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6 mice
Neuropharmacolog~Vol. 26, No. 5, pp. 445-452, 1987 Printed in Great Britain. All rights reserved
0028-3908/87 $3.00 + 0.00
Copyright 0 1987Pergamon Journals Ltd
DIFFERENCES IN BEHAVIORAL RESPONSES TO OXOTREMORINE AND PHYSOSTIGMINE IN NEW ZEALAND BLACK (NZB/BINJ) AND C57BL/6 MICE K. C. RETZ*, M. J. FORSTER, N. FRANTZ and H. LAL Department of Pha~acology, Texas College of Osteopathic Medicine, Camp Bowie at Montgomery, Fort Worth, TX 76107-2690, U.S.A. (Accepted 2 May 1986)
Summary-The NZB/BINJ (NZB) mice are an autoimmune-prone strain, known to develop brain-reactive antibodies in serum at much earlier chronological ages than normal mice. Measurement of locomotor activity in 8-10 month old C57BL/6 ((37) mice following the administration of either oxotremo~ne or physostigmine, revealed a biphasic response consisting of inhibition at small doses, but increased motor activity at large doses. In contrast, age-matched NZB mice exhibited little inhibition at the smaller doses, but had much greater increases in activity after the larger doses. Similarly, when compared to C57 mice, NZB mice were less sensitive to oxotremorine-induced salivation, diarrhea and visible tremors. Moreover, oxotremorine-induced hypothermia occurred at smaller doses in C57 mice than in NZB mice and was of a greater magnitude. Thus, at an age when NZB mice possess high levels of brain-reactive antibodies, and exhibit impai~ent in tests of learning/memory, these mice also show diminished responses in several tests of ~hoiinomimetic-indu~ behavior and physiologicai alterations. Key words: NZB/BlNJ mice, C57BL/6 mice, oxotremorine, physostigmine, locomotor activity, brainreactive antibodies.
The fo~ation of brain-reactive antibodies (BRA) in is a concomitant of normal aging in humans (Elizan, Casals and Yahr, 1983; Ingram, Phegan and Blumenthal, 1974; Nandy, 1978), subhuman primates (Nandy, 1981) rats (Feden, Baldinger, Miller-Soule and Blumenthal, 1979) and mice (Biumenthat, Young, Wozniak and Finger, 1984; Nandy, 1972; Nandy, Lal, Bennett and Bennett, 1983; Threat& Nandy and Fritz, 1972). Brain-reactive antibodies are also found with abnormally high frequency in patients with Alzheimer’s disease (Bahmanyar, MoreauDubois, Brown, Cathala and Gajdusek, 1983; Sotelo, Gibbs and Gajdusek, 1980; Nandy, 1978) and are associated with several disease states involving neuropsychopathology (Baldinger and Blumenthal, 1982; Bergsma and Goldstein, 1978). These findings have led to the consideration of immune mechanisms in relation to the etiology of aging-associated dysfunctions of the CNS (Lal, Forster and Nandy, 1985; La1 and Forster, 1986; Nandy, 1985; Film, Luine, Reisberg, Amador, McEwen and Zabriskie, 1985). Recently, studies in this laboratory have suggested a correlation between the appearance of brainreactive antibodies and deficits in learning by normally aging mice (La1 and Forster, 1986; La1 et al., 1985) and in NZB mice, a genetically autoimmuneprone strain known to develop serum brain-reactive antibodies at much earlier chronological ages as compared to the normal strains studied (Forster,
*To whom correspondence
should be addressed.
Retz, Popper and La], 1985; La1 and Forster, 1986; Lal et al., 1985; Nandy et al., 1983). These findings add support to the link between brain-reactive antibodies and behavioral dysfunction, and have raised the possibility that mature NZB mice exhibit neurological alterations similar to those which normally occur in old age, or in pathological states associated with aging. The possibility that NZB mice show aged-like neurological alterations, led to the present investigation of the question of whether mature NZB mice exhibit alterations in central cholinergic mechanisms. There are many lines of evidence which suggest that acetylcholine-mediated processes are compromised or altered in aging rodents (Bartus, Dean, Beer and Lippa, 1982; Coyle, Price and DeLong, 1983; Lippa, Loullis, Rotrosen, Cordasco, Critchett and Joseph, 1985; Bartus, Dean, Pontecorvo and Flicker, 1985; Retz and Lal, 1984). Moreover, some age-related alterations in cholinergic function can be detected pharmacologically, as abnormal behavioral responses to cholinomimetic drugs. For example, in mature rodents, muscarinic agonists produce biphasic effects upon locomotor activity (Fibiger, Lynch and Cooper, 1971; Mason, Roberts and Fibiger, 1978; Hughes and Trowland, 1976), reduce body temperature (Clark and Clark, 1980) and produce analgesia via central mechanisms (Ringdahl, Ehler and Jenden, 1982). In aged rodents, several studies have reported alterations in sensitivity to these components of agonist response, including the motor (Finch, Marshalf and Randall, 1981; Ingram and Brennan, 1984), thermo-
445
K. C.
446
lytic (Pedigo, Minor and Krumrei, 1984; Ferguson, Turner, Cooper and Veale, 1985) and analgesic (Pedigo et af., 1984) effects. In the light of this knowledge, a pharmacological approach was used to test the hypothesis that acetylcholine-mediated processes are altered in mature NZB mice. In the present studies 8-10 month-old NZB and C57BL/6 mice have been compared by observing the motor, autonomic and thermolytic effects of oxotremorine, a direct-acting muscarinic agonist and the effects of physostigmine, an indirectacting cholinomimetic which inhibits acetylcholinesterase. It was expected that mature NZB mice would show abnormal responses in motor behavior, autonomic events and temperature regulation when challenged with the cholinomimetic drugs.
METHODS
kTZ
et al.
Oxotremorine and physostigmine were administered to separate groups of each strain just prior to testing, with each group given drug receiving doses of the drug on separate test days in tandem with a matching session in which vehicle was administered. Tests were spaced 2-4 days apart, with half of the animals in each group receiving vehicle on a given test day. Each animal was observed in the same chamber during sessions of vehicle and drug administration for a given drug and dose. During each test, the mice were observed and the presence of the following was noted: diarrhea, salivation and visible tremors. Measurement of hypothermia induced by oxotremorine
In a separate series of experiments, the magnitude of hypothermia induced by oxotremorine was measured. Colonic temperatures were measured at 15 and 0 min before administration and 15, 30, 45, 60, 90, 120, 150 and 180 min after oxotremorine (Model TH-6 thermometer; Bailey Instruments (Sensortech); Saddlebrook, New Jersey).
Animals
Male C57BL/6J mice and NZB/BlNJ mice were obtained from Jackson Laboratories (Bar Harbor, Maine) at 2-3 months age and housed in the College Vivarium until 8-10 months old. The colony room was maintained at 23 & 1°C under a normal 12 hr light-dark cycle, beginning at 0800 hr. The mice had unlimited access to food and water. Drug administration
All drugs were dissolved in normal saline solution and administered by intraperitoneal injection (lOml/kg) in the doses indicated in the text. Oxotremorine (Aldrich Chemical Co., Milwaukee, Wisconsin) was administered in the “free” form. Physostigmine sulfate (Sigma Chemical Co., St. Louis, Missouri) was administered as the sulfate with doses calculated and expressed as the “free” drug present. Measurement behavior
Statistical analysis
Parametric data from experiments involving two or more factors were subjected to Analysis of Variance (ANOVA) with Drug group (where appropriate) and Strain as between-groups factors and Doses and Sampling interval as within-groups factors. Analysis of frequency data, i.e. numbers of animals showing behavioral responses, was made using a Chi-squared test at the 0.05 significance level.
RESULTS
Motor activity
A preliminary inspection of the data revealed two overall trends: (a) after vehicle, the C57BL/6 mice had higher levels of activity than the NZB mice (Fig. 1); and (b) the expected biphasic effects of oxo-
of motor activity and assessment of
Observation and testing of motor activity took place in six clear, polypropylene chambers (28 cm L x 19 cm W x 13 cm H) with stainless steel wire lids, each located above one sensor of an Electronic Activity Monitor (EAM) (Stoelting Co., Chicago, Illinois). In this apparatus, activity is measured in terms of the numbers of movements exceeding a fixed amplitude and above 1- or 10 Hz frequency cutoffs, detected through disruption of a radio frequency capacitance field by the animal. Activity counts from the slow (2 1 Hz) and fast (2 10 Hz) channels were monitored for 30 min using a sensitivity of 15% full scale for both channels. The 15% sensitivity setting resulted in zero counts of background activity when no animals were on the activity monitors.
