/kg/day was offered. Details of this method and typical dose consumption have been published 6. Rats randomly assigned as controls received undrugged saccharin water for the same duration as their FZPtreated counterparts, In order to follow the development of, and recovery from tolerance, various treatment schedules were studied and compared to results in control rats. Treated rats were studied immediately upon completing 3, 7 or 28 days of treatment, and 2 or 7 days after completing the 28-day treatment. Rats weighing 250-320 g at the end of chronic treatment were anesthetized with chloral hydrate
Correspondence: H.C. Rosenberg, Department of Pharmacology, Medical College of Ohio, C.S. 10008, Toledo, OH 43699, U.S.A.
0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
345 (400 mg/kg, i.p.). The femoral artery and vein were cannulated for blood pressure measurement and FZP administration, respectively. In the event that fluctuations in depth of anesthesia might affect the results, anesthesia was maintained throughout the experiment by a constant intravenous infusion of chloral hydrate beginning 30 rain after initial chloral hydrate injection. The dose was 2.2 mg/kg/min for control rats, and those tested 48 h or 7 days after 4 weeks of treatment. Other treatment groups received 2.5 mg/kg/min. These doses were sufficient to prevent spontaneous and reflex movements during the experiment. Body temperature was maintained between 36 and 38 °C using a Deltaphase isothermal pad. Control rats were interspersed with treated rats throughout the study. The techniques used for extracellular, single unit recording have been previously described 1'17. Single barrel glass capillary electrodes, 3 - 8 Mr2 resistance, were filled with a 1% solution of Pontamine sky blue dye in 2 M NaCI. Electrical activity was recorded using a high-input impedance amplifier (World Precision Instruments Model DAM-5A), and monitored on an oscilloscope. Action potential frequency recorded from a single neuron was plotted as a histogram by a strip chart recorder. Each SNpr cell was monitored for 5 min. After this baseline period, cumulative intravenous FZP doses were administered. Doses ranging from 0.125 to 4 mg/kg were administered every 2 min so that each increment doubled the previously administered dose, as described by Ross et al. 13. Drug effect for each dose was quantified by the average frequency of the cell for the 2 min post-injection, expressed as a percent of baseline. Only one cell was studied per rat. The recording site was marked by passing current through the electrode to eject the dye. Only those experiments with a recording site histologically verified to be in SNpr were included in this study. To assess the amount of drug present, brain BZlike activity was measured by radioreceptor assay. Rats were sacrificed by decapitation immediately upon completing FZP treatment for 3 days, 1 week and 4 weeks. To evaluate the amount o f FZP in the brain during infusion of the drug, cumulative doses of FZP were given to 4 chloral hydrate anesthetized control rats, as described above. Two min after a total dose of 2 mg/kg (which produced approximately
I O0 ,i,~i " "'-o.
LJ <
x
•
40-
2o 0.1
i 1.0
10
CUMULATIVE FZP DOSE ( r n g / k g )
Fig. 1. Cumulative log dose-response curves of the effects of intravenous flurazepam (FZP) on SNpr single unit activity. Data are presented for control rats (n = 16, O) and rats tested after 4 weeks of FZP treatment. Treated rats were tested immediately upon completing treatment (n = 12, Q) or after stopping treatment for 2 days (n = 13, z~) or 7 days (n = 9, A). FZP was administered every 2 rain so that each increment doubled the previously administered dose ~3. The average frequency for the 2-rain period following FZP injection was expressed as a percent of initial baseline frequency. Only one cell per rat was studied for drug response. Vertical lines represent S.E.M.
