- Email: [email protected]
N mouse strain
Behavioural Brain Research 155 (2004) 283–289
Research report
A behavioural characterisation of the FVB/N mouse strain Perdita L. Pugh∗ , Sharlin F. Ahmed, Martin I. Smith, Neil Upton, A. Jacqueline Hunter Neurology and GI CEDD, GlaxoSmithKline, Third Avenue, Harlow, Essex CM19 5AW, UK Received 8 April 2004; accepted 30 April 2004 Available online 5 June 2004
Abstract The use of transgenic models in scientific research has made an enormous contribution to our understanding of the causes and symptoms of many diseases, including neurodegenerative conditions such as Alzheimer’s Disease (AD) and Parkinson’s Disease (PD). In the creation of transgenic models of neurodegenerative disease, effects of the background strain of the animal on the resulting genotype must be taken into consideration. This is particularly true for behavioural studies in which the background strain of the mouse may mask the phenotype of the genetic manipulation. Here, the behaviour of two mouse strains used in transgenic models, FVB/N and C57BL6/J, were compared. Studies of circadian wheel activity, cognition and aggression revealed considerable phenotypic differences between strains. These data also indicate that the FVB/N strain is not appropriate as a background strain in the behavioural assessment of transgenic mouse models. © 2004 Elsevier B.V. All rights reserved. Keywords: FVB/N; Transgenic; Alzheimer; Circadian; Strain
1. Introduction Gene targeted approaches are becoming increasingly important in the investigation of neurological diseases. Spontaneous and induced mouse mutations have been used to explore the involvement of various genes in neuronal function, however, specific targeted deletions or overexpression of known genes of interest are most popularly used [21,22]. These can interrogate the genetic basis of age-related progressive diseases such as AD. Some inbred strains are amenable to embryonic stem cell manipulation, for example 129/Sv [30] and C57BL6 are often used as the background in behavioural studies [7,33]. The FVB/N mouse has become popular for the generation of transgenic animals [1,31] and is increasingly used as the background strain for in vivo studies investigating aspects of disease. More recently, this strain has been popular in the study of genes implicated in the aetiology of AD [5,17,28,34]. It is important to consider the effect of the background strain per se as this may mask or alter the behavioural phenotype [6,9,18]. Recent literature indicates the FVB/N strain exhibits specific traits that could limit its ∗
Corresponding author. Tel.: +44 1279 622935; fax: +44 1279 622211. E-mail address: Pippa L [email protected] (P.L. Pugh).
0166-4328/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2004.04.021
use in disease models using genetically manipulated animals [16,15,32]. The FVB/N strain expresses the retinal degenerative (rd) mutation; however, the extent to which this directly affects the behaviour of the strain has not been investigated. Previous literature [27,32] suggests that the FVB/N strain is impaired in the Morris Watermaze, a test of spatial learning and memory, and it is thought that this impairment could be due to the already degenerating network of photoreceptors—rods and cones. By 9 weeks of age, mice homozygous for the rd mutation have obliteration of rods whilst total degeneration of cones may take up to 18 months [23]. Hence, the rd mutation may be responsible for the deficit seen in FVB/N mice in the watermaze. However, studies investigating visual mechanisms involved in regulation of the biological clock have revealed additional behavioural traits of mice bearing the rd mutation. In vivo models involving the use of mice with inherited retinal degenerative (rd) allele mutations [3] demonstrate that with degeneration of rod and cone photoreceptors, rd mice, although blind to visual images, are capable of maintaining a regular circadian rhythm using light. In the absence of melanopsin an apparent total loss of photoentrainment, pupillary responses and other non-image forming responses is exhibited [2,13,20].
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P.L. Pugh et al. / Behavioural Brain Research 155 (2004) 283–289
This indicates the possibility for two distinct visual systems; one to be ‘image-forming’ to provide a visual image of the surroundings and a ‘non image-forming’ system that can detect changes in light but is not used for image construction. Studies exploring the ‘non image-forming’ system have revealed specific photosensing retinal ganglion cell (RGC) groups that contain photopigments able to directly sense light [8]. Photopigments such as melanopsin [24–26] appear to be localised in RGCs projecting to the suprachiasmatic nucleus (SCN), a centre involved in the control of mammalian circadian rhythms [10–12]. Although many studies investigate strain comparisons of simple behaviours such as rearing, grooming and exploration, fewer studies have examined more complex behaviours, especially with regard to the FVB/N strain [15,32]. Disturbed sleep patterns, increased antisocial behaviour and cognitive decline are all present in the wide range of behavioural alterations seen in AD. Therefore, in this study we assessed circadian rhythm, aggression and the spatial cognitive phenotype of the FVB/N strain, comparing the performance of this strain with that of C57BL6/J, a strain popularly used in behavioural assays.
