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A Rodent Model of Spontaneous Stereotypy
Physiology & Behavior, Vol. 66, No. 2, pp. 355–363, 1999 © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/99/$–see front matter
PII S0031-9384(98)00303-5
A Rodent Model of Spontaneous Stereotypy: Initial Characterization of Developmental, Environmental, and Neurobiological Factors SUSAN B. POWELL,*1 HOWARD A. NEWMAN,* JANE F. PENDERGAST† AND MARK H. LEWIS* *Departments of Psychiatry and Psychology, P.O. Box 100256, University of Florida, Gainesville, FL 32610, and †Department of Statistics, University of Florida, Gainesville, FL 32610 Received 24 October 1997; Accepted 26 October 1998 POWELL, S. B., H. A. NEWMAN, J. F. PENDERGAST AND M. H. LEWIS. A mouse model of spontaneous stereotypy: Initial characterization of developmental, environmental, and neurobiological factors. PHYSIOL BEHAV 66(2) 355– 363, 1999.—Stereotypies are patterns of motor behavior that are repetitive, excessive, topographically invariant, and that lack any obvious function or purpose. In humans, stereotyped behaviors are associated with psychiatric, neurological, and developmental disorders. In animals, stereotypy has been frequently associated with adverse environmental circumstances and often related to alterations in striatal dopamine. To assess the development of stereotyped behaviors and to test the hypothesis that these behaviors are associated with environmental restriction, deer mice were housed in either standard laboratory cages or larger, enriched cages, and the development of stereotypy was followed from weaning over a 17-week period. Standardcaged deer mice engaged in stereotyped behaviors at a higher rate and developed these behaviors more quickly when compared to animals in enriched caging. Additionally, enriched caging was associated with higher rates of patterned running, whereas jumping and backward somersaulting were typically observed in standard cages. In addition, there was a significant effect of litter, but no effect of sex or cage, on the time to develop stereotypy. No differences were found in the density of either striatal D1 or D2 dopamine receptors or the concentration of striatal dopamine or its metabolites as a function of rearing condition or as a function of whether the animals developed stereotypy. These results characterize the development of stereotypies in this species, demonstrate the importance of environmental conditions in the genesis of stereotypy, and suggest that alterations in striatal dopamine content or dopamine receptor density do not account for the expression of stereotyped behaviors in this model. © 1999 Elsevier Science Inc. Repetitive behavior
Dopamine
Deer mice
Enrichment
social or maternal deprivation exhibit stereotyped behaviors (e.g., body rocking, tail biting) (4,20), as do other species that experience confinement and movement restraint (21,26,38). The expression of such behaviors (e.g., route tracing, chain chewing, cribbing) in zoo and farm animals are considered a major animal welfare concern by veterinarians and animal caretakers, as they are thought to represent the animal’s response to an inadequate environment (13,24). The empirical evidence for the role of environmental restriction in the stereotypy of zoo and farm animals is relatively sparse and weak (18,45). In the laboratory, several species exhibit spontaneous stereotypies (absent a specific environmental or pharmacological
STEREOTYPIES are sequences of motor behavior that are repetitive, topographically invariant, often rhythmical, and apparently purposeless (4,14,27). In humans, stereotypy (e.g., body rocking, head rolling) has been considered an important feature of psychopathology as well as being associated with a variety of neurological and developmental disorders such as mental retardation and autism (2,6,9,22,30). Despite the high occurrence of stereotypy in these clinical populations, little is known about the pathophysiology and pharmacological treatment of these behavior disorders. Stereotypic behaviors have also been associated with adverse environmental circumstances in a large number of animal species (34,35). Nonhuman primates that experience early
1To
Striatum
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355
356 challenge) whether raised in captivity or caught in the wild [for review, see (27)]. For example, trapped bank voles (Clethrionomys glareolus) develop stereotyped jumping, backwards somersaulting, and patterned running when housed in standard rodent cages (40). When housed under enriched conditions (e.g., larger cage size, addition of nest materials, hiding places, twigs on which to climb), substantially fewer bank voles developed stereotypy compared to bank voles housed in standard cages (50). Ödberg (41) has also observed a decrease in the number of bank voles developing stereotypy through the addition of enrichment objects to the cage. These are among the few studies that have empirically demonstrated the relationship between environmental restriction and the development of stereotypy. Stereotyped body rocking appears relatively early (ca. 29 days of age) in the behavioral repertoire of chimpanzees raised in isolation with a stationary surrogate mother, but is not observed in chimpanzees raised in isolation with a moving surrogate mother (37). Studies of rodents indicate that stereotypies in response to standard laboratory caging appear early during ontogeny with wire gnawing in ICR mice (56) and digging in gerbils (54) developing by 30 days of age. As suggested by these investigators, understanding the developmental time course of stereotypy in deer mice will be important for the characterization of the model and subsequent studies on sensitive periods of susceptibility and the neurobiological basis of the behavior. Information about the neurobiological basis of stereotyped behavior has generally relied on models of drug-induced stereotypy (27). It is now well established that stereotyped patterns of behavior can be induced in a number of mammalian species following administration of dopamine agonists and drugs that alter nigrostriatal dopamine function (10,17,30,44). For example, dopamine or dopamine agonists injected directly into the striatum induce stereotyped behaviors in rats [e.g., (16)]. Induction of stereotyped behavior by application of GABA agonists to the substantia nigra pars reticulata supports the importance of the nigrostriatal circuitry and its output pathways (48). Dopamine agonist-induced stereotypy can be blocked by dopamine receptor antagonists such as haloperidol (46), by inhibiting the synthesis of dopamine using a-methyl-p-tyrosine (51) or by destroying dopamine-containing neurons with the neurotoxicant 6-hydroxydopamine (6OHDA) (12). The issue of whether the neurobiological mechanisms underlying drug-induced stereotypies versus spontaneous stereotypies are the same is very much an open question (34,36,53). Few studies of environmentally produced stereotypies have addressed the neurobiology of such repetitive behaviors (14). Exceptions include the finding that decreased tyrosine hydroxylase immunoreactivity in the striatum and substantia nigra was observed in rhesus monkeys that developed stereotypy following social isolation early in development (33). These monkeys also showed an increased behavioral sensitivity to an acute dose of apomorphine, suggestive of dopamine receptor supersensitivity (28). The development of repetitive oral behaviors in early weaned piglets has been associated with decreases in HVA and DOPAC, the major metabolites of dopamine, in the putamen, and nucleus accumbens (19,49), and increases in D2 dopamine receptors in the caudate nucleus (49). In individuals who have mental retardation, stereotyped behavior has been associated with decreases in spontaneous blink rate (8) and plasma HVA concentrations (29), suggesting hypodopaminergic function. Studies examining captivity-induced stereotypies of bank voles have suggested an important role for dopamine systems
POWELL ET AL. in the mediation of these repetitive behaviors (23). Stereotyped jumping in bank voles was decreased by a-methyl-ptyrosine, an inhibitor of tyrosine hydroxylase, and increased by L-dopa, the precursor to dopamine (42). These drug effects were not solely due to a generalized effect on motor activity as both drug treatments failed to affect other activities. The dopamine-b-hydroxylase (DBH) inhibitor fusaric acid had no significant effects on stereotyped behavior in these animals, suggesting the importance of dopamine but not norepinephrine (42). The literature on spontaneous stereotypy has been primarily descriptive due, at least in part, to the species being studied. A mouse model of spontaneous stereotypy would lend itself to examination of basic neural mechanisms involved in the expression of stereotyped behavior observed in clinical populations. Deer mice, when housed under standard laboratory conditions, develop high rates of spontaneous stereotyped motor behavior (3). The current study was designed to characterize the specific forms of stereotyped behavior displayed by deer mice, describe the developmental trajectory for the stereotyped behaviors, and determine the upper and lower age limits for the initial expression of these behaviors. To test the environmental restriction hypothesis, a series of observations to assess the effects of environmental enrichment on stereotypy and track the development of stereotypies in animals housed in standard and enriched cages was conducted. It was hypothesized that stereotyped behavior was associated with decreased concentrations of dopamine and its metabolites and an increase in dopamine receptors in the corpus striatum. METHODS
Animals Deer mice (Peromyscus maniculatus bairdii) were housed in a standard colony room kept at 248C and maintained on a 16/8-h light/dark cycle, with lights off at 0930 h. At the time of weaning (23 days of age), deer mice were randomly assigned to either standard (n 5 16) or enriched (n 5 15) caging. Standard caging involved either two or three same sex mice in a standard laboratory mouse cage (29 3 18 3 13 cm) with rodent chow and water available ad lib. Four cages contained three animals, and two cages contained only two animals. The discrepancy in number of animals per cage was due to an unequal number of males and females and the death of one animal. Enriched caging involved housing three same-sex mice in a larger cage (51 3 41 3 22 cm) equipped with a running wheel, habit trails, small enclosures for nesting or hiding, nesting material, and sunflower seeds inside the cage. Rodent chow and water were located on the cage top and available ad lib. The objects within the cage were changed and rearranged weekly. A commercially available blonde hair dye was applied to the animals’ fur every 4 weeks to identify each mouse in a given cage. A second cohort of deer mice (n 5 17) was housed in the same manner as described above in both standard (n 5 9) and enriched (n 5 8) cages. These animals were included in the analysis of dopamine receptors and monoamine concentrations in striatum. Observational Procedures Behavioral observations were conducted twice daily at approximately 1030 and 1430 h every other day (approximately three times per week) for 17 weeks. Deer mice were observed during their dark cycle only as previous studies with this colony of mice have shown marked diurnal variation, with the mice engaging in very little activity during the light cycle (3).
