clonic seizures in captive born bank voles (Clethrionomys glareolus)

Applied Animal Behaviour Science 118 (2009) 84–90

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Applied Animal Behaviour Science journal homepage: www.elsevier.com/locate/applanim

Handling-induced tonic/clonic seizures in captive born bank voles (Clethrionomys glareolus) Bryan Schønecker * University of Copenhagen, Department of Biology, The Animal Behaviour Group, Tagensvej 16, DK 2200 Copenhagen N, Denmark

A R T I C L E I N F O

A B S T R A C T

Article history: Accepted 16 February 2009

Handling-induced seizures observed among 23 of 333 captive born bank voles was characterized by tonic/clonic convulsions, occasionally accompanied by an apparent loss of consciousness. Seizures were never observed among wild caught voles (N = 71). Median age for first observation of seizures was 157 days. Median latency to onset following mild handling was 12.6 s and median time to resumption of normal behaviour after arrest of convulsions was 28.05 s. Consecutive daily tests to provoke seizures indicated that more seizures were elicitated on the first day of testing (25.6%) compared to the following 4 testdays (8.7%). Incidence of seizure prone (SP) voles declined from 10.2% in F1 to 5.1% in F2 with no sex bias. A possible explanation for this decline could be that all F1 voles (N = 118) descended from non-stereotyping (N-Ster) parents where the majority of F2 voles (n = 138) descended from two stereotyping (Ster) parents: incidence of SP voles were five times higher among offspring from N-Ster parents than Ster parents (10–11% vs. 2.2%; p < 0.0001). However, the development of stereotypic behaviours did not affect seizure proneness. Roughly one-third of the captive born voles developed diabetes. However, the disease did not affect seizure proneness. SP voles were distributed among the litters (n = 60) in accordance with the negative binomial distribution, which indicate a ‘‘lumped’’ distribution. The proportion of SP voles which had SP full siblings, was significantly higher than the proportion of non-SP voles having SP full siblings (15/23 vs. 30/310, p = 0.0001), which, taken together, suggest the possibility for future establishment of lines differing in seizure proneness. ß 2009 Elsevier B.V. All rights reserved.

Keywords: Bank voles Animal model Seizures Hyperglycemia Heritability Stereotypic behaviours

1. Introduction Bank voles (Clethrionomys glareolus; the second largest genus within the subfamily Arvicolinae) have been known for decades to develop various types of captivity-induced stereotypic behaviours in high frequencies (see e.g. Sørensen and Randrup, 1986). A previous definition of stereotypies focused on features such as ‘‘morphologically identic’’ movements, which are ‘‘repeated regularly’’ and

* Present address: University of Copenhagen, Faculty of Life Sciences, Department of Large Animal Sciences, The Ethology Group, Grønnega˚rdsvej 8, 1870 Frederiksberg C, Denmark. Tel.: +45 36 96 07 63. E-mail address: [email protected]. 0168-1591/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.applanim.2009.02.024

¨ dberg, 1978) seem ‘‘unusual’’, ‘‘useless’’ or ‘‘purposeless’’ (O but an undisputed definition is still not available (Rushen and Mason, 2006). The predominant type of stereotypy among Danish voles are backward somersaults (Schoenecker et al., 2000; Sørensen, 1987) whereas voles originating from Sweden (Sørensen, 1987) and United ¨ dberg, 1986) for the most part engage in highKingdom (O speed jumps on the hind legs, typically in a corner of the cage. Less frequently observed types include running in circles (also known as ‘‘pacing’’) and a special ‘‘windscreen wiper movement’’ where the vole sways from side to side up against a wall, standing on the hind legs (Schoenecker ¨ dberg, 1986). As a rule of et al., 2000; Sørensen, 1987; O thumb, stereotypies only develop among captive born bank voles (F-voles); of 176 wild caught voles (P-voles),