50% reduction in neuronal discharge rate), the rats were decapitated, and the brains were collected. Whole brains were removed and frozen (-70 °C) until the time of the assay. Assays were based on the displacement of 2 nM [3H]flunitrazepam specifically bound to freshly prepared synaptosomal membranes of rat whole brain. Procedures for preparing ethanol extracts, preparation of synaptosomal membranes and the radioreceptor assay have previously been described in detail 11,16. The overall analysis for single unit studies was carried out using a two-factor repeated measures A N O V A . Individual planned comparisons between control and each treated group to evaluate the effect of cumulative FZP administration were made by repeated measures A N O V A using the Bonferroni test, which required P < 0.01 for significance 5. Baseline frequencies were compared using a one-way ANOVA. The baseline discharge rates of SNpr neurons ranged from 10 to 53 Hz (mean +_ S.E.M. = 25.68 _+ 1.16). In none of the treatment groups was the baseline discharge frequency significantly different (P = 0.27) from that in control rats (28.53 _+ 2.74 Hz). FZP significantly inhibited SNpr neuron firing rate in a dose-dependent manner (P ~< 0.0001), as previous-
346 ly observed 13. The overall treatment effect did not reach statistical significance (P = 0.054). However, tolerance was shown by a significant overall interaction between F Z P dose infused and treatment group (P = 0.014). The probe of the overall interaction by A N O V A revealed a significant shift of the d o s e - r e sponse curves at some but not other time points, in rats treated for 4 weeks and tested 0 h after removal of drugged water, the dose-response curve was significantly shifted to the right of the control curve (Fig. 1), showing tolerance to FZP (P = 0.006). FZP administered 48 h post-4-week treatment produced less suppression of neuronal activity than in control rats (Fig. 1), showing that tolerance was still present 48 h after treatment had ended (P = 0.006). Rats tested 7 days post-4-week treatment were no longer tolerant (P = 0.38), indicating that the reduced sensitivity to FZP, as measured by suppression of SNpr neuronal activity, had largely reversed a week after the 4-week F Z P treatment. Rats tested immediately after 3 days of chronic treatment did not exhibit tolerance to FZP (Fig. 2), as indicated by a non-significant shift in the dose-response curve (P = 0.67). However, the dose-response curve of rats tested immediately after 1 week of 100 mg/kg FZP treatment was significantly shifted to the right of control (P = 0.002). Using this chloral hydrate infusion method, there was no significant difference in initial mean arterial pressure across groups (P t> 0.24). There was also no difference in mean arterial pressure before and after intravenous cumulative FZP administration in any treatment group ( P / > 0.20) except rats tested after 3 day treatment. The drop in mean arterial pressure in this group did not appear to change the sensitivity of these neurons to FZP, as compared to other treatment groups (Figs. 1 and 2). Brain BZ-like activity was measured by radioreceptor assay after 3 days, 1 week and 4 weeks of chronic treatment. Activity for rats tested after 3 days of treatment with 100 mg/kg (n = 6) was equivalent to a concentration of 1.67 ktM FZP. Rats receiving 1 week of treatment with 100 mg/kg (n = 6) had the equivalent of a concentration of 1.06 uM. These values were not significantly different (P = 0.06). After 4 weeks of treatment (100 mg/kg for 1 week, 150 mg/kg for 3 weeks), BZ-like activity was equivalent to a concentration of 3.44 uM. A time course for the
100
(D z
13< 60 z w
< ~3
40-
20 0,1
j 1.0
_
_
q 10
CUMULATIVE FZP DOSE ( m g / k g )
