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Mouse strain variation in maximal electroshock seizure threshold
Brain Research 936 (2002) 82–86 www.elsevier.com / locate / bres
Research report
Mouse strain variation in maximal electroshock seizure threshold Thomas N. Ferraro a , *, Gregory T. Golden a,b , George G. Smith a,b , Denis DeMuth a,b , Russell J. Buono a , Wade H. Berrettini a a
Center for Neurobiology and Behavior, Department of Psychiatry, University of Pennsylvania, 415 Curie Blvd., Philadelphia, PA 19104 -6140, USA b Department of Veteran’ s Affairs Medical Center, Coatesville, PA 19320, USA Accepted 15 January 2002
Abstract Maximal electroshock seizure threshold (MEST) is a classical measure of seizure sensitivity with a wide range of experimental applications. We determined MEST in nine inbred mouse strains and one congenic strain using a procedure in which mice are given one shock per day with an incremental (1 mA) current increase in each successive trial until a maximal seizure (tonic hindlimb extension) is elicited. C57BL / 6J and DBA / 2J mice exhibited the highest and lowest MEST, respectively, with the values of other strains falling between these two extremes. The relative rank order of MEST values by inbred strain (highest to lowest) is as follows: C57BL / 6J.CBA / J5C3H / HeJ.A / J.Balb / cJ5129 / SvIMJ5129 / SvJ.AKR / J.DBA / 2J. Results of experiments involving a single electroconvulsive shock given to separate groups of mice at different current intensities suggest that determination of MEST by the method used is not affected by repeated sub-maximal seizures. Overall, results document a distinctive mouse strain distribution pattern for MEST. Additionally, low within strain variability suggests that environmental factors which affect quantification of MEST are readily controlled under the conditions of this study. We conclude that MEST represents a useful tool for dissecting the multifactorial nature of seizure sensitivity in mice. 2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Epilepsy: human studies and animal models Keywords: Electroshock seizure; Mouse; Complex trait; Strain difference
1. Introduction Characterization of specific traits among inbred strains of mice serves as a foundation to facilitate identification of underlying genetic influences. Historically, complex (polygenic) traits have been less amenable to classical genetic studies compared to Mendelian traits [24]. An important reason for this discrepancy is the relatively smaller individual influences of genes involved in complex trait determination and the great variation evident in the traits themselves, much of which is due to environmental factors. Thus, the very nature of multifactorial traits compromises the precision with which they can be quantified [12] and hinders their genetic dissection. Studies of mouse strain differences in seizure suscep*Corresponding author. Tel.: 11-215-573-4581; fax: 11-215-5732041. E-mail address: [email protected] (T.N. Ferraro).
tibility have focused primarily on responses to the administration of various pharmacological agents. Classical chemoconvulsants such as pentylenetetrazol, kainic acid, and strychnine have been examined [17,22] as well as less standard drugs [19,25]. Strain surveys have been limited in breadth; however, a few strains have been common to a number of studies. In particular, DBA / 2 and C57BL / 6 mice have been well-studied with regard to their differential susceptibility to chemically-elicited seizures [3,10,13,15] and generally are regarded as seizure sensitive and resistant, respectively. In addition to pharmacological means, seizures can be elicited through various physical methods including those involving vestibular stimuli [21], auditory stimuli [16,18] and progressive compression in high oxygen environments [20]. Of the physical stimuli used to induce experimental seizures to date, electroconvulsive shock (ECS) may be the most common. Although ECS was introduced decades ago [27], it is still recognized as a valid and reliable means of
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )02565-9
T.N. Ferraro et al. / Brain Research 936 (2002) 82 – 86
studying seizure mechanisms, particularly those related to genetics [11] as well as those relevant to the development of anticonvulsant drugs [29]. The present study was undertaken to provide a foundation of baseline information for future studies on the genetic influences involved in determining electrical seizure thresholds and response to anticonvulsant drugs. Results of the strain survey are in general agreement with those from a recent study [11], including characterization of DBA / 2 and C57BL / 6 as prototypical seizure sensitive and resistant strains, respectively [3,8,11].
2. Material and methods
2.1. Animals Inbred strains of mice including 129 / SvIMJ, 129 / SvJ, A / J, AKR / J, BALB / cJ, C3H / HeJ, C57BL / 6J, CBA / J, and DBA / 2J as well as the congenic strain B6.D2MTV7a / Ty were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice (male, 4–6 weeks of age at time of arrival in the laboratory) were housed three or four per cage and maintained on a 12-h light, 12-h dark schedule. Food and water were freely available throughout the course of the study. Following their arrival to the laboratory from the vendor, mice were housed for at least 2 weeks prior to the initiation of seizure tests. Experiments were approved by Animal Care and Use Committees overseeing each participating laboratory.