B. Schønecker / Applied Animal Behaviour Science 118 (2009) 84–90

kept for a significant time in captivity, just two developed stereotypies in the form of backward somersaults and pacing after 356 and 179 days respectively (Schønecker, unpublished observation). I first noted seizures during ad hoc observations made while cleaning cages in my first colony of Danish bank voles consisting of 92 wild caught (P-generation) voles and 518 captive born (F-generations) voles. These seizures typically occurred within the first minute after transfer to a new clean cage and a general pattern seemed to be that situations involving presumed fear, frustration and novelty preceded the seizures. In total 10 F-voles was observed having seizures where seizures were not once observed among P-voles. However, apart from noting each episode of seizures, no systematic screenings were performed. The main purpose of my second colony (described below) was to further validate prior findings of diabetes among polydipsic bank voles made by Irene Vejgaard Sørensen, Tonny Freimanis and me in the late nineties (Freimanis et al., 2003; Schoenecker et al., 2000). The results of the study was that polydipsic/hyperglycemic Danish bank voles were indeed suffering from severe type 1 diabetes associated with antibodies against the Ljungan virus in their pancreas, and antibodies against GAD65/IA-2/Insulin (Niklasson et al., 2003). It was further shown that the titres of antibodies against Ljungan virus and GAD65 in sera from wild caught bank voles were markedly increased after roughly a month in captivity compared to the levels immediately after capture. The protocol involved weekly measurements of weight gains/losses from time of capture/weaning and the following 6 months during which the vole was placed in a cold glass jar on a scale. This procedure turned out to be effective in inducing seizures in susceptible voles compared to preliminary tests using auditory stimuli (e.g. clapping hands), which were ineffectual. Observations of handling-induced seizures have on one previous occasion been described in the largest genus of the Arvicolinae. According to Bronson and de la Rosa (1994) both laboratory born, and some wild caught meadow voles (Microtus pennsylvanicus) from central Pennsylvania, USA, developed seizures involving a genetic component and apparent inability to elicitation by olfactory and auditory stimuli. However, handling-induced seizures in the genus Clethrionomys have to my knowledge never before been published. The purpose of this paper is therefore to describe the seizures; measure fundamentals such as incidence, age of first observation and any hereditary aspects, such as a more than chance of encountering seizure prone (SP) voles in certain litters. Considering prior findings of a proconvulsant effect of hyperglycemia in the electroshock-, the bicuculline- and the flurothyl seizure animal models (Koltai and Minker, 1975; Schwechter et al., 2003; Tutka et al., 1998), effect of diabetes on seizure proneness among the voles will be investigated. It has furthermore been observed that pre-natal stress in rats lead to increased levels of anxiety in adulthood (Valle´e et al., 1997), and in another rat model of epilepsy, pre-natal stress increased the rate of kindled seizure development in infant rats and adult males (Edwards et al., 2002). Other studies have

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shown persistent effects on hippocampus resulting from post-natal life stressors like maternal deprivation (Mirescu et al., 2004) and, again, that post-natal stress lowered the threshold for kindled seizures effecting female rats more than males (Salzberg et al., 2007). Stressors affecting captive born F1 female bank voles used in this study, both from captivity in general and the imposed company of a stereotyping male, could therefore be thought to influence female levels of glucocorticoids and other hormones, thereby affecting their unborn pups directly across the placental barrier. Post-natal stress from a Ster mother could likewise be thought to influence the pups negatively. It has in fact been noted how stereotypic females on occasion would engage in stereotypies with a pup in the mouth (Sørensen and Randrup, 1986) and it is not uncommon for a female exhibiting pacing to run for up to half an hour in the preferred path, seemingly indifferent to any pups that might be lying there (personal observation). Consequently the parental status as either a Ster or a N-Ster will be examined for effects on seizure proneness in the resultant offspring. As mentioned above wild caught voles from my first colony did not show any signs of seizures and the reason could be that proneness to develop seizures are dependant on the interaction of significant stressors/discrete stimuli from the captive environment during a sensitive period before adulthood. It is consequently predicted that a low level of seizures will be found among the wild caught voles in this study and, since both captive born generations have been housed similarly with random breeding, their level of seizures will be higher than that of the wild caught voles and not affected by generation. However, among the captive born bank voles, the expectation will be a relatively low level of seizures among offspring from NSter and a higher level among offspring from Ster due to the assumption that Ster mothers ‘‘stresses’’ pre-weaned pups the most. Lastly, this initial validation will investigate to what extent SP bank voles show refractoriness to postictal induction of further seizures (also known as postictal seizure protection), a phenomenon previously described in rats and mice (Fochtmann, 1998; Herberg and Rose, 1994). 2. Methods 2.1. Animals Three groups of voles are analyzed in this study: a wild caught parental (P) stock (N = 71) and the first two captive born generations (F1 and F2; N = 118 and 215 respectively). Wild caught voles were captured on the island of Zealand (Denmark) in late autumn, 2000, and the colony was kept in the laboratory as previously described (Schoenecker et al., 2000). The only difference in this study was that all pups were weaned at the age of 21 days instead of between 25 and 53 days. Briefly, the voles were kept under 12 h light conditions (0800–2000 h) and housed individually in small barren cages of transparent plastic (13.5 cm  16.0 cm  22.5 cm) supplied with a woodcutting bed. Maintenance and observations of the voles present in the colony at any given time typically

