Br. J. exp. Path. (1976) 57, 742
MOTOR ACTIVITY CHANGES IN SCRAPIE-AFFECTED MICE A. J. SUCKLING, S. BATEMAN, C. B. WALDRON, H. E. WEBB AND R. H. KIMBERLIN* From the Rayne Institute, St Thomas' Hospital, London SE1 7EH, and the Agricultural Research Council*, Institute for Research on Animal Diseases, Compton, Newbury, Berks. RG16 ONN Received for publication July 22, 1976
Summary.-Measurements of spontaneous motor activity using Animex equipment have been made throughout the incubation period in mice developing scrapie. A progressive fall in activity has been noted from an early stage in the disease process well before clinical signs of scrapie were evident. The initial fall was followed by an upsurge in activity at about the time when clinical signs of disease develop.
WE HAVE SHOWN previously that Animex motility measuring equipment registers a transient fall in spontaneous motor activity in the absence of clinical signs 5 days after infection of mice with an avirulent strain of Semliki forest virus (Suckling, Bateman and Webb, 1975). Since activity changes could be detected during the course of this acute disease process we felt that our technique might be of use in detecting activity changes at an early stage in slower, or chronic disease processes. The slow, progressive infection of mice with scrapie was chosen as our model system. Mice inoculated with scrapie-infectious material pass through a phase lasting several months before the clinical signs of scrapie appear but during much of this period it is well known that histological and biochemical changes are progressing within the central nervous system (Fraser, 1976; Kimberlin, 1976). Behavioural tests have demonstrated abnormalities at an early stage in scrapie, the results of emergence tests showing that emergence times were significantly slower than controls at 35-40% of the incubation period (Heitzman and Corp, 1968), although open-field testing has shown no effect attributable to scrapie (Savage and Field, 1965). In this paper we report investigations into the changes in spontaneous motor activity
which occur during the scrapie disease process. MATERIALS AND METHODS Production of scrapie-infected mice. Scrapie was produced in male Swiss/A2G mice by the intracerebral inoculation of 0-03 ml of a 10-4 homogenate in saline of a brain pool derived from mice clinically affected with the Chandler strain of scrapie. This inoculum contained 103 4ICLD50. Control animals were similarly inoculated but with normal mouse brain homogenate. To determine the length of the incubation period as accurately as possible another 2 groups of 20-25 mice, 1 infected and 1 control, were kept separately and observed visually for the development of the clinical signs of scrapie. These mice also provided samples for studies of the development of histological lesions. Experimental procedure.-Groups of 3 mice were placed in each of 6 plastic cages, 3 cages containing infected, and 3 control mice. The apparatus used to monitor activity was an Animex motility meter type S.E. (LKB Instruments Ltd) connected via a multiplexer to 6 external detectors as described previously (Suckling et al., 1975). Counts were recorded for 1 min out of 6 throughout the experimental period and the accumulated results punched on paper tape in computer-compatible form. The data was processed using a Varian 620/L-100 computer, providing numerical daily totals of activity, " night " activity and a graphical representation of the 24-h pattern of activity. The basic data were subsequently smoothed using a digital filter programme and plotted against time. The environment within the
MOTOR ACTIVITY CHANGES IN SCRAPIE-AFFECTED MICE
room containing the experimental animals was controlled only insofar as a 12-h-oin, 12-h-off lighting regime was imposed. Trial experiments showed that restricting the access of personnel to the animal room caused little alteration in the total counts recorded. Tap water was given ad lib. and the amount consumed by the mice was determined at intervals of 2 or 3 days.
743
in all groups by 148 days. The mean ± s.e. mean for the incubation period, estimated by the time of first definite appearance of characteristic incoordination and ataxia was 141 ± 1 days (n
23).
The control level of tap water conwas between 4-1 and 4-5 g per mouse per day and a significant reduction RESULTS in consumption in the infected compared No illness was apparent in either of the with control groups (P < 0.01) was first visually observed groups (and also in the observed at 114 days post-inoculation Animex test groups) until 133 days after (- 810% of the incubation period) although inoculation and the disease was evident a less significant decrease was observed
sumption
15-
10-
,'A/rX x
inoculation
0
SI 1
0
60
120
180
days after inoculation FIG. 1 Total activity per day throughout the experiment, Cages 3 and 4 only, 3 mice per cage. Mice in Cage 3 were inoculated with normal brain homogenate, those in Cage 4 with scrapie brain homogenate. Data are computer-smoothed and the regression lines computer-plotted.