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mA with each successive daily trial until a maximal seizure was observed. The initial current level for C57BL / 6J mice was 40 mA since previous work had shown this strain to have a relatively high MEST [8]. Seizures were elicited at all current intensities used with lower intensities producing facial and forelimb clonus and higher intensities producing generalized seizures. The sequence of events which defines a maximal seizure response involves tonic forelimb flexion, tonic hindlimb flexion, tonic hindlimb extension and hindlimb clonus. Mice were euthanized by cervical dislocation under CO 2 anesthesia immediately after the session during which a maximal seizure was elicited. In experiment 2, DBA / 2J and C57BL / 6J mice were given a single shock at a specific current level between 15 and 75 mA (n55–10 mice per setting) and scored for the presence or absence of a maximal seizure. For all seizure tests, the ECS apparatus was equipped with earclip electrodes and the pulse train generator was used in a square-wave mode. Shocks were delivered at constant current with a frequency of 60 Hz, a pulse width of 0.4 ms and a duration of 0.2 s.
2.3. Data analysis For experiment 1, inbred strain MEST values were taken as the arithmetic mean and were evaluated using one-factor ANOVA. Between-group differences were evaluated using Newman–Keuls post-hoc test. For experiment 2, the relationship between current intensity and induction of a maximal seizure was analyzed by linear regression and calculation of Pearson’s correlation coefficient.
2.2. Seizure testing In experiment 1, maximal electroshock seizure threshold (MEST) was determined as described previously [8] using a constant current electroshock unit (model [7801, Ugo Basile, Varese, Italy). Mice (n58–12 per strain) were tested once per day beginning at age 8–12 weeks. Initially, current level was set at 20 mA and it was increased by 1
3. Results Results of the strain survey for MEST are shown in Table 1. There is a wide range of MEST values observed among the 10 strains tested and a highly significant strain effect is documented (F5297, P,10 250 ). MEST values
Table 1 Maximal electroshock seizure threshold values for inbred mouse strains Strain
Mean a
S.D.b
N
Confidence interval overlap c
DBA / 2J AKR / J B6.D2-MTV7a / Ty 129 / SvIMJ 129 / SvJ BALB / cJ A/J C3H / HeJ CBA / J C57BL / 6J
24.4 33.5 37.9 39.5 40.7 41.0 47.4 56.1 57.2 72.2
2.0 3.3 3.1 3.5 2.7 4.2 4.6 3.8 4.4 5.5
12 8 8 11 12 9 8 9 10 12
None MTV7a / Ty AKR / J, 129 / SvJ, 129 / SvIMJ, Balb / cJ MTV7a / Ty, 129 / SvJ, Balb / cJ MTV7a / Ty, 129 / SvIMJ, Balb / cJ 129 / SvIMJ, 129 / SvJ None CBA / J C3H / HeJ None
a
Mean MEST values are given in mA and were determined as described in Section 2.2 for experiment 1. Standard deviation of the mean. c ANOVA with Newman–Keuls post-hoc test. One-way ANOVA for strain effect: F5297; P,10 250 . Confidence interval overlaps were evaluated at the P,0.01 level. b
T.N. Ferraro et al. / Brain Research 936 (2002) 82 – 86
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Table 2 Maximal seizure response to a single electroshock in DBA / 2J and C57BL / 6J mice: effect of current intensity Current (mA)
DBA / 2J (n)
C57BL / 6J (n)
15 20 25 30 35 40 45 50 55 60 65 70 75
0 (5) 0 (10) 0.6 (10) 1.0 (5) – – – – – – – – –
– – – 0 0 (10) 0.1 (10) 0.2 (10) 0.2 (10) 0.2 (10) 0.4 (10) 0.2 (5) 0.4 (5) 1.0 (5)
Mice (male age 8–12 weeks) were given a single electroshock at the indicated current level. Values represent the fraction of the group responding with a maximal seizure (tonic hindlimb extension). Numbers in parentheses indicate the number of mice tested. Correlation between current and fraction with maximal seizure: DBA / 2J: r50.95, n54, P50.05; C57BL / 6J: r50.80, n59, P50.01.