Fig. 2. Cumulative FZP log dose-response curves of the effects of intravenous FZP on SNpr single unit activity. Data were plotted as described in Fig. 1. Data are presented for control rats (n = 16, O) and rats tested immediately upon completing 3 days (n = 10, O) or 7 days (n = 1l, A) of FZP treatment. disappearance of residual drug after this chronic treatment has been described elsewhere u. Four control rats were given a cumulative F Z P dose of 2 mg/kg over 8 min, i.e. the first 5 injections of the FZP infusion used during electrophysiological recording. Brain tissue from these rats was found to contain enough BZ to be equivalent to a FZP concentration of 14.8 (+ 2.2) ktM. Thus, the amount of residual F Z P or active metabolites u in treated rats was probably insufficient to directly suppress SNpr discharge frequency. In this study, tolerance developed maximally between the third and seventh day of treatment, and remained essentially unchanged for at least 2 days after treatment was stopped. This time course was similar to that of tolerance to the anticonvulsant effect of diazepam in rats given the same chronic F Z P treatment 9"11. This chronic FZP treatment is also known to cause down-regulation of BZ receptors 6'7, which was shown to be most pronounced in SNpr Is. However, the time course for this down-regulation was quite different from that of tolerance measured in the present study, so that this does not appear to be a mechanism for tolerance measured in the SNpr. Some comparisons can be made with the results of a recent study in which rats received chronic treatment with diazepam released from a subcutaneous reservoir. The effects of intravenous diazepam on SNpr neuronal discharge rate, and the potentiation of G A B A by microiontophoretically-applied FZP were measured 19. Tolerance was found after only
347 one day of exposure to diazepam. Tolerance reversed rapidly, within 24 h. Thus, tolerance in SNpr appeared to require the continued presence of diazepam (or perhaps a metabolite). In contrast, tolerance measured in SNpr in the present study was still present 2 days after stopping chronic treatment, at which time brain levels of BZ (including metabolites) had declined to virtually zero II. Interestingly, the time course for producing tolerance during chronic FZP treatment, measured in SNpr (Fig. 1), was very similar to the time course of tolerance in rats treated by the diazepam reservoir technique, measured by the action of diazepam against bicuculline-induced seizures 4. Also, during treatment with diazepam reservoirs, the time course for production and for reversal of subsensitivity to iontophoretically-applied ),-aminobutyric acid in dorsal raphe neurons 4 was remarkably similar to the time course of tolerance measured in SNpr in the present study. It is not clear why the two different treatments produced these differing results in SNpr. A n y future attempt to address this point would need to consider the roles of active metabolites, including desmethyldiazepam, and the rather potent and long-acting desalkyl-FZP. A n o t h e r consideration is the difference in chronic treatment regimens, which results in a relatively greater brain dia-
zepam-like activity with the FZP treatment used in the present study (equivalent to about 2 - 4 times that produced with the diazepam reservoir technique; cf. refs. 4, 11, 15). Though BZs have direct effects in the SNpr 12't4'19,
1 Bunney, B.S., Waiters, J.R., Roth, R.H. and Aghajanian, G.K., Dopaminergic neurons: effects of antipsychotic drugs and amphetamines on single cell activity, J. Pharmacol. Exp. Ther., 185 (1973) 560-571. 2 Christensen, J.D., Tolerance development with chlordiazepoxide in relation to the plasma levels of the parent compound and its main metabolites in mice, Acta Pharmacol. Toxicol., 33 (1973) 262-272. 3 Cook, L. and Sepinwall, J., Behavioral analysis of the effects and mechanisms of action of benzodiazepines. In E. Costa and P. Greengard (Eds.), Advances in Biochemical Psychopharmacology, Vol. 14, Mechanism of Action of Benzodiazepines, Raven, New York, 1975, pp. 1-27. 4 Gonsalves, S.F. and Gallager, D.W., Time course for development of anticonvulsant tolerance and GABAergic subsensitivity after chronic diazepam, Brain Research, 405 (1987) 94-99. 5 Kirk, R.E., Experimental Design: Procedures for the Behavioral Sciences, Brooks/Cole, Monterey, 1982. 6 Rosenberg, H.C. and Chiu, T.H., Tolerance during chronic benzodiazepine treatment associated with decreased receptor density, Eur. J. Pharmacol., 70 (1981) 453-460. 7 Rosenberg. H.C. and Chiu. T.H., Regional specificity of benzodiazepine receptor down-regulation during chronic treatment of rats with flurazepam, Neurosci. Lett.. 24 (1981) 49-52.