744
A. J. SUCKLING ET AL.
1200
1800
0000 0600 1100 time of day Fig 2.-Graphical rcpresentation of the 24 h activity recorded from Cages 1 (scrapie-infected) and 2 (control) 165 days after inoculation. Verticle axis represents spontaneous motor activity on an arbitrary but linear scale which was computer-plotted. The 'night' period accounted for 95% of the total activity in Cage I and 63% of the total activity in Cage 2.
earlier. This result is similar to the decrease in water consumption found in A 2G mice after intracerebral infection with the ME7 strain of scrapie (Outram, 1972). In those cages containing scrapieinfected mice a gradual fall in total counts per day was observed (Fig. 1) and after performing least squares linear regression analysis on the data from post-inoculation Days 20 to 130, correlation coefficients of -0-72, -0-51 and 0-86 were produced. No such fall in activity was observed in data derived from the cages containing mice inoculated with normal brain and the correlation coefficients of the data from these cages were + 0-01, 0-15 and -0-33. This fall in counts was pronounced well before the onset at 60-65%O of the incubation period of histological lesions in the CNS. In addition, the scrapie-infected mice
TABLE. The Increase in the Proportion of Total Counts Registered in the " night " Period in Mice Inoculated with Scrapie Compared with Normal Brain Homogenates
Cage no. Control 2
Weeks after inoculation when " Fraction of counts control " period values were registered during " control " night exceeded by 2 s.d. or more period
3 5
0.72±0.05* 0 * 74±0 * 05 0 * 78±0 04
Scrapie-infected 1
0 * 69±0 02
none none none
13, 16, 17, 20 onwardst 4 0 74±0 05 16, 19, 21 onwards 6 0 70±0 03 19 onwards * Mean ± s.d. (n = 15) of values derived from total weekly recordings of each cage from 5 weeks before to 10 weeks after inoculation (the " control period). t Recordings continued to Week 25.
MOTOR ACTIVITY CHANGES IN SCRAPIE-AFFECTED MICE
showed a gradual increase in the proportion of counts derived from the " night " period (Table). At about 141 days after inoculation a substantial increase in the total counts was observed, due largely to an increase in the proportion of " night " counts. During the clinical phase of the disease it was noted that apparently moribund mice (20 days post-clinical) unexpectedly showed daily totals of activity very close to normal but their activities were almost entirely confined to the period of darkness (Fig. 2). DISCUSSION
Of the changes recorded in scrapieinfected mice before clinical signs develop, the earliest are the diminished drinking response to various solutions (Outram, 1972) and increased emergence times in some behavioural studies (Heitzman and Corp, 1968). In another spongiform encephalopathy, experimental CreutzfeldJakob disease in the chimpanzee, early EEG abnormalities have been recorded (Cathala et al., 1974). Many of these different types of functional change can occur as early as 30 to 35 % of the incubation period and their nature indicates that the primary lesion in scrapie is central, rather than peripheral, in origin. Early motor activity changes are probably also a manifestation of a central change in function. It has been suggested that a primary functional change in scrapie may be a gradual deterioration in the operation of a particular class of synapse perhaps with a change in neutrotransmitter levels (Outram, 1972). A gradual fall in spontaneous motor activity could result from a dysfunction of this type. Our findings provide an interesting comparison with the results obtained from our previous work with Semliki forest virus (Suckling et al., 1975). Here the fall in total activity was accompanied by a reduction in the proportion of counts occurring during the " night " period.
745
In scrapie, at about 141 days after inoculation, a substantial increase in the proportion of " night " counts was re-
corded. This observation quantitates the previous observed excitable phase in behaviour which occurs around the time of development of clinical signs of disease (Dickinson, Meikle and Fraser, 1968). The marked rise in proportion of " night " counts may also provide a method for objectively assessing the scrapie incubation period, particularly with agent-strain, mouse-strain combinations which are not easy to " score " subjectively. At our present state of knowledge, however, attributing causes for these different activity changes represents a considerable problem. A major difficulty common to all investigations concerning the experimental pathology of slow virus diseases is that only biological assays are commonly available for monitoring infectivity. Although changes in activity may not be caused specifically by scrapie infection, the method may go some way towards producing an early warning of a successful infection. At present there are limitations to our technique, firstly because only the lighting conditions are controlled and secondly because an intracerebral route of inoculation was used. Trial experiments have shown that recovery from an intracerebral inoculation, in terms of motor activity, may take up to 7 days and there is also the possibility of long-term modifications of behavioural patterns. However, since our results show significant changes under the present conditions, attention to these details in further experiments will provide a more sensitive technique which may be applied to the problems of the pathogenesis of scrapie and other slow, or clinically inapparent, virus diseases.