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lasted between 2 and 4 h/day and the voles were provided with free access to standard rat chow and water. Single mating pairs, involving only P-voles caught in different non-adjacent locations, were established in larger enriched cages (14.5 cm  21.5 cm  37.5 cm) supplied with a woodcutting bed, toilet paper, and paper rolls. Unrelated single mating pairs (no full- or half siblings) from F1 were chosen randomly and housed in larger enriched cages as described above. Breeding males were always returned to small barren cages immediately after it was observed that a female had given birth. A F3 (N = 131) was bred with the specific intend to investigate heritability of seizures (selective breeding for and against proneness to seizure) but due to logistic constraints, the entire colony had to be terminated when the cohort was 2–3 months old. At the time of termination, no F3 had showed signs of seizures and these voles are excluded from this study. Wild caught voles spend 1–253 days in captivity between capture and exit from the colony whereas their offspring were maintained in the colony for 45–837 days after birth (see Table 1A for additional information). ‘‘Exit’’ could be caused by a number of reasons: sudden death without obvious cause; euthanasia by CO2 sedation followed by cervical translocation; death as a result of accidents/fights or, for some of the wild caught, release to the exact place of capture. Animal care and use conformed to institutional policies and guidelines exercised by the University of Copenhagen, Denmark. 2.2. Classification Voles were classified as diabetic if their average daily water intake was in excess of 21 ml at least for a months (arbitrary defined as polydipsia) since previous studies had demonstrated a correlation between this definition of polydipsia, and measurements of glucosuria, hyperglycemia, hyperlipemia and destroyed beta cells (Freimanis et al., 2003; Niklasson et al., 2003; Schoenecker et al., 2000; Schønecker et al., submitted). If repetitive behaviours such as backward somersaults, high-speed jumping, pacing in circles or windscreen wiper movements was observed in bouts of at least 5 repetitions, the vole was classified as stereotypic (Ster) according to previous definitions (Schoenecker and Heller, 2000). Voles, which showed no signs of stereotypic behaviours were designated non-stereotypic (N-Ster). Voles that were observed having a seizure at least once were classified as seizure-prone (SP). Alternatively, voles were classified as seizure-resistant (SR). 2.3. Basic observations Observations included life-long measurements of average daily water intake (to screen for diabetes), onset ages of first observation of Ster and seizures. Additional basic observations included ages at deaths/exits, data concerning fecundity, breeding protocols etc. 2.4. Weighing protocol Weekly weight gain/losses were measured for the first 6 months after capture/weaning. The protocol involved

Table 1 Basic demographics (A) and ages at first observed seizures (B). Median and interquartile range (25; 50; 75) is used to describe lengths of captivity and age for first observation of seizures. Results of statistical comparisons are seen in (B). (A)

Captivity (days) n (M/F/Total) n litters n SP (M/F) % SP M % SP F % SP M + F

P

F1

F2

105; 113; 192 30/41/71 N.A. 0/0 0 0 0

140; 251; 274 61/57/118 19 6/6 9.8 10.5 10.2

454; 519; 560 100/115/215 41 6/5 6.0 4.3 5.1

(B)

M + F (days) M (days) F (days) n M/F p (age M vs. F)

F1

F2

F1 + F2

p (F1 vs. F2)

93; 150; 165 92; 109; 146 153; 165; 604 6/6 0.0538

168; 277; 435 250; 422; 436 133; 272; 330 6/5 0.4102

113; 157; 427 102; 152; 422 144; 173; 437 12/11 0.3718

0.0963 0.0163 0.8551

Note: Abbreviations used are P (wild caught voles), F1 and F2 (first and second generation born in captivity), SP (SP voles), M (male) and F (female).