for DBA / 2J mice are significantly lower than all other strains tested whereas values for C57BL / 6J mice are significantly higher. There are several strains in the intermediate range that form subgroups having similar MEST values as indicated by overlap of confidence intervals. Thus, the two 129 substrains and the BALB / cJ strain have nearly identical values as do the C3H / HeJ and CBA / J strains. Results of experiment 2 are shown in Table 2 and demonstrate the relationship between current intensity and induction of a maximal seizure. Their is a statistically significant correlation both for DBA / 2J (r50.95, P50.05, n54) and C57BL / 6J (r50.80, P50.01, n59) strains. Additionally, there is a close correspondence between the MEST determined for DBA / 2J and C57BL / 6J mice in experiment 1 (Table 1) and the current intensity required to elicit a maximal seizure in the majority of mice tested with a single electroshock (Table 2).
4. Discussion We have used a classical electroshock seizure test involving stepwise increments of electrical current [27] to establish a mouse strain-specific distribution pattern for MEST. Of the nine common inbred strains studied, C57BL / 6J and DBA / 2J exhibited the highest and lowest thresholds, respectively. These data confirm previous reports on the relative sensitivity of these two strains to electrically-induced seizures [3,8,11] and parallel previous reports on their relative sensitivity to pharmacologicallyinduced seizures [3,10,13]. Quantitative trait loci (QTL) mapping studies indicate that a genetic locus on distal chromosome 1 explains a large part of the difference
between C57BL / 6 and DBA / 2 mice in susceptibility to seizures induced by both chemical [7,9] and electrical [6] stimuli. This hypothesis is supported by the relatively low MEST value exhibited by congenic B6.D2-Mtv7a mice (Table 1) which have a DBA / 2-derived segment of distal chromosome 1 introgressed on a C57BL / 6 genetic background [6,26]. In spite of significant differences in methodological approaches, comparison of strain survey results reported here with those from a recent study [11] reveal considerable correspondence regarding strain-specific maximal seizure responses. Not surprisingly, C57BL / 6 and DBA / 2 strains represent the most extreme responding strains in both studies. Additionally, both studies find that AKR / J and 129 / SvIMJ strains have a relatively low threshold for maximal seizure and C3H / HeJ and CBA / J strains have a relatively high threshold. Notable differences between the studies involve the responses of BALB and A / J mice. These two strains are characterized by relatively low MEST values in the present study but are reported as relatively resistant to maximal seizures by Frankel et al. [11]. One factor which may contribute to this discrepancy, at least regarding BALB mice, is the fact that the substrains used in the two studies are different. We used the BALB / cJ strain whereas Frankel et al. [11] used BALB / cByJ. Just as likely, discordance between studies in responses of A / J (as well as BALB) mice may be related to differences in the way the electroshock stimulus was administered since we used auricular (earclip) electrodes whereas Frankel et al. [11] used corneal electrodes. Previous work suggests that transauricular stimulation preferentially activates brainstem structures whereas transcorneal stimulation preferentially activates forebrain structures [2], the former being more relevant to induction of tonic seizures [1]. Another major difference between studies is that whereas Frankel et al. determined strainspecific seizure thresholds by testing groups of animals at different current intensities (every animal tested only once) [11], we used a repeated-seizure testing paradigm. Thus, in spite of significant differences in procedure, there is overall good correspondence between our results and those of Frankel et al. [11] regarding relative MEST among inbred strains of mice. Ultimately, the most likely explanation for the modest differences in strain response between our study and that of Frankel et al. [11] may involve simple undefined factors related to the unique housing and laboratory environments and experimental manipulations. Related to this, even within the Frankel et al. study itself in which seven inbred strains were tested in two different laboratory sites that were carefully matched with regard to experimental protocol, there was one strain, 129S3 / SvImJ, which showed a significantly different rank order [11]. Previous reports have shown that the effect of repeated seizure activity on seizure threshold is subject to the influence of various experimental factors including the method of inducing seizures, the time course of seizure induction and the total number of seizures induced, to
T.N. Ferraro et al. / Brain Research 936 (2002) 82 – 86
