SvJ mice

Brain Research Bulletin 116 (2015) 16–24

Contents lists available at ScienceDirect

Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull

Research report

Behaviour and prefrontal protein differences in C57BL/6N and 129 X1/SvJ mice Xiaofan Zhang a,b , Qi Li b , Naikei Wong c , Min Zhang a , Wei Wang a , Bitao Bu a , Grainne Mary McAlonan b,d,∗ a

Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (HUST), China Department of Psychiatry, University of Hong Kong, Hong Kong Special Administrative Region c Department of Chemistry, University of Hong Kong, Hong Kong Special Administrative Region d Department of Forensic and Neurodevelopmental Sciences, The Institute of Psychiatry, King’s College London, UK b

a r t i c l e

i n f o

Article history: Received 7 April 2015 Received in revised form 11 May 2015 Accepted 12 May 2015 Available online 21 May 2015 Keywords: C57BL/6N 129X1/SvJ Prepulse inhibition Amphetamine test Proteomics

a b s t r a c t Experimental animals provide valuable opportunities to establish aetiological mechanisms and test new treatments for neurodevelopmental psychiatric conditions. However, it is increasingly appreciated that inter-strain differences cannot be neglected in the experimental design. In addition, the importance of including females in preclinical – but also clinical – research is now recognised. Here, we compared behaviour and prefrontal protein differences in male and female C57BL/6N and 129X1/SvJ mice as both are commonly used experimental rodents. Relative to 129X1/SvJ mice, both sexes of C57BL/6N mice had weaker sensorimotor gating, measured in the prepulse inhibition (PPI) of startle paradigm, and were more sensitive to amphetamine challenge in the open field. The pattern of protein expression in the prefrontal cortex of C57BL6N mice was also clearly distinct from 129X1/SvJ mice. Proteins differentially expressed were those associated with oxidative metabolism, receptor protein signalling, cell communication and signal transduction and energy pathways. We suggest that the C57BL/6N mouse may usefully proxy features of the neurodevelopmental disorders and could have application in pre-translational screening of new therapeutic approaches. The 129X1/SvJ strain in contrast, might be better suited to experimental studies of causal risk factors expected to lower PPI and increase amphetamine sensitivity. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Neurodevelopmental disorders such as schizophrenia and autism are common and debilitating, but their underlying causes remain unclear and treatment approaches are limited. For example, interpretation of post-mortem studies (Bogerts et al., 1985) is complicated because such disorders usually present much earlier (Jablensky, 2000; Rutter, 1978). Thus, it is difficult to control for tissue changes related to ageing, treatment, and cause of death.

Abbreviations: PPI, prepulse inhibition; mPFC, medial prefrontal cortex; PLSDA, partial least squares-discriminant analysis; SOD2, superoxide dismutase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PDH, pyruvate dehydrogenase; GLAST, astrocyte glutamate transporter; PKC, protein kinase C; OAT, ornithine aminotransferase. ∗ Corresponding author at: Department of Forensic and Neurodevelopmental Sciences. Institute of Psychiatry, King’s College London, De Crespigny Park, Denmark Hill, London SE5 8AF, UK. Tel.: +44 20 7848 0831. E-mail address: [email protected] (G.M. McAlonan). http://dx.doi.org/10.1016/j.brainresbull.2015.05.003 0361-9230/© 2015 Elsevier Inc. All rights reserved.

Accordingly, animal models are called for to facilitate experimental control and help clarify potential pathophysiological mechanisms. Animal models of neuropsychiatric disorders can be generated through multiple approaches, including selective breeding, genetic engineering, brain lesions and environmental manipulations (Nestler and Hyman, 2010). However, it is now appreciated that the genetic background of the animal, which contributes to their behavioural profile, may interact with the effects of pharmacological or genetic manipulations. Although, the importance of considering differences in the ‘wild-type’ behavioural phenotype of experimental in-bred strains of experimental mice is increasingly recognised, the majority of previous studies have investigated male mice exclusively (Belknap et al., 1993; Bryant et al., 2008; Hefner et al., 2008; Owen et al., 1997) Also, potential differences in brain protein expression across mouse inbred strains, which are likely to influence the final behavioural expression of an environmental or genetic manipulation, have received limited attention. In the current study, we first examined in vivo behavioural differences in male and female C57BL/6N and 129X1/SvJ mice as