8 Rosenberg, H.C. and Chiu, T.H., Time course for development of benzodiazepine tolerance and physical dependence, Neurosci. Biobehav. Rev., 9 (1985) 123-131. 9 Rosenberg, H.C., Chiu, T.H. and Tietz, E.I., Studies on the mechanism of tolerance to benzodiazepines. In H. Frey, W.P. Koella, W. Fr6scher and H. Meinardi (Eds.), Tolerance to Beneficial and Adverse Effects of Antiepileptic Drugs, Raven, New York, 1986, pp. 53-61. 10 Rosenberg, H.C., Smith, S. and Chiu, T.H., Benzodiazepine-specific and non-specific tolerance following chronic flurazepam treatment. Life Sci., 32 (1983) 279-285. 11 Rosenberg, H.C., Tietz, E.I. and Chiu, T.H., Tolerance to the anticonvulsant action of benzodiazepines: relationship to decreased receptor density, Neuropharmacology, 24 (1985) 639-644. 12 Rosenberg, H.C., Tietz, E.I. and Chiu, T.H., Anticonvulsant activity of a benzodiazepine injected in substantia nigra pars reticulata, Soc. Neurosci. Abstr., 11 (1985) 272. 13 Ross, R., Waszczak, B.L., Lee, E.K. and Walters, J.R., Effect of benzodiazepines on single unit activity in the substantia nigra pars reticulata, Life Sci., 31 (1982) 1025-1035. 14 Tietz, E.I. and Rosenberg, H.C., Behavioral measurement of benzodiazepine tolerance and GABAergic subsensitivity in substantia nigra pars reticulata, Brain Research, 438 (1988) 41-51. 15 Tietz, E.I., Rosenberg, H.C. and Chiu, T.H., Autoradio-
B Z induced suppression of SNpr neuronal activity and the changes with chronic treatment may not be mediated within SNpr. There was a strong similarity between the time course of tolerance measured in the present study and that found in previous experiments using the same 9'~1 or some other treatment regimens 4. This similarity supports the idea that changes measured in SNpr may be related to the anticonvulsant activity of systemically administered BZs and the tolerance which develops to their anticonvulsant action. The authors wish to thank William M. Gilliam for his skillful technical assistance. FZP was a generous gift of Hoffmann-La Roche. This work was supported by D H H S Grant R01-DA02194. J.L.T. was supported in part by the Academic Challenge Program of the Ohio Board of Regents. Preliminary reports of this work have previously been presented: Fed. Proc., 45 (1986) 675, and Soc, Neurosci. Abstr., 12 (1986) 668.
348
graphic localization of benzodiazepine receptor downregulation, J. Pharmacol. Exp. Ther., 236 (1986)284-29Z 16 Tyma, J.L., Rosenbcrg, }t.C. and Chiu, T.tl., Radiorcceptor assay of benzodiazepines m cerebrospinal fluid during chronic flurazcpam lrcatment in cats. tSt,. ,I l)tlarmaco/., 1(15 (1984) 301-308. 17 Waszczak, B.L., Eng, N. and Waiters, ,I.R., Effects of
muscimol and picrotoxin on single unit activity of substantia nigra neurons, Brain Research, 188 (1980) 185-1'47.
18 Waszczak, B.L., Lee, E.K. and Waiters. J.R., f:;flccts ol anticonvulsant drugs on suhstantia nigra par~, rcticulata ncurons, J. Pharmaco]. Kvp. Yher., 239 (1986)f',{16-61 l. 19 Wilson, M.A. and Oallager, I).W., Effects of chronic diazepam exposure on GABA scnsitiviD and on benzodiazcpine potentiation of GABA-mcdiatcd responses or"substautia nigra pars reticulata neurons of rats, Eur. ,I Pharmaco[. 136 (1987) 333-343.
Short Communications
Effects of chronic flurazepam treatment on firing rate of rat substantia nigra pars reticulata neurons Jennifer L. Tyma, Howard C. Rosenberg, Elizabeth I. Tietz and T.H. Chiu Department of Pharmacology, Medical College of Ohio, Toledo, OH 43699 (U. S. A. )
(Accepted 15 March 1988) Key words: Benzodiazepine: Tolerance: Substantia nigra pars reticulata; Flurazepam
The effect of a benzodiazepine, flurazepam, on the spontaneous activity of neurons in the pars reticulata of the substantia nigra was studied in chloral hydrate anesthetized rats. Flurazepam produced a dose-related suppression of neuronal activity. In rats that were chronically treated with flurazepam, tolerance to flurazepam was present after 7 and 28 days, but not after only 3 days of treatment. Tolerance persisted at least 2, but not 7 days after 4 weeks of chronic treatment.