lifting the vole from its home cage using a transparent jar of glass and placing the jar on an electronic scale. After approximately 30 s the weight had been noted and the vole returned to its home cage in the jar. Before next use, the jar was rinsed in cold water to remove any urine, faeces or dirt left by the former visitor. 2.5. Tests to investigate whether SP voles show some level of postictal seizure protection (‘‘PSP1’’, ‘‘PSP2’’ and ‘‘Follow up’’) All available SP voles (N = 13; n male/female (F1): 4/3; n male/female (F2): 2/4) participated in two postictal seizure protection tests (‘‘PSP1’’, ‘‘PSP2’’) and one ‘‘Follow-up’’ test. They had not been selected in any way, apart from their seizure proneness and availability, and ranged between 195 and 827 days of age at the first test-day (mean  S.D.: 593.2  191.5). The voles had been observed in seizures for 1–7 times (median = 3) prior to their first test, which took place between 62 and 735 days (mean  S.D.: 313.4  235.5 days) after the individual voles have had their first observed seizure. The three tests consisted of one daily provocation using a modified weighing protocol in which the vole stayed in the jar for up to 180 s before it was returned to the home cage. If a seizure began within 180 s, the time was noted with a standard stopwatch and the vole stayed in the jar until the seizures, subjectively assessed, ceased (typically after 15– 25 s). Then the vole was transferred to its home cage and time to resumption of normal behaviours was measured from the instant the vole touched the floor of the home cage. ‘‘Normal behaviour’’ could e.g. be upright position, uninhibited locomotion, normal attention to surroundings etc (see Sørensen (1987) for a thorough ethogram). The PSP1 test consisted of a daily attempt/vole to induce seizures on 5 consecutive days.

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The PSP2 test was carried out 2 months after PSP1 and involved five daily tests. However, due to a logistical problem the voles were not tested on day 2, so the five tests took place over a period of 6 days. The Follow-up test was a 1-day only test and was carried out 7 months after the PSP1 test. All tests took place between 16.30 and 17.00 and the total number of test was 143 (11 tests  13 voles). Only data from the first day in the three tests have been used to estimate latency to onset and resumption of normal behaviour (10 pairs of measurements) in order to counteract any influence from previous attempts to elicit seizures. The 13 voles produced 19 recordings in total of latency to seizure (range 0–7 recordings/vole) and 16 recordings of time to recover (range 0–5 recordings/vole). Evidently not all SP voles developed seizures every time they were placed in the jar; a minority started when the cage lid was removed – others after having jumped out of the jar. Any measurements/estimations of such events have been excluded since they either did not occur in the proper settings for these tests. 2.6. Statistics Data addressing weight, ages and measurements in relation to the lengths of seizures were first tested for normality with Kolmogorov–Smirnov one-sample tests and F-tests for even variances. Mean  S.D. were used to describe central tendencies if data were normally distributed (Method); otherwise medians and 25/75 quartiles were used (Table 1A and B). Since data did not meet the requirements for parametric testings, even after transformations, they were analyzed using the non-parametric Mann–Whitney U-tests for pair wise comparing (3.1; 3.2; Table 1B). Chi square tests (with Yates continuity correction factor when n < 20) were used in all pair wise (2  2) tests involving nominal data (3.2; 3.4; 3.5; 3.6 and 3.7). A one-group Chi square was used as Goodness of Fit test to see if the observed (Obs) frequencies of SP voles among the litters in F1, F2 and F1/F2 combined fitted a negative binomial distribution (3.4; Table 2). Expected (Exp) data derived from the negative binomial distribution was calculated using a HP 15C. Cells were merged in the Exp columns from the 4+ ends until no expected frequencies were below one. Correspondent columns (Obs) were merged in Table 2 Dispersion of seizure prone (SP) voles in the litters of the first (F1) and second (F2) generation of captive born bank voles. Observed (Obs) frequencies of litters containing 0, 1, 2, 3, 4 or more SP voles/litter in F1, F2 and F1/F2 combined are tabulated. Expected (Exp) values are derived from the negative binomial distribution, due to dispersion indexes (variance/mean) between 1.796 (F1) and 1.683 (F2). N SP/Litter