DEVELOPMENT OF STEREOTYPY IN DEER MICE Each cage was observed for 5 min at each of the two time periods. Each 5-min observation period was divided into 5-s intervals. During each 5-s scoring interval the occurrence of specific topographies of stereotyped behavior was recorded for individual animals. From preliminary observations of the animals, three distinct topographies of stereotyped behaviors were observed and operationally defined: jumping, backward somersaulting, and route tracing or patterned running. Similar behavior patterns have been observed in captive bank voles (50). To be considered a stereotypy, the behavior had to occur more than once within the 5-s interval. Interrater agreement across topographies of stereotyped behavior as computed using Cohen’s kappa averaged 0.83 (SD 5 0.15). The second cohort of animals was observed in a similar manner, but only for 2 weeks before being killed. These animals were not included in the analysis of behavioral data, as they were only used for determination of monoamines and dopamine receptors. Homogenate Radioligand Binding At the end of the 17-week period, animals were killed by cervical dislocation followed by decapitation and brains were rapidly removed, snap frozen in isopentane, and stored at 2808C until time of assay. Estimates of the density of D1 and D2 dopamine receptor sites in the corpus striatum (caudate nucleus and putamen) were determined in animals in both standard and enriched caging. At the time of assay, individual striata were homogenized in a volume of ice cold 50 mM HEPES buffer (pH 7.4; 48C), using Teflon-glass homogenizers (Eberbach, Ann Arbor, MI), to equal a concentration of approximately 1.0 mg wet weight/mL. At this point a 300-mL aliquot of homogenate was removed for HPLC analysis. The remaining tissue was centrifuged at 27,000 3 g for 10 min, the supernatant discarded, and the pellet resuspended in 5 mL ice cold buffer and centrifuged again. The final pellet was suspended at a concentration of approximately 1.0 mg wet weight/mL. Assay tubes (1 mL final volume) were incubated at 378C for 20 min. Binding of 1.0 nM 3H-SCH23390 was used to assess the density of D1 receptors with unlabeled SCH23390 at a concentration of 10 mM to define nonspecific binding. Ketanserin tartrate (500 nM) was used to displace binding of SCH23390 to 5-HT2 receptors. Binding of 1.0 nM 3H-spiperone was used to determine the density of D2 receptors with unlabeled domperidone (10 mM) to define nonspecific binding. Ketanserin tartrate (500 nM) was used to displace binding of spiperone to 5-HT2 receptors. Binding was terminated by filtering with 15 mL ice-cold buffer on a Skatron cell harvester (Skatron INC, Sterling, VA) using glass fiber filter mats (Skatron #7034, Sterling, VA). Filters were then allowed to dry and 3.0 mL of Scintiverse E (Fischer Scientific Co., Fair Lawn, NJ) was added. After shaking for 30 min, radioactivity was determined on a LKB Rack Beta liquid scintillation counter. Tissue protein levels were estimated using a BCA spectrophotometric assay and microplate reader with absorbance set at 562 nm. Estimates of the density of dopamine receptors were computed at the given concentration of radiolabeled drug used for the two receptor subtypes.
357 ence electrode. Chromatographic separations were performed using a deltabond stainless steel column (150 mm 3 4.6 mm i.d.) packed with 3 mm C18 bonded microparticulate silica (Keystone Scientific, INC, Bellafonte, PA). The mobile phase consisted of 95 mM Na2HPO4 containing 27 mM citric acid, 0.038% sodium octyl sulfate (SOS), and 13% methanol, with a final pH of 3.4 and a flow rate of 0.80 mL/min. Standard curves for the quantification of all compounds [dopamine (DA), serotonin (5-HT), homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA)] were prepared by analyzing a series of standard solutions containing a fixed amount of the internal standard 3,4-dihydroxybenzylamine (DHBA) and varying amounts of each compound. The slopes of the standard curves obtained by linear regression were routinely greater than or equal to 0.99. At the time of the radioligand binding assay, 240 mL of perchloric acid was added to the 300 mL of striatal homogenate. Samples were vortexed, then centrifuged for 10 min at 13,200 rpm, supernatant collected, and both supernatant and pellet were stored at 2808C until assay (for HPLC and protein assays, respectively). Supernatant (450 mL) was removed and 50 mL of DHBA (20 ng/mL) was added. The solution was filtered (0.2 mm nylon acrodisc), and 100 mL of the solution was injected onto the column. The concentration of monoamines and their metabolites in these unknown brain samples were determined from the internalized standard curve and the ratios of the particular compounds to DHBA. The tissue pellet was sonicated in 500 mL mobile phase buffer using an ultrasonic cell disrupter (Heatsystems, Farmingdale, NY; setting 1) for protein determination. Tissue protein levels were estimated using a BCA spectrophotometric assay and microplate reader with absorbance set at 562 nm. RESULTS
Development of Stereotypy and Effects of Enrichment Table 1 lists the topographies and operational definitions of stereotyped behaviors that were observed in both standard cages and enriched cages. The percentage of intervals in which any of the three topographies of stereotypy occurred was calculated for each animal by week. The mean weekly percentages of intervals of total stereotypy over the 17-week observation period across the two housing conditions are depicted in Fig. 1. The effect of housing condition, sex, week, cage (fixed effects), and litter (random effect) on stereotypy was assessed using a general linear mixed model analysis of variance with a first-order autoregressive [AR(1)] correlation structure within animal over time (GLMM: PROC MIXED in SAS). In addition to the main effects, the following interactions were also included in the model: housing condition by sex, sex by week, TABLE 1 STEREOTYPED BEHAVIORS IN DEER MICE IN STANDARD AND ENRICHED CAGING
Repetitive jumping
HPLC Analysis of Monoamines and Metabolites The concentrations of monoamines and metabolites from striata were quantified using an HPLC procedure with electrochemical detection. Differences in specific compounds between specified experimental conditions were determined quantitatively using amperometric detection of the column effluent with a potential of 10.75 V versus an Ag/AgCl refer-
Backward somersault
Patterned running
Rearing in one of the four corners of the cage and repeatedly jumping on his/her hind paws A somersault in a backward direction with or without assistance from the cagetop or side Repetitive route tracing or circling of the cage in a clear pattern
358
FIG. 1. Effects of housing condition on the percent of intervals of total stereotypy over the 17th week experimental period.