Test interval
(min)
Fig. I. Overall strain differences in motor activity of 8-10 month old C57BL/6 and NZB mice. The overall slow (2 1 Hz) motor activity and fast motor activity (a 10 Hz), expressed as total counts/5 min, occurring in the time intervals O-5, 5-15 and 15-30 min after the administration of control and drug is plotted as mean + SEM (N = 6). The slow motor activity data are shown on the left panel and the
fast motor actlvlty ones
are shown on the right panel.
Effects of cholinergic drugs in NZB
tremorine and physostigmine were observed in C57BL/6 mice, using the low and high frequency channels of the Electronic Activity Monitor apparatus. To provide a meaningful comparison in subsequent analysis, activity after administration of drugs was calculated as a percentage of the baseline level and the strains were compared within separate dose ranges, matching the 2 phases of the response to the drug in C57BL/6 mice. The effects of the smaller doses of oxotremorine (i.e. 0,002~.0Smg/kg) and physostigmine (i.e. 0.01-0.32 mg/kg) were analyzed as the action of decreasing the motor activity. Similarly, the effects of the larger doses of oxotremorine (i.e. 0.054.32 mg/kg) and physostigmine (i.e. 0.16-0.64 mg/kg) were analyzed as action of increasing the motor activity. The changes in “slow” 2 1 Hz and “fast” > 10 Hz motor activity observed after both drugs yielded similar patterns of effect. However, slow motor activity showed a greater sensitivity during the decreasing phase of motor activity and fast activity showed a greater sensitivity during the increasing phase of motor activity. These findings are consistent with literature, suggesting that relatively small doses of cholinomimetics reduce locomotor activity or produce catalepsy (Fibiger et al., 1971; Mason et al., 1978; Hughes and Trowland, 1976), whereas larger doses induce high-frequency muscle tremor (Gonzalez, 1984; Ringdahl et al., 1982). In particular, Gonzalez (1984) has noted that the high and low frequency components of the motor response to physostigminc can be distinguished with a modined Stoelting Electronic Activity Monitor. Therefore, the present authors have presented analyses of the “slow” data when examining decreasing effects on motor activity and analyses of the “fast” data when examining increasing effects on motor activity. Sessions with vehicle. Mean slow (left) and fast (right) counts of motor activity after giving the vehicle in all activity experiments are shown in Figure 1 as a function of the three sampling intervals. Drug group (physosti~ine or oxotremo~ne), Strain, Sampling interval and the dose to which a given session with vehicle was matched (designated Dose) were the factors considered in a separate ANOVA conducted on each activity measure. Analyses supported the overall observation that NZB mice had significantfy lower levels of slow (F(1,20) = 12.31, P < 0,002s) and fast @‘(1,20)=6.31, P x0.01) activity when compared to C57BL/6 mice, with both strains showing decreases in activity within the session (t;s(Z, 40) > 28.0, Ps < 0.01). Activity during sessions with vehicle did not vary consistently as a function of Drug group or Dose, and higher-order effects gave little indication that these variables were influenced by Strain. With one exception, both analyses failed to indicate significant higher-order interactions involving Strain, Dose and Drug. A marginally significant Strain by Dose (F(3,60) = 3.09, P < 0.05) interaction was obtained in the analysis of slow
mice
Oxotremofine (mg/kgf
447
Physosfigmine (mg/kg)
Fig. 2. Strain differences in the slow motor activity of 8-10 month old C57BL/6 and NZB mice after the administration of oxotremorine and physostigmine-total observations. The slow (> 1Hz) motor activity after the administration of oxotremorine (left panel) or physostigmine (right panel) is expressed as a percentage mean of control activity with saline, occurring from O-30 min after the administration of the drugs (N = 6).
activity, although the pattern of strain differences which contributed to this interaction was not similar to the dose-related strain differences in the effects of oxotremorine or physosti~ine on slow activity, described in subsequent sections. Activity-decreasing phase. Slow (3 1 Hz) motor activity for the dose range reducing activity is shown in Figure 2 for oxotremorine (left) and physostigmine (right) as a function of strain and dose. Separate analyses were conducted for the groups given physostigmine and oxotremorine, with Strain, Dose and Sampling interval as factors. As seen in Figure 2, there was a dose-dependent reduction in the activity of C57BL/6 mice following the administration of both oxotremorine (0.02-0.05 mg/kg) and physostigmine (0.01-0.32 mg/kg). The activity of the NZB mice was almost never decreased (although it was sometimes increased) within those ranges. This difference between the strains was reflected in effects of Strain (Kr(l, 10) > 13.0, significant Ps < 0.005) in both analyses, and in a Strain by Dose interaction (F(3,30) = 4.06, P < 0.025) in the analysis for oxotremorine. Figure 3 shows the time course of slow (> 1 Hz) motor activity after the administration of oxotremorine (top panels) and physostigmine (lower panels) within the testing sessions as a function of Dose and Strain (C57BL/6, left; NZB, right). For C57BL/6 mice, the reduction of activity after injections of 0.01, 0.02 and 0.05 mg/kg oxotremorine was evident by the first 5 min after testing, and it remained depressed throughout the 30min session. A similar effect was seen after injections of 0.04, 0.16 and 0.32mg/kg physostigmine, with the exception that reduction of activity was not evident until after .5min with this drug. In contrast to the effects in C57BL/6 mice, the activity of NZB mice tended to increase as a function of time after injections of oxotremorine or physostigmine although this trend was erratic and did not have a consistent relationship with the dose of either drug. The trend for increasing
K. c. RET2
448
o-5
m-30
5-f5
o-5
5-15
15-X)
Test interval Fig. 3. Strain differences in the slow motor activity of 840 month old C57BL/6 and NZB mice after the administration of oxotremorine and physostigmine-interval observations. The slow (> 1Hz) motor activity after the administration of oxotremorine (upper panels) or physostigmine (lower panels) is expressed as a mean of percentage control activity with saline, occurring from D-5, 5-15 and 15-30 min after the administration of drugs (N = 6). The data for CS?BL/6J mice are shown on the left panels and the data for the NZB mice are shown on the right panels.
et al.
Activity-increasingphase. The dose ranges for oxotremorine (0.05, 0.1 and 0.32 mg/kg, left) and physostigmine (0.16, 0.32, and 0.64 mg/kg, right) which increased activity are shown in Figure 4 for NZB and C57BL/6 mice. Analyses of these data, involved the same factors considered previously for the decreasing phase of activity. As Figure 4 shows, fast activity after administration of both drugs showed an overall increase as a function of Dose (Fs(2,20) > 33.0, Ps < O.OOl),with much greater dose-related increases occurring in the NZB strain (Fs(1, lo)> 35.0, Ps < 0.001). The marked difference between the strains with increasing doses resulted in significant Strain x Dose interactions (Fs(2,20) > 10.0, Ps < 0.001) in both analyses. Analysis of the data for oxotremorine also indicated an effect of sampling interval (F(2,20) = 40.1, P < 0.001), as well as significant higher-order interactions of Strain and Dose with Sampling interval (all Fs > 4.0, Ps < 0.01). As can be seen in Figure 5, the major source of these interactions was the fact that increases in fast activity after 0.32mgjkg were of much greater magnitude in the NZB mice as compared to the C57BL/6 mice. In contrast to oxotremorine, overall increases in fast activity within sessions were not evident after the administration of physostigmine (F < l.O), although the high level of activity of 0.64 mg/kg-treated NZB mice during the first 5 min of testing contributed to a marginal Dose by Interval interaction (F(4,40) = 2.62, P < 0.05). It I
activity in the NZB mice was most evident after the administration of oxotremorine and contributed to significant 2 and 3-way interactions involving Strain, Dose and Sampling interval (all Fs > 4.0, all Ps < 0.025) in that analysis. The trend was less clear for physostigmine, although it contributed to a significant Strain by Sampling interval interaction [F(2,20) = 6.29, P < 0.011.
4200
800
‘5
6oo
g
400
NZB
t
1000
3
I C57BL/6 t
-
0 0.05
-
A 0.32
A
100
0.64
t
0.05
0.1
0.32 0.16
Oxotrernorine (Wlql)
0.32
0.64
P
OXOtnmori~img/kpl
t
‘~~ o-5
5-15
15-X)
o-5
5-15
15-3
Physosiigmine lmglkg)
Fig. 4. Strain differences in the fast motor activity of 8-10 month old C57BLj6 and NZB mice after the administration of oxotremorine and physostigmine-total observations. The fast (3 10 Hz) motor activity after the administration of oxotremorine (left panel) or physostigmine (right panel) is expressed as a mean percentage of control activity with saline, occurring from O-30 min after the administration of drugs FSEM (N = 6).