We should like to thank St Thomas' Hospital Endowment Fund and the Multiple Sclerosis Society for the financial support which made this work possible.
746
A. J. SUCKLING ET AL. REFERENCES
CATHALA, F., COURT, L., ROHMER, F., GAJDUSEK, D. C., GIBBS, C. J., JR & CASTAIGNE, P. (1974) Experimental Transmission of Creutzfeldt-Jakob Disease to a Chimpanzee, with an Electroencephalographic Study using Indwelling Electrodes. In Proc. Xth Int. Congr. Neurology. Eds. A. Subirana, J. M. Espadaler and E. H. Burrows. Amsterdam: Exerpta Medica. p. 3 1. DICKINSON, A. G., MEIKLE, V. M. H. & FRASER, XI. (1968) Identification of a Gene which Controls the Incubation Period of Some Strains of Scrapie Agent in Mice. J. comp. Path., 78, 293. FRASER, H. (1976) The Pathology of Natural and Experimental Scrapie. In Slow Virus Diseases of Animals and Man. Ed. R. H. Kimberlijl, Frontiers of Biology, 44, 267.
HEITZMAN, R. J. & CORP, C. R. (1968) Behaviour in Emergence and Openfield Tests of Normal and Scrapie-affected Mice. Res. vet. Sci., 9, 600. KIMBERLIN, R. H. (1976) Biochemical and Behavioural Changes in Scrapie. In Slow Virus Diseases of Animals and Man. Ed. R. H. Kimberlin, Frontiers of Biology, 44, 307. OUTRAM, G. W. (1972) Changes in Drinking and Feeding Habits of Mice with Experimental Scrapie. J. comp. Path., 82, 415. SAVAGE, R. D. & FIELD, E. J. (1965) Brain Damage and Emotional Behaviour: the Effects of Scrapie on the Emotional Responses of Mice. J. Anim. Behav., 13, 443. SUCKLING, A. J., BATEMAN, S. & WEBB, H. E. (1975) A New Method of Monitoring Sub-clinical Virus Infections of the Central Nervous System of Mice. Br. J. exp. Path., 56, 287.
MOTOR ACTIVITY CHANGES IN SCRAPIE-AFFECTED MICE A. J. SUCKLING, S. BATEMAN, C. B. WALDRON, H. E. WEBB AND R. H. KIMBERLIN* From the Rayne Institute, St Thomas' Hospital, London SE1 7EH, and the Agricultural Research Council*, Institute for Research on Animal Diseases, Compton, Newbury, Berks. RG16 ONN Received for publication July 22, 1976
Summary.-Measurements of spontaneous motor activity using Animex equipment have been made throughout the incubation period in mice developing scrapie. A progressive fall in activity has been noted from an early stage in the disease process well before clinical signs of scrapie were evident. The initial fall was followed by an upsurge in activity at about the time when clinical signs of disease develop.
WE HAVE SHOWN previously that Animex motility measuring equipment registers a transient fall in spontaneous motor activity in the absence of clinical signs 5 days after infection of mice with an avirulent strain of Semliki forest virus (Suckling, Bateman and Webb, 1975). Since activity changes could be detected during the course of this acute disease process we felt that our technique might be of use in detecting activity changes at an early stage in slower, or chronic disease processes. The slow, progressive infection of mice with scrapie was chosen as our model system. Mice inoculated with scrapie-infectious material pass through a phase lasting several months before the clinical signs of scrapie appear but during much of this period it is well known that histological and biochemical changes are progressing within the central nervous system (Fraser, 1976; Kimberlin, 1976). Behavioural tests have demonstrated abnormalities at an early stage in scrapie, the results of emergence tests showing that emergence times were significantly slower than controls at 35-40% of the incubation period (Heitzman and Corp, 1968), although open-field testing has shown no effect attributable to scrapie (Savage and Field, 1965). In this paper we report investigations into the changes in spontaneous motor activity
which occur during the scrapie disease process. MATERIALS AND METHODS Production of scrapie-infected mice. Scrapie was produced in male Swiss/A2G mice by the intracerebral inoculation of 0-03 ml of a 10-4 homogenate in saline of a brain pool derived from mice clinically affected with the Chandler strain of scrapie. This inoculum contained 103 4ICLD50. Control animals were similarly inoculated but with normal mouse brain homogenate. To determine the length of the incubation period as accurately as possible another 2 groups of 20-25 mice, 1 infected and 1 control, were kept separately and observed visually for the development of the clinical signs of scrapie. These mice also provided samples for studies of the development of histological lesions. Experimental procedure.-Groups of 3 mice were placed in each of 6 plastic cages, 3 cages containing infected, and 3 control mice. The apparatus used to monitor activity was an Animex motility meter type S.E. (LKB Instruments Ltd) connected via a multiplexer to 6 external detectors as described previously (Suckling et al., 1975). Counts were recorded for 1 min out of 6 throughout the experimental period and the accumulated results punched on paper tape in computer-compatible form. The data was processed using a Varian 620/L-100 computer, providing numerical daily totals of activity, " night " activity and a graphical representation of the 24-h pattern of activity. The basic data were subsequently smoothed using a digital filter programme and plotted against time. The environment within the
MOTOR ACTIVITY CHANGES IN SCRAPIE-AFFECTED MICE
room containing the experimental animals was controlled only insofar as a 12-h-oin, 12-h-off lighting regime was imposed. Trial experiments showed that restricting the access of personnel to the animal room caused little alteration in the total counts recorded. Tap water was given ad lib. and the amount consumed by the mice was determined at intervals of 2 or 3 days.