and sex by housing condition. Animals were nested within the litter effect, as well as within the cage effect. A square root transformation of the raw data was used to better meet the underlying assumptions of the model. The analysis was conducted on the amount of total stereotypy averaged across 1-week periods. Deer mice in standard cages engaged in higher rates of stereotyped behavior than did deer mice in enriched cages, F(1, 448) 5 3.79, p 5 0.05. There was also a significant effect of time with the amount of stereotypy increasing over successive weeks, F(16, 448) 5 3.84, p , 0.001, and a significant interaction between experimental condition and time, F(16, 448) 5 2.09, p 5 0.008. There were no effects of cage, F(7, 448) 5 0.66, p 5 0.70, or sex, F(1, 448) 5 0.94, p 5 0.33, on stereotypy. Animals were considered nested within the random litter effect, as well as within the fixed cage effect. Because the litter effect is a random effect, there is no F-test to assess its importance within the model. The percent of animals engaging in stereotyped behavior (collapsing across topographies) observed in each of the two housing conditions over the 17-week period is presented in Fig. 2. Animals were judged to have stereotypy in a given week if they engaged in repetitive behavior in greater than 5% of the intervals. As can be seen in Fig. 2, animals housed in standard cages developed stereotypies at a faster rate than did animals housed in enriched cages. To test whether environmental condition resulted in a differential rate of development of stereotypy, an extension of logistic regression was used. A generalized estimating equations approach was used to fit a logistic regression model that could account for the correlation within animal across time (31). The logit probability of exhibiting stereotypy was modeled as a function of experimental condition, time, litter, and sex effects. Cage was excluded from this analysis as a second random variable could not be accommodated in this model, and it was shown previously to have no effect on level of stereotypy. Animals housed in standard cages developed stereotypy at a significantly faster rate than did animals raised in enriched cages (z 5 21.84, p 5 0.033, one tailed). As we were interested in a preliminary investigation of the relative importance of genetic and/or early environmental experience on the
POWELL ET AL.
FIG. 2. Effects of housing condition on the percent of animals developing stereotypy over the 17th week experimental period.
development of stereotypy, the effect of litter on the rate of development of stereotypy was included in the model. There was an overall effect of litter on the rate of development of stereotypy, x2(8) 5 20.64, p 5 0.0082, but no effect of sex (z 5 1.24, p 5 0.22). To compare the number of animals that developed stereotypy in each condition, a stricter criterion was applied. An animal was judged to be stereotypic if the repetitive behavior occurred in greater than 5% of the intervals/week for 2 consecutive weeks. Although stereotyped behavior was observed in 62.5% (10 of 16) of animals housed in standard cages versus 46.7% (7 of 15) of animals housed in environmental enrichment cages (Table 2), these proportions were not significantly different, x2(1) 5 0.275, p 5 0.60. Although the number of animals exhibiting stereotypy was not significantly different between the housing conditions, the stereotyped behaviors exhibited by the two groups were of different forms. Table 2 indicates the number of mice in each condition judged to have developed each topography of stereotypy (jumping, backwards somersaulting, and patterned running). Significantly more deer mice housed in standard cages (7/ 16) developed repetitive jumping than did deer mice housed in enriched cages (0/15) (Fisher’s exact probability test, p 5 0.007). Forty percent (6/15) of deer mice housed in enriched cages developed patterned running versus only 6.3% (1/16) of TABLE 2 NUMBER OF ANIMALS DEVELOPING STEREOTYPIES IN STANDARD AND ENRICHED HOUSING CONDITIONS
Overall (all topographies) Jumping Backward somersaulting Patterned running
Standard Cages (n 5 16)
Enriched Cages (n 5 15)
p Values*
10 7 3 1
7 0 2 6
0.60 0.007 1.0 0.037
*Fisher’s exact probability test.
DEVELOPMENT OF STEREOTYPY IN DEER MICE deer mice housed in standard cages (Fisher’s exact probability test, p 5 0.04). The number of mice developing backwards somersaulting did not differ between the two housing conditions (Fisher’s exact probability test, p 5 1.0) (Figs. 3, 4, and 5). Considering that each topography developed in only a subset of animals, further analyses included only those animals, which were judged through the previous criterion to have developed specific topographies. Deer mice in standard cages engaged in higher rates of backward somersaulting (25.7 6 2.54% of intervals) versus deer mice housed in enriched cages (6.8 6 2.14% of intervals). Of those animals judged to engage in patterned running, animals in the enriched condition did so at higher rates (13.8 6 15.1% of intervals) than did the one animal in the standard cage condition (2.9% of intervals). No mice in enriched caging were judged to have developed repetitive jumping. Inferential statistics were not applied to these results due to the small sample sizes. Within the standard cages, there was a different developmental rate for somersaulting and jumping. As seen in Fig. 6, stereotyped jumping occurred in a greater percentage of animals housed in standard cages and developed earlier than backward somersaulting.
359
FIG. 4. Effects of housing on the percent of intervals in which patterned running was observed over the 17th week experimental period.
Analysis of Striatal Dopamine and Dopamine Receptors Multiple linear regressions were conducted to determine if the linear combination of the frequency of stereotypy during the last week of observations, experimental condition, sex, cage, and litter accounted for the relative density of dopamine receptors and the concentration of monoamines and monoamine metabolites. Cage and litter, when both in the model, presented problems with mulitcollinearity; thus, cage was subsequently left out of the model because it was previously shown not to have an effect on stereotypy and the analysis was repeated. The linear combination of these variables did not predict D1 or D2 dopamine receptor densities or monoamine and metabolite concentrations (r2 range: 0.03–0.10). There were no effects of housing condition, percentage of stereotypy, sex, or litter on either D1 or D2 dopamine receptor densities or monoamine and monoamine metabolite concentrations. When litter was removed from the model, there were still no effects of housing condition, stereotypy, or sex on either D1 or
FIG. 3. Effects of housing on the percent of intervals in which repetitive jumping was observed over the 17th week experimental period.
D2 dopamine receptor densities or monoamine and monoamine metabolite concentrations. The effects of stereotypy status (stereotypy/no stereotypy) and housing condition on D1 and D2 dopamine receptors and monoamine and monoamine metabolites were analyzed using 2 3 2 analyses of variance (ANOVA). Stereotypy status was based on the criteria used in the analysis of number of mice developing stereotypy above (.5% of the intervals for 2 consecutive weeks). Table 3 indicates the binding of [3H]-SCH23390 and [3H]-spiperone to D1 and D2 dopamine receptors, respectively, in striata of deer mice. There were no effects of housing condition or stereotypy status on either D1 or D2 receptor densities. There were also no significant housing condition by stereotypy status interactions for D1 or D2 dopamine receptors. The concentrations of monoamines and monoamine metabolites in striata are shown in Table 4. These levels of
FIG. 5. Effects of housing on the percent of intervals in which backwards somersaulting was observed over the 17th week experimental period.
360
POWELL ET AL. TABLE 4 CONCENTRATION (ng/mg PROTEIN) OF DOPAMINE, HVA, DOPAC, AND SEROTONIN IN STRIATUM OF DEER MICE
Group
n
Dopamine (ng/mg pr) Avg (SD)
Stereotypy Standard caging Enriched caging No stereotypy Standard caging Enriched caging
22 14 8 16 6 10
134.6 (31.1) 129.0 (36.6) 144.3 (15.8) 137.2 (27.6) 138.7 (25.7) 136.3 (30.1)
DOPAC* (ng/mg pr) Avg (SD)
HVA† (ng/mg pr) Avg (SD)
Serotonin (ng/mg pr) Avg (SD)
16.9 (5.7) 9.58 (4.4) 3.3 (1.1) 16.8 (6.6) 9.19 (5.1) 3.3 (1.0) 17.1 (3.8) 10.26 (2.9) 3.4 (1.2) 15.4 (4.3) 7.84 (2.4) 4.6 (4.2) 16.2 (4.9) 6.65 (1.9) 3.26 (1.8) 15.0 (4.1) 8.55 (2.5) 5.5 (5.0)
*3,4-Dihydroxyphenylacetic acid (DOPAC). †Homovanillic acid (HVA).