Chronic benzodiazepine (BZ) exposure has been shown to produce tolerance to the sedative, muscle relaxant and anticonvulsant actions of BZs 2"3'6"8-11. Changes produced by chronic treatment, such as alterations in the G A B A / B Z receptor complex or its function, may be the basis for tolerance to various BZ actions. For example, with the chronic BZ treatment used in the present study a temporal relationship was found between B Z receptor down-regulation and tolerance to ataxia produced by large doses of flurazepam (FZP) 6 or diazepam i°. This treatment also produced tolerance to diazepam's anticonvulsant activity, though the time course differed from that of receptor down-regulation H. This study investigated the possibility that tolerance to BZs could be measured by the action of BZs to suppress spontaneous activity of neurons in the pars reticulata of substantia nigra (SNpr) 13"18. The SNpr appears to be a useful site for studying the acute and chronic actions of BZs 12-15'18'19. Using the method described by Ross et al. 13, dose-response data were recorded during intravenous infusion of a water-soluble BZ, FZP, in control and chronically treated rats. Once tolerance was found, the time course for its production and reversal was deter-
mined and compared to similar information from studies of tolerance measured behaviorally, and studies of receptor regulation. It was thought that comparison of the rates of development and reversal of these p h e n o m e n a might provide some insight as to possible mechanisms of tolerance. Male S p r a g u e - D a w l e y rats were given FZP according to an established chronic treatment protocol 6, in which a saccharin-flavored F Z P solution was provided as the sole source of drinking water. During the first week, the FZP concentration was adjusted to provide 100 mg/kg/day. During weeks 2 - 4 , 150 mg/kg/day was offered. Details of this method and typical dose consumption have been published 6. Rats randomly assigned as controls received undrugged saccharin water for the same duration as their FZPtreated counterparts, In order to follow the development of, and recovery from tolerance, various treatment schedules were studied and compared to results in control rats. Treated rats were studied immediately upon completing 3, 7 or 28 days of treatment, and 2 or 7 days after completing the 28-day treatment. Rats weighing 250-320 g at the end of chronic treatment were anesthetized with chloral hydrate
Correspondence: H.C. Rosenberg, Department of Pharmacology, Medical College of Ohio, C.S. 10008, Toledo, OH 43699, U.S.A.
0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
345 (400 mg/kg, i.p.). The femoral artery and vein were cannulated for blood pressure measurement and FZP administration, respectively. In the event that fluctuations in depth of anesthesia might affect the results, anesthesia was maintained throughout the experiment by a constant intravenous infusion of chloral hydrate beginning 30 rain after initial chloral hydrate injection. The dose was 2.2 mg/kg/min for control rats, and those tested 48 h or 7 days after 4 weeks of treatment. Other treatment groups received 2.5 mg/kg/min. These doses were sufficient to prevent spontaneous and reflex movements during the experiment. Body temperature was maintained between 36 and 38 °C using a Deltaphase isothermal pad. Control rats were interspersed with treated rats throughout the study. The techniques used for extracellular, single unit recording have been previously described 1'17. Single barrel glass capillary electrodes, 3 - 8 Mr2 resistance, were filled with a 1% solution of Pontamine sky blue dye in 2 M NaCI. Electrical activity was recorded using a high-input impedance amplifier (World Precision Instruments Model DAM-5A), and monitored on an oscilloscope. Action potential frequency recorded from a single neuron was plotted as a histogram by a strip chart recorder. Each SNpr cell was monitored for 5 min. After this baseline period, cumulative intravenous FZP doses were administered. Doses ranging from 0.125 to 4 mg/kg were administered every 2 min so that each increment doubled the previously administered dose, as described by Ross et al. 13. Drug effect for each dose was quantified by the average frequency of the cell for the 2 min post-injection, expressed as a percent of baseline. Only one cell was studied per rat. The recording site was marked by passing current through the electrode to eject the dye. Only those experiments with a recording site histologically verified to be in SNpr were included in this study. To assess the amount of drug present, brain BZlike activity was measured by radioreceptor assay. Rats were sacrificed by decapitation immediately upon completing FZP treatment for 3 days, 1 week and 4 weeks. To evaluate the amount o f FZP in the brain during infusion of the drug, cumulative doses of FZP were given to 4 chloral hydrate anesthetized control rats, as described above. Two min after a total dose of 2 mg/kg (which produced approximately
I O0 ,i,~i " "'-o.
