Age-dependent enhancement of diazepam sensitivity is accelerated in New Zealand black mice

Life Sciences, Vol. 38, pp. 1433-1439 Printed in the U.S.A.

Pergamon Press

AGE-DEPENDENT ENHANCEMENT OF DIAZEPAM SENSITIVITY IS ACCELERATED IN NEW ZEALAND BLACK MICE

Michael J. Forster, Konrad C. Retz, Mark D. Popper, and Harbans Lal Department of Pharmacology, Texas College of Osteopathic Medicine, Fort Worth, Texas, 76107 (Received in final form February 6, 1986)

Summary Separate age groups of C57BL/6 and autoimmune New Zealand Black (NZB) mice were compared for diazepam-induced ataxia and barbiturateinduced loss of r i g h t i n g r e f l e x . Between 1 and 3 months of age, both strains showed a similar age-related decrease in ED50 f o r diazepaminduced ataxia. However, between 3 and 12 months the decrease in ED50 was markedly greater in NZB mice. In contrast, age-related increases in the durations of loss of r i g h t i n g r e f l e x following hexobarbital or barbital were similar in both strains. The results suggest that NZB mice show r e l a t i v e l y accelerated age-related increases in s e n s i t i v i t y to benzodiazepine, but not to barbiturates. Immune mechanisms have been considered in r e l a t i o n to the e t i o l o g y of CNS dysfunctions associated with aging (1,2,3) and with numerous disease states involving neuropsychopathology (3,4,5). The involvement of immune factors in CNS dysfunctions is suggested by the high incidence of b r a i n - r e a c t i v e a n t i bodies (BRA) in sera of e l d e r l y individuals (6,7,8), Alzheimer's disease pat i e n t s (8,9,10) and persons with neurological disorders (4,5,9). Additional support comes from the recently i d e n t i f i e d association between BRA and behavioral abnormalities in senescent animals (1,2,11,12). Serum BRA are found more frequently with aging in mice (11,13,14), rats (15), and nonhuman primates (16) with "normal" life-spans. New Zealand Black (NZB) mice, a s h o r t - l i v e d autoimmune s t r a i n , begin to accumulate BRA e a r l i e r (by about I to 3 months of age) and attain levels comparable to senescent (2425 month-old) C57BL/6 mice by approximately 12 months of age (1,2,11). In addition to t h e i r e a r l i e r accumulation of BRA, young NZB mice e x h i b i t certain behavioral (1,2,11,12,17) and morphological (18) abnormalities which are chara c t e r i s t i c of senescent mice. These findings suggest that age-related CNS a l t e r a t i o n s in young NZB mice could be similar to those which occur in senescence for mice with longer l i f e spans. I f CNS a l t e r a t i o n s in young NZB mice and senescent mice are similar, young NZB mice should also show senescence-like a l t e r a t i o n s in pharmacologic sensitivity. For example, senescent humans (19,20) and rodents (21,22) e x h i b i t enhanced sedation following benzodiazepines when compared to younger subjects, e f f e c t s which cannot be f u l l y explained by age differences in benzodiazepine pharmacokinetics (19). In order to determine whether young NZB mice develop senescence-like s e n s i t i v i t y to benzodiazepine, we compared age differences in diazepam-induced ataxia in NZB and C57BL/6 mice. I t was expected that NZB mice would show enhancement of diazepam s e n s i t i v i t y at younger ages when compared to C57BL/6 mice, in accordance with t h e i r e a r l i e r development of BRA and other 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.