2. Methods 2.1. Animals All experiments were conducted according to the requirements of the United Kingdom Animals (Scientific Procedures) Act (1986) and conformed to GlaxoSmithKline ethical standards. Due to the nature of the procedures, separate cohorts of FVB/N and C57BL6/J mice were obtained from Charles River, UK. Each cohort was used to assess behaviour in either the Morris water maze (male and female, n = 8) circadian running wheels (males only, n = 6) or resident–intruder paradigm (males only, n = 10). Mice were housed in individual cages and maintained under a standard 12:12 h light:dark cycle with food and water available ad libitum. Testing began when the mice were approximately 12 weeks of age. 2.2. Circadian wheel activity Running wheel activity was used as a measure of circadian activity. Mice were singly housed in cages (35 cm × 19 cm × 13 cm) containing food, water and metal running wheel (23 cm diameter) with magnetic switches connected to a data acquisition system (ClockLab, Actimetrics, USA). Cages were housed in light boxes with an internal light source that could be altered. Mice were maintained on a 12–12 h light–dark cycle (lights on-lights off; 8.00–20.00 h) and left for 1–2 weeks to acclimatise before data collection. Following two weeks baseline data collection, mice received a 4 h phase advance in the light:dark cycle. Wheel running activity was recorded in 5-min time
bins using in-house software and activity recorded as actograms. 2.3. Morris water maze A mouse water maze consisting of a white perspex pool 120 cm in diameter and 50 cm high was filled with water and made opaque by addition of a latex compound (Warner Jenkinson Europe Ltd., Norfolk, UK). A platform (10 cm clear perspex disk) was anchored to the floor and placed in the centre of a quadrant of the pool. Various screens and coloured posters surrounded the pool to act as visual cues. A video camera was suspended above the pool and linked to an image analyser (HVS, Oxford, UK), which tracked the mice during each trial. A PC stored data on path length, heading angle, latency and percentage time spent in each quadrant. Each mouse received three days training with a visible platform located in the centre of quadrant number 4. Each mouse was given four trials a day, from each starting position, for three days. Between each of the four trials the mouse was given an inter-trial interval (ITI) of no shorter than ten minutes. During the ITI the mouse was placed into a heat box (Beta Medical & Scientific (Datesand Ltd.), Manchester) set at 33 ◦ C. Trial duration was 60 s and each mouse was placed into the water or onto the platform by hand and always removed from the apparatus by training the mice to sit on an extended sieve. After completion of the visible platform acquisition, each mouse was given four trials per day for five consecutive days, with a hidden platform placed in quadrant number 2. On the fifth day each mouse underwent a single probe trial immediately after completion of acquisition, in which the platform was removed and the mouse began each trial from position number 4 directly opposite the platform position. Retention tests were also performed on the 4th and 7th days after acquisition. 2.4. Resident–intruder paradigm A singly housed ‘intruder’ was placed into the home cage of a singly housed ‘resident’ and in this study the intruder was always of the same genotype as the resident. Pilot studies concluded that this would potentially provide equal aggression levels for the opponents and reduce the variability (data not shown). The behaviour of the mice was recorded on videotape for a period of five minutes. During this time, various parameters were measured. Cage orientated (rearing and digging), partner orientated (ano-genital sniffing) and self orientated (grooming) frequencies were recorded. Offence (frequency, duration and latency to attack) and defence (latency and frequency of defence posture) were also recorded. 2.5. Data analysis Statistical analyses were conducted using StatisticaTM (Statsoft, Inc.). ANOVA was performed to compare the
P.L. Pugh et al. / Behavioural Brain Research 155 (2004) 283–289 Table 1 Baseline activity for C57BL6/J and FVB/N male mice
Light phase Dark phase
C57BL6/J
FVB/N
209.6 ± 57.4 36005.7 ± 2754.4
8040.3 ± 2895∗ 27813.6 ± 5822.1
Data represented as mean ± S.E.M. of five consecutive 24 h periods. Symbol ∗ indicates P < 0.05 vs. C57BL6/J strain in the light phase.
effects of genotype and sex on each of the behavioural variables, where P < 0.05 data was considered to be significantly different. Post hoc simultaneous comparisons were carried out using the Sheffe test. Where appropriate, non-parametric analysis was conducted using the Mann–Whitney U-test.
3. Results
285
3.2. Morris water maze Repeated measures ANOVA indicated that FVB/N mice exhibited a significantly higher path length than C57BL6/J mice, during acquisition of both the visible [F(5,42) = 80.40, P ≤ 0.001] (Fig. 2) and hidden platform [F(5,42) = 96.80, P ≤ 0.001]. This suggests FVB/N mice may have a visual impairment due to their inability, over time, to find a clearly visible platform. There is no evidence that the FVB/N mice are learning the task over repeated trials as exemplified by the shortening path length of the C57BL6/J strain. FVB/N mice spent significantly [F(5,42) = 10.56, P ≤ 0.001] less time in the platform quadrant than C57BL6/J mice which were able to remember the location of the hidden platform (Fig. 3). The performance of the FVB/N strain is at chance level at each retention trial indicating the strain was unable to remember the location of a hidden platform after repeated acquisition trials.