Fig. 5. Strain differences in the fast motor activity of 8-10 month old C57BL/6 and NZB mice after the administration of oxotremo~ne and physosti~in~interva~ observations. The fast (> 10 Hz) motor activity after the administration of oxotremorine (upper panels) or physostigmine (lower panels) is expressed as a mean percentage of control activity with saline, occurring from O-5, 5-l 5 and 15-30 min after the administration of drugs rt SEM (N = 6). The data for C57BL/6 mice are shown on the left panels and the data for the NZB mice are shown on the right panels.
Effects of cholinergic drugs in NZB mice should be noted that although the administration of physodgmine resulted in small, dose-related increases in the fast activity of C57BL/6 mice, the actual points fell below baseline for saline. Only the NZB mice tested after the 0.64mg/kg dose of physostigmine showed fast activity which exceeded the baseline for vehicle. Autonomic effects
The frequencies of mice exhibiting diarrhea, salivation or visible tremors are shown in Table 1 as a function of Drug, Dose and Strain. After the administration of oxotremorine, the tendency was for the C57BL/6 mice to show diarrhea, salivation and visible tremors at smaller doses than did NZB mice. When Chi-squared tests were performed on the data within the 0.02-0.32 mg/kg dose range, statistical significance was observed only with salivation (x*(3) = 8.55, P < 0.05). The same tendencies were not as clear after injections of physostigmine (see 0.160.64 mg/kg). Little or no diarrhea was observed after injections of physostigmine in either strain. When compared with the effects produced by oxotremorine, there was a less prominent tendency for the C57BL/6 mice to show salivation and visible tremors at a smaller dose of physostigmine than that needed for NZB mice. However, neither of these tendencies was of a magnitude sufficient to produce significance with the Chi-squared test. Death occurred in 17% of the C57BL/6 mice and in 50% of the NZB mice at the 0.64mg/kg dose of physostigmine. For this reason, larger doses were not examined. Oxotremorine-induced
hypothermia
Both NZB and C57BL/6 mice showed a doserelated drop in temperature expressed as total degrees of hypothermia and maximal hypothermia, Table 2. The drop in temperature relative to the effects of the smallest dose was first evident at O.O4mg/kg for C57BL/6 mice in terms of both measures of hypothermia. In contrast, in the NZB mice, significant Table 2. Oxotremorine-induced
Dose tmeika) Saline 0.01 0.02 0.04 0.08 0.16
C57BLi6 2.0 10.1 7.8 19.2 42.0 69.9
f 1.3 + 1.4 i 1.2 ? 1.9 _+ 3.5 f 1.1
Table 1. The percentage of animals exhibiting diarrhea, salivation and visible tremors after the administration of oxotremorine and physostigmine in C57BL/6 and NZB mice Diarrhea
Salivation
Visible tremors
Dose C57
(mg/kg) 0.002 0.01 0.02 0.05 0.10 0.32 0.01 0.04 0.16 0.32 0.64
es7
NZB
NZB
0 0 17 50 100 83
0 0 0 17 67 67
O,Xotremoriae 0 0 0 0 17 0 100 0 100 0 100 100
0 0 17 0 0
0 0 0 0 0
fhysosfigmine 0 0 0 0 17 0 0 0 83 83
c57
NZB
0 0 0 33 100 100
0 0 0 0 0 100
0 0 0 50 100
0 0 0 0 100
Groups of 6 mice were observed for the presence of diarrhea, salivation and visible tremors during the 30min period after administration of drug, while the animals were in the observation chamber. Mortality occurred only after the 0.64 mg/kg dose of physostigmine; C57 = 17%, NZB = 50%. (See Results section for statistical analysis of the data).
hypothermia was first evident at 0.08 mg/kg in terms of total degrees and at 0.16 mg/kg for maximal hypothermia. The discrepancy between the 2 measures was related to the fact that the response of the C57BL/6 mice tended to have a more dramatic inflection at the time of maximal hypothermia than did the response of the NZB mice (not shown), as well as exhibiting an apparent correlation between maximal hypothermia and time of occurrence. Overall, oxotremorine produced a much greater hypothermia in C57BL/6 mice as compared with NZB mice (see Table 2). With both measures the only exception was after the O.O2mg/kg dose, where the hypothermia produced in NZB mice was greater than that produced in C57BL/6 mice. DISCUSSION
The effects of oxotremorine and physostigmine on motor activity, autonomic responses and fall in tem-
hypothermia
Total degrees of hypothermia
449
in C57BL/6
and NZB mice
Maximal hypothermia (time of occurrence)
NZB
C57BLl6
15.8 _+2.0”’ 13.1 k2.1 15.7 + 1.5’1’ 12.1 * 1.1** 17.4& 1.9’1’ 38.4 + 3.8***
0.4 f 0.3 (120) 1.3 f 0.1 (120) 1.4iO.3(15) 4.9 * 0.3 (30) 7.7 _+0.7 (60) 10.8 f 0.2 (90)
NZB 2.1 * 0.3 2.6 _+0.7 2.4 + 0.3 1.9 -+ 0.2 2.6 f 0.3 5.8 + 0.6
(120)*** (120) (90)’ (120)“1 (90)*** (60)“’
Colonic temperatures (“C) were measured at 15 and 0 min before administration and 15, 30, 45, 60, 90, 120, 150 and 180 min after administration of drug. Total degrees of hypothermia was calculated as the sum of (mean pre-administration temperature -post-administration temperature at each of the 8 time points) and is expressed as mean + SEM; N = 15. The maximal hypothermia, mean + SEM (and time of occurrence in parenthesis), is indicated in the two right columns. The mean pre-administration temperatures for C57BL/6 mice ranged from 37.8 k O.I”C to 38.7 f O.l”C and for NZB mice ranged from 37.8 + 0.3”C to 38.4 _+0.2”C. Significantly different from C57BL/6 mice using student’s r-test, ‘P < 0.05; **p < 0.01; l**p c 0.001.
450
K. C. RETzet ai.
perature in 8-month old CS7BL/6 mice yielded the expected patterns reported for adult rodents after the administration of cholinomimetic agents. On the other hand, NZB mice exhibited a number of abnormal responses to these chohnomimetic agents. In C57BL/6 mice, smaller doses of each drug depressed locomotor activity (Fibiger et al., 1971; Mason et al., 1978; Hughes and Trowland, 1976), whereas larger doses tended to potentiate high-frequency activity (Gonzalez, 1984; Ringdahl ef al., 1982), accompanied by observable tremors. While the overall patterns produced by the administration of oxotremorine and physostigmine were similar in C57 mice, some differences in the effect of each drug were evident. For oxotremorine, autonomic effects appeared at smaller doses than observable tremor, a pattern consistent with published ED, values for salivation, 0.19 & 0.07 jtmols/kg and tremors, 0.27 & 0.05 pmots/kg as measured in Swiss-Webster mice (Ringdahl et al., 1982). For physostigmine, fewer autonomic effects were observed and these usually occurred after doses that were equal to or less than those which also produced tremor. Also, potentiation of high frequency motor activity of CS7 mice was of smaller magnitude after the administration of physostigmine (50% control) than after that of oxotremorine (260% of control). The NZB mice failed to show a significant reduction of locomotor (low frequency) activity with any of the doses of drug and showed much greater high frequency activity than did the C57BL/6 mice within the tremorogenic dose range. The absence of the reducing effects of chohnomimetics on locomotor activity in the NZB mice is relevant to studies indicating that old C57BLj6 mice are more resistant to the “cataleptic” effects of the cholinergic agonist, pilocarpine (Finch et al., 1981; see Ingram and Brennan, 1984). Thus, 8-10 month-old NZB mice may he similar to much older CS7BL/6 mice in this regard. Although catalepsy, per se, was not measured in the present studies, many of the smatl doses of the drugs were observed to immobilize CSfBL,/6 mice. Moreover, the absence of a reduction in locomotor activity in the NZB mice indicates that in this strain, catalepsy was not present after the administration of either of the cholinomimetics. The fact that locomotor activity after injections of vehicle was moderately less in NZB than C57BL~6 mice cannot explain this lack of reduction in cholinomimeticinduced locomotor activity in the NZB mice. For example, reliable, dose-dependent reductions in the locomotor activity of 8 month-old NZB mice have been observed after non-catafeptic doses of the dopaminergic antagonist, haloperidol (unpublished and thus, the lower baseline activity of the NZB mice does not seem to represent a “floor” effect. The lack of effects of the cholinomimetics in reducing activity would also not appear to be related to strain differences in the pharmacokinetics of the agents, insofar as the NZB mice showed much greater in-