FIG. 6. Percent of deer mice raised in standard cages exhibiting jumping and backwards somersaulting over the 17th week experimental period.
monoamines and metabolites are comparable to those reported by Lin and Pivorun (32) in the same species. The major metabolite of serotonin, 5-hydroxyindolacetic acid (5-HIAA), was undetectable in the striatum. Monoamine and metabolite concentrations did not differ between the two housing conditions or the two stereotypy groups, nor were there any significant housing condition by stereotypy status interactions. There were also no differences in metabolite (DOPAC, HVA)-to-dopamine ratios as a function of stereotypy status or housing condition. DISCUSSION
The current study provided an initial assessment of the development of spontaneous stereotypies in deer mice, including differences in the trajectories for specific topographies (jumping vs. backward somersaulting). These data indicate that enriching the environment had a substantial effect on the amount and type of stereotyped behavior expressed and the time course of its development. Stereotyped behavior, primarily patterned running, was observed in enriched cages, albeit expressed later in development than the jumping and backward somersaulting observed in standard cages. The increased range of motor activities available may explain the delayed development of stereotypy in animals raised in enriched cages. TABLE 3 DENSITY (fmol/mg PROTEIN) OF D1 (3H-SCH23390) AND D2 (3H-SPIPERONE) DOPAMINE RECEPTORS IN STRIATUM OF DEER MICE 1.0 nM 3H-SCH23390 (fmol/mg protein)
1.0 nM 3H-Spiperone (fmol/mg protein)
Group
n
Avg
SD
Avg
SD
Stereotypy Standard caging Enriched caging No stereotypy Standard caging Enriched caging
22 14 8 16 6 10
1005.3 983.8 1042.9 958.4 920.5 981.1
180.9 211.7 111.1 145.0 99.6 167.3
262.1 276.3 237.1 234.9 228.0 239.1
122.1 150.8 36.7 47.2 31.8 55.7
Although individual animal data were not presented, the developmental trajectory for stereotypy varied between animals. Comparing jumping to backward somersaulting in standard cages suggests that somersaulting develops later during ontogeny than does jumping. This differential rate of development may relate to the animals’ stage of physical development with backward somersaulting requiring greater motor competence [e.g., (5)] and could account for some of the variability in the rate of development observed between animals. The appearance of stereotypies relatively early in development in deer mice is consistent with observations in bank voles. Odberg (41) reported that in bank voles stereotypy developed at about 20 days of age and becomes well developed within 10 days. The degree of enrichment used in the present study was not sufficient to completely prevent the development of stereotyped behavior. A significantly larger and/or more complex housing area would be more likely to prevent the development of stereotypy and support the notion that stereotypies observed in deer mice in standard laboratory cages is associated with environmental restriction. Larger living environments might be particularly important for wild-type muroid rodents such as deer mice, which display a high level of locomotor activity and maintain a relatively large home range in the wild [estimated range 242–3,000 square meters; (55)]. With the exception of the work with bank voles (11,50), the relationship between cage size and complexity and the development of stereotypy has not been empirically analyzed. Other investigations of the relationship between cage size and stereotypy have focused on changes in the amount of stereotypy performed in relation to changes in cage size in adult animals (7,15). In the study by Berkson et al. (7), chimpanzees were separated from their mothers at birth and raised in small cages during the first 2-1/2 years of life. As adult animals (4–5 years of age), these chimpanzees engaged in more stereotyped behavior when temporarily placed in small enclosures. Similarly, wild-born and laboratory-reared rhesus monkeys (Macaca mulatta) display higher rates of stereotyped behavior when placed in small versus large enclosures (15,43). Both of these studies provide support for the movement restraint hypothesis of stereotypy, but neither of them address the critical environmental components associated with the development of stereotypy. The current study assessed the effects of both increased cage size and environmental complexity on the ontogeny of stereotypy. Identifying what specific aspects of the environment are critical for the prevention of stereotypy (e.g., cage size, enrichment objects, social density) may be necessary for a better understanding of the role of the environment
DEVELOPMENT OF STEREOTYPY IN DEER MICE in the development or prevention of stereotypy (27). Indeed, Ödberg (41) found that the enrichment material in the cage was more effective than a larger cage size in decreasing the number of bank voles developing stereotypy. The emergence of different topographies between the standard and enriched cage conditions suggests the importance of environmental constraints associated with the development of repetitive behavior patterns. Mason (34) and Dantzer (14) have proposed that stereotyped behaviors may develop from the normal behaviors appropriate to the particular environmental context. Although occurring in a small percentage of the animals, Wurbel et al. (56) describe the development of jumping in ICR mice as originating from exploratory behavior at the cage walls, beginning with mice rearing in the corners, stretching their bodies, and sniffing toward the top of the cage. Similar behavioral patterns were observed during our observations of deer mice. The fact that patterned running was the most prevalent form of stereotypy in the enriched cages may have been due to the physical arrangement of the cages. It should be stressed, however, that there was ample space in the enriched cages for stereotyped jumping (which occurred at a low rate), and a small number of animals did exhibit backward somersaulting in the enriched cages. Thus, the substantial decrease in jumping and somersaulting, by far the most prevalent forms of stereotypy observed in standard cages, was not due to physical impediment of the expression of these behaviors. Considering the high degree of interindividual variability, an interesting and as-yet unanswered question in the study of spontaneous stereotypy is the relative importance of genetic factors associated with the behavior. A genetic component to the development of stereotypy has been suggested in bank voles (40). Our findings support an effect of litter, independent of experimental condition, on the time to develop stereotypy. This litter effect suggests a potentially important role for genetic and/or early environmental factors in the rate of development of stereotyped behaviors. Manipulating prenatal and postnatal maternal environments (e.g., crossfostering pups between stereotypy and nonstereotypy mothers) will be necessary to disentangle the relative importance of genetic and early environmental factors in the development of stereotypy. Our findings indicate that males and females developed stereotypy at similar rates, which has also been reported in bank voles (40) and laboratory mice (56). No differences in the concentration of dopamine and its metabolites were observed as a function of stereotypy. Similarly, the density of D1 and D2 dopamine receptors did not differ as a function of the amount of stereotypy, nor did they differ between the two housing conditions. These findings are inconsistent with the decrease in dopamine metabolite con-
361 centration and increase in dopamine receptor density observed in piglets developing nonnutritive oral behavior after being denied the opportunity to suckle (19,49). Previous work on drug-induced stereotypy has suggested the importance of the nigrostriatal dopamine system in the expression of stereotyped behaviors. Stereotypies in these models, although similar to the stereotypies observed in deer mice in terms of being repetitive, are quite different in their topography. For example, administration of high doses of dopamine agonists to rats primarily induces focused sniffing, gnawing, and licking of the cage floor (10). Although not discounting the importance of nigrostriatal dopamine in the mediation of stereotypy in deer mice, it may be critical to examine other dopaminergic pathways as well. For example, mesolimbic dopamine may be particularly important in the expression of locomotor stereotypies (25). For example, intraaccumbens injections of amphetamine have also been reported to induce stereotypy (1), and work on schedule-induced polydipsia has suggested the importance of the nucleus accumbens in the acquisition of these behaviors (47). An association between stereotypy and dopamine receptor supersensitivity has been made in other animal models of stereotyped behavior. Rhesus monkeys that develop stereotypies following complete social isolation early in development are behaviorally more sensitive to the effects of dopamine agonists (28). Rats with neurotoxic lesions of the dopamine system using 6-OHDA are more sensitive to the induction of stereotypy following administration of direct-acting dopamine agonists (52). These observations of behavioral supersensitivity are generally attributed to an upregulation of dopamine receptors in terminal fields of dopamine-producing neurons. The failure to observe a difference in dopamine receptor number in the current study, however, does not rule out the possibility that stereotypy is associated with dopamine receptor supersensitivity. Behavioral supersensitivity has been observed in other models without an increase in the density of D1 or D2 dopamine receptors (39). As Ödberg et al. (41) suggest, the literature on drug-induced stereotypy has not been very well integrated with the literature on spontaneous stereotypies developing in conditions of environmental restriction. Similarly, there is little integration of the findings from studies of repetitive behaviors in animals and stereotypies observed in clinical populations. Such integration would provide a better understanding of the neurobiology of repetitive motor behavior in general and potentially contribute to treatment strategies for clinical populations. Thus, development of a rodent model of spontaneous stereotypy that appears to have a variety of similarities with spontaneous stereotypies in other animal species and in humans may provide us with a means to address some of these questions.