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senescence-like characteristics. To determine the specificity of age-related changes in benzodiazepine sensitivity, age difference~;:in barbiturate sensitivi t y were also examined. A preliminary report of this~work has appeared in abstract form (23). Methods Animals: Animals were separate groups of 8-15 male NZB/BINJ and C57BL/6J mice (Mus musculus) aged 1-2, 2-4, 5-7, or 11-13 months, obtained from Jackson Laborator--ies ~ r Harbor, ME) when 1 month old. Mice were housed in the Texas College of Osteopathic Medicine mouse colony in clear 36 X 31X 17 cm polycarbonate cages (7-12 per cage) until the time of testing. The mice had access to food and water ad lib. The colony room was maintained at 22~1° C, on a normal light-dark cycle beginning at 0700 hrs. Tests were conducted during the light portion of the cycle. Drugs: Diazepam (Ro 5-2857; Roche) was suspended in 0.9% saline; 10% propylene glycol (with 1 drop of Tween-80 added per 20 ml total volume) and given in doses of 0.64, 1.25, 2.5, 5.0, 10.0, 20.0, or 40.0 mg/kg. Hexobarbital sodium (120 mg/kg; Sigma Chemical Co.) and barbital sodium (300 mg/kg; Sigma Chemical Co.) were dissolved in 0.9% saline. All drugs were injected IP in a volume of 10 ml/kg. In diazepam studies, each mouse of each age group received all doses of the drug, with a washout period of at least 3 days between treatments. Measurement of Ataxia: The latency of mice to f a l l from a gnurled, steel cylinder (3-cm dia) rotating at 3.33 rpm (Wahlquist Instruments) was recorded to a criterion of 120 seconds. Ataxia was considered as failure to meet this criterion in a single test. Mice were tested for ataxia just prior to each drug treatment, and drug effects were assessed in a second rotarod test conducted 30 min thereafter. In the time-course studies, rotarod tests were administered to the same mice at pre-determined times following drug injections. Loss of rightin 9 reflex: ~parate groups of mice aged 1-2, 5-7, or 11-13 months were tested for onset and duration of loss of righting reflex following hexobarbital or barbital according to a method outlined previously (24). Following the f i r s t 2-min period without locomotion, each mouse was tested for righting a b i l i t y at 2-min intervals. Onset latency was designated as the time interval between injection and the beginning of the f i r s t interval during which righting failed to occur. Duration of loss of the righting reflex, discussed as "sleep time", was the time interval between onset of reflex loss and spontaneous righting. Results Diazepam-induced ataxia: Diazepam-induced ataxia is shown in Fig. I, expressed as the percent mice (probit conversion) f a i l i n g to remain on the rotating cylinder for 120 sec. When tested prior to drug injections, all mice of all ages remained on the rotating cylinder for the f u l l 120 sec. When tested 30-min following diazepam, there was a dose-dependent increase in the percentages of mice showing ataxia (motor impairment) in all age groups of each strain. In addition, there was an overall shift to the l e f t in the diazepam dose-effect curves of each strain between the ages of 1-2 and 11-13 months. While there was l i t t l e effect of strain between the ages of 1-2 and 2-4 months, the decrease in EDSO (25) for ataxia in NZB mice after 2-4 months was more marked than the change for C57BL/6 mice. There was at least a lO-fold decrease in ED50 between the ages of 2-4

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and 11-13 months for NZB mice, as opposed to roughly a 2-fold shift for C57BL/6 mice (see inset of Fig. 1). Parametric analysis supported the observation of differential changes in response of the strains with age. When rotarod latencies of the 2-4, 5-7, and 11-13 month-old groups which had received a common diazepam dose (5 mg/kg) were considered in a 2-way Analysis of Variance (26), a significant Age X Strain interaction [F(2,60)=17.6, p<0.0005] was obtained.

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DIAZEPAM(mg/kg) FIG. 1 Percent mice (probit scale) showing motor impairment (% falling before 120 sec) as a function of age (top to bottom), diazepam dose (log scale), and strain. The inset shows EDSO values (log scale) with g9% confidence intervals as a function of strain and age.

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Time-course of diazepam-induced ataxia: Fig. 2 shows recovery from ataxia For 5-7 month-old mice following 20 mg/kg diazepam, expressed as the percentage of each strain recovering at predetermined times before or following injection. All mice recovered from ataxia within 4 hours, with a trend toward faster recovery among NZB mice as compared to the C57BL/6 mice. However, a ~X]2 analysis failed to indicate a significant difference between the strains ( ~2=3.0, p
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Percentages of 5-7 month-old NZB and C57BL/6 mice unimpaired (% remaining for 120 sec) prior to (PRE) and following 20 mg/kg diazepam (N=12 for each strain). Barbiturate-induced loss of rightin 9 reflex: The results of t--he-ba-rbiturate e ~ e n t s are summarized in Fig. 3. The data represented within each panel were considered in separate 2-way Analyses of Variance with Strain and Age as factors. Although sleep time following barbital was longer than that following hexobarbital, the patterns of sleep time as a Function of age and strain were similar for the barbiturates (top panels, Fig. 3). Sleep time following each barbiturate showed overall increase with age [Fs(2,48)>63.4, ps5.8, ps
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Body weight (lower l e f t , Fig. 3) showed an overall increase with age in both strains [F(2,48)=228.0, p
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FIG. 3 Age-related changes in barbiturate effects and body weights in NZB and C57BL/6 mice. The upper panels show sleep times (duration of loss of righting reflex) following hexobarbital (120 mg/kg, l e f t ) or barbital (300 mg/kg, right) as a function of age and strain. (Note different ordinate scales for hexobarbital and barbital). Barbital onset latency (latency to loss of righting reflex) and mean body weight are shown in the lower panels ( l e f t to right) as a function of age and strain. Discussion When compared at 1-2 and 2-4 months, NZB and C57BL/6 mice had similar responsiveness to diazepam and showed parallel age-related increases in sensit i v i t y . This finding suggested that there was l i t t l e genetic difference in diazepam s e n s i t i v i t y between the two inbred strains at these young ages. A period of rapid increase in diazepam s e n s i t i v i t y occurred in the NZB mice between 3 and 12 months, age-related increases in C57BL/6 mice being less marked during that period. These differential sensitivity changes in the strains may well r e f l e c t strain/age-dependent differences in CNS-mediated processes underlying drug effects. The time-course studies provided no indication that pharmacokinetic factors were involved, as there was no significant strain difference in onset or duration of ataxia at 5-7 or 12 months following 20 mg/kg diazepam. Yet, at both of these ages NZB mice showed ataxia at lower diazepam doses when compared to C57BL/6 mice. The period of enhanced diazepam s e n s i t i v i t y of NZB mice does not appear to be related to a nonspecific increase in sensitivity to sedative agents, per se. While there was an age-related increase in barbiturate-induced loss of righting reflex in both strains, there was no period of relatively accelerated increase