creasing effects on motor activity than did C57 mice, within the larger dose ranges of both oxotremorine and physostigmine. Strain differences in the peripheral autonomic and centrai thermolytic effects of oxotremorine were consistent with the effects on locomotor activity in that they also suggest reduced sensitivity to muscarinic agents in NZB mice. After the administration of oxotremorine, C57BL/6 mice exhibited significant salivation, with diarrhea and a drop in temperature, at much smaller doses than did NZB mice. On the other hand, the motor effects of the largest doses of oxotremorine and physostigmine were inconsistent, suggesting instead, that the NZB mice were more sensitive to both agents. The existence of this apparent paradox depends upon how the data for the fast motor activity are interpreted. The NZB mice had substantially greater fast activity (expressed as a percentage of baseline) than the C57BL/6 mice, at doses of oxotremorine and physostigmine which produced visible tremors in the C57BL/6 mice (see Fig. 4, Table If. However, it was clear that the NZB mice did not show increases in fast activity at smaller doses than the C57 mice, which would be expected if the NZB mice had greater sensitivity to the drugs. Thus, the fact that NZB mice show more (higher frequency) tremors may be independent of sensitivity to the drugs, per se, and may instead involve strain differences in the frequency ~m~nents of the tremors. This conclusion is supported by the data, which suggest that C57BL/6 mice show visible tremors at doses much smaller than in NZB mice (see Table 1). The fact that fast (high frequency) locomotor activity was markedly increased in the NZB mice at doses for which tremors were not observed, suggests that tremors in the C57BL/6 mice were of a fower (observable) frequency, whereas the tremors in NZB mice may be of a much higher frequency and not always detected by observation. Because it is not clear how the frequency of tremor might be related to dose of drug, no f&m conclusions can be reached regarding the sensitivity of NZB and C57BL/6 mice to the tremerogenic effects of the cholinergic agents. While resistance to the effects of the cholinergic agents in reducing locomotor activity in NZB mice appears to be consistent with the effects found only in older mice of other strains, it is not clear that an overall reduced ~ns~ti~ty to chofinergie agents is characteristic of aging in rodents. In particular, the findings of studies of cholinomimetic effects on the regulation of temperature in aged rats reveal some inconsistency. For example, Pedigo et al. (1984) observed that senescent Fisher 344 rats showed increased sensitivity to the b~~othe~jc and antinociceptive effects of oxotremorine, whereas Martin, Fuchs and Harting (1985) recently reported that maximal hypothermia was unchanged after the administration of oxotremorine in senescent rats of two different strains. Although the decrease in the the~oiy~c response of PlIZB mice to oxotremorine
Effects of cholinergic drugs in NZB mice
observed here is not in agreement with the studies just cited, other investigators have reported that 15-18-month old Sprague-Dawley rats do show decreased sensitivity to the thermolytic effects of the cholinergic agonist, carbachol (Ferguson et al., 1985) when compared to 3-5 month olds. Because the present authors were unaware of any studies of the thermolytic effects of cholinomimetics in aged C57BL/6 mice, it was difficult to evaluate the “aged-like” character of reduced sensitivity to cholinomimetics in NZB mice. It is interesting that mature NZB mice exhibited some abnormalities of the response to cholinergic drugs which were “aged-like” in character. However, to say that these strain differences are related to age-related autoimmune processes in NZB mice is an interpretation requiring caution. Whether or not the abnormalities of 8-month-old NZB mice represent the end points of age-dependent changes in response to drugs was not determined in the present studies; nor was it possible to verify that similar end points would be reached in senescent C57BL/6 mice. Hence, neurological differences between these strains may be related to either deterioration, i.e. age-dependent, or inborn, i.e. strain-dependent, abnormalities. Previous developmental analyses of the effects of anxiolytics and sedative-hypnotics have supported the former interpretation with respect to the NZB strain. The studies indicated that NZB mice exhibit similar, but more rapid age-related changes in sensitivity to diazepam (Forster, Retz, Popper and Lal, 1986) and ethanol (Retz, Forster, Ashford and Lal, 1985; Retz, Forster, Popper and Lal, 1985). In both cases, the behavioral responses of very young (l-3 month old) NZB and C57BL/6 mice were similar, but they differed markedly at more advanced ages. However, the extent to which abnormalities of responses to cholinergic drugs follow this pattern will require further study. Regardless of whether the abnormalities of NZB mice involve age-dependent alterations, they imply abnormalities in neural function involving the cholinergic system. This finding is consistent with one recent anatomical study of the brains of NZB mice, which suggested that NZB mice have lower densities of cholinergic cell packing in their basal forebrain (Zilles, 1985). The extent to which this difference represents a loss of cells with age is not known, although such a loss would distinguish NZB mice from C57BL/6 mice of advanced age which show no apparent loss of cholinergic cells in this region (Hornberger, Buell, Flood, McNeil1 and Coleman, 1985). In any case, the present findings do suggest that anatomical and biochemical studies of the brains of NZB mice may yield interesting abnormalities of cholinergic function, which may have direct relevance to immune-mediated aging processes. Acknowledgements-This research was supported by a Texas College of Osteopathic Medicine grant 34144 (K.C.R.) and a U.S.P.H.S. grant AGO3623 (H.L.). Pre-
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liminary portions of the results were presented in abstract form: Fed. Proc. 43: 624 (1984). The authors wish to thank Subir Paul for his technical assistance. The secretarial assistance of Diana Bemstine is gratefully appreciated. REFERENCES
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Bergsma D. and Goldstein A. L. (Eds) (1978) Neurochemical and Immunologic Components in Schizophrenia. Liss, New York. Blumenthal H. T., Young D., Wozniak D. and Finger S. (1984) Effects of age, brain damage and stress on antibody binding to the brain. J. Geronf. 39: 552-560. Clark W. G. and Clark Y. L. (1980) Changes in body temperature after administration of acetylcholine, histamine, morphine, prostaglandins, and related agents. Neurosci. Biobehav. Rev. 4: 175-240.
Coyle J. T., Price D. L. and DeLong M. R. (1983) Alzheimer’s disease: A disorder of cortical cholinergic innervation. Science 219: 1184-l 190. Elizan T. S., Casals J. and Yahr M. D. (1983) Antineurofilament antibodies in postencephalitic and idiopathic Parkinson’s disease. J. Neurol. Sci. 59: 341-347. Feden G., Baldinger A., Miller-Soule D. and Blumenthal H. T. (1979) An in vivo and in vitro study of an agingrelated neuron cytoplasmic binding antibody in male Fischer rats. J. Geronf. 34: 651660. Ferguson A. V., Turner S. L., Cooper K. E. and Veale W. L. (1985) Neurotransmitter effects on body temperature are modified with increasing age. Physiol. Behuv. 34: 977-981.
Fibiger H. C., Lynch G. S. and Cooper H. P. (1971) A biphasic action of central cholinergic stimulation on behavioral arousal in the rat. Psychopharmacologia 20: 366-382.
Fillit H., Luine V. N., Reisberg B. Amador R., McEwen B. and Zabriskie J. B. (1985) Studies of the specificity of Antibrain antibodies in Alzheimer’s disease. In: Senile Dementia of rhe Alzheimer Type (Hutton J. T. and Kenny A. D., Eds), pp. 307-318. Liss, New York. Finch C. E., Marshall J. F. and Randall P. K. (1981) Aging and basal ganglia function. Ann. Rev. Geronr. Geriat. 2: 49-90. Forster M. J., Retz K. C., Popper M. D. and La1 H. (1985) Age-related deterioration of learning ability is accelerated in autoimmune mouse strains. Sot. Neurosci. Absrr. 11: 722. Forster M. J., Retz K. C., Popper M. D. and La1 H. (1986) Age-dependent enhancement of diazepam sensitivity is accelerated in New Zealand Black mice. L@ Sci. 38: 1433-1439. Gonzalez L. P. (1984) Quantitative analysis of physostigmine-induced changes in behavior. Pharmac. Biochem. Behav. 21: 551-554.
Huahes R. N. and Trowland R. (1976) Physostigrnine effects 0%activity and reactions to novelty: L& &i.i9: 793-796. Homberger J. C., Buell S. J., Flood D. G., McNeil1 T. H. and Coleman P. D. (1985) Stability of numbers but not
size of mouse forebrain cholinergic neurons to 53 months. Neurobiol. Aging 6: 269-275.