743
in all groups by 148 days. The mean ± s.e. mean for the incubation period, estimated by the time of first definite appearance of characteristic incoordination and ataxia was 141 ± 1 days (n
23).
The control level of tap water conwas between 4-1 and 4-5 g per mouse per day and a significant reduction RESULTS in consumption in the infected compared No illness was apparent in either of the with control groups (P < 0.01) was first visually observed groups (and also in the observed at 114 days post-inoculation Animex test groups) until 133 days after (- 810% of the incubation period) although inoculation and the disease was evident a less significant decrease was observed
sumption
15-
10-
,'A/rX x
inoculation
0
SI 1
0
60
120
180
days after inoculation FIG. 1 Total activity per day throughout the experiment, Cages 3 and 4 only, 3 mice per cage. Mice in Cage 3 were inoculated with normal brain homogenate, those in Cage 4 with scrapie brain homogenate. Data are computer-smoothed and the regression lines computer-plotted.
744
A. J. SUCKLING ET AL.
1200
1800
0000 0600 1100 time of day Fig 2.-Graphical rcpresentation of the 24 h activity recorded from Cages 1 (scrapie-infected) and 2 (control) 165 days after inoculation. Verticle axis represents spontaneous motor activity on an arbitrary but linear scale which was computer-plotted. The 'night' period accounted for 95% of the total activity in Cage I and 63% of the total activity in Cage 2.
earlier. This result is similar to the decrease in water consumption found in A 2G mice after intracerebral infection with the ME7 strain of scrapie (Outram, 1972). In those cages containing scrapieinfected mice a gradual fall in total counts per day was observed (Fig. 1) and after performing least squares linear regression analysis on the data from post-inoculation Days 20 to 130, correlation coefficients of -0-72, -0-51 and 0-86 were produced. No such fall in activity was observed in data derived from the cages containing mice inoculated with normal brain and the correlation coefficients of the data from these cages were + 0-01, 0-15 and -0-33. This fall in counts was pronounced well before the onset at 60-65%O of the incubation period of histological lesions in the CNS. In addition, the scrapie-infected mice
TABLE. The Increase in the Proportion of Total Counts Registered in the " night " Period in Mice Inoculated with Scrapie Compared with Normal Brain Homogenates
Cage no. Control 2
Weeks after inoculation when " Fraction of counts control " period values were registered during " control " night exceeded by 2 s.d. or more period
3 5
0.72±0.05* 0 * 74±0 * 05 0 * 78±0 04
Scrapie-infected 1
0 * 69±0 02
none none none
13, 16, 17, 20 onwardst 4 0 74±0 05 16, 19, 21 onwards 6 0 70±0 03 19 onwards * Mean ± s.d. (n = 15) of values derived from total weekly recordings of each cage from 5 weeks before to 10 weeks after inoculation (the " control period). t Recordings continued to Week 25.