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in the NZB strain paralleling that obtained with diazepam. In faqt, the findings suggest that barbiturate sensitivity increased more rapidly With age in C57BL/6 mice, 5-7 month-old C57BL/6 mice showing longer sleeping times than age-matched NZB mice. Thus, the mechanisms involved in accelerated diazepam sensitivity increases in NZB mice are probably different from those contributing to age differences in barbiturate sensitivity. I t is not yet clear whether the age-related changes in barbiturate sensit i v i t y involve pharmacokinetic or pharmacodynamic mechanisms. Hepatic drug metabolism is not a significant factor because barbital, a barbiturate which is excreted largely as the unchanged drug, produced the same age-dependent pattern of effect as hexobarbital, a barbiturate inactivated largely through hepatic metabolism. On the other hand, there was no age-related decrease in barbital onset latency, an effect which would be expected i f pharmacodynamic factors contributed to the age-related increase in barbiturate sensitivity. Because doses were calculated on the basis of total body mass, age-related increases in barbiturate sensitivity may be attributable to changes in ratios of brain/body weight or lean/fat body mass (19). However, there was no clear relationship between body weight and barbiturate effects (see Fig. 3). For example, body weights of C57BL/6 mice increased markedly between 5-7 and 11-13 months, with l i t t l e corresponding change in barbiturate effect. Conversely, NZB mice showed l i t t l e change in weight during this period, but exhibited an increase in barbiturate sensitivity. Although the diazepam sensitivity increases in young NZB mice could involve mechanisms similar to those underlying senescence-related changes in mice with longer life-spans, the effect could also involve maturation-related processes. Reliable age-related increases in diazepam sensitivity occurred for both NZB and C57BL/6 strains between i and 12 months of age. This trend was similar for both strains between 1 and 3 months, though accelerated in NZB mice between the ages of 3 and 12 months. Whereas the age groups tested cover the major portion of the life-span of male NZB mice (27), the age groups of C57BL/6 tested were insufficient to determine whether the maturation-related increases (between 1 and 6 months) in sensitivity are continuous with those expected to occur during post-maturity aging (21,22). Senescence- and maturation-related sensitivity increases could involve fundamentally different mechanisms, or could represent maturational changes in benzodiazepine-relevant CNS mechanisms which begin in periadolescence, and continue throughout maturity and senescence (28,29). S t i l l another possibility is that decreased diazepam s e n s i t i v i t y is peculiar to periadolescence, reflecting ontogenetic discontinuity in development of relevant CNS mechanisms (30). Although the mechanisms involved in accelerated benzodiazepine sensitivity changes of NZB mice are not yet clear, the changes are noteworthy by virtue of their temporal relationship with immunologic characteristics of this strain which may reflect CNS aging processes. By 6 months of age, NZB mice develop levels of BRA not attained by C57BL/6 mice until about 18 months (1,2,11). The temporal correspondence between early BRA development and behavioral deterioration in the NZB mice (1,2,11) is consistent with the hypothesis that agerelated CNS deterioration is accelerated in this strain. The present findings add support to this hypothesis, indicating that BRA development is also associated with a period of accelerated increase in diazepam s e n s i t i v i t y , an alteration similar to that shown by senescent humans and animals.

Acknowledgments This research was p a r t i a l l y supported by U.S.P.H.S. grant AG03623. The authors thank Hoffmann-La Roche for their donation of diazepam.

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