name several. In general, however, there appears to be a biphasic effect of seizure activity on seizure threshold with a short-term (several hours) increase [4] and a long-term decrease [5,28,30]. The latter effect is consistent with a ‘kindling’ phenomenon in which a constant daily subconvulsive electrical (or chemical) stimulus results in the development of a seizure over time [14]. In contrast, there is some evidence to support the concept that repeated electroconvulsive shock increases seizure threshold in humans [23]. In order to investigate the possibility that the relatively high MEST of the C57BL / 6J strain determined in experiment 1 is related to the greater number of generalized (but sub-maximal) seizures to which mice from this strain are subjected during the threshold test, we treated groups of C57BL / 6J mice with a single, variableintensity electroshock in experiment 2. Results documented a significant correlation between current level and the presence of maximal seizure (Table 2) with less than 20% of mice exhibiting a maximal seizure more than two standard deviations below the group mean suggesting that the relatively high mean MEST for C57BL / 6J mice determined in experiment 1 is not an artifact of the repeated-seizure testing paradigm. A similar correspondence between experiments using repeated or single electroshock testing was seen for the DBA / 2J strain such that all mice tested in the single electroshock paradigm experienced a seizure at a current intensity within 5 mA of the group mean MEST as determined in experiment 1 (Table 2). In experiment 2, the observation that a small number of C57BL / 6J mice exhibited a maximal seizure at a current level many standard deviations below the group mean MEST suggests that there may be a subtle environmental interaction between repeated sub-maximal seizure activity and MEST. In summary, we have established a strain-specific distribution pattern for MEST using a series of common inbred mouse strains. The relatively wide range of responses between strains and the extent to which these responses clearly distinguish several subgroups of strains suggests a rich resource for mapping and identifying genes that control seizure threshold. The low within strain variability associated with quantification of MEST reflects minimal uncontrolled environmental influence under the conditions used in this study and enhances the potential utility of this seizure-related parameter as an end-point for future QTL and gene mapping experiments.
Acknowledgements This work was supported by NIH award NS39516.
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[2] R.A. Browning, D.K. Nelson, Variation in threshold and pattern of electroshock-induced seizures in rats depending on site of stimulation, Life Sci. 37 (1985) 2205–2211. [3] F.L. Engstrom, D.M. Woodbury, Seizure susceptibility in DBA and C57 mice: the effects of various convulsants, Epilepsia 29 (1988) 389–395. [4] C.F. Essig, Frequency of repeated electroconvulsions and the acquisition rate of a tolerance-like response, Exp. Neurol. 25 (1969) 571–574. [5] R.J. Ferland, C.D. Applegate, Decreased brainstem seizure thresholds and facilitated seizure propagation in mice exposed to repeated flurothyl-induced generalized forebrain seizures, Epilepsy Res. 30 (1998) 49–62. [6] T.N. Ferraro, G.T. Golden, G.G. Smith, R.L. Longman, R.L. Snyder, D. DeMuth, I. Szpilzak, N. Mulholland, E. Eng, F.W. Lohoff, R.J. Buono, W.H. Berrettini, Quantitative genetic study of maximal electroshock seizure threshold in mice: evidence for a major seizure susceptibility locus on distal chromosome 1, Genomics 75 (2001) 35–42. [7] T.N. Ferraro, G.T. Golden, G.G. Smith, P. St Jean, N.J. Schork, N. Mulholland, C. Ballas, J. Schill, R.J. Buono, W.H. Berrettini, Mapping loci for pentylenetetrazol-induced seizure susceptibility in mice, J. Neurosci. 19 (1999) 6733–6739. [8] T.N. Ferraro, G.T. Golden, R. Snyder, M. Laibinis, G.G. Smith, R.J. Buono, W.H. Berrettini, Genetic influences on electrical seizure threshold, Brain Res. 813 (1998) 207–210. [9] T.N. Ferraro, G.T. Golden, G.G. Smith, N.J. Schork, P. St Jean, C. Ballas, H. Choi, W.H. Berrettini, Mapping murine loci for seizure response to kainic acid, Mamm. Genome 8 (1997) 200–208. [10] T.N. Ferraro, G.T. Golden, G.G. Smith, W.H. Berrettini, Differential susceptibility to seizures induced by systemic kainic acid treatment in mature DBA / 2J and C57BL / 6J mice, Epilepsia 36 (1995) 301– 307. [11] W.N. Frankel, L. Taylor, B. Beyer, B.L. Tempel, H.S. White, Electroconvulsive thresholds of inbred mouse strains, Genomics 74 (2001) 306–312. [12] W.N. Frankel, Taking stock of complex trait genetics in mice, Trends Genet. 11 (1995) 471–477. [13] R.K. Freund, R.J. Marley, J.M. Wehner, Differential sensitivity to bicuculline in three inbred mouse strains, Brain Res. Bull. 18 (1986) 657–662. [14] G.V. Goddard, D.C. McIntyre, C.K. Leech, A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol. 25 (1969) 295–330. [15] G.T. Golden, T.N. Ferraro, G.G. Smith, R.L. Snyder, N.L. Jones, W.H. Berrettini, Acute cocaine-induced seizures: differential sensitivity of six inbred mouse strains, Neuropsychopharmacology 24 (2001) 291–299. [16] C.S. Hall, Genetic differences in fatal audiogenic seizures between two inbred strains of house mouse, J. Hered. 