3.1. Circadian wheel activity 3.3. Resident–intruder paradigm C57BL6/J mice were hypoactive during the light phase with an increase in overall activity during the dark phase, characteristic of normal behaviour (Table 1). FVB/N mice demonstrated greater activity during the light phase [F(1,10) = 7.31, P ≤ 0.05] however peak activity during the dark period did not reach the levels displayed by the C57BL6/J strain. C57BL6/J mice also demonstrated good entrainment to a 12:12 h L:D cycle and re-entrainment to a phase advance of 4 h (Fig. 1A). However FVB/N mice exhibited a fragmented and arrhythmic pattern of activity with no entrainment to a 12:12 h L:D cycle (Fig. 1B). Baseline activity data and representative wheel running traces suggest the FVB/N strain exhibit abnormal circadian behaviour. Overall activity is random and relatively non-specific over light and dark phases of a 24-h period compared with C57BL6/J mice.
FVB/N mice showed a significantly higher frequency [F(1,28) = 21.56, P < 0.001] and duration of offense [F(1,28) = 31.6, P < 0.001] than C57BL6/J mice (Fig. 4a and b) with residents showing greater offense than intruders [F(1,28) = 9.37, P < 0.0048] for both strains. FVB/N mice displayed significantly greater defense frequency than C57BL6/J mice [F(1,28) = 20.78, P < 0.001] (Fig. 5) relating to the increased frequency of aggressive bouts displayed by resident FVB/N mice.
4. Discussion Behavioural assessment of a transgenic mouse phenotype can be greatly influenced by the background strain [9,18,6].
Fig. 1. Representative traces of wheel running activity in male mice, open bar and closed bar corresponding to light and dark phase, respectively. Dot on (A) indicates 4 h phase advance. (A) C57BL6/J mice re-entrain to a phase advance, (B) FVB/N mice illustrate arrhythmic pattern to activity.
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FVB male FVB female 1600 BL6 male
*
1400
* 1200
BL6 female
*
Length (cm)
1000 800 600 400 200 0 1
2
3
Day
Fig. 2. Path length to visible platform. Data shown as mean ± S.E.M. for each session of training (four trials), n = 8. Symbol ∗ indicates P < 0.001 compared with either male or female C57BL6/J mice.
Fig. 3. Percent target quadrant time, 0, 4 and 7 days post acquisition of hidden platform. Data shown as mean ± S.E.M. Symbol ∗ indicates P < 0.05 vs. female FVB/N, ∗∗ indicates P < 0.01 vs. male and female FVB/N.
P.L. Pugh et al. / Behavioural Brain Research 155 (2004) 283–289
287
Fig. 4. (a) Frequency of offensive bouts shown as mean ± SEM (n = 10 per group). ∗ indicates P < 0.05 vs. all other groups. (b) Duration of attack, ∗ indicates P < 0.001 vs. all groups. Data shown as mean ± S.E.M.
Previous studies [15,32] have investigated the effects of the FVB/N strain on behaviours such as locomotor activity and the watermaze. The strain carries the rd mutation [21] for retinal degeneration and this has been reported to affect behaviour in tests of cognition. Our investigation indicates that the FVB/N strain may additionally suffer impairment in using light to regulate circadian rhythm. We found C57BL6/J mice entrained to a 12:12 h light: dark cycle and were able to re-entrain to a 4 h phase advance, however, FVB/N animals showed a fragmented and arrhythmic activity pattern, with increased activity during the light phase. They were also unable to re-entrain to the phase advance. This suggests that the FVB/N strain not only
has impairment in the classical photoreceptor system used for forming visual images due to the effects of the rd mutation but that it is additionally impaired in sensing light and maintaining a regular circadian rhythm. This may be due to damage of the RGC groups that communicate directly with the SCN and are responsible for photoentrainment, or a loss of the photopigment melanopsin [10–12] that is suggested to be important in detecting changes in light intensity in the environment [13,20]. Work reviewed by Berson [3] using mice with a degeneration of classical photoreceptors showed they were as efficient as unaffected mice at maintaining a circadian rhythm. Since the discovery of melanopsin [24,25] and a subset of RGC groups in the mouse inner retina [26] it is
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Fig. 5. Frequency of defensive bouts, ∗ indicates P < 0.001 vs. all groups. Data shown as mean ± S.E.M.