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PII S0031-9384(98)00303-5
A Rodent Model of Spontaneous Stereotypy: Initial Characterization of Developmental, Environmental, and Neurobiological Factors SUSAN B. POWELL,*1 HOWARD A. NEWMAN,* JANE F. PENDERGAST† AND MARK H. LEWIS* *Departments of Psychiatry and Psychology, P.O. Box 100256, University of Florida, Gainesville, FL 32610, and †Department of Statistics, University of Florida, Gainesville, FL 32610 Received 24 October 1997; Accepted 26 October 1998 POWELL, S. B., H. A. NEWMAN, J. F. PENDERGAST AND M. H. LEWIS. A mouse model of spontaneous stereotypy: Initial characterization of developmental, environmental, and neurobiological factors. PHYSIOL BEHAV 66(2) 355– 363, 1999.—Stereotypies are patterns of motor behavior that are repetitive, excessive, topographically invariant, and that lack any obvious function or purpose. In humans, stereotyped behaviors are associated with psychiatric, neurological, and developmental disorders. In animals, stereotypy has been frequently associated with adverse environmental circumstances and often related to alterations in striatal dopamine. To assess the development of stereotyped behaviors and to test the hypothesis that these behaviors are associated with environmental restriction, deer mice were housed in either standard laboratory cages or larger, enriched cages, and the development of stereotypy was followed from weaning over a 17-week period. Standardcaged deer mice engaged in stereotyped behaviors at a higher rate and developed these behaviors more quickly when compared to animals in enriched caging. Additionally, enriched caging was associated with higher rates of patterned running, whereas jumping and backward somersaulting were typically observed in standard cages. In addition, there was a significant effect of litter, but no effect of sex or cage, on the time to develop stereotypy. No differences were found in the density of either striatal D1 or D2 dopamine receptors or the concentration of striatal dopamine or its metabolites as a function of rearing condition or as a function of whether the animals developed stereotypy. These results characterize the development of stereotypies in this species, demonstrate the importance of environmental conditions in the genesis of stereotypy, and suggest that alterations in striatal dopamine content or dopamine receptor density do not account for the expression of stereotyped behaviors in this model. © 1999 Elsevier Science Inc. Repetitive behavior
Dopamine
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Enrichment
social or maternal deprivation exhibit stereotyped behaviors (e.g., body rocking, tail biting) (4,20), as do other species that experience confinement and movement restraint (21,26,38). The expression of such behaviors (e.g., route tracing, chain chewing, cribbing) in zoo and farm animals are considered a major animal welfare concern by veterinarians and animal caretakers, as they are thought to represent the animal’s response to an inadequate environment (13,24). The empirical evidence for the role of environmental restriction in the stereotypy of zoo and farm animals is relatively sparse and weak (18,45). In the laboratory, several species exhibit spontaneous stereotypies (absent a specific environmental or pharmacological
STEREOTYPIES are sequences of motor behavior that are repetitive, topographically invariant, often rhythmical, and apparently purposeless (4,14,27). In humans, stereotypy (e.g., body rocking, head rolling) has been considered an important feature of psychopathology as well as being associated with a variety of neurological and developmental disorders such as mental retardation and autism (2,6,9,22,30). Despite the high occurrence of stereotypy in these clinical populations, little is known about the pathophysiology and pharmacological treatment of these behavior disorders. Stereotypic behaviors have also been associated with adverse environmental circumstances in a large number of animal species (34,35). Nonhuman primates that experience early
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whom requests for reprints should be addressed. E-mail: [email protected]
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356 challenge) whether raised in captivity or caught in the wild [for review, see (27)]. For example, trapped bank voles (Clethrionomys glareolus) develop stereotyped jumping, backwards somersaulting, and patterned running when housed in standard rodent cages (40). When housed under enriched conditions (e.g., larger cage size, addition of nest materials, hiding places, twigs on which to climb), substantially fewer bank voles developed stereotypy compared to bank voles housed in standard cages (50). Ödberg (41) has also observed a decrease in the number of bank voles developing stereotypy through the addition of enrichment objects to the cage. These are among the few studies that have empirically demonstrated the relationship between environmental restriction and the development of stereotypy. Stereotyped body rocking appears relatively early (ca. 29 days of age) in the behavioral repertoire of chimpanzees raised in isolation with a stationary surrogate mother, but is not observed in chimpanzees raised in isolation with a moving surrogate mother (37). Studies of rodents indicate that stereotypies in response to standard laboratory caging appear early during ontogeny with wire gnawing in ICR mice (56) and digging in gerbils (54) developing by 30 days of age. As suggested by these investigators, understanding the developmental time course of stereotypy in deer mice will be important for the characterization of the model and subsequent studies on sensitive periods of susceptibility and the neurobiological basis of the behavior. Information about the neurobiological basis of stereotyped behavior has generally relied on models of drug-induced stereotypy (27). It is now well established that stereotyped patterns of behavior can be induced in a number of mammalian species following administration of dopamine agonists and drugs that alter nigrostriatal dopamine function (10,17,30,44). For example, dopamine or dopamine agonists injected directly into the striatum induce stereotyped behaviors in rats [e.g., (16)]. Induction of stereotyped behavior by application of GABA agonists to the substantia nigra pars reticulata supports the importance of the nigrostriatal circuitry and its output pathways (48). Dopamine agonist-induced stereotypy can be blocked by dopamine receptor antagonists such as haloperidol (46), by inhibiting the synthesis of dopamine using a-methyl-p-tyrosine (51) or by destroying dopamine-containing neurons with the neurotoxicant 6-hydroxydopamine (6OHDA) (12). The issue of whether the neurobiological mechanisms underlying drug-induced stereotypies versus spontaneous stereotypies are the same is very much an open question (34,36,53). Few studies of environmentally produced stereotypies have addressed the neurobiology of such repetitive behaviors (14). Exceptions include the finding that decreased tyrosine hydroxylase immunoreactivity in the striatum and substantia nigra was observed in rhesus monkeys that developed stereotypy following social isolation early in development (33). These monkeys also showed an increased behavioral sensitivity to an acute dose of apomorphine, suggestive of dopamine receptor supersensitivity (28). The development of repetitive oral behaviors in early weaned piglets has been associated with decreases in HVA and DOPAC, the major metabolites of dopamine, in the putamen, and nucleus accumbens (19,49), and increases in D2 dopamine receptors in the caudate nucleus (49). In individuals who have mental retardation, stereotyped behavior has been associated with decreases in spontaneous blink rate (8) and plasma HVA concentrations (29), suggesting hypodopaminergic function. Studies examining captivity-induced stereotypies of bank voles have suggested an important role for dopamine systems
POWELL ET AL. in the mediation of these repetitive behaviors (23). Stereotyped jumping in bank voles was decreased by a-methyl-ptyrosine, an inhibitor of tyrosine hydroxylase, and increased by L-dopa, the precursor to dopamine (42). These drug effects were not solely due to a generalized effect on motor activity as both drug treatments failed to affect other activities. The dopamine-b-hydroxylase (DBH) inhibitor fusaric acid had no significant effects on stereotyped behavior in these animals, suggesting the importance of dopamine but not norepinephrine (42). The literature on spontaneous stereotypy has been primarily descriptive due, at least in part, to the species being studied. A mouse model of spontaneous stereotypy would lend itself to examination of basic neural mechanisms involved in the expression of stereotyped behavior observed in clinical populations. Deer mice, when housed under standard laboratory conditions, develop high rates of spontaneous stereotyped motor behavior (3). The current study was designed to characterize the specific forms of stereotyped behavior displayed by deer mice, describe the developmental trajectory for the stereotyped behaviors, and determine the upper and lower age limits for the initial expression of these behaviors. To test the environmental restriction hypothesis, a series of observations to assess the effects of environmental enrichment on stereotypy and track the development of stereotypies in animals housed in standard and enriched cages was conducted. It was hypothesized that stereotyped behavior was associated with decreased concentrations of dopamine and its metabolites and an increase in dopamine receptors in the corpus striatum. METHODS
Animals Deer mice (Peromyscus maniculatus bairdii) were housed in a standard colony room kept at 248C and maintained on a 16/8-h light/dark cycle, with lights off at 0930 h. At the time of weaning (23 days of age), deer mice were randomly assigned to either standard (n 5 16) or enriched (n 5 15) caging. Standard caging involved either two or three same sex mice in a standard laboratory mouse cage (29 3 18 3 13 cm) with rodent chow and water available ad lib. Four cages contained three animals, and two cages contained only two animals. The discrepancy in number of animals per cage was due to an unequal number of males and females and the death of one animal. Enriched caging involved housing three same-sex mice in a larger cage (51 3 41 3 22 cm) equipped with a running wheel, habit trails, small enclosures for nesting or hiding, nesting material, and sunflower seeds inside the cage. Rodent chow and water were located on the cage top and available ad lib. The objects within the cage were changed and rearranged weekly. A commercially available blonde hair dye was applied to the animals’ fur every 4 weeks to identify each mouse in a given cage. A second cohort of deer mice (n 5 17) was housed in the same manner as described above in both standard (n 5 9) and enriched (n 5 8) cages. These animals were included in the analysis of dopamine receptors and monoamine concentrations in striatum. Observational Procedures Behavioral observations were conducted twice daily at approximately 1030 and 1430 h every other day (approximately three times per week) for 17 weeks. Deer mice were observed during their dark cycle only as previous studies with this colony of mice have shown marked diurnal variation, with the mice engaging in very little activity during the light cycle (3).