F1

F2

F1 + F2

Obs

Exp

Obs

Exp

Obs

Exp

0 1 2 3 4 4+

12 4 2 0 1 0

11.932 4.199 1.672 0.684 0.285 0.228

34 4 2 1 0 0

33.415 5.330 1.504 0.492 0.164 0.095

46 8 4 1 1 0

45.194 9.740 3.179 1.155 0.441 0.291

Total litters

19

19.000

41

41.000

60

60.000

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addition so matched pairs could be analyzed using the onegroup Chi square. That resulted in four pairs being compared in F1; three pairs in F2 and four pairs in F1/F2 combined. Significance level was a priori set at 0.05 and tests were twotailed and corrected for ties when needed (Siegel and Castellan, 1988). 3. Results 3.1. Description of a typical seizure A seizure typically commenced with jerks with the vibrissae, gradually increasing in magnitude, and/or in some cases repetitive eye blinking/squinting and ear twitching. The vole would then turn quickly in one or the other direction, arch the back with the neck bend backwards and start pawing in the air with both front legs in a rearing position, the mouth typically wide open. After approximately 5–10 s the eyes could start rolling, displaying on occasions only the sclera in the eyes. In other instances a sclera lining was visible around the entire iris. The vole then tilted onto its side and lay, twitching the two legs on the upper side rapidly. Mostly the convulsions ceased after 15–20 s after which time the vole was returned to its home cage. The vole typically lied motionless with almost imperceptible breathing when returned to the home cage. Often it began moving rather abruptly, with at sudden jump. In one vole, the sclera was still visible for 10 min after normal behaviour (e.g. upright position, uninhibited locomotion, normal attention to surroundings) had been resumed. The seizures seemed to irritate the eyes of some voles since they could spend minutes thereafter rubbing their eyes. Special ‘‘chirping’’ vocalisations and chattering of teeth could on some occasions precede/accompany a seizure. Median latency to seizures after placement in the glass jar was 12.6 s (25/75 quartiles = 10.5/28.6 s: n = 10) and median time for recovery and resumption of normal behaviour was 28.05 s (25/75 quartiles = 19.2/77.4 s; n = 10). There were neither significant sex (U  16; n1 = 6; n2 = 4; p  0.3938) or generation related (U  13; n1 = 7; n2 = 3; p  0.5688) differences in these measurements, nor were there any, subjectively assessed, sex/generation related differences in appearance of the seizures. 3.2. Incidence of seizures and age of first observation Table 1A shows that seizures were never observed among 71 wild caught voles subjected to the same treatment as the two laboratory born generations. Overall incidence of seizures was 10.2% (F1) declining to 5.1% in F2 (Chi = 3.026; d.f. = 1; p = 0.082). There were no sex differences concerning proneness to develop seizures or the ages at first observation of seizures in either F1 or F2, nor did the median age differ between the generations (statistical results presented in Table 1B). However, F2-males were observed in seizures significantly later than F1-males (U = 33; n1 = 6; n2 = 6; p = 0.0163) but no such differences were observed among females. The median age at first observation of seizures was 157 days (n = 23).

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3.3. Daily attempts to induce seizures indicative of refractory period Testing 13 SP voles once a day for 5 days (PSP1) resulted in provocation of seizures among 4, 2, 3, 0 and 1 of the tested voles respectively. The corresponding number of seizures induced among the same 13 SP voles during the second postictal seizure protection test (PSP2) was 2, 2, 1, 0, and 0 respectively. During a 1-day test follow-up test four of the 13 SP voles were provoked into a seizure. The fraction of seizures observed on the first test-day alone (39 tests resulting in a total of 10 seizures: 25.6%) appeared higher than the sum of seizures observed during test days 2–5 put together (104 tests resulting in 9 seizures: 8.7%). However, data were insufficient for a Friedman trend analysis regarding latencies to seizures or resumption of normal behaviours. 3.4. Clustering of SP voles in certain litters SP voles from F1, F2 and the generations combined (F1 + F2) occurred among the 60 litters in accordance with the negative binomial distribution (Table 2: Goodness of Fit: Chi  0.588; p  0.7452 in the three cases). The probability of a litter including one or more SP voles was 14/60 (F1 + F2: 23.3%) whereas the probability of finding two, or more, SP voles in a litter already including one SP vole was 6/14 (42.9%; Chi = 2.194; d.f. = 1; p = 0.1386). 3.5. Incidence of SP full/half siblings related to the SP and SR voles As seen in Table 3, full siblings with a history of seizures were present almost seven times more often among the 23 SP voles than among the 310 SR voles (65.2% vs. 9.7% respectively; Chi = 56.512; d.f. = 1; p = 0.0001). There were no significant differences in the number of SP half siblings to the voles in the SP and SR groups that had indeed half siblings (67% vs. 42% respectively; Chi = 2.717; d.f. = 1; p = 0.0993). 3.6. Parental proneness to engage in stereotypies (Ster) relates to offspring proneness to develop seizures All F1 (n = 118) in this study descended from two nonstereotyping (N-Ster) parents and of these offspring, twelve (10.2%) were classified as SP. Among F2, 63 voles descended from two N-Ster parents (n SP/% SP = 7/11.1%) Table 3 Presence (+) and absence ( ) of seizure prone (SP) full- and half siblings to members of the seizure prone (SP) voles and seizure-resistant (SR) voles subgroups of captive born bank voles. Voles, which did not have any half siblings, are designated (N/A). Data expressed as n voles (%).