postulated that a second system is responsible for mediating non-image photoreceptive tasks such as regulating the circadian rhythm. Hence, it is possible that the FVB/N strain may display a genetically endogenous impairment in both visual image-forming and non-image forming processes. This severe combined visual impairment will have strong implications on the behaviour of the strain, particularly in behavioural assays relying on spatial awareness. Numerous studies [18,32] have included analysis of FVB/N mice in the morris watermaze and have ascertained that the strain is unable to find the position of a platform even when cued. Our data support these findings, as the FVB/N strain was unable to locate a visual platform. This is in contrast to C57BL6/J mice, which illustrated a rapid decrease in path length to the platform indicating they learnt and remembered its location over the three day acquisition period. C57BL6/J mice were also able to successfully remember the location of a hidden platform as indicated by the percentage time spent in the platform quadrant. As expected, this memory diminished over time although still remained over chance levels seven days post acquisition. The FVB/N strain can also show cognitive impairment in non-visual tasks such as fear conditioning [4,19]. FVB/N mice display impaired performance during contextually conditioned fear tasks in which vision is thought not to be involved, based on data from C3H rd mice and the congenic strain C3 [4]. The FVB/N strain may, therefore, have an initial cognitive impairment that is accentuated in assays relying on visual stimuli such as the watermaze. A disrupted sleep-wake cycle may also have an effect on natural spontaneous behaviours. Evidence suggests rapid eye movement (REM) sleep deprivation can increase aggressiveness in rats [14,29] implicating the importance of the circadian rhythm in maintaining social behaviours. The
FVB/N strain is reported to display elevated aggressive behaviours towards opponents such as decreased attack latency and increased tail rattling [16]. Our assessment supports these findings with FVB/N mice displaying a higher duration of offensive behaviour compared with C57BL6/J. In this protocol, unlike that of previous studies [15], resident animals were placed against same strain intruders and were singly housed prior to the experiment. This protocol has resulted in the FVB/N animals displaying very high levels of defensive behaviours in both resident and intruder mice. This suggests that each offensive bout made by animals of the FVB/N strain may have been of a higher intensity than that seen in the C57BL6/J mice. In conclusion, the FVB/N strain, although highly suitable for constructing transgenics, is not a feasible background strain for certain aspects of behavioural analysis. The unusual circadian rhythm patterns in combination with their apparent impairment in detecting visual images makes this strain particularly inappropriate for use in behavioural studies where circadian rhythm may have an impact on the phenotype of the animal. This is also true of studies, which require spatial awareness, for example in the Morris watermaze or T maze tests in order to assess spatial learning and memory. These studies have illustrated that the transgenic phenotype of a mouse may be influenced by the background strain; in this case FVB/N. Background strain selection for transgene manipulation is, therefore, an important consideration when behavioural phenotyping is required. References [1] Auerbach AB, Norinsky R, Ho W, Losos K, Guo Q, Chatterjee S, et al. Strain dependent differences in the efficiency of transgenic mouse production. Transgenic Res 2002;12:59–69.
P.L. Pugh et al. / Behavioural Brain Research 155 (2004) 283–289 [2] Bellingham J, Foster RG. Opsins and mammalian photoentrainment. Cell Tissue Res 2002;309:57–71. [3] Berson DM. Strange vision: ganglion cells as circadian photoreceptors. Trends in Neurosci 2003;26:314–20. [4] Bolivar VJ, Poller O, Flaherty L. Inbred strain variation in contextual and cued fear conditioning behaviour. Mamm Genome 2001;12:651– 6. [5] Carlson GA, Borchelt DR, Dake A, Turner S, Danielson V, Coffin JD, et al. Genetic modification of the phenotypes produced by amyloid precursor protein overexpression in transgenic mice. Hum Mol Genet 1997;6:1951–9. [6] Crawley JN, Belknap JK, Collins A, Crabbe JC, Frankel W, Henderson N, et al. Behavioural phenotypes of inbred mouse strains: implications and recommendations for molecular studies. Psychopharmacology 1997;132:107–24. [7] Dockstader CL, van der Kooy D. Mouse strain differences in opiate reward learning are explained by differences in anxiety, not reward or learning. J Neurosci 2001;21:9077–81. [8] He S, Dong W, Deng Q, Weng S, Sun W. Seeing more clearly: recent advances in understanding retinal circuitry. Science 2003;32:408–11. [9] Gerlai R. Gene-targeting studies of mammalian behaviour: is it the mutation or the background phenotype? Trends Neurosci 1996;19:177–81. [10] Gooley JJ, Lu J, Chou TC, Scammell TE, Saper CB. Melanopsin in cells of origin of the retinohypothalamic tract. Nat Neurosci 2001;4:1165. [11] Gooley JJ, Lu J, Fischer D, Saper CB. A broad role for melanopsin in nonvisual photoreception. J Neurosci 2003;23:7093–106. [12] Hannibal J, Fahrenkrug J. Melanopsin: a novel photopigment involved in the photoentrainment of the brain’s biological clock? Ann Med 2002;34:401–7. [13] Hattar S, Lucas RJ, Mrosovsky N, Thompson S, Douglas RH, Hankins MW, et al. Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice. Nature 2003;424:76–81. [14] Hicks RA, Moore JD, Hayes C, Phillips N, Hawkins J. REM sleep deprivation increases aggressiveness in male rats. Phys Behav 1979;22:1097–100. [15] Kim S, Lee S, Ryu S, Suk J, Park C. Comparative analysis of the anxiety-related behaviours in four inbred mice. Behav Processes 2002;60:181–90. [16] Mineur YS, Crusio WE. Behavioural and neuroanatomical characterization of FVB/N inbred mice. Brain Res Bull 2002;57:41–7. [17] Moechars D, Lorent K, Dewachter I, Baekelandt V, Strooper B, Van Leuven F. Transgenic mice expressing an alpha-secretion mutant of the amyloid precursor protein in the brain develop a progressive CNS disorder. Behav Brain Res 1998;95:55–64. [18] Nguyen PV, Gerlai R. Behavioural and physiological characterisation of inbred mouse strains: prospects for elucidating the molecular mechanisms of mammalian learning and memory. Genes Brain Behav 2002;1:72–81.