X. Zhang et al. / Brain Research Bulletin 116 (2015) 16–24

both are commonly used to generate animal models (Crawley, 1996; Gerlai, 1996). We focused on prepulse inhibition of startle (PPI) and response to an amphetamine challenge in the open field. PPI is a measure of sensorimotor gating (Braff and Geyer, 1990), and impairment has been postulated to contribute to repetitive behaviours (Perry et al., 2007), sensory flooding and cognitive fragmentation in neurodevelopmental disorders (McGhie and Chapman, 1961). The response to amphetamine challenge in an open field provides a measure of sensitivity of the dopamine system, and an increased amphetamine response is thought to simulate dopaminergic over activity in schizophrenia (Meltzer, 1980). We hypothesized that C57BL/6N mice would have lower PPI and be more sensitive to amphetamine than 129X1/SvJ mice, consistent with a previous study in closely ‘related’ C57BL/6J and 129X1/SvJ inbred mouse strains (Ralph et al., 2001). Next we used 2D gel techniques in a preliminary study of protein expression in the prefrontal cortex of each strain. Frontal lobe abnormalities are associated with PPI impairment and increased sensitivity to amphetamine (Flores et al., 1996; Li et al., 2009); therefore we hypothesized that any strain differences in behaviours would be accompanied by a difference in the expression of proteins within networks implicated in neurodevelopmental disorders. 2. Material and methods 2.1. Animals The 52, 7-week-old (young adult) mice [C57BL/6N = 26 (female = 13, male = 13); 129X1/SvJ = 26 (female = 13, male = 13)] used in this study were obtained from and housed in the Laboratory Animal Unit (LAU) at the University of Hong Kong. The experimental protocol was approved by the Committee on the Use of Live Animals for Teaching and Research, University of Hong Kong (CULATR case No: 2624-12). Ten male and 10 female mice from each strain were tested in PPI and open field and amphetamine challenge paradigms. Three male and three female mice from each strain not tested (free of influences on protein expression), were sacrificed for a 2D gel-proteomics study. Tissue from mice tested in behavioural studies was used to validate 2D gel results in selected proteins using western blot. The behaviour holding room was maintained at 21 ◦ C and, unlike the general areas of the LAU, had a reversed day–night cycle (light on: 7 PM–7 AM). Mice were therefore acclimatized for a minimum of 1 week before any testing. All behavioural tests were conducted in the dark phase of the light–dark cycle. 2.2. Behavioural tests 2.2.1. PPI The whole-body startle responses of mice were measured in mouse startle chambers supplied by SR-LAB (San Diego Instruments, San Diego, CA, USA). Mice were placed in the startle chamber and left undisturbed for 10 min before the PPI test began. The PPI paradigm followed a standard protocol (Li et al., 2009; Meyer et al., 2005). Startle stimuli and background stimuli (NS) comprised white noise. The background stimulus was 65 dB. The 3× 40 ms pulse intensities were 100 dB, 110 dB and 120 dB; the 3× 20 ms prepulse intensities were 6 dB, 12 dB or 18 dB above background. The first block (6 trials) had 2 of each pulse-alone condition to habituate and stabilize the startle response. These trials were not included in the analysis. The test session included 10 blocks of 16 trials in pseudorandom order. Each block comprised 3 prepulse-alone trials (+6, +12 or +18 dB), 3 pulse-alone trials (100, 110 or 120 dB), nine combinations of prepulse-pulse trials (3 prepulse options × 3 pulse options), and 1 no-stimulus (background, NS) trial. The test session