Ingram C. R., Phegan K. J. and Blumenthal H. T. (1974) Significance of an aging-linked neuron binding gamma globulin fraction of human sera. J. Geront. 29: 20-27. Ingram D. K. and Brennan M. J. (1984) Animal behavioral models in biogerontology. Neurobiol. Aging 5: 63-66. La1 H.. Forster M. J. and Nandv K. (1985) Immunoloaic factors related to cognitive/behavioral dysfunctions -in aging. In: Senile Dementia of the .Ilzheimer Type: Early Diagnosis, Neuropathology and Animal Models, Advances in Applied Neurological Sciences, Vol. 2. (Gispen W. H.
and Traber J., Eds), pp. 343-354. Springer, Berlin. La1 H. and Forster M. J. (1986) Cognitive disorders related to immune dysfunction: Novel animal models for drug development. Drug Devel. Res. 7: 195-208. Lippa A. S., Loullis C. C., Rotrosen J. Cordasco D. M., Critchett D. J. and Joseph J. A. (1985) Conformational changes in muscarinic receptors may produce diminished cholinergic neurotransmission and memory deficits in aged rats. Neurobiol. Aging 6: 317-323. Martin J. R., Fuchs A. and Harting J. (1985) Drug induced hypothermia in adult and senescent rats. Neurobiol. Aging 6: 121-124.
Mason S. T., Roberts D. C. S. and Fibiger H. C. (1978) Noradrenergic influences in catalepsy. Psychopharmacology 60: 53-51.
Nandy K. (1972) Brain-reactive antibodies in mouse serum as a function of age. J. Geront. 21: 1733177. Nandy K. (1978) Brain-reactive antibodies in aging and senile dementia. In: Alzheimer’s disease-Senile dementia and related disorders (Katzman R., Terry R. and Bick K., Eds), pp. 503-512. Raven Press, New York. Nandy K. (1981) Brain-reactive antibodies in aging nonhuman primates. Mech. Ageing Deu. 16: 141-146. Nandy K. (1985) Immunopathology of aging and dementia. In: Senile Dementia of the Alzheimer Type (Hutton J. T. and Kenny A. D., Eds), pp. 293-305. Liss, New York.
Nandy K., La1 H., Bennett M. and Bennett D. (1983) Correlation between a learning disorder and elevated brain-reactive antibodies in aged C57BL/6 and young NZB mice. Life Sci. 33: 1499-1503. Pedigo N. W. Jr, Minor L. D. and Krumrei T. N. (1984) Cholinergic drug effects and brain muscarinic receptor binding in aged rats. Neurobiol. Aging. 5: 221-233. Retz K. C., Forster M. J., Ashford N. J. and La1 H. (1985) Accelerated enhancement of sensitivity to ethanol in aging New Zealand Black (NZB/BINJ) mice. Sot. Neurosci. Abstr. 11: 727.
Retz K. C., Forster M. J., Popper M. D. and La1 H. (1985) Neurochemical and pharmacological studies suggest potential mechanisms for impaired task performance in the aging NZB mouse. J. Neurochem. 44: ~124. Retz K. C. and La1 H. (1984) Cholinergic neuropsychopharmacology and neuropathology of dementias. In: Central Cholinergic Mechanisms and Adaptive Dysfunctions (Singh M. M., Warburton D. M. and La1 I-I.,
Eds). vv. 335-352. Plenum Press. New York. Ringdahl B., Ehler F. J. and JendenD. J. (1982) Muscarinic activity and receptor binding of the enantiomers of aceclidine and its methiodide. Molec. Pharmac. 21: I.
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and Traber J., Eds), pp. 355-365. Springer, Berlin.
0028-3908/87 $3.00 + 0.00
Copyright 0 1987Pergamon Journals Ltd
DIFFERENCES IN BEHAVIORAL RESPONSES TO OXOTREMORINE AND PHYSOSTIGMINE IN NEW ZEALAND BLACK (NZB/BINJ) AND C57BL/6 MICE K. C. RETZ*, M. J. FORSTER, N. FRANTZ and H. LAL Department of Pha~acology, Texas College of Osteopathic Medicine, Camp Bowie at Montgomery, Fort Worth, TX 76107-2690, U.S.A. (Accepted 2 May 1986)
Summary-The NZB/BINJ (NZB) mice are an autoimmune-prone strain, known to develop brain-reactive antibodies in serum at much earlier chronological ages than normal mice. Measurement of locomotor activity in 8-10 month old C57BL/6 ((37) mice following the administration of either oxotremo~ne or physostigmine, revealed a biphasic response consisting of inhibition at small doses, but increased motor activity at large doses. In contrast, age-matched NZB mice exhibited little inhibition at the smaller doses, but had much greater increases in activity after the larger doses. Similarly, when compared to C57 mice, NZB mice were less sensitive to oxotremorine-induced salivation, diarrhea and visible tremors. Moreover, oxotremorine-induced hypothermia occurred at smaller doses in C57 mice than in NZB mice and was of a greater magnitude. Thus, at an age when NZB mice possess high levels of brain-reactive antibodies, and exhibit impai~ent in tests of learning/memory, these mice also show diminished responses in several tests of ~hoiinomimetic-indu~ behavior and physiologicai alterations. Key words: NZB/BlNJ mice, C57BL/6 mice, oxotremorine, physostigmine, locomotor activity, brainreactive antibodies.
The fo~ation of brain-reactive antibodies (BRA) in is a concomitant of normal aging in humans (Elizan, Casals and Yahr, 1983; Ingram, Phegan and Blumenthal, 1974; Nandy, 1978), subhuman primates (Nandy, 1981) rats (Feden, Baldinger, Miller-Soule and Blumenthal, 1979) and mice (Biumenthat, Young, Wozniak and Finger, 1984; Nandy, 1972; Nandy, Lal, Bennett and Bennett, 1983; Threat& Nandy and Fritz, 1972). Brain-reactive antibodies are also found with abnormally high frequency in patients with Alzheimer’s disease (Bahmanyar, MoreauDubois, Brown, Cathala and Gajdusek, 1983; Sotelo, Gibbs and Gajdusek, 1980; Nandy, 1978) and are associated with several disease states involving neuropsychopathology (Baldinger and Blumenthal, 1982; Bergsma and Goldstein, 1978). These findings have led to the consideration of immune mechanisms in relation to the etiology of aging-associated dysfunctions of the CNS (Lal, Forster and Nandy, 1985; La1 and Forster, 1986; Nandy, 1985; Film, Luine, Reisberg, Amador, McEwen and Zabriskie, 1985). Recently, studies in this laboratory have suggested a correlation between the appearance of brainreactive antibodies and deficits in learning by normally aging mice (La1 and Forster, 1986; La1 et al., 1985) and in NZB mice, a genetically autoimmuneprone strain known to develop serum brain-reactive antibodies at much earlier chronological ages as compared to the normal strains studied (Forster,
*To whom correspondence
should be addressed.
Retz, Popper and La], 1985; La1 and Forster, 1986; Lal et al., 1985; Nandy et al., 1983). These findings add support to the link between brain-reactive antibodies and behavioral dysfunction, and have raised the possibility that mature NZB mice exhibit neurological alterations similar to those which normally occur in old age, or in pathological states associated with aging. The possibility that NZB mice show aged-like neurological alterations, led to the present investigation of the question of whether mature NZB mice exhibit alterations in central cholinergic mechanisms. There are many lines of evidence which suggest that acetylcholine-mediated processes are compromised or altered in aging rodents (Bartus, Dean, Beer and Lippa, 1982; Coyle, Price and DeLong, 1983; Lippa, Loullis, Rotrosen, Cordasco, Critchett and Joseph, 1985; Bartus, Dean, Pontecorvo and Flicker, 1985; Retz and Lal, 1984). Moreover, some age-related alterations in cholinergic function can be detected pharmacologically, as abnormal behavioral responses to cholinomimetic drugs. For example, in mature rodents, muscarinic agonists produce biphasic effects upon locomotor activity (Fibiger, Lynch and Cooper, 1971; Mason, Roberts and Fibiger, 1978; Hughes and Trowland, 1976), reduce body temperature (Clark and Clark, 1980) and produce analgesia via central mechanisms (Ringdahl, Ehler and Jenden, 1982). In aged rodents, several studies have reported alterations in sensitivity to these components of agonist response, including the motor (Finch, Marshalf and Randall, 1981; Ingram and Brennan, 1984), thermo-
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K. C.
446
lytic (Pedigo, Minor and Krumrei, 1984; Ferguson, Turner, Cooper and Veale, 1985) and analgesic (Pedigo et af., 1984) effects. In the light of this knowledge, a pharmacological approach was used to test the hypothesis that acetylcholine-mediated processes are altered in mature NZB mice. In the present studies 8-10 month-old NZB and C57BL/6 mice have been compared by observing the motor, autonomic and thermolytic effects of oxotremorine, a direct-acting muscarinic agonist and the effects of physostigmine, an indirectacting cholinomimetic which inhibits acetylcholinesterase. It was expected that mature NZB mice would show abnormal responses in motor behavior, autonomic events and temperature regulation when challenged with the cholinomimetic drugs.