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[19] Owen EH, Logue SF, Rasmussen DL, Wehner JM. Assessment of learning by the Morris Water Task and fear conditioning in inbred mouse strains and F1 hybrids: implications of genetic background for single gene mutations and quantitative trait loci analyses. Neuroscience 1997;80:1087–99. [20] Panda S, Provencio I, Tu DC, Pires SS, Rollag MD, Castrucci AM, et al. Melanopsin is required for non-image-forming photic responses in blind mice. Science 2003;301:525–7. [21] Picciotto MR. Knockout mouse models used to study neurobiological systems. Crit Rev Neurobiol 1999;13:103–49. [22] Picciotto MR, Wickman K. Using knockout and transgenic mice to study neurophysiology and behaviour. Physiol Rev 1998;78:1131– 63. [23] Provencio I, Cooper HM, Foster RG. Retinal projections in mice with inherited retinal degeneration: implications for circadian photoentrainment. J Comp Neurol 1998;395:417–39. [24] Provencio I, Jiang G, De rip WJ, Hayes WP, Rollag MD. Melanopsin: an opsin in melanophores. Proc Natl Acad Sci USA 1998;95: 340–5. [25] Provencio I, Rodriguez IR, Jiang G, Hayes WP, Moreira EF, Rollag MD. A novel human opsin in the inner retina. J Neurosci 2000;20:600–5. [26] Provencio I, Rollag MD, Castrucci AM. Photoreceptive net in the mammalian retina. This mesh of cells may explain how some blind mice can still tell day from night. Nature 2002;415:493. [27] Royle SJ, Collins FC, Rupniak HT, Barnes JC, Anderson R. Behavioural analysis and susceptibility to CNS injury of four inbred strains of mice. Brain Res 1999;816:337–49. [28] Schauwecker PE. Modulation of cell death by mouse genotype: differential vulnerability to excitatory amino acid-induced lesions. Exp Neurol 2002;178:219–35. [29] Sloan MA. The effects of deprivation of rapid eye movement (REM) sleep on maze learning and aggression in the albino rat. J Psychiat Res 1972;9:101–11. [30] Simpson EM, Linder CC, Sargent EE, Davisson MT, Mobraaten LE, Sharp JJ. Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice. Nat Genet 1997;16:19–27. [31] Taketo M, Schroeder AC, Mobraaten LE, Gunning KB, Hanten G, Fox RR, et al. FVB/N: An inbred mouse strain preferable for transgenic analyses. Proc Natl Acad Sci USA 1991;88:2065–9. [32] Voikar V, Koks S, Vasar E, Rauvala H. Strain and gender differences in the behaviour of mouse lines commonly used in transgenic studies. Physiol Behav 2001;72:271–81. [33] Wolfer DP, Muller U, Stagliar M, Lipp HP. Assessing the effects of the 129/Sv genetic background on swimming navigation learning in transgenic mutants: a study using mice with a modified beta-amyloid precursor protein gene. Brain Res 1997;771:1–13. [34] Zhang F, Eckman C, Younkin S, Hsiao KK, Iadecola C. Increased susceptibility to ischaemic brain damage in transgenic mice overexpressing the amyloid precursor protein. J Neurosci 1997;17:7655– 61.
Research report
A behavioural characterisation of the FVB/N mouse strain Perdita L. Pugh∗ , Sharlin F. Ahmed, Martin I. Smith, Neil Upton, A. Jacqueline Hunter Neurology and GI CEDD, GlaxoSmithKline, Third Avenue, Harlow, Essex CM19 5AW, UK Received 8 April 2004; accepted 30 April 2004 Available online 5 June 2004
Abstract The use of transgenic models in scientific research has made an enormous contribution to our understanding of the causes and symptoms of many diseases, including neurodegenerative conditions such as Alzheimer’s Disease (AD) and Parkinson’s Disease (PD). In the creation of transgenic models of neurodegenerative disease, effects of the background strain of the animal on the resulting genotype must be taken into consideration. This is particularly true for behavioural studies in which the background strain of the mouse may mask the phenotype of the genetic manipulation. Here, the behaviour of two mouse strains used in transgenic models, FVB/N and C57BL6/J, were compared. Studies of circadian wheel activity, cognition and aggression revealed considerable phenotypic differences between strains. These data also indicate that the FVB/N strain is not appropriate as a background strain in the behavioural assessment of transgenic mouse models. © 2004 Elsevier B.V. All rights reserved. Keywords: FVB/N; Transgenic; Alzheimer; Circadian; Strain
1. Introduction Gene targeted approaches are becoming increasingly important in the investigation of neurological diseases. Spontaneous and induced mouse mutations have been used to explore the involvement of various genes in neuronal function, however, specific targeted deletions or overexpression of known genes of interest are most popularly used [21,22]. These can interrogate the genetic basis of age-related progressive diseases such as AD. Some inbred strains are amenable to embryonic stem cell manipulation, for example 129/Sv [30] and C57BL6 are often used as the background in behavioural studies [7,33]. The FVB/N mouse has become popular for the generation of transgenic animals [1,31] and is increasingly used as the background strain for in vivo studies investigating aspects of disease. More recently, this strain has been popular in the study of genes implicated in the aetiology of AD [5,17,28,34]. It is important to consider the effect of the background strain per se as this may mask or alter the behavioural phenotype [6,9,18]. Recent literature indicates the FVB/N strain exhibits specific traits that could limit its ∗
Corresponding author. Tel.: +44 1279 622935; fax: +44 1279 622211. E-mail address: Pippa L [email protected] (P.L. Pugh).