(+) SP full siblings ( ) SP full siblings (+) SP half siblings ( ) SP half siblings (N/A) SP half siblings

SP voles (n = 23)

SR voles (n = 310)

15 8 8 4 11

30 280 55 76 179

(65.2) (34.8) (34.8) (17.4) (47.8)

(9.7) (90.3) (17.7) (24.5) (57.7)

and 138 were descendants from two Ster parents (n SP/% SP = 3/2.2%). The overall incidence of SP voles in these three lines differed significantly (Chi = 8.505; d.f. = 2; p = 0.0142) and while there was no statistically difference between F1 and F2 offspring from two N-Ster parents (Chi = 0.039; d.f. = 1; p = 0.8439), offspring in F2 from two Ster parents showed significant less proneness to develop seizures than offspring from two N-Ster parents in both F2 (Chi = 7.308; d.f. = 1; p = 0.0069) and F1 (Chi = 7.372; d.f. = 1; p = 0.0066). 3.7. Effect of diabetes and performance of Ster on seizure proneness Diabetic voles occurred with the statistically same frequency in both the SP (N = 23) and SR (N = 310) groups (n/% (SP vs. SR) = 6/26% vs. 101/33%; Chi = 0.414, d.f. = 1, p = 0.5199). Likewise, there seemed to be no effect of the performance of stereotypes on seizure proneness since Ster were found in equal frequencies in the two groups (n/% (SP vs. SR) = 14/61% vs. 170/55%: Chi = 0.315; d.f. = 1; p = 0.5746). 4. Discussion This report describes how laboratory born Danish bank voles can be induced into displaying seizures by mild handling. Without the aid of electrographic data, the nature of these seizures is at present unresolved; what can be said at this point in time is that the seizures observed ranged in severity from only mild jerks and twitching of vibrissae (stage 1–2 on Racine’s 5-point scale of seizures; Racine, 1972) to severe tonic/clonic convulsions, rearings, loss of postural control (stage 5) and, in some instances, accompanied by an apparent loss of consciousness. Ad hoc attempts to induce seizures by loud clap of hands in front of voles, which had previously experienced seizures, proved unsuccessful, paralleling the findings of Bronson and de la Rosa (1994), which showed that the observed seizures among American meadow voles (Microtus pennsylvanicus) could not be elicited by auditory provocations. If SP voles experienced daily attempts to provoke seizures for 5 days, the ratio of provoked seizures/tested voles on the first test-day (26%) appeared higher than the ratio for the next 4 test-days combined (9%) suggestive of a refractory period of at least a day. Furthermore, some bank voles showed a lowered threshold, or facilitation, after the initial seizure, which typically was observed as a result of cage change or during the weighing protocol. After 3–5 observed seizures it was sometimes possible to provoke a seizure in the home cage just by moving it. Available data could indicate that proneness to develop seizures is influenced by a genetic component given the significant clustering of SP voles in certain litters. SP full siblings were almost seven times more frequently encountered among voles from the SP cohort. Further (non-significant) support for this assumption comes from the observation that prior presence of a SP vole in a litter almost doubles the possibility of a second vole being prone