METHODS
kTZ
et al.
Oxotremorine and physostigmine were administered to separate groups of each strain just prior to testing, with each group given drug receiving doses of the drug on separate test days in tandem with a matching session in which vehicle was administered. Tests were spaced 2-4 days apart, with half of the animals in each group receiving vehicle on a given test day. Each animal was observed in the same chamber during sessions of vehicle and drug administration for a given drug and dose. During each test, the mice were observed and the presence of the following was noted: diarrhea, salivation and visible tremors. Measurement of hypothermia induced by oxotremorine
In a separate series of experiments, the magnitude of hypothermia induced by oxotremorine was measured. Colonic temperatures were measured at 15 and 0 min before administration and 15, 30, 45, 60, 90, 120, 150 and 180 min after oxotremorine (Model TH-6 thermometer; Bailey Instruments (Sensortech); Saddlebrook, New Jersey).
Animals
Male C57BL/6J mice and NZB/BlNJ mice were obtained from Jackson Laboratories (Bar Harbor, Maine) at 2-3 months age and housed in the College Vivarium until 8-10 months old. The colony room was maintained at 23 & 1°C under a normal 12 hr light-dark cycle, beginning at 0800 hr. The mice had unlimited access to food and water. Drug administration
All drugs were dissolved in normal saline solution and administered by intraperitoneal injection (lOml/kg) in the doses indicated in the text. Oxotremorine (Aldrich Chemical Co., Milwaukee, Wisconsin) was administered in the “free” form. Physostigmine sulfate (Sigma Chemical Co., St. Louis, Missouri) was administered as the sulfate with doses calculated and expressed as the “free” drug present. Measurement behavior
Statistical analysis
Parametric data from experiments involving two or more factors were subjected to Analysis of Variance (ANOVA) with Drug group (where appropriate) and Strain as between-groups factors and Doses and Sampling interval as within-groups factors. Analysis of frequency data, i.e. numbers of animals showing behavioral responses, was made using a Chi-squared test at the 0.05 significance level.
RESULTS
Motor activity
A preliminary inspection of the data revealed two overall trends: (a) after vehicle, the C57BL/6 mice had higher levels of activity than the NZB mice (Fig. 1); and (b) the expected biphasic effects of oxo-
of motor activity and assessment of
Observation and testing of motor activity took place in six clear, polypropylene chambers (28 cm L x 19 cm W x 13 cm H) with stainless steel wire lids, each located above one sensor of an Electronic Activity Monitor (EAM) (Stoelting Co., Chicago, Illinois). In this apparatus, activity is measured in terms of the numbers of movements exceeding a fixed amplitude and above 1- or 10 Hz frequency cutoffs, detected through disruption of a radio frequency capacitance field by the animal. Activity counts from the slow (2 1 Hz) and fast (2 10 Hz) channels were monitored for 30 min using a sensitivity of 15% full scale for both channels. The 15% sensitivity setting resulted in zero counts of background activity when no animals were on the activity monitors.
Test interval
(min)
Fig. I. Overall strain differences in motor activity of 8-10 month old C57BL/6 and NZB mice. The overall slow (2 1 Hz) motor activity and fast motor activity (a 10 Hz), expressed as total counts/5 min, occurring in the time intervals O-5, 5-15 and 15-30 min after the administration of control and drug is plotted as mean + SEM (N = 6). The slow motor activity data are shown on the left panel and the
fast motor actlvlty ones
are shown on the right panel.
Effects of cholinergic drugs in NZB
tremorine and physostigmine were observed in C57BL/6 mice, using the low and high frequency channels of the Electronic Activity Monitor apparatus. To provide a meaningful comparison in subsequent analysis, activity after administration of drugs was calculated as a percentage of the baseline level and the strains were compared within separate dose ranges, matching the 2 phases of the response to the drug in C57BL/6 mice. The effects of the smaller doses of oxotremorine (i.e. 0,002~.0Smg/kg) and physostigmine (i.e. 0.01-0.32 mg/kg) were analyzed as the action of decreasing the motor activity. Similarly, the effects of the larger doses of oxotremorine (i.e. 0.054.32 mg/kg) and physostigmine (i.e. 0.16-0.64 mg/kg) were analyzed as action of increasing the motor activity. The changes in “slow” 2 1 Hz and “fast” > 10 Hz motor activity observed after both drugs yielded similar patterns of effect. However, slow motor activity showed a greater sensitivity during the decreasing phase of motor activity and fast activity showed a greater sensitivity during the increasing phase of motor activity. These findings are consistent with literature, suggesting that relatively small doses of cholinomimetics reduce locomotor activity or produce catalepsy (Fibiger et al., 1971; Mason et al., 1978; Hughes and Trowland, 1976), whereas larger doses induce high-frequency muscle tremor (Gonzalez, 1984; Ringdahl et al., 1982). In particular, Gonzalez (1984) has noted that the high and low frequency components of the motor response to physostigminc can be distinguished with a modined Stoelting Electronic Activity Monitor. Therefore, the present authors have presented analyses of the “slow” data when examining decreasing effects on motor activity and analyses of the “fast” data when examining increasing effects on motor activity. Sessions with vehicle. Mean slow (left) and fast (right) counts of motor activity after giving the vehicle in all activity experiments are shown in Figure 1 as a function of the three sampling intervals. Drug group (physosti~ine or oxotremo~ne), Strain, Sampling interval and the dose to which a given session with vehicle was matched (designated Dose) were the factors considered in a separate ANOVA conducted on each activity measure. Analyses supported the overall observation that NZB mice had significantfy lower levels of slow (F(1,20) = 12.31, P < 0,002s) and fast @‘(1,20)=6.31, P x0.01) activity when compared to C57BL/6 mice, with both strains showing decreases in activity within the session (t;s(Z, 40) > 28.0, Ps < 0.01). Activity during sessions with vehicle did not vary consistently as a function of Drug group or Dose, and higher-order effects gave little indication that these variables were influenced by Strain. With one exception, both analyses failed to indicate significant higher-order interactions involving Strain, Dose and Drug. A marginally significant Strain by Dose (F(3,60) = 3.09, P < 0.05) interaction was obtained in the analysis of slow
mice
Oxotremofine (mg/kgf
447
Physosfigmine (mg/kg)
Fig. 2. Strain differences in the slow motor activity of 8-10 month old C57BL/6 and NZB mice after the administration of oxotremorine and physostigmine-total observations. The slow (> 1Hz) motor activity after the administration of oxotremorine (left panel) or physostigmine (right panel) is expressed as a percentage mean of control activity with saline, occurring from O-30 min after the administration of the drugs (N = 6).
activity, although the pattern of strain differences which contributed to this interaction was not similar to the dose-related strain differences in the effects of oxotremorine or physosti~ine on slow activity, described in subsequent sections. Activity-decreasing phase. Slow (3 1 Hz) motor activity for the dose range reducing activity is shown in Figure 2 for oxotremorine (left) and physostigmine (right) as a function of strain and dose. Separate analyses were conducted for the groups given physostigmine and oxotremorine, with Strain, Dose and Sampling interval as factors. As seen in Figure 2, there was a dose-dependent reduction in the activity of C57BL/6 mice following the administration of both oxotremorine (0.02-0.05 mg/kg) and physostigmine (0.01-0.32 mg/kg). The activity of the NZB mice was almost never decreased (although it was sometimes increased) within those ranges. This difference between the strains was reflected in effects of Strain (Kr(l, 10) > 13.0, significant Ps < 0.005) in both analyses, and in a Strain by Dose interaction (F(3,30) = 4.06, P < 0.025) in the analysis for oxotremorine. Figure 3 shows the time course of slow (> 1 Hz) motor activity after the administration of oxotremorine (top panels) and physostigmine (lower panels) within the testing sessions as a function of Dose and Strain (C57BL/6, left; NZB, right). For C57BL/6 mice, the reduction of activity after injections of 0.01, 0.02 and 0.05 mg/kg oxotremorine was evident by the first 5 min after testing, and it remained depressed throughout the 30min session. A similar effect was seen after injections of 0.04, 0.16 and 0.32mg/kg physostigmine, with the exception that reduction of activity was not evident until after .5min with this drug. In contrast to the effects in C57BL/6 mice, the activity of NZB mice tended to increase as a function of time after injections of oxotremorine or physostigmine although this trend was erratic and did not have a consistent relationship with the dose of either drug. The trend for increasing
K. c. RET2
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o-5
m-30
5-f5
o-5
5-15
15-X)
Test interval Fig. 3. Strain differences in the slow motor activity of 840 month old C57BL/6 and NZB mice after the administration of oxotremorine and physostigmine-interval observations. The slow (> 1Hz) motor activity after the administration of oxotremorine (upper panels) or physostigmine (lower panels) is expressed as a mean of percentage control activity with saline, occurring from D-5, 5-15 and 15-30 min after the administration of drugs (N = 6). The data for CS?BL/6J mice are shown on the left panels and the data for the NZB mice are shown on the right panels.
et al.