0166-4328/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2004.04.021
use in disease models using genetically manipulated animals [16,15,32]. The FVB/N strain expresses the retinal degenerative (rd) mutation; however, the extent to which this directly affects the behaviour of the strain has not been investigated. Previous literature [27,32] suggests that the FVB/N strain is impaired in the Morris Watermaze, a test of spatial learning and memory, and it is thought that this impairment could be due to the already degenerating network of photoreceptors—rods and cones. By 9 weeks of age, mice homozygous for the rd mutation have obliteration of rods whilst total degeneration of cones may take up to 18 months [23]. Hence, the rd mutation may be responsible for the deficit seen in FVB/N mice in the watermaze. However, studies investigating visual mechanisms involved in regulation of the biological clock have revealed additional behavioural traits of mice bearing the rd mutation. In vivo models involving the use of mice with inherited retinal degenerative (rd) allele mutations [3] demonstrate that with degeneration of rod and cone photoreceptors, rd mice, although blind to visual images, are capable of maintaining a regular circadian rhythm using light. In the absence of melanopsin an apparent total loss of photoentrainment, pupillary responses and other non-image forming responses is exhibited [2,13,20].
284
P.L. Pugh et al. / Behavioural Brain Research 155 (2004) 283–289
This indicates the possibility for two distinct visual systems; one to be ‘image-forming’ to provide a visual image of the surroundings and a ‘non image-forming’ system that can detect changes in light but is not used for image construction. Studies exploring the ‘non image-forming’ system have revealed specific photosensing retinal ganglion cell (RGC) groups that contain photopigments able to directly sense light [8]. Photopigments such as melanopsin [24–26] appear to be localised in RGCs projecting to the suprachiasmatic nucleus (SCN), a centre involved in the control of mammalian circadian rhythms [10–12]. Although many studies investigate strain comparisons of simple behaviours such as rearing, grooming and exploration, fewer studies have examined more complex behaviours, especially with regard to the FVB/N strain [15,32]. Disturbed sleep patterns, increased antisocial behaviour and cognitive decline are all present in the wide range of behavioural alterations seen in AD. Therefore, in this study we assessed circadian rhythm, aggression and the spatial cognitive phenotype of the FVB/N strain, comparing the performance of this strain with that of C57BL6/J, a strain popularly used in behavioural assays.
2. Methods 2.1. Animals All experiments were conducted according to the requirements of the United Kingdom Animals (Scientific Procedures) Act (1986) and conformed to GlaxoSmithKline ethical standards. Due to the nature of the procedures, separate cohorts of FVB/N and C57BL6/J mice were obtained from Charles River, UK. Each cohort was used to assess behaviour in either the Morris water maze (male and female, n = 8) circadian running wheels (males only, n = 6) or resident–intruder paradigm (males only, n = 10). Mice were housed in individual cages and maintained under a standard 12:12 h light:dark cycle with food and water available ad libitum. Testing began when the mice were approximately 12 weeks of age. 2.2. Circadian wheel activity Running wheel activity was used as a measure of circadian activity. Mice were singly housed in cages (35 cm × 19 cm × 13 cm) containing food, water and metal running wheel (23 cm diameter) with magnetic switches connected to a data acquisition system (ClockLab, Actimetrics, USA). Cages were housed in light boxes with an internal light source that could be altered. Mice were maintained on a 12–12 h light–dark cycle (lights on-lights off; 8.00–20.00 h) and left for 1–2 weeks to acclimatise before data collection. Following two weeks baseline data collection, mice received a 4 h phase advance in the light:dark cycle. Wheel running activity was recorded in 5-min time
bins using in-house software and activity recorded as actograms. 2.3. Morris water maze A mouse water maze consisting of a white perspex pool 120 cm in diameter and 50 cm high was filled with water and made opaque by addition of a latex compound (Warner Jenkinson Europe Ltd., Norfolk, UK). A platform (10 cm clear perspex disk) was anchored to the floor and placed in the centre of a quadrant of the pool. Various screens and coloured posters surrounded the pool to act as visual cues. A video camera was suspended above the pool and linked to an image analyser (HVS, Oxford, UK), which tracked the mice during each trial. A PC stored data on path length, heading angle, latency and percentage time spent in each quadrant. Each mouse received three days training with a visible platform located in the centre of quadrant number 4. Each mouse was given four trials a day, from each starting position, for three days. Between each of the four trials the mouse was given an inter-trial interval (ITI) of no shorter than ten minutes. During the ITI the mouse was placed into a heat box (Beta Medical & Scientific (Datesand Ltd.), Manchester) set at 33 ◦ C. Trial duration was 60 s and each mouse was placed into the water or onto the platform by hand and always removed from the apparatus by training the mice to sit on an extended sieve. After completion of the visible platform acquisition, each mouse was given four trials per day for five consecutive days, with a hidden platform placed in quadrant number 2. On the fifth day each mouse underwent a single probe trial immediately after completion of acquisition, in which the platform was removed and the mouse began each trial from position number 4 directly opposite the platform position. Retention tests were also performed on the 4th and 7th days after acquisition. 