38 (1947) 2–6. [17] A.E. Kosobud, J.C. Crabbe, Genetic correlations among inbred strain sensitivities to convulsions induced by 9 convulsant drugs, Brain Res. 526 (1990) 8–16. [18] B. Martin, C. Marchaland, J. Phillips, G. Chapouthier, C. Spach, R. Motta, Recombinant congenic strains of mice from B10.D2 and DBA / 2: their contribution to behavior genetic research and application to audiogenic seizures, Behav. Genet. 22 (1992) 685–701. [19] B. Martin, C. Desforges, G. Chapouthier, Comparisons between patterns of convulsions induced by two beta-carbolines in 10 inbred strains of mice, Neurosci. Lett. 133 (1991) 73–76. [20] R.D. McCall, D. Frierson, Evidence that two loci predominantly determine the difference in susceptibility to the high pressure neurologic syndrome type I seizure in mice, Genetics 99 (1981) 285–307. [21] M.L. Rise, W.N. Frankel, J.M. Coffin, T.N. Seyfried, Genes for epilepsy mapped in the mouse, Science 253 (1991) 669–673. [22] K. Schlesinger, W.O. Boggan, B.J. Griek, Pharmacogenetic correlates of pentylenetetrazol and electroconvulsive seizure thresholds in mice, Psychopharmacologia 13 (1968) 181–188.
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[23] A.I. Scott, H. Boddy, The effect of repeated bilateral electroconvulsive therapy on seizure threshold, J. ECT 16 (2000) 244–251. [24] S.D. Tanksley, Mapping polygenes, Annu. Rev. Genet. 27 (1993) 205–233. [25] B.A. Taylor, Genetic analysis of susceptibility to isoniazid-induced seizures in mice, Genetics 83 (1976) 373–377. [26] B.A. Taylor, W.N. Frankel, A new strain congenic for the Mtv-7 / Mls-1 locus of mouse chromosome 1, Immunogenetics 38 (1993) 235–237. [27] J.E.P. Toman, E.A. Swinyard, L.S. Goodman, Properties of maximal seizures and their alteration by anticonvulsant drugs and other agents, J. Neurophysiol. 9 (1946) 231–239.
[28] K.H. Tress, L.J. Herberg, Permanent reduction in seizure threshold resulting from repeated electrical stimulation, Exp. Neurol. 37 (1972) 347–359. [29] H.S. White, S.D. Brown, J.H. Woodhead, G.A. Skeen, H.H. Wolf, Topiramate enhances GABA-mediated chloride flux and GABAevoked chloride currents in murine brain neurons and increases seizure threshold, Epilepsy Res. 28 (1997) 167–179. [30] B.Y. Wong, S.L. Moshe, Mutual interactions between repeated flurothyl convulsions and electrical kindling, Epilepsy Res. 1 (1987) 159–164.
Research report
Mouse strain variation in maximal electroshock seizure threshold Thomas N. Ferraro a , *, Gregory T. Golden a,b , George G. Smith a,b , Denis DeMuth a,b , Russell J. Buono a , Wade H. Berrettini a a
Center for Neurobiology and Behavior, Department of Psychiatry, University of Pennsylvania, 415 Curie Blvd., Philadelphia, PA 19104 -6140, USA b Department of Veteran’ s Affairs Medical Center, Coatesville, PA 19320, USA Accepted 15 January 2002
Abstract Maximal electroshock seizure threshold (MEST) is a classical measure of seizure sensitivity with a wide range of experimental applications. We determined MEST in nine inbred mouse strains and one congenic strain using a procedure in which mice are given one shock per day with an incremental (1 mA) current increase in each successive trial until a maximal seizure (tonic hindlimb extension) is elicited. C57BL / 6J and DBA / 2J mice exhibited the highest and lowest MEST, respectively, with the values of other strains falling between these two extremes. The relative rank order of MEST values by inbred strain (highest to lowest) is as follows: C57BL / 6J.CBA / J5C3H / HeJ.A / J.Balb / cJ5129 / SvIMJ5129 / SvJ.AKR / J.DBA / 2J. Results of experiments involving a single electroconvulsive shock given to separate groups of mice at different current intensities suggest that determination of MEST by the method used is not affected by repeated sub-maximal seizures. Overall, results document a distinctive mouse strain distribution pattern for MEST. Additionally, low within strain variability suggests that environmental factors which affect quantification of MEST are readily controlled under the conditions of this study. We conclude that MEST represents a useful tool for dissecting the multifactorial nature of seizure sensitivity in mice. 2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Epilepsy: human studies and animal models Keywords: Electroshock seizure; Mouse; Complex trait; Strain difference
1. Introduction Characterization of specific traits among inbred strains of mice serves as a foundation to facilitate identification of underlying genetic influences. Historically, complex (polygenic) traits have been less amenable to classical genetic studies compared to Mendelian traits [24]. An important reason for this discrepancy is the relatively smaller individual influences of genes involved in complex trait determination and the great variation evident in the traits themselves, much of which is due to environmental factors. Thus, the very nature of multifactorial traits compromises the precision with which they can be quantified [12] and hinders their genetic dissection. Studies of mouse strain differences in seizure suscep*Corresponding author. Tel.: 11-215-573-4581; fax: 11-215-5732041. E-mail address: [email protected] (T.N. Ferraro).