Activity-increasingphase. The dose ranges for oxotremorine (0.05, 0.1 and 0.32 mg/kg, left) and physostigmine (0.16, 0.32, and 0.64 mg/kg, right) which increased activity are shown in Figure 4 for NZB and C57BL/6 mice. Analyses of these data, involved the same factors considered previously for the decreasing phase of activity. As Figure 4 shows, fast activity after administration of both drugs showed an overall increase as a function of Dose (Fs(2,20) > 33.0, Ps < O.OOl),with much greater dose-related increases occurring in the NZB strain (Fs(1, lo)> 35.0, Ps < 0.001). The marked difference between the strains with increasing doses resulted in significant Strain x Dose interactions (Fs(2,20) > 10.0, Ps < 0.001) in both analyses. Analysis of the data for oxotremorine also indicated an effect of sampling interval (F(2,20) = 40.1, P < 0.001), as well as significant higher-order interactions of Strain and Dose with Sampling interval (all Fs > 4.0, Ps < 0.01). As can be seen in Figure 5, the major source of these interactions was the fact that increases in fast activity after 0.32mgjkg were of much greater magnitude in the NZB mice as compared to the C57BL/6 mice. In contrast to oxotremorine, overall increases in fast activity within sessions were not evident after the administration of physostigmine (F < l.O), although the high level of activity of 0.64 mg/kg-treated NZB mice during the first 5 min of testing contributed to a marginal Dose by Interval interaction (F(4,40) = 2.62, P < 0.05). It I
activity in the NZB mice was most evident after the administration of oxotremorine and contributed to significant 2 and 3-way interactions involving Strain, Dose and Sampling interval (all Fs > 4.0, all Ps < 0.025) in that analysis. The trend was less clear for physostigmine, although it contributed to a significant Strain by Sampling interval interaction [F(2,20) = 6.29, P < 0.011.
4200
800
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6oo
g
400
NZB
t
1000
3
I C57BL/6 t
-
0 0.05
-
A 0.32
A
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0.64
t
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0.1
0.32 0.16
Oxotrernorine (Wlql)
0.32
0.64
P
OXOtnmori~img/kpl
t
‘~~ o-5
5-15
15-X)
o-5
5-15
15-3
Physosiigmine lmglkg)
Fig. 4. Strain differences in the fast motor activity of 8-10 month old C57BLj6 and NZB mice after the administration of oxotremorine and physostigmine-total observations. The fast (3 10 Hz) motor activity after the administration of oxotremorine (left panel) or physostigmine (right panel) is expressed as a mean percentage of control activity with saline, occurring from O-30 min after the administration of drugs FSEM (N = 6).
Fig. 5. Strain differences in the fast motor activity of 8-10 month old C57BL/6 and NZB mice after the administration of oxotremo~ne and physosti~in~interva~ observations. The fast (> 10 Hz) motor activity after the administration of oxotremorine (upper panels) or physostigmine (lower panels) is expressed as a mean percentage of control activity with saline, occurring from O-5, 5-l 5 and 15-30 min after the administration of drugs rt SEM (N = 6). The data for C57BL/6 mice are shown on the left panels and the data for the NZB mice are shown on the right panels.
Effects of cholinergic drugs in NZB mice should be noted that although the administration of physodgmine resulted in small, dose-related increases in the fast activity of C57BL/6 mice, the actual points fell below baseline for saline. Only the NZB mice tested after the 0.64mg/kg dose of physostigmine showed fast activity which exceeded the baseline for vehicle. Autonomic effects
The frequencies of mice exhibiting diarrhea, salivation or visible tremors are shown in Table 1 as a function of Drug, Dose and Strain. After the administration of oxotremorine, the tendency was for the C57BL/6 mice to show diarrhea, salivation and visible tremors at smaller doses than did NZB mice. When Chi-squared tests were performed on the data within the 0.02-0.32 mg/kg dose range, statistical significance was observed only with salivation (x*(3) = 8.55, P < 0.05). The same tendencies were not as clear after injections of physostigmine (see 0.160.64 mg/kg). Little or no diarrhea was observed after injections of physostigmine in either strain. When compared with the effects produced by oxotremorine, there was a less prominent tendency for the C57BL/6 mice to show salivation and visible tremors at a smaller dose of physostigmine than that needed for NZB mice. However, neither of these tendencies was of a magnitude sufficient to produce significance with the Chi-squared test. Death occurred in 17% of the C57BL/6 mice and in 50% of the NZB mice at the 0.64mg/kg dose of physostigmine. For this reason, larger doses were not examined. Oxotremorine-induced
hypothermia
Both NZB and C57BL/6 mice showed a doserelated drop in temperature expressed as total degrees of hypothermia and maximal hypothermia, Table 2. The drop in temperature relative to the effects of the smallest dose was first evident at O.O4mg/kg for C57BL/6 mice in terms of both measures of hypothermia. In contrast, in the NZB mice, significant Table 2. Oxotremorine-induced
Dose tmeika) Saline 0.01 0.02 0.04 0.08 0.16
C57BLi6 2.0 10.1 7.8 19.2 42.0 69.9
f 1.3 + 1.4 i 1.2 ? 1.9 _+ 3.5 f 1.1
Table 1. The percentage of animals exhibiting diarrhea, salivation and visible tremors after the administration of oxotremorine and physostigmine in C57BL/6 and NZB mice Diarrhea
Salivation
Visible tremors
Dose C57
(mg/kg) 0.002 0.01 0.02 0.05 0.10 0.32 0.01 0.04 0.16 0.32 0.64
es7
NZB
NZB
0 0 17 50 100 83
0 0 0 17 67 67
O,Xotremoriae 0 0 0 0 17 0 100 0 100 0 100 100
0 0 17 0 0
0 0 0 0 0
fhysosfigmine 0 0 0 0 17 0 0 0 83 83
c57
NZB
0 0 0 33 100 100
0 0 0 0 0 100
0 0 0 50 100
0 0 0 0 100
Groups of 6 mice were observed for the presence of diarrhea, salivation and visible tremors during the 30min period after administration of drug, while the animals were in the observation chamber. Mortality occurred only after the 0.64 mg/kg dose of physostigmine; C57 = 17%, NZB = 50%. (See Results section for statistical analysis of the data).
hypothermia was first evident at 0.08 mg/kg in terms of total degrees and at 0.16 mg/kg for maximal hypothermia. The discrepancy between the 2 measures was related to the fact that the response of the C57BL/6 mice tended to have a more dramatic inflection at the time of maximal hypothermia than did the response of the NZB mice (not shown), as well as exhibiting an apparent correlation between maximal hypothermia and time of occurrence. Overall, oxotremorine produced a much greater hypothermia in C57BL/6 mice as compared with NZB mice (see Table 2). With both measures the only exception was after the O.O2mg/kg dose, where the hypothermia produced in NZB mice was greater than that produced in C57BL/6 mice. DISCUSSION
The effects of oxotremorine and physostigmine on motor activity, autonomic responses and fall in tem-
hypothermia
Total degrees of hypothermia
449
in C57BL/6
and NZB mice
Maximal hypothermia (time of occurrence)
NZB
C57BLl6
15.8 _+2.0”’ 13.1 k2.1 15.7 + 1.5’1’ 12.1 * 1.1** 17.4& 1.9’1’ 38.4 + 3.8***
0.4 f 0.3 (120) 1.3 f 0.1 (120) 1.4iO.3(15) 4.9 * 0.3 (30) 7.7 _+0.7 (60) 10.8 f 0.2 (90)
NZB 2.1 * 0.3 2.6 _+0.7 2.4 + 0.3 1.9 -+ 0.2 2.6 f 0.3 5.8 + 0.6
(120)*** (120) (90)’ (120)“1 (90)*** (60)“’
Colonic temperatures (“C) were measured at 15 and 0 min before administration and 15, 30, 45, 60, 90, 120, 150 and 180 min after administration of drug. Total degrees of hypothermia was calculated as the sum of (mean pre-administration temperature -post-administration temperature at each of the 8 time points) and is expressed as mean + SEM; N = 15. The maximal hypothermia, mean + SEM (and time of occurrence in parenthesis), is indicated in the two right columns. The mean pre-administration temperatures for C57BL/6 mice ranged from 37.8 k O.I”C to 38.7 f O.l”C and for NZB mice ranged from 37.8 + 0.3”C to 38.4 _+0.2”C. Significantly different from C57BL/6 mice using student’s r-test, ‘P < 0.05; **p < 0.01; l**p c 0.001.