2.4. Resident–intruder paradigm A singly housed ‘intruder’ was placed into the home cage of a singly housed ‘resident’ and in this study the intruder was always of the same genotype as the resident. Pilot studies concluded that this would potentially provide equal aggression levels for the opponents and reduce the variability (data not shown). The behaviour of the mice was recorded on videotape for a period of five minutes. During this time, various parameters were measured. Cage orientated (rearing and digging), partner orientated (ano-genital sniffing) and self orientated (grooming) frequencies were recorded. Offence (frequency, duration and latency to attack) and defence (latency and frequency of defence posture) were also recorded. 2.5. Data analysis Statistical analyses were conducted using StatisticaTM (Statsoft, Inc.). ANOVA was performed to compare the
P.L. Pugh et al. / Behavioural Brain Research 155 (2004) 283–289 Table 1 Baseline activity for C57BL6/J and FVB/N male mice
Light phase Dark phase
C57BL6/J
FVB/N
209.6 ± 57.4 36005.7 ± 2754.4
8040.3 ± 2895∗ 27813.6 ± 5822.1
Data represented as mean ± S.E.M. of five consecutive 24 h periods. Symbol ∗ indicates P < 0.05 vs. C57BL6/J strain in the light phase.
effects of genotype and sex on each of the behavioural variables, where P < 0.05 data was considered to be significantly different. Post hoc simultaneous comparisons were carried out using the Sheffe test. Where appropriate, non-parametric analysis was conducted using the Mann–Whitney U-test.
3. Results
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3.2. Morris water maze Repeated measures ANOVA indicated that FVB/N mice exhibited a significantly higher path length than C57BL6/J mice, during acquisition of both the visible [F(5,42) = 80.40, P ≤ 0.001] (Fig. 2) and hidden platform [F(5,42) = 96.80, P ≤ 0.001]. This suggests FVB/N mice may have a visual impairment due to their inability, over time, to find a clearly visible platform. There is no evidence that the FVB/N mice are learning the task over repeated trials as exemplified by the shortening path length of the C57BL6/J strain. FVB/N mice spent significantly [F(5,42) = 10.56, P ≤ 0.001] less time in the platform quadrant than C57BL6/J mice which were able to remember the location of the hidden platform (Fig. 3). The performance of the FVB/N strain is at chance level at each retention trial indicating the strain was unable to remember the location of a hidden platform after repeated acquisition trials.
3.1. Circadian wheel activity 3.3. Resident–intruder paradigm C57BL6/J mice were hypoactive during the light phase with an increase in overall activity during the dark phase, characteristic of normal behaviour (Table 1). FVB/N mice demonstrated greater activity during the light phase [F(1,10) = 7.31, P ≤ 0.05] however peak activity during the dark period did not reach the levels displayed by the C57BL6/J strain. C57BL6/J mice also demonstrated good entrainment to a 12:12 h L:D cycle and re-entrainment to a phase advance of 4 h (Fig. 1A). However FVB/N mice exhibited a fragmented and arrhythmic pattern of activity with no entrainment to a 12:12 h L:D cycle (Fig. 1B). Baseline activity data and representative wheel running traces suggest the FVB/N strain exhibit abnormal circadian behaviour. Overall activity is random and relatively non-specific over light and dark phases of a 24-h period compared with C57BL6/J mice.
FVB/N mice showed a significantly higher frequency [F(1,28) = 21.56, P < 0.001] and duration of offense [F(1,28) = 31.6, P < 0.001] than C57BL6/J mice (Fig. 4a and b) with residents showing greater offense than intruders [F(1,28) = 9.37, P < 0.0048] for both strains. FVB/N mice displayed significantly greater defense frequency than C57BL6/J mice [F(1,28) = 20.78, P < 0.001] (Fig. 5) relating to the increased frequency of aggressive bouts displayed by resident FVB/N mice.
4. Discussion Behavioural assessment of a transgenic mouse phenotype can be greatly influenced by the background strain [9,18,6].
Fig. 1. Representative traces of wheel running activity in male mice, open bar and closed bar corresponding to light and dark phase, respectively. Dot on (A) indicates 4 h phase advance. (A) C57BL6/J mice re-entrain to a phase advance, (B) FVB/N mice illustrate arrhythmic pattern to activity.
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FVB male FVB female 1600 BL6 male
*
1400
* 1200
BL6 female
*
Length (cm)
1000 800 600 400 200 0 1
2
3
Day
Fig. 2. Path length to visible platform. Data shown as mean ± S.E.M. for each session of training (four trials), n = 8. Symbol ∗ indicates P < 0.001 compared with either male or female C57BL6/J mice.