LJ <
x
•
40-
2o 0.1
i 1.0
10
CUMULATIVE FZP DOSE ( r n g / k g )
Fig. 1. Cumulative log dose-response curves of the effects of intravenous flurazepam (FZP) on SNpr single unit activity. Data are presented for control rats (n = 16, O) and rats tested after 4 weeks of FZP treatment. Treated rats were tested immediately upon completing treatment (n = 12, Q) or after stopping treatment for 2 days (n = 13, z~) or 7 days (n = 9, A). FZP was administered every 2 rain so that each increment doubled the previously administered dose ~3. The average frequency for the 2-rain period following FZP injection was expressed as a percent of initial baseline frequency. Only one cell per rat was studied for drug response. Vertical lines represent S.E.M.
50% reduction in neuronal discharge rate), the rats were decapitated, and the brains were collected. Whole brains were removed and frozen (-70 °C) until the time of the assay. Assays were based on the displacement of 2 nM [3H]flunitrazepam specifically bound to freshly prepared synaptosomal membranes of rat whole brain. Procedures for preparing ethanol extracts, preparation of synaptosomal membranes and the radioreceptor assay have previously been described in detail 11,16. The overall analysis for single unit studies was carried out using a two-factor repeated measures A N O V A . Individual planned comparisons between control and each treated group to evaluate the effect of cumulative FZP administration were made by repeated measures A N O V A using the Bonferroni test, which required P < 0.01 for significance 5. Baseline frequencies were compared using a one-way ANOVA. The baseline discharge rates of SNpr neurons ranged from 10 to 53 Hz (mean +_ S.E.M. = 25.68 _+ 1.16). In none of the treatment groups was the baseline discharge frequency significantly different (P = 0.27) from that in control rats (28.53 _+ 2.74 Hz). FZP significantly inhibited SNpr neuron firing rate in a dose-dependent manner (P ~< 0.0001), as previous-
346 ly observed 13. The overall treatment effect did not reach statistical significance (P = 0.054). However, tolerance was shown by a significant overall interaction between F Z P dose infused and treatment group (P = 0.014). The probe of the overall interaction by A N O V A revealed a significant shift of the d o s e - r e sponse curves at some but not other time points, in rats treated for 4 weeks and tested 0 h after removal of drugged water, the dose-response curve was significantly shifted to the right of the control curve (Fig. 1), showing tolerance to FZP (P = 0.006). FZP administered 48 h post-4-week treatment produced less suppression of neuronal activity than in control rats (Fig. 1), showing that tolerance was still present 48 h after treatment had ended (P = 0.006). Rats tested 7 days post-4-week treatment were no longer tolerant (P = 0.38), indicating that the reduced sensitivity to FZP, as measured by suppression of SNpr neuronal activity, had largely reversed a week after the 4-week F Z P treatment. Rats tested immediately after 3 days of chronic treatment did not exhibit tolerance to FZP (Fig. 2), as indicated by a non-significant shift in the dose-response curve (P = 0.67). However, the dose-response curve of rats tested immediately after 1 week of 100 mg/kg FZP treatment was significantly shifted to the right of control (P = 0.002). Using this chloral hydrate infusion method, there was no significant difference in initial mean arterial pressure across groups (P t> 0.24). There was also no difference in mean arterial pressure before and after intravenous cumulative FZP administration in any treatment group ( P / > 0.20) except rats tested after 3 day treatment. The drop in mean arterial pressure in this group did not appear to change the sensitivity of these neurons to FZP, as compared to other treatment groups (Figs. 1 and 2). Brain BZ-like activity was measured by radioreceptor assay after 3 days, 1 week and 4 weeks of chronic treatment. Activity for rats tested after 3 days of treatment with 100 mg/kg (n = 6) was equivalent to a concentration of 1.67 ktM FZP. Rats receiving 1 week of treatment with 100 mg/kg (n = 6) had the equivalent of a concentration of 1.06 uM. These values were not significantly different (P = 0.06). After 4 weeks of treatment (100 mg/kg for 1 week, 150 mg/kg for 3 weeks), BZ-like activity was equivalent to a concentration of 3.44 uM. A time course for the
100
(D z
13< 60 z w
< ~3
40-
20 0,1
j 1.0
_
_
q 10
CUMULATIVE FZP DOSE ( m g / k g )