MOTOR ACTIVITY CHANGES IN SCRAPIE-AFFECTED MICE
showed a gradual increase in the proportion of counts derived from the " night " period (Table). At about 141 days after inoculation a substantial increase in the total counts was observed, due largely to an increase in the proportion of " night " counts. During the clinical phase of the disease it was noted that apparently moribund mice (20 days post-clinical) unexpectedly showed daily totals of activity very close to normal but their activities were almost entirely confined to the period of darkness (Fig. 2). DISCUSSION
Of the changes recorded in scrapieinfected mice before clinical signs develop, the earliest are the diminished drinking response to various solutions (Outram, 1972) and increased emergence times in some behavioural studies (Heitzman and Corp, 1968). In another spongiform encephalopathy, experimental CreutzfeldJakob disease in the chimpanzee, early EEG abnormalities have been recorded (Cathala et al., 1974). Many of these different types of functional change can occur as early as 30 to 35 % of the incubation period and their nature indicates that the primary lesion in scrapie is central, rather than peripheral, in origin. Early motor activity changes are probably also a manifestation of a central change in function. It has been suggested that a primary functional change in scrapie may be a gradual deterioration in the operation of a particular class of synapse perhaps with a change in neutrotransmitter levels (Outram, 1972). A gradual fall in spontaneous motor activity could result from a dysfunction of this type. Our findings provide an interesting comparison with the results obtained from our previous work with Semliki forest virus (Suckling et al., 1975). Here the fall in total activity was accompanied by a reduction in the proportion of counts occurring during the " night " period.
745
In scrapie, at about 141 days after inoculation, a substantial increase in the proportion of " night " counts was re-
corded. This observation quantitates the previous observed excitable phase in behaviour which occurs around the time of development of clinical signs of disease (Dickinson, Meikle and Fraser, 1968). The marked rise in proportion of " night " counts may also provide a method for objectively assessing the scrapie incubation period, particularly with agent-strain, mouse-strain combinations which are not easy to " score " subjectively. At our present state of knowledge, however, attributing causes for these different activity changes represents a considerable problem. A major difficulty common to all investigations concerning the experimental pathology of slow virus diseases is that only biological assays are commonly available for monitoring infectivity. Although changes in activity may not be caused specifically by scrapie infection, the method may go some way towards producing an early warning of a successful infection. At present there are limitations to our technique, firstly because only the lighting conditions are controlled and secondly because an intracerebral route of inoculation was used. Trial experiments have shown that recovery from an intracerebral inoculation, in terms of motor activity, may take up to 7 days and there is also the possibility of long-term modifications of behavioural patterns. However, since our results show significant changes under the present conditions, attention to these details in further experiments will provide a more sensitive technique which may be applied to the problems of the pathogenesis of scrapie and other slow, or clinically inapparent, virus diseases.
We should like to thank St Thomas' Hospital Endowment Fund and the Multiple Sclerosis Society for the financial support which made this work possible.
746
A. J. SUCKLING ET AL. REFERENCES
CATHALA, F., COURT, L., ROHMER, F., GAJDUSEK, D. C., GIBBS, C. J., JR & CASTAIGNE, P. (1974) Experimental Transmission of Creutzfeldt-Jakob Disease to a Chimpanzee, with an Electroencephalographic Study using Indwelling Electrodes. In Proc. Xth Int. Congr. Neurology. Eds. A. Subirana, J. M. Espadaler and E. H. Burrows. Amsterdam: Exerpta Medica. p. 3 1. DICKINSON, A. G., MEIKLE, V. M. H. & FRASER, XI. (1968) Identification of a Gene which Controls the Incubation Period of Some Strains of Scrapie Agent in Mice. J. comp. Path., 78, 293. FRASER, H. (1976) The Pathology of Natural and Experimental Scrapie. In Slow Virus Diseases of Animals and Man. Ed. R. H. Kimberlijl, Frontiers of Biology, 44, 267.
HEITZMAN, R. J. & CORP, C. R. (1968) Behaviour in Emergence and Openfield Tests of Normal and Scrapie-affected Mice. Res. vet. Sci., 9, 600. KIMBERLIN, R. H. (1976) Biochemical and Behavioural Changes in Scrapie. In Slow Virus Diseases of Animals and Man. Ed. R. H. Kimberlin, Frontiers of Biology, 44, 307. OUTRAM, G. W. (1972) Changes in Drinking and Feeding Habits of Mice with Experimental Scrapie. J. comp. Path., 82, 415. SAVAGE, R. D. & FIELD, E. J. (1965) Brain Damage and Emotional Behaviour: the Effects of Scrapie on the Emotional Responses of Mice. J. Anim. Behav., 13, 443. SUCKLING, A. J., BATEMAN, S. & WEBB, H. E. (1975) A New Method of Monitoring Sub-clinical Virus Infections of the Central Nervous System of Mice. Br. J. exp. Path., 56, 287.