Fig. 3. Percent target quadrant time, 0, 4 and 7 days post acquisition of hidden platform. Data shown as mean ± S.E.M. Symbol ∗ indicates P < 0.05 vs. female FVB/N, ∗∗ indicates P < 0.01 vs. male and female FVB/N.
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Fig. 4. (a) Frequency of offensive bouts shown as mean ± SEM (n = 10 per group). ∗ indicates P < 0.05 vs. all other groups. (b) Duration of attack, ∗ indicates P < 0.001 vs. all groups. Data shown as mean ± S.E.M.
Previous studies [15,32] have investigated the effects of the FVB/N strain on behaviours such as locomotor activity and the watermaze. The strain carries the rd mutation [21] for retinal degeneration and this has been reported to affect behaviour in tests of cognition. Our investigation indicates that the FVB/N strain may additionally suffer impairment in using light to regulate circadian rhythm. We found C57BL6/J mice entrained to a 12:12 h light: dark cycle and were able to re-entrain to a 4 h phase advance, however, FVB/N animals showed a fragmented and arrhythmic activity pattern, with increased activity during the light phase. They were also unable to re-entrain to the phase advance. This suggests that the FVB/N strain not only
has impairment in the classical photoreceptor system used for forming visual images due to the effects of the rd mutation but that it is additionally impaired in sensing light and maintaining a regular circadian rhythm. This may be due to damage of the RGC groups that communicate directly with the SCN and are responsible for photoentrainment, or a loss of the photopigment melanopsin [10–12] that is suggested to be important in detecting changes in light intensity in the environment [13,20]. Work reviewed by Berson [3] using mice with a degeneration of classical photoreceptors showed they were as efficient as unaffected mice at maintaining a circadian rhythm. Since the discovery of melanopsin [24,25] and a subset of RGC groups in the mouse inner retina [26] it is
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Fig. 5. Frequency of defensive bouts, ∗ indicates P < 0.001 vs. all groups. Data shown as mean ± S.E.M.
postulated that a second system is responsible for mediating non-image photoreceptive tasks such as regulating the circadian rhythm. Hence, it is possible that the FVB/N strain may display a genetically endogenous impairment in both visual image-forming and non-image forming processes. This severe combined visual impairment will have strong implications on the behaviour of the strain, particularly in behavioural assays relying on spatial awareness. Numerous studies [18,32] have included analysis of FVB/N mice in the morris watermaze and have ascertained that the strain is unable to find the position of a platform even when cued. Our data support these findings, as the FVB/N strain was unable to locate a visual platform. This is in contrast to C57BL6/J mice, which illustrated a rapid decrease in path length to the platform indicating they learnt and remembered its location over the three day acquisition period. C57BL6/J mice were also able to successfully remember the location of a hidden platform as indicated by the percentage time spent in the platform quadrant. As expected, this memory diminished over time although still remained over chance levels seven days post acquisition. The FVB/N strain can also show cognitive impairment in non-visual tasks such as fear conditioning [4,19]. FVB/N mice display impaired performance during contextually conditioned fear tasks in which vision is thought not to be involved, based on data from C3H rd mice and the congenic strain C3 [4]. The FVB/N strain may, therefore, have an initial cognitive impairment that is accentuated in assays relying on visual stimuli such as the watermaze. A disrupted sleep-wake cycle may also have an effect on natural spontaneous behaviours. Evidence suggests rapid eye movement (REM) sleep deprivation can increase aggressiveness in rats [14,29] implicating the importance of the circadian rhythm in maintaining social behaviours. The
FVB/N strain is reported to display elevated aggressive behaviours towards opponents such as decreased attack latency and increased tail rattling [16]. Our assessment supports these findings with FVB/N mice displaying a higher duration of offensive behaviour compared with C57BL6/J. In this protocol, unlike that of previous studies [15], resident animals were placed against same strain intruders and were singly housed prior to the experiment. This protocol has resulted in the FVB/N animals displaying very high levels of defensive behaviours in both resident and intruder mice. This suggests that each offensive bout made by animals of the FVB/N strain may have been of a higher intensity than that seen in the C57BL6/J mice. In conclusion, the FVB/N strain, although highly suitable for constructing transgenics, is not a feasible background strain for certain aspects of behavioural analysis. The unusual circadian rhythm patterns in combination with their apparent impairment in detecting visual images makes this strain particularly inappropriate for use in behavioural studies where circadian rhythm may have an impact on the phenotype of the animal. This is also true of studies, which require spatial awareness, for example in the Morris watermaze or T maze tests in order to assess spatial learning and memory. These studies have illustrated that the transgenic phenotype of a mouse may be influenced by the background strain; in this case FVB/N. Background strain selection for transgene manipulation is, therefore, an important consideration when behavioural phenotyping is required. References [1] Auerbach AB, Norinsky R, Ho W, Losos K, Guo Q, Chatterjee S, et al. Strain dependent differences in the efficiency of transgenic mouse production. Transgenic Res 2002;12:59–69.
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