DEVELOPMENT OF STEREOTYPY IN DEER MICE Each cage was observed for 5 min at each of the two time periods. Each 5-min observation period was divided into 5-s intervals. During each 5-s scoring interval the occurrence of specific topographies of stereotyped behavior was recorded for individual animals. From preliminary observations of the animals, three distinct topographies of stereotyped behaviors were observed and operationally defined: jumping, backward somersaulting, and route tracing or patterned running. Similar behavior patterns have been observed in captive bank voles (50). To be considered a stereotypy, the behavior had to occur more than once within the 5-s interval. Interrater agreement across topographies of stereotyped behavior as computed using Cohen’s kappa averaged 0.83 (SD 5 0.15). The second cohort of animals was observed in a similar manner, but only for 2 weeks before being killed. These animals were not included in the analysis of behavioral data, as they were only used for determination of monoamines and dopamine receptors. Homogenate Radioligand Binding At the end of the 17-week period, animals were killed by cervical dislocation followed by decapitation and brains were rapidly removed, snap frozen in isopentane, and stored at 2808C until time of assay. Estimates of the density of D1 and D2 dopamine receptor sites in the corpus striatum (caudate nucleus and putamen) were determined in animals in both standard and enriched caging. At the time of assay, individual striata were homogenized in a volume of ice cold 50 mM HEPES buffer (pH 7.4; 48C), using Teflon-glass homogenizers (Eberbach, Ann Arbor, MI), to equal a concentration of approximately 1.0 mg wet weight/mL. At this point a 300-mL aliquot of homogenate was removed for HPLC analysis. The remaining tissue was centrifuged at 27,000 3 g for 10 min, the supernatant discarded, and the pellet resuspended in 5 mL ice cold buffer and centrifuged again. The final pellet was suspended at a concentration of approximately 1.0 mg wet weight/mL. Assay tubes (1 mL final volume) were incubated at 378C for 20 min. Binding of 1.0 nM 3H-SCH23390 was used to assess the density of D1 receptors with unlabeled SCH23390 at a concentration of 10 mM to define nonspecific binding. Ketanserin tartrate (500 nM) was used to displace binding of SCH23390 to 5-HT2 receptors. Binding of 1.0 nM 3H-spiperone was used to determine the density of D2 receptors with unlabeled domperidone (10 mM) to define nonspecific binding. Ketanserin tartrate (500 nM) was used to displace binding of spiperone to 5-HT2 receptors. Binding was terminated by filtering with 15 mL ice-cold buffer on a Skatron cell harvester (Skatron INC, Sterling, VA) using glass fiber filter mats (Skatron #7034, Sterling, VA). Filters were then allowed to dry and 3.0 mL of Scintiverse E (Fischer Scientific Co., Fair Lawn, NJ) was added. After shaking for 30 min, radioactivity was determined on a LKB Rack Beta liquid scintillation counter. Tissue protein levels were estimated using a BCA spectrophotometric assay and microplate reader with absorbance set at 562 nm. Estimates of the density of dopamine receptors were computed at the given concentration of radiolabeled drug used for the two receptor subtypes.
357 ence electrode. Chromatographic separations were performed using a deltabond stainless steel column (150 mm 3 4.6 mm i.d.) packed with 3 mm C18 bonded microparticulate silica (Keystone Scientific, INC, Bellafonte, PA). The mobile phase consisted of 95 mM Na2HPO4 containing 27 mM citric acid, 0.038% sodium octyl sulfate (SOS), and 13% methanol, with a final pH of 3.4 and a flow rate of 0.80 mL/min. Standard curves for the quantification of all compounds [dopamine (DA), serotonin (5-HT), homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA)] were prepared by analyzing a series of standard solutions containing a fixed amount of the internal standard 3,4-dihydroxybenzylamine (DHBA) and varying amounts of each compound. The slopes of the standard curves obtained by linear regression were routinely greater than or equal to 0.99. At the time of the radioligand binding assay, 240 mL of perchloric acid was added to the 300 mL of striatal homogenate. Samples were vortexed, then centrifuged for 10 min at 13,200 rpm, supernatant collected, and both supernatant and pellet were stored at 2808C until assay (for HPLC and protein assays, respectively). Supernatant (450 mL) was removed and 50 mL of DHBA (20 ng/mL) was added. The solution was filtered (0.2 mm nylon acrodisc), and 100 mL of the solution was injected onto the column. The concentration of monoamines and their metabolites in these unknown brain samples were determined from the internalized standard curve and the ratios of the particular compounds to DHBA. The tissue pellet was sonicated in 500 mL mobile phase buffer using an ultrasonic cell disrupter (Heatsystems, Farmingdale, NY; setting 1) for protein determination. Tissue protein levels were estimated using a BCA spectrophotometric assay and microplate reader with absorbance set at 562 nm. RESULTS
Development of Stereotypy and Effects of Enrichment Table 1 lists the topographies and operational definitions of stereotyped behaviors that were observed in both standard cages and enriched cages. The percentage of intervals in which any of the three topographies of stereotypy occurred was calculated for each animal by week. The mean weekly percentages of intervals of total stereotypy over the 17-week observation period across the two housing conditions are depicted in Fig. 1. The effect of housing condition, sex, week, cage (fixed effects), and litter (random effect) on stereotypy was assessed using a general linear mixed model analysis of variance with a first-order autoregressive [AR(1)] correlation structure within animal over time (GLMM: PROC MIXED in SAS). In addition to the main effects, the following interactions were also included in the model: housing condition by sex, sex by week, TABLE 1 STEREOTYPED BEHAVIORS IN DEER MICE IN STANDARD AND ENRICHED CAGING
Repetitive jumping
HPLC Analysis of Monoamines and Metabolites The concentrations of monoamines and metabolites from striata were quantified using an HPLC procedure with electrochemical detection. Differences in specific compounds between specified experimental conditions were determined quantitatively using amperometric detection of the column effluent with a potential of 10.75 V versus an Ag/AgCl refer-
Backward somersault
Patterned running
Rearing in one of the four corners of the cage and repeatedly jumping on his/her hind paws A somersault in a backward direction with or without assistance from the cagetop or side Repetitive route tracing or circling of the cage in a clear pattern
358
FIG. 1. Effects of housing condition on the percent of intervals of total stereotypy over the 17th week experimental period.