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to develop seizures. Consequently, the results obtained in this study suggest the possibility of establishing separate lines, differing in proneness to develop seizures. The above-mentioned study concerning American meadow voles (Bronson and de la Rosa, 1994) did observe seizures among some of the wild caught voles. However, in this study no wild caught bank voles developed seizures in sharp contrast to their captive born offspring. Seizures were likewise never observed among the 92 wild caught voles from my first colony, kept in captivity for a comparable period, nor were they noted among members of yet another Danish wild caught cohort, I maintained in captivity, albeit for a considerable shorter period of time (16.3  5.7 days; N = 86). Maybe this difference between wild caught and captive born voles regarding proneness to develop seizures represent a parallel to the development of stereotypies? As in the case of handling-induced seizures, captivityinduced stereotypies is practically never seen among wild caught voles (see e.g. Schoenecker et al., 2000; Sørensen and Randrup, 1986) and the proneness to develop these types of behaviours seem to some extent to be subject to genetic control both in bank voles (Schoenecker and Heller, 2000) and in other rodents, e.g. the African striped mouse (Schwaibold and Pillay, 2001). While an actual explanation of these differences in seizure proneness between wild caught and captive born bank voles is unknown at present, a first approach to investigate the issue could be to focus on a probable interaction between early pre- and post-natal environmental factors (social, behavioural and housing) and subsequent development of certain brain regions and neurological pathways of relevance to both seizures and stereotypies. As an example, both gamma-aminobutyric acid (GABA) and serotonin have been implicated in the development of seizures, stereotypies and mood- and anxiety disorders (Aliev and Kryzhanovskii, 1979; Griebel, 1995; Isaac, 2005; Kanner and Balabanov, 2002; Pilc and Nowak, 2005; Schoenecker and Heller, 2003; Wong et al., ¨ dberg and Meers, 1998). Furthermore, it was found 2003; O that increasing the level of serotonin in the human brain increases the level of GABA in the occipital cortex (Bhagwagar et al., 2004). As mentioned in the introduction, important differences between voles caught in the wild, and those bred in captivity is likely to be founded during the period of gestation and weaning. In addition to the possibility of prenatal stressors affecting the pups, there is not much doubt that bank voles descending from stereotypic females are subject to a post-natal treatment they would otherwise not encounter if born in the wild. Considering that it took 96 days on average for Danish voles to develop stereotypies (Schoenecker et al., 2000) after a life of isolation in small barren cages, combined with an estimated lifespan of 60– 100 days in the wild (obtained in a study based on wild living voles from a Polish island; Bujalska, 1975), it seems likely that practically no wild born pup will ever experience nursing from a stereotypic mother. Considering the above, one of the expectations of this study was that wild caught voles would exhibit the lowest proneness to develop seizures; laboratory born offspring from N-Ster would exhibit an intermediate proneness,

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and offspring from stereotypic parents the highest proneness. It was therefore contrary to expectations to find that offspring from Ster seemed to experience a factor five reduction in seizure proneness compared to offspring from N-Ster. A possible parallel to the paradoxical result from this study might be found in an earlier study focussing on effect of various types of stress (maternal separation and water immersion), on subsequent development of type 1 diabetes in bank voles (Freimanis et al., 2003). The most potent stressor of these two was subjecting pre-weaning voles to water immersion. Lowfrequency stress (one treatment/week until the pups were weaned at age 21 days) increased the incidence of type 1 diabetes in adulthood where high-frequency stress in the same period (one treatment/day until weaning) reduced the incidence markedly. It could be that offspring from a stereotypic mother in this study experiences severe stressors (e.g. irregular and/or reduced levels of nursing, exposure to cold, maybe even physical traumas) at a daily basis in their pre-weaning period. For adults the result is strongly decreased proneness to develop seizures, paralleling the impact of high-frequency stressors in the Freimanis et al. (2003) study on later proneness to develop type 1 diabetes. It was furthermore to be expected that the seizures observed among bank voles in this study to some degree would be affected by the voles’ classification as either diabetic, or non-diabetic since prior work with other seizure models had demonstrated proconvulsant effects of hyperglycemia (Koltai and Minker, 1975; Schwechter et al., 2003; Tutka et al., 1998). However, as evident from the results, diabetes and concomitant hyperglycemia seems to have no facilitating effect on the bank voles’ proneness to experience a seizure. Finally it should be noted that bank voles are wellknown reservoirs and vectors for viruses, some of which are zoonoses, and other suspected zoonoses (Niklasson et al., 2007; Olsson et al., 2003). Virus might be implicated in human febrile seizures too (Chung and Wong, 2007) and a significant number of voles from this particular colony had previously been tested positive for antibodies against the Ljungan virus (Niklasson et al., 2003). Several potential regions of sequence similarities and cross-reactivity between GAD65 autoantibodies and mouse or human anti-Ljungan virus antibodies have previously been demonstrated (Niklasson and Lernmark, 2003) so a proposal for an alternative hypothesis would be that a Ljungan-like virus to some extent could be implicated in the development of seizure proneness among these voles. The method of which could e.g. be an autoimmune reaction directed against GAD65 subsequent to infection with Ljungan virus (‘‘molecular mimicry’’) interacting with the captivity-induced stress, certain genes, sensitive periods for normal brain development in sub-adult voles and later deprivation of opportunities for normal interactions with conspecifics. In support of this hypothesis is the observation that GAD65 / mutant mice previously have been described to develop seizures (Kash et al., 1997), which correspond well with the multiple papers associating disturbances in GABAergic neurotransmission with human epilepsy (see e.g. Majewska, 1992).