Fig. 2. Cumulative FZP log dose-response curves of the effects of intravenous FZP on SNpr single unit activity. Data were plotted as described in Fig. 1. Data are presented for control rats (n = 16, O) and rats tested immediately upon completing 3 days (n = 10, O) or 7 days (n = 1l, A) of FZP treatment. disappearance of residual drug after this chronic treatment has been described elsewhere u. Four control rats were given a cumulative F Z P dose of 2 mg/kg over 8 min, i.e. the first 5 injections of the FZP infusion used during electrophysiological recording. Brain tissue from these rats was found to contain enough BZ to be equivalent to a FZP concentration of 14.8 (+ 2.2) ktM. Thus, the amount of residual F Z P or active metabolites u in treated rats was probably insufficient to directly suppress SNpr discharge frequency. In this study, tolerance developed maximally between the third and seventh day of treatment, and remained essentially unchanged for at least 2 days after treatment was stopped. This time course was similar to that of tolerance to the anticonvulsant effect of diazepam in rats given the same chronic F Z P treatment 9"11. This chronic FZP treatment is also known to cause down-regulation of BZ receptors 6'7, which was shown to be most pronounced in SNpr Is. However, the time course for this down-regulation was quite different from that of tolerance measured in the present study, so that this does not appear to be a mechanism for tolerance measured in the SNpr. Some comparisons can be made with the results of a recent study in which rats received chronic treatment with diazepam released from a subcutaneous reservoir. The effects of intravenous diazepam on SNpr neuronal discharge rate, and the potentiation of G A B A by microiontophoretically-applied FZP were measured 19. Tolerance was found after only
347 one day of exposure to diazepam. Tolerance reversed rapidly, within 24 h. Thus, tolerance in SNpr appeared to require the continued presence of diazepam (or perhaps a metabolite). In contrast, tolerance measured in SNpr in the present study was still present 2 days after stopping chronic treatment, at which time brain levels of BZ (including metabolites) had declined to virtually zero II. Interestingly, the time course for producing tolerance during chronic FZP treatment, measured in SNpr (Fig. 1), was very similar to the time course of tolerance in rats treated by the diazepam reservoir technique, measured by the action of diazepam against bicuculline-induced seizures 4. Also, during treatment with diazepam reservoirs, the time course for production and for reversal of subsensitivity to iontophoretically-applied ),-aminobutyric acid in dorsal raphe neurons 4 was remarkably similar to the time course of tolerance measured in SNpr in the present study. It is not clear why the two different treatments produced these differing results in SNpr. A n y future attempt to address this point would need to consider the roles of active metabolites, including desmethyldiazepam, and the rather potent and long-acting desalkyl-FZP. A n o t h e r consideration is the difference in chronic treatment regimens, which results in a relatively greater brain dia-
zepam-like activity with the FZP treatment used in the present study (equivalent to about 2 - 4 times that produced with the diazepam reservoir technique; cf. refs. 4, 11, 15). Though BZs have direct effects in the SNpr 12't4'19,
1 Bunney, B.S., Waiters, J.R., Roth, R.H. and Aghajanian, G.K., Dopaminergic neurons: effects of antipsychotic drugs and amphetamines on single cell activity, J. Pharmacol. Exp. Ther., 185 (1973) 560-571. 2 Christensen, J.D., Tolerance development with chlordiazepoxide in relation to the plasma levels of the parent compound and its main metabolites in mice, Acta Pharmacol. Toxicol., 33 (1973) 262-272. 3 Cook, L. and Sepinwall, J., Behavioral analysis of the effects and mechanisms of action of benzodiazepines. In E. Costa and P. Greengard (Eds.), Advances in Biochemical Psychopharmacology, Vol. 14, Mechanism of Action of Benzodiazepines, Raven, New York, 1975, pp. 1-27. 4 Gonsalves, S.F. and Gallager, D.W., Time course for development of anticonvulsant tolerance and GABAergic subsensitivity after chronic diazepam, Brain Research, 405 (1987) 94-99. 5 Kirk, R.E., Experimental Design: Procedures for the Behavioral Sciences, Brooks/Cole, Monterey, 1982. 6 Rosenberg, H.C. and Chiu, T.H., Tolerance during chronic benzodiazepine treatment associated with decreased receptor density, Eur. J. Pharmacol., 70 (1981) 453-460. 7 Rosenberg. H.C. and Chiu. T.H., Regional specificity of benzodiazepine receptor down-regulation during chronic treatment of rats with flurazepam, Neurosci. Lett.. 24 (1981) 49-52.