tibility have focused primarily on responses to the administration of various pharmacological agents. Classical chemoconvulsants such as pentylenetetrazol, kainic acid, and strychnine have been examined [17,22] as well as less standard drugs [19,25]. Strain surveys have been limited in breadth; however, a few strains have been common to a number of studies. In particular, DBA / 2 and C57BL / 6 mice have been well-studied with regard to their differential susceptibility to chemically-elicited seizures [3,10,13,15] and generally are regarded as seizure sensitive and resistant, respectively. In addition to pharmacological means, seizures can be elicited through various physical methods including those involving vestibular stimuli [21], auditory stimuli [16,18] and progressive compression in high oxygen environments [20]. Of the physical stimuli used to induce experimental seizures to date, electroconvulsive shock (ECS) may be the most common. Although ECS was introduced decades ago [27], it is still recognized as a valid and reliable means of
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )02565-9
T.N. Ferraro et al. / Brain Research 936 (2002) 82 – 86
studying seizure mechanisms, particularly those related to genetics [11] as well as those relevant to the development of anticonvulsant drugs [29]. The present study was undertaken to provide a foundation of baseline information for future studies on the genetic influences involved in determining electrical seizure thresholds and response to anticonvulsant drugs. Results of the strain survey are in general agreement with those from a recent study [11], including characterization of DBA / 2 and C57BL / 6 as prototypical seizure sensitive and resistant strains, respectively [3,8,11].
2. Material and methods
2.1. Animals Inbred strains of mice including 129 / SvIMJ, 129 / SvJ, A / J, AKR / J, BALB / cJ, C3H / HeJ, C57BL / 6J, CBA / J, and DBA / 2J as well as the congenic strain B6.D2MTV7a / Ty were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice (male, 4–6 weeks of age at time of arrival in the laboratory) were housed three or four per cage and maintained on a 12-h light, 12-h dark schedule. Food and water were freely available throughout the course of the study. Following their arrival to the laboratory from the vendor, mice were housed for at least 2 weeks prior to the initiation of seizure tests. Experiments were approved by Animal Care and Use Committees overseeing each participating laboratory.
83
mA with each successive daily trial until a maximal seizure was observed. The initial current level for C57BL / 6J mice was 40 mA since previous work had shown this strain to have a relatively high MEST [8]. Seizures were elicited at all current intensities used with lower intensities producing facial and forelimb clonus and higher intensities producing generalized seizures. The sequence of events which defines a maximal seizure response involves tonic forelimb flexion, tonic hindlimb flexion, tonic hindlimb extension and hindlimb clonus. Mice were euthanized by cervical dislocation under CO 2 anesthesia immediately after the session during which a maximal seizure was elicited. In experiment 2, DBA / 2J and C57BL / 6J mice were given a single shock at a specific current level between 15 and 75 mA (n55–10 mice per setting) and scored for the presence or absence of a maximal seizure. For all seizure tests, the ECS apparatus was equipped with earclip electrodes and the pulse train generator was used in a square-wave mode. Shocks were delivered at constant current with a frequency of 60 Hz, a pulse width of 0.4 ms and a duration of 0.2 s.