and sex by housing condition. Animals were nested within the litter effect, as well as within the cage effect. A square root transformation of the raw data was used to better meet the underlying assumptions of the model. The analysis was conducted on the amount of total stereotypy averaged across 1-week periods. Deer mice in standard cages engaged in higher rates of stereotyped behavior than did deer mice in enriched cages, F(1, 448) 5 3.79, p 5 0.05. There was also a significant effect of time with the amount of stereotypy increasing over successive weeks, F(16, 448) 5 3.84, p , 0.001, and a significant interaction between experimental condition and time, F(16, 448) 5 2.09, p 5 0.008. There were no effects of cage, F(7, 448) 5 0.66, p 5 0.70, or sex, F(1, 448) 5 0.94, p 5 0.33, on stereotypy. Animals were considered nested within the random litter effect, as well as within the fixed cage effect. Because the litter effect is a random effect, there is no F-test to assess its importance within the model. The percent of animals engaging in stereotyped behavior (collapsing across topographies) observed in each of the two housing conditions over the 17-week period is presented in Fig. 2. Animals were judged to have stereotypy in a given week if they engaged in repetitive behavior in greater than 5% of the intervals. As can be seen in Fig. 2, animals housed in standard cages developed stereotypies at a faster rate than did animals housed in enriched cages. To test whether environmental condition resulted in a differential rate of development of stereotypy, an extension of logistic regression was used. A generalized estimating equations approach was used to fit a logistic regression model that could account for the correlation within animal across time (31). The logit probability of exhibiting stereotypy was modeled as a function of experimental condition, time, litter, and sex effects. Cage was excluded from this analysis as a second random variable could not be accommodated in this model, and it was shown previously to have no effect on level of stereotypy. Animals housed in standard cages developed stereotypy at a significantly faster rate than did animals raised in enriched cages (z 5 21.84, p 5 0.033, one tailed). As we were interested in a preliminary investigation of the relative importance of genetic and/or early environmental experience on the
POWELL ET AL.
FIG. 2. Effects of housing condition on the percent of animals developing stereotypy over the 17th week experimental period.
development of stereotypy, the effect of litter on the rate of development of stereotypy was included in the model. There was an overall effect of litter on the rate of development of stereotypy, x2(8) 5 20.64, p 5 0.0082, but no effect of sex (z 5 1.24, p 5 0.22). To compare the number of animals that developed stereotypy in each condition, a stricter criterion was applied. An animal was judged to be stereotypic if the repetitive behavior occurred in greater than 5% of the intervals/week for 2 consecutive weeks. Although stereotyped behavior was observed in 62.5% (10 of 16) of animals housed in standard cages versus 46.7% (7 of 15) of animals housed in environmental enrichment cages (Table 2), these proportions were not significantly different, x2(1) 5 0.275, p 5 0.60. Although the number of animals exhibiting stereotypy was not significantly different between the housing conditions, the stereotyped behaviors exhibited by the two groups were of different forms. Table 2 indicates the number of mice in each condition judged to have developed each topography of stereotypy (jumping, backwards somersaulting, and patterned running). Significantly more deer mice housed in standard cages (7/ 16) developed repetitive jumping than did deer mice housed in enriched cages (0/15) (Fisher’s exact probability test, p 5 0.007). Forty percent (6/15) of deer mice housed in enriched cages developed patterned running versus only 6.3% (1/16) of TABLE 2 NUMBER OF ANIMALS DEVELOPING STEREOTYPIES IN STANDARD AND ENRICHED HOUSING CONDITIONS
Overall (all topographies) Jumping Backward somersaulting Patterned running
Standard Cages (n 5 16)
Enriched Cages (n 5 15)
p Values*
10 7 3 1
7 0 2 6
0.60 0.007 1.0 0.037
*Fisher’s exact probability test.
DEVELOPMENT OF STEREOTYPY IN DEER MICE deer mice housed in standard cages (Fisher’s exact probability test, p 5 0.04). The number of mice developing backwards somersaulting did not differ between the two housing conditions (Fisher’s exact probability test, p 5 1.0) (Figs. 3, 4, and 5). Considering that each topography developed in only a subset of animals, further analyses included only those animals, which were judged through the previous criterion to have developed specific topographies. Deer mice in standard cages engaged in higher rates of backward somersaulting (25.7 6 2.54% of intervals) versus deer mice housed in enriched cages (6.8 6 2.14% of intervals). Of those animals judged to engage in patterned running, animals in the enriched condition did so at higher rates (13.8 6 15.1% of intervals) than did the one animal in the standard cage condition (2.9% of intervals). No mice in enriched caging were judged to have developed repetitive jumping. Inferential statistics were not applied to these results due to the small sample sizes. Within the standard cages, there was a different developmental rate for somersaulting and jumping. As seen in Fig. 6, stereotyped jumping occurred in a greater percentage of animals housed in standard cages and developed earlier than backward somersaulting.
359
FIG. 4. Effects of housing on the percent of intervals in which patterned running was observed over the 17th week experimental period.
Analysis of Striatal Dopamine and Dopamine Receptors Multiple linear regressions were conducted to determine if the linear combination of the frequency of stereotypy during the last week of observations, experimental condition, sex, cage, and litter accounted for the relative density of dopamine receptors and the concentration of monoamines and monoamine metabolites. Cage and litter, when both in the model, presented problems with mulitcollinearity; thus, cage was subsequently left out of the model because it was previously shown not to have an effect on stereotypy and the analysis was repeated. The linear combination of these variables did not predict D1 or D2 dopamine receptor densities or monoamine and metabolite concentrations (r2 range: 0.03–0.10). There were no effects of housing condition, percentage of stereotypy, sex, or litter on either D1 or D2 dopamine receptor densities or monoamine and monoamine metabolite concentrations. When litter was removed from the model, there were still no effects of housing condition, stereotypy, or sex on either D1 or
FIG. 3. Effects of housing on the percent of intervals in which repetitive jumping was observed over the 17th week experimental period.
D2 dopamine receptor densities or monoamine and monoamine metabolite concentrations. The effects of stereotypy status (stereotypy/no stereotypy) and housing condition on D1 and D2 dopamine receptors and monoamine and monoamine metabolites were analyzed using 2 3 2 analyses of variance (ANOVA). Stereotypy status was based on the criteria used in the analysis of number of mice developing stereotypy above (.5% of the intervals for 2 consecutive weeks). Table 3 indicates the binding of [3H]-SCH23390 and [3H]-spiperone to D1 and D2 dopamine receptors, respectively, in striata of deer mice. There were no effects of housing condition or stereotypy status on either D1 or D2 receptor densities. There were also no significant housing condition by stereotypy status interactions for D1 or D2 dopamine receptors. The concentrations of monoamines and monoamine metabolites in striata are shown in Table 4. These levels of
FIG. 5. Effects of housing on the percent of intervals in which backwards somersaulting was observed over the 17th week experimental period.
360
POWELL ET AL. TABLE 4 CONCENTRATION (ng/mg PROTEIN) OF DOPAMINE, HVA, DOPAC, AND SEROTONIN IN STRIATUM OF DEER MICE
Group
n
Dopamine (ng/mg pr) Avg (SD)
Stereotypy Standard caging Enriched caging No stereotypy Standard caging Enriched caging
22 14 8 16 6 10
134.6 (31.1) 129.0 (36.6) 144.3 (15.8) 137.2 (27.6) 138.7 (25.7) 136.3 (30.1)
DOPAC* (ng/mg pr) Avg (SD)
HVA† (ng/mg pr) Avg (SD)
Serotonin (ng/mg pr) Avg (SD)
16.9 (5.7) 9.58 (4.4) 3.3 (1.1) 16.8 (6.6) 9.19 (5.1) 3.3 (1.0) 17.1 (3.8) 10.26 (2.9) 3.4 (1.2) 15.4 (4.3) 7.84 (2.4) 4.6 (4.2) 16.2 (4.9) 6.65 (1.9) 3.26 (1.8) 15.0 (4.1) 8.55 (2.5) 5.5 (5.0)
*3,4-Dihydroxyphenylacetic acid (DOPAC). †Homovanillic acid (HVA).