450
K. C. RETzet ai.
perature in 8-month old CS7BL/6 mice yielded the expected patterns reported for adult rodents after the administration of cholinomimetic agents. On the other hand, NZB mice exhibited a number of abnormal responses to these chohnomimetic agents. In C57BL/6 mice, smaller doses of each drug depressed locomotor activity (Fibiger et al., 1971; Mason et al., 1978; Hughes and Trowland, 1976), whereas larger doses tended to potentiate high-frequency activity (Gonzalez, 1984; Ringdahl ef al., 1982), accompanied by observable tremors. While the overall patterns produced by the administration of oxotremorine and physostigmine were similar in C57 mice, some differences in the effect of each drug were evident. For oxotremorine, autonomic effects appeared at smaller doses than observable tremor, a pattern consistent with published ED, values for salivation, 0.19 & 0.07 jtmols/kg and tremors, 0.27 & 0.05 pmots/kg as measured in Swiss-Webster mice (Ringdahl et al., 1982). For physostigmine, fewer autonomic effects were observed and these usually occurred after doses that were equal to or less than those which also produced tremor. Also, potentiation of high frequency motor activity of CS7 mice was of smaller magnitude after the administration of physostigmine (50% control) than after that of oxotremorine (260% of control). The NZB mice failed to show a significant reduction of locomotor (low frequency) activity with any of the doses of drug and showed much greater high frequency activity than did the C57BL/6 mice within the tremorogenic dose range. The absence of the reducing effects of chohnomimetics on locomotor activity in the NZB mice is relevant to studies indicating that old C57BLj6 mice are more resistant to the “cataleptic” effects of the cholinergic agonist, pilocarpine (Finch et al., 1981; see Ingram and Brennan, 1984). Thus, 8-10 month-old NZB mice may he similar to much older CS7BL/6 mice in this regard. Although catalepsy, per se, was not measured in the present studies, many of the smatl doses of the drugs were observed to immobilize CSfBL,/6 mice. Moreover, the absence of a reduction in locomotor activity in the NZB mice indicates that in this strain, catalepsy was not present after the administration of either of the cholinomimetics. The fact that locomotor activity after injections of vehicle was moderately less in NZB than C57BL~6 mice cannot explain this lack of reduction in cholinomimeticinduced locomotor activity in the NZB mice. For example, reliable, dose-dependent reductions in the locomotor activity of 8 month-old NZB mice have been observed after non-catafeptic doses of the dopaminergic antagonist, haloperidol (unpublished and thus, the lower baseline activity of the NZB mice does not seem to represent a “floor” effect. The lack of effects of the cholinomimetics in reducing activity would also not appear to be related to strain differences in the pharmacokinetics of the agents, insofar as the NZB mice showed much greater in-
creasing effects on motor activity than did C57 mice, within the larger dose ranges of both oxotremorine and physostigmine. Strain differences in the peripheral autonomic and centrai thermolytic effects of oxotremorine were consistent with the effects on locomotor activity in that they also suggest reduced sensitivity to muscarinic agents in NZB mice. After the administration of oxotremorine, C57BL/6 mice exhibited significant salivation, with diarrhea and a drop in temperature, at much smaller doses than did NZB mice. On the other hand, the motor effects of the largest doses of oxotremorine and physostigmine were inconsistent, suggesting instead, that the NZB mice were more sensitive to both agents. The existence of this apparent paradox depends upon how the data for the fast motor activity are interpreted. The NZB mice had substantially greater fast activity (expressed as a percentage of baseline) than the C57BL/6 mice, at doses of oxotremorine and physostigmine which produced visible tremors in the C57BL/6 mice (see Fig. 4, Table If. However, it was clear that the NZB mice did not show increases in fast activity at smaller doses than the C57 mice, which would be expected if the NZB mice had greater sensitivity to the drugs. Thus, the fact that NZB mice show more (higher frequency) tremors may be independent of sensitivity to the drugs, per se, and may instead involve strain differences in the frequency ~m~nents of the tremors. This conclusion is supported by the data, which suggest that C57BL/6 mice show visible tremors at doses much smaller than in NZB mice (see Table 1). The fact that fast (high frequency) locomotor activity was markedly increased in the NZB mice at doses for which tremors were not observed, suggests that tremors in the C57BL/6 mice were of a fower (observable) frequency, whereas the tremors in NZB mice may be of a much higher frequency and not always detected by observation. Because it is not clear how the frequency of tremor might be related to dose of drug, no f&m conclusions can be reached regarding the sensitivity of NZB and C57BL/6 mice to the tremerogenic effects of the cholinergic agents. While resistance to the effects of the cholinergic agents in reducing locomotor activity in NZB mice appears to be consistent with the effects found only in older mice of other strains, it is not clear that an overall reduced ~ns~ti~ty to chofinergie agents is characteristic of aging in rodents. In particular, the findings of studies of cholinomimetic effects on the regulation of temperature in aged rats reveal some inconsistency. For example, Pedigo et al. (1984) observed that senescent Fisher 344 rats showed increased sensitivity to the b~~othe~jc and antinociceptive effects of oxotremorine, whereas Martin, Fuchs and Harting (1985) recently reported that maximal hypothermia was unchanged after the administration of oxotremorine in senescent rats of two different strains. Although the decrease in the the~oiy~c response of PlIZB mice to oxotremorine
Effects of cholinergic drugs in NZB mice
observed here is not in agreement with the studies just cited, other investigators have reported that 15-18-month old Sprague-Dawley rats do show decreased sensitivity to the thermolytic effects of the cholinergic agonist, carbachol (Ferguson et al., 1985) when compared to 3-5 month olds. Because the present authors were unaware of any studies of the thermolytic effects of cholinomimetics in aged C57BL/6 mice, it was difficult to evaluate the “aged-like” character of reduced sensitivity to cholinomimetics in NZB mice. It is interesting that mature NZB mice exhibited some abnormalities of the response to cholinergic drugs which were “aged-like” in character. However, to say that these strain differences are related to age-related autoimmune processes in NZB mice is an interpretation requiring caution. Whether or not the abnormalities of 8-month-old NZB mice represent the end points of age-dependent changes in response to drugs was not determined in the present studies; nor was it possible to verify that similar end points would be reached in senescent C57BL/6 mice. Hence, neurological differences between these strains may be related to either deterioration, i.e. age-dependent, or inborn, i.e. strain-dependent, abnormalities. Previous developmental analyses of the effects of anxiolytics and sedative-hypnotics have supported the former interpretation with respect to the NZB strain. The studies indicated that NZB mice exhibit similar, but more rapid age-related changes in sensitivity to diazepam (Forster, Retz, Popper and Lal, 1986) and ethanol (Retz, Forster, Ashford and Lal, 1985; Retz, Forster, Popper and Lal, 1985). In both cases, the behavioral responses of very young (l-3 month old) NZB and C57BL/6 mice were similar, but they differed markedly at more advanced ages. However, the extent to which abnormalities of responses to cholinergic drugs follow this pattern will require further study. Regardless of whether the abnormalities of NZB mice involve age-dependent alterations, they imply abnormalities in neural function involving the cholinergic system. This finding is consistent with one recent anatomical study of the brains of NZB mice, which suggested that NZB mice have lower densities of cholinergic cell packing in their basal forebrain (Zilles, 1985). The extent to which this difference represents a loss of cells with age is not known, although such a loss would distinguish NZB mice from C57BL/6 mice of advanced age which show no apparent loss of cholinergic cells in this region (Hornberger, Buell, Flood, McNeil1 and Coleman, 1985). In any case, the present findings do suggest that anatomical and biochemical studies of the brains of NZB mice may yield interesting abnormalities of cholinergic function, which may have direct relevance to immune-mediated aging processes. Acknowledgements-This research was supported by a Texas College of Osteopathic Medicine grant 34144 (K.C.R.) and a U.S.P.H.S. grant AGO3623 (H.L.). Pre-
N.P. 26,5--O
451
liminary portions of the results were presented in abstract form: Fed. Proc. 43: 624 (1984). The authors wish to thank Subir Paul for his technical assistance. The secretarial assistance of Diana Bemstine is gratefully appreciated. REFERENCES
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