2.3. Data analysis For experiment 1, inbred strain MEST values were taken as the arithmetic mean and were evaluated using one-factor ANOVA. Between-group differences were evaluated using Newman–Keuls post-hoc test. For experiment 2, the relationship between current intensity and induction of a maximal seizure was analyzed by linear regression and calculation of Pearson’s correlation coefficient.
2.2. Seizure testing In experiment 1, maximal electroshock seizure threshold (MEST) was determined as described previously [8] using a constant current electroshock unit (model [7801, Ugo Basile, Varese, Italy). Mice (n58–12 per strain) were tested once per day beginning at age 8–12 weeks. Initially, current level was set at 20 mA and it was increased by 1
3. Results Results of the strain survey for MEST are shown in Table 1. There is a wide range of MEST values observed among the 10 strains tested and a highly significant strain effect is documented (F5297, P,10 250 ). MEST values
Table 1 Maximal electroshock seizure threshold values for inbred mouse strains Strain
Mean a
S.D.b
N
Confidence interval overlap c
DBA / 2J AKR / J B6.D2-MTV7a / Ty 129 / SvIMJ 129 / SvJ BALB / cJ A/J C3H / HeJ CBA / J C57BL / 6J
24.4 33.5 37.9 39.5 40.7 41.0 47.4 56.1 57.2 72.2
2.0 3.3 3.1 3.5 2.7 4.2 4.6 3.8 4.4 5.5
12 8 8 11 12 9 8 9 10 12
None MTV7a / Ty AKR / J, 129 / SvJ, 129 / SvIMJ, Balb / cJ MTV7a / Ty, 129 / SvJ, Balb / cJ MTV7a / Ty, 129 / SvIMJ, Balb / cJ 129 / SvIMJ, 129 / SvJ None CBA / J C3H / HeJ None
a
Mean MEST values are given in mA and were determined as described in Section 2.2 for experiment 1. Standard deviation of the mean. c ANOVA with Newman–Keuls post-hoc test. One-way ANOVA for strain effect: F5297; P,10 250 . Confidence interval overlaps were evaluated at the P,0.01 level. b
T.N. Ferraro et al. / Brain Research 936 (2002) 82 – 86
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Table 2 Maximal seizure response to a single electroshock in DBA / 2J and C57BL / 6J mice: effect of current intensity Current (mA)
DBA / 2J (n)
C57BL / 6J (n)
15 20 25 30 35 40 45 50 55 60 65 70 75
0 (5) 0 (10) 0.6 (10) 1.0 (5) – – – – – – – – –
– – – 0 0 (10) 0.1 (10) 0.2 (10) 0.2 (10) 0.2 (10) 0.4 (10) 0.2 (5) 0.4 (5) 1.0 (5)
Mice (male age 8–12 weeks) were given a single electroshock at the indicated current level. Values represent the fraction of the group responding with a maximal seizure (tonic hindlimb extension). Numbers in parentheses indicate the number of mice tested. Correlation between current and fraction with maximal seizure: DBA / 2J: r50.95, n54, P50.05; C57BL / 6J: r50.80, n59, P50.01.
for DBA / 2J mice are significantly lower than all other strains tested whereas values for C57BL / 6J mice are significantly higher. There are several strains in the intermediate range that form subgroups having similar MEST values as indicated by overlap of confidence intervals. Thus, the two 129 substrains and the BALB / cJ strain have nearly identical values as do the C3H / HeJ and CBA / J strains. Results of experiment 2 are shown in Table 2 and demonstrate the relationship between current intensity and induction of a maximal seizure. Their is a statistically significant correlation both for DBA / 2J (r50.95, P50.05, n54) and C57BL / 6J (r50.80, P50.01, n59) strains. Additionally, there is a close correspondence between the MEST determined for DBA / 2J and C57BL / 6J mice in experiment 1 (Table 1) and the current intensity required to elicit a maximal seizure in the majority of mice tested with a single electroshock (Table 2).