FIG. 6. Percent of deer mice raised in standard cages exhibiting jumping and backwards somersaulting over the 17th week experimental period.
monoamines and metabolites are comparable to those reported by Lin and Pivorun (32) in the same species. The major metabolite of serotonin, 5-hydroxyindolacetic acid (5-HIAA), was undetectable in the striatum. Monoamine and metabolite concentrations did not differ between the two housing conditions or the two stereotypy groups, nor were there any significant housing condition by stereotypy status interactions. There were also no differences in metabolite (DOPAC, HVA)-to-dopamine ratios as a function of stereotypy status or housing condition. DISCUSSION
The current study provided an initial assessment of the development of spontaneous stereotypies in deer mice, including differences in the trajectories for specific topographies (jumping vs. backward somersaulting). These data indicate that enriching the environment had a substantial effect on the amount and type of stereotyped behavior expressed and the time course of its development. Stereotyped behavior, primarily patterned running, was observed in enriched cages, albeit expressed later in development than the jumping and backward somersaulting observed in standard cages. The increased range of motor activities available may explain the delayed development of stereotypy in animals raised in enriched cages. TABLE 3 DENSITY (fmol/mg PROTEIN) OF D1 (3H-SCH23390) AND D2 (3H-SPIPERONE) DOPAMINE RECEPTORS IN STRIATUM OF DEER MICE 1.0 nM 3H-SCH23390 (fmol/mg protein)
1.0 nM 3H-Spiperone (fmol/mg protein)
Group
n
Avg
SD
Avg
SD
Stereotypy Standard caging Enriched caging No stereotypy Standard caging Enriched caging
22 14 8 16 6 10
1005.3 983.8 1042.9 958.4 920.5 981.1
180.9 211.7 111.1 145.0 99.6 167.3
262.1 276.3 237.1 234.9 228.0 239.1
122.1 150.8 36.7 47.2 31.8 55.7
Although individual animal data were not presented, the developmental trajectory for stereotypy varied between animals. Comparing jumping to backward somersaulting in standard cages suggests that somersaulting develops later during ontogeny than does jumping. This differential rate of development may relate to the animals’ stage of physical development with backward somersaulting requiring greater motor competence [e.g., (5)] and could account for some of the variability in the rate of development observed between animals. The appearance of stereotypies relatively early in development in deer mice is consistent with observations in bank voles. Odberg (41) reported that in bank voles stereotypy developed at about 20 days of age and becomes well developed within 10 days. The degree of enrichment used in the present study was not sufficient to completely prevent the development of stereotyped behavior. A significantly larger and/or more complex housing area would be more likely to prevent the development of stereotypy and support the notion that stereotypies observed in deer mice in standard laboratory cages is associated with environmental restriction. Larger living environments might be particularly important for wild-type muroid rodents such as deer mice, which display a high level of locomotor activity and maintain a relatively large home range in the wild [estimated range 242–3,000 square meters; (55)]. With the exception of the work with bank voles (11,50), the relationship between cage size and complexity and the development of stereotypy has not been empirically analyzed. Other investigations of the relationship between cage size and stereotypy have focused on changes in the amount of stereotypy performed in relation to changes in cage size in adult animals (7,15). In the study by Berkson et al. (7), chimpanzees were separated from their mothers at birth and raised in small cages during the first 2-1/2 years of life. As adult animals (4–5 years of age), these chimpanzees engaged in more stereotyped behavior when temporarily placed in small enclosures. Similarly, wild-born and laboratory-reared rhesus monkeys (Macaca mulatta) display higher rates of stereotyped behavior when placed in small versus large enclosures (15,43). Both of these studies provide support for the movement restraint hypothesis of stereotypy, but neither of them address the critical environmental components associated with the development of stereotypy. The current study assessed the effects of both increased cage size and environmental complexity on the ontogeny of stereotypy. Identifying what specific aspects of the environment are critical for the prevention of stereotypy (e.g., cage size, enrichment objects, social density) may be necessary for a better understanding of the role of the environment
DEVELOPMENT OF STEREOTYPY IN DEER MICE in the development or prevention of stereotypy (27). Indeed, Ödberg (41) found that the enrichment material in the cage was more effective than a larger cage size in decreasing the number of bank voles developing stereotypy. The emergence of different topographies between the standard and enriched cage conditions suggests the importance of environmental constraints associated with the development of repetitive behavior patterns. Mason (34) and Dantzer (14) have proposed that stereotyped behaviors may develop from the normal behaviors appropriate to the particular environmental context. Although occurring in a small percentage of the animals, Wurbel et al. (56) describe the development of jumping in ICR mice as originating from exploratory behavior at the cage walls, beginning with mice rearing in the corners, stretching their bodies, and sniffing toward the top of the cage. Similar behavioral patterns were observed during our observations of deer mice. The fact that patterned running was the most prevalent form of stereotypy in the enriched cages may have been due to the physical arrangement of the cages. It should be stressed, however, that there was ample space in the enriched cages for stereotyped jumping (which occurred at a low rate), and a small number of animals did exhibit backward somersaulting in the enriched cages. Thus, the substantial decrease in jumping and somersaulting, by far the most prevalent forms of stereotypy observed in standard cages, was not due to physical impediment of the expression of these behaviors. Considering the high degree of interindividual variability, an interesting and as-yet unanswered question in the study of spontaneous stereotypy is the relative importance of genetic factors associated with the behavior. A genetic component to the development of stereotypy has been suggested in bank voles (40). Our findings support an effect of litter, independent of experimental condition, on the time to develop stereotypy. This litter effect suggests a potentially important role for genetic and/or early environmental factors in the rate of development of stereotyped behaviors. Manipulating prenatal and postnatal maternal environments (e.g., crossfostering pups between stereotypy and nonstereotypy mothers) will be necessary to disentangle the relative importance of genetic and early environmental factors in the development of stereotypy. Our findings indicate that males and females developed stereotypy at similar rates, which has also been reported in bank voles (40) and laboratory mice (56). No differences in the concentration of dopamine and its metabolites were observed as a function of stereotypy. Similarly, the density of D1 and D2 dopamine receptors did not differ as a function of the amount of stereotypy, nor did they differ between the two housing conditions. These findings are inconsistent with the decrease in dopamine metabolite con-
361 centration and increase in dopamine receptor density observed in piglets developing nonnutritive oral behavior after being denied the opportunity to suckle (19,49). Previous work on drug-induced stereotypy has suggested the importance of the nigrostriatal dopamine system in the expression of stereotyped behaviors. Stereotypies in these models, although similar to the stereotypies observed in deer mice in terms of being repetitive, are quite different in their topography. For example, administration of high doses of dopamine agonists to rats primarily induces focused sniffing, gnawing, and licking of the cage floor (10). Although not discounting the importance of nigrostriatal dopamine in the mediation of stereotypy in deer mice, it may be critical to examine other dopaminergic pathways as well. For example, mesolimbic dopamine may be particularly important in the expression of locomotor stereotypies (25). For example, intraaccumbens injections of amphetamine have also been reported to induce stereotypy (1), and work on schedule-induced polydipsia has suggested the importance of the nucleus accumbens in the acquisition of these behaviors (47). An association between stereotypy and dopamine receptor supersensitivity has been made in other animal models of stereotyped behavior. Rhesus monkeys that develop stereotypies following complete social isolation early in development are behaviorally more sensitive to the effects of dopamine agonists (28). Rats with neurotoxic lesions of the dopamine system using 6-OHDA are more sensitive to the induction of stereotypy following administration of direct-acting dopamine agonists (52). These observations of behavioral supersensitivity are generally attributed to an upregulation of dopamine receptors in terminal fields of dopamine-producing neurons. The failure to observe a difference in dopamine receptor number in the current study, however, does not rule out the possibility that stereotypy is associated with dopamine receptor supersensitivity. Behavioral supersensitivity has been observed in other models without an increase in the density of D1 or D2 dopamine receptors (39). As Ödberg et al. (41) suggest, the literature on drug-induced stereotypy has not been very well integrated with the literature on spontaneous stereotypies developing in conditions of environmental restriction. Similarly, there is little integration of the findings from studies of repetitive behaviors in animals and stereotypies observed in clinical populations. Such integration would provide a better understanding of the neurobiology of repetitive motor behavior in general and potentially contribute to treatment strategies for clinical populations. Thus, development of a rodent model of spontaneous stereotypy that appears to have a variety of similarities with spontaneous stereotypies in other animal species and in humans may provide us with a means to address some of these questions.
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