8 Rosenberg, H.C. and Chiu, T.H., Time course for development of benzodiazepine tolerance and physical dependence, Neurosci. Biobehav. Rev., 9 (1985) 123-131. 9 Rosenberg, H.C., Chiu, T.H. and Tietz, E.I., Studies on the mechanism of tolerance to benzodiazepines. In H. Frey, W.P. Koella, W. Fr6scher and H. Meinardi (Eds.), Tolerance to Beneficial and Adverse Effects of Antiepileptic Drugs, Raven, New York, 1986, pp. 53-61. 10 Rosenberg, H.C., Smith, S. and Chiu, T.H., Benzodiazepine-specific and non-specific tolerance following chronic flurazepam treatment. Life Sci., 32 (1983) 279-285. 11 Rosenberg, H.C., Tietz, E.I. and Chiu, T.H., Tolerance to the anticonvulsant action of benzodiazepines: relationship to decreased receptor density, Neuropharmacology, 24 (1985) 639-644. 12 Rosenberg, H.C., Tietz, E.I. and Chiu, T.H., Anticonvulsant activity of a benzodiazepine injected in substantia nigra pars reticulata, Soc. Neurosci. Abstr., 11 (1985) 272. 13 Ross, R., Waszczak, B.L., Lee, E.K. and Walters, J.R., Effect of benzodiazepines on single unit activity in the substantia nigra pars reticulata, Life Sci., 31 (1982) 1025-1035. 14 Tietz, E.I. and Rosenberg, H.C., Behavioral measurement of benzodiazepine tolerance and GABAergic subsensitivity in substantia nigra pars reticulata, Brain Research, 438 (1988) 41-51. 15 Tietz, E.I., Rosenberg, H.C. and Chiu, T.H., Autoradio-
B Z induced suppression of SNpr neuronal activity and the changes with chronic treatment may not be mediated within SNpr. There was a strong similarity between the time course of tolerance measured in the present study and that found in previous experiments using the same 9'~1 or some other treatment regimens 4. This similarity supports the idea that changes measured in SNpr may be related to the anticonvulsant activity of systemically administered BZs and the tolerance which develops to their anticonvulsant action. The authors wish to thank William M. Gilliam for his skillful technical assistance. FZP was a generous gift of Hoffmann-La Roche. This work was supported by D H H S Grant R01-DA02194. J.L.T. was supported in part by the Academic Challenge Program of the Ohio Board of Regents. Preliminary reports of this work have previously been presented: Fed. Proc., 45 (1986) 675, and Soc, Neurosci. Abstr., 12 (1986) 668.
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graphic localization of benzodiazepine receptor downregulation, J. Pharmacol. Exp. Ther., 236 (1986)284-29Z 16 Tyma, J.L., Rosenbcrg, }t.C. and Chiu, T.tl., Radiorcceptor assay of benzodiazepines m cerebrospinal fluid during chronic flurazcpam lrcatment in cats. tSt,. ,I l)tlarmaco/., 1(15 (1984) 301-308. 17 Waszczak, B.L., Eng, N. and Waiters, ,I.R., Effects of
muscimol and picrotoxin on single unit activity of substantia nigra neurons, Brain Research, 188 (1980) 185-1'47.
18 Waszczak, B.L., Lee, E.K. and Waiters. J.R., f:;flccts ol anticonvulsant drugs on suhstantia nigra par~, rcticulata ncurons, J. Pharmaco]. Kvp. Yher., 239 (1986)f',{16-61 l. 19 Wilson, M.A. and Oallager, I).W., Effects of chronic diazepam exposure on GABA scnsitiviD and on benzodiazcpine potentiation of GABA-mcdiatcd responses or"substautia nigra pars reticulata neurons of rats, Eur. ,I Pharmaco[. 136 (1987) 333-343.