4. Discussion We have used a classical electroshock seizure test involving stepwise increments of electrical current [27] to establish a mouse strain-specific distribution pattern for MEST. Of the nine common inbred strains studied, C57BL / 6J and DBA / 2J exhibited the highest and lowest thresholds, respectively. These data confirm previous reports on the relative sensitivity of these two strains to electrically-induced seizures [3,8,11] and parallel previous reports on their relative sensitivity to pharmacologicallyinduced seizures [3,10,13]. Quantitative trait loci (QTL) mapping studies indicate that a genetic locus on distal chromosome 1 explains a large part of the difference
between C57BL / 6 and DBA / 2 mice in susceptibility to seizures induced by both chemical [7,9] and electrical [6] stimuli. This hypothesis is supported by the relatively low MEST value exhibited by congenic B6.D2-Mtv7a mice (Table 1) which have a DBA / 2-derived segment of distal chromosome 1 introgressed on a C57BL / 6 genetic background [6,26]. In spite of significant differences in methodological approaches, comparison of strain survey results reported here with those from a recent study [11] reveal considerable correspondence regarding strain-specific maximal seizure responses. Not surprisingly, C57BL / 6 and DBA / 2 strains represent the most extreme responding strains in both studies. Additionally, both studies find that AKR / J and 129 / SvIMJ strains have a relatively low threshold for maximal seizure and C3H / HeJ and CBA / J strains have a relatively high threshold. Notable differences between the studies involve the responses of BALB and A / J mice. These two strains are characterized by relatively low MEST values in the present study but are reported as relatively resistant to maximal seizures by Frankel et al. [11]. One factor which may contribute to this discrepancy, at least regarding BALB mice, is the fact that the substrains used in the two studies are different. We used the BALB / cJ strain whereas Frankel et al. [11] used BALB / cByJ. Just as likely, discordance between studies in responses of A / J (as well as BALB) mice may be related to differences in the way the electroshock stimulus was administered since we used auricular (earclip) electrodes whereas Frankel et al. [11] used corneal electrodes. Previous work suggests that transauricular stimulation preferentially activates brainstem structures whereas transcorneal stimulation preferentially activates forebrain structures [2], the former being more relevant to induction of tonic seizures [1]. Another major difference between studies is that whereas Frankel et al. determined strainspecific seizure thresholds by testing groups of animals at different current intensities (every animal tested only once) [11], we used a repeated-seizure testing paradigm. Thus, in spite of significant differences in procedure, there is overall good correspondence between our results and those of Frankel et al. [11] regarding relative MEST among inbred strains of mice. Ultimately, the most likely explanation for the modest differences in strain response between our study and that of Frankel et al. [11] may involve simple undefined factors related to the unique housing and laboratory environments and experimental manipulations. Related to this, even within the Frankel et al. study itself in which seven inbred strains were tested in two different laboratory sites that were carefully matched with regard to experimental protocol, there was one strain, 129S3 / SvImJ, which showed a significantly different rank order [11]. Previous reports have shown that the effect of repeated seizure activity on seizure threshold is subject to the influence of various experimental factors including the method of inducing seizures, the time course of seizure induction and the total number of seizures induced, to
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name several. In general, however, there appears to be a biphasic effect of seizure activity on seizure threshold with a short-term (several hours) increase [4] and a long-term decrease [5,28,30]. The latter effect is consistent with a ‘kindling’ phenomenon in which a constant daily subconvulsive electrical (or chemical) stimulus results in the development of a seizure over time [14]. In contrast, there is some evidence to support the concept that repeated electroconvulsive shock increases seizure threshold in humans [23]. In order to investigate the possibility that the relatively high MEST of the C57BL / 6J strain determined in experiment 1 is related to the greater number of generalized (but sub-maximal) seizures to which mice from this strain are subjected during the threshold test, we treated groups of C57BL / 6J mice with a single, variableintensity electroshock in experiment 2. Results documented a significant correlation between current level and the presence of maximal seizure (Table 2) with less than 20% of mice exhibiting a maximal seizure more than two standard deviations below the group mean suggesting that the relatively high mean MEST for C57BL / 6J mice determined in experiment 1 is not an artifact of the repeated-seizure testing paradigm. A similar correspondence between experiments using repeated or single electroshock testing was seen for the DBA / 2J strain such that all mice tested in the single electroshock paradigm experienced a seizure at a current intensity within 5 mA of the group mean MEST as determined in experiment 1 (Table 2). In experiment 2, the observation that a small number of C57BL / 6J mice exhibited a maximal seizure at a current level many standard deviations below the group mean MEST suggests that there may be a subtle environmental interaction between repeated sub-maximal seizure activity and MEST. In summary, we have established a strain-specific distribution pattern for MEST using a series of common inbred mouse strains. The relatively wide range of responses between strains and the extent to which these responses clearly distinguish several subgroups of strains suggests a rich resource for mapping and identifying genes that control seizure threshold. The low within strain variability associated with quantification of MEST reflects minimal uncontrolled environmental influence under the conditions used in this study and enhances the potential utility of this seizure-related parameter as an end-point for future QTL and gene mapping experiments.
Acknowledgements This work was supported by NIH award NS39516.
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