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5. Conclusion The seizures observed in captive Danish bank voles could in most cases resemble generalised seizures with indications of a facilitative component and a refractory period following a seizure. Based on available evidence, hyperglycemia does not seem to be a part of the etiology. My data support the notion of a genetic component affecting proneness to develop seizures and stresses the importance of pre-weaning conditions. Acknowledgements I would like to thank Knud Erik Heller (University of Copenhagen, Department of Biology, Denmark) for providing the necessary logistics to house and maintain the colonies described in this paper. I would further like to thank Jan Ladewig and Bjo¨rn Forkman, both from the University of Copenhagen, Department of Large Animal Sciences, Denmark for their comments on early drafts and general support. The study was supported in part by funds from the Danish Natural Science Research Council, A˚ke Lernmark (University of Washington, USA) and Apodemus AB (Swedish Research company), neither of which have had any influence on either the study, analysis or interpretations/presentation. References Aliev, M.N., Kryzhanovskii, G.N., 1979. Experimental stereotypy induced by disturbance of GABA-ergic mechanisms in the caudate nuclei. Bull. Exp. Biol. Med. 87, 315–318. Bhagwagar, Z., Wylezinska, M., Taylor, M., Jezzard, P., Matthews, P.M., Cowen, P.J., 2004. Increased brain GABA concentrations following acute administration of a selective serotonin reuptake inhibitor. Am. J. Psychiatry 161, 368–370. Bronson, F.H., de la Rosa, J., 1994. Tonic-clonic convulsions in meadow voles. Physiol. Behav. 56, 683–685. Bujalska, G., 1975. Reproduction and mortality of bank voles and the changes in the size of an island population. Acta Theriol. (Warsz) 20, 41–56. Chung, B., Wong, V., 2007. Relationship between five common viruses and febrile seizure in children. Arch. Dis. Child. 92, 589–593. Edwards, H.E., Dortok, D., Tam, J., Won, D., Burnham, W.M., 2002. Prenatal stress alters seizure thresholds and the development of kindled seizures in infant and adult rats. Horm. Behav. 42, 437–447. Griebel, G., 1995. 5-Hydroxytryptamine-interacting drugs in animal models of anxiety disorders: more than 30 years of research. Pharmacol. Ther. 65, 319–395. Fochtmann, L.J., 1998. Genetic approaches to the neurobiology of electroconvulsive therapy. J. ECT 14, 206–219. Freimanis, T., Heller, K.E., Schønecker, B., Bildsøe, M., 2003. Effects of postnatal stress on the development of type 1 diabetes in bank voles (Clethrionomys glareolus). Int. J. Exp. Diabesity Res. 4, 21–25. Herberg, L.J., Rose, I.C., 1994. Kindled epileptic seizures, postictal refractoriness, status epilepticus, and electrical self-stimulation. Neurosci. Biobehav. Rev. 18, 411–420. Isaac, M., 2005. Serotonergic 5-HT2C receptors as a potential therapeutic target for the design antiepileptic drugs. Curr. Top. Med. Chem. 5, 59–67. Kanner, A.M., Balabanov, A., 2002. Depression and epilepsy: how closely related are they? Neurology 58, 27–39. Kash, S.F., Johnson, R.S., Tecott, L.H., Noebels, J.L., Mayfield, R.D., Hanahan, D., Baekkeskov, S., 1997. Epilepsy in mice deficient in the 65-kDa isoform of glutamic acid decarboxylase. Proc. Natl. Acad. Sci. U.S.A. 94, 14060–14065. Koltai, M., Minker, E., 1975. Changes of electro-shock seizure threshold in alloxan diabetic rats. Experientia 31, 1369.

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