17

ended with a final block of 6 pulse-alone trials. The whole session lasted approximately 45 min. Startle responses were collected by SR-LAB (San Diego Instruments, San Diego, CA, USA). PPI was taken as the reduction in pulseinduced startle on prepulse pulse trials relative to pulse alone trials. The proportional reduction of the startle amplitude (%PPI), based on the non-transformed startle response, was calculated as [(Rpulse alone − Rprepulse pulse)/Rpulse alone] × 100%, where “R” was the individual mean response of the startle reaction. Data were analysed using a General Linear Model running in SPSS20 as reported previously (Li et al., 2009; Meyer et al., 2005). Briefly this involved a 3[Pulse(100, 110 or 120 dB)] × 3[Prepulse(71, 77 or 83 dB)] × (C57BL/6N or 129X1/SvJ) × 2[Sex(female or male)] repeated measures design, in which three pulse conditions and 3 prepulse conditions were the with-in group factors; Strain and sex were the between group factors. 2.2.2. Open field and amphetamine challenge test Open field exploration took place in a 40 cm × 40 cm rectangular white arenas. The mouse was gently placed in the centre of the arena, and allowed to explore for 1 h. The mouse was then briefly removed, and saline administered subcutaneously to control for non-specific effects of injection on activity. The arena was cleaned with 70% ethanol; the mouse was placed back in the arena and allowed to explore for another 30 min. The mouse was again briefly removed and received an intraperitoneal injection of 2.5 mg/kg damphetamine (calculated as the salt, Sigma–Aldrich, Switzerland) dissolved in a 0.9% NaCl solution in a volume of 5 ml/kg. The arena was cleaned again and the mouse returned for another hour. Mouse locomotor activities were tracked by EthoVision video tracking system (EthoVision XT 7.0, Noldus). Locomotor activity at baseline and in response to saline and amphetamine was analysed by a 2[Strain (C57BL/6N or 129X1/SvJ) × 2[Sex(female or male)] × n [Time(5 min time bins)] repeated measures General Linear Model, where n = 12, 6 and 12 respectively. 2.3. Proteomics study Tissue from 12 mice [C57BL/6N = 26 (F = 3, M = 3); 129X1/SvJ = 26 (F = 3, M = 3)], without exposure to testing, was included in a 2D proteomic gel analysis. Animals were sacrificed by decapitation and brains dissected, immediately frozen and stored at −80 ◦ C. Medial prefrontal cortex (mPFC) was thawed and homogenized in 2D lysis buffer (7 M urea, 2 M thiourea, 2% CHAPS, 10 mmol DTT, 0.5% IPG buffer) for 30 s, then centrifuged at 15,000 × g for 15 min to remove cell debris. Supernatant for 2-DE was collected and quantified with protein assay kit (Pierce® 660 nm Protein Assay, Thermo Scientific). The 2D procedure followed the protocol from Bio-Rad Laboratories (Bio-Rad Laboratories, Inc.), with minor modifications. Rehydration and first dimension were carried out using Bio-Rad isoelectric focusing (IEF) separation (Bio-Rad IEF system). The 350 ␮l rehydration buffer containing 180 ␮g protein was loaded on 18 cm, pH 4–7, IPG DryStrips (Bio-Rad). IEF conditions were: 50 V, 18 h, 1000 V, 1 h; 3000 V, 3 h, 8000 V, 6 h. After the IEF run was complete, the IPG strips were equilibrated for 2× 20 min in the equilibration buffer containing 50 mM Tris, 6 M urea, 30% glycerol, 2% SDS. The first equilibration was performed in the equilibration buffer with 1.0% (w/v) DTT followed by a second equilibration with 2.5% (w/v) iodoacetamide. The strips were subsequently subjected to a secondary dimensional separation by 12% SDS-PAGE (Bio-Rad vertical system). The SDS-PAGE was performed at 10 mA/gel for 30 min, and then 45 mA/gel, until the dye front reached the bottom of gels. After fixing in 50% ethanol, 12% acetate solution and staining with silver nitrate, gels were scanned at 300 dpi resolution and the images

18

X. Zhang et al. / Brain Research Bulletin 116 (2015) 16–24

Fig. 1. PPI in C57BL/6N and 129X1/SvJ mice. Error bars refer to ±SEM. (A1) Reactivity to pulse alone in C57BL/6N and 129X1/SvJ mice. (A2) PPI in C57BL/6N and 129X1/SvJ mice. (B) Sex differences in PPI in C57BL/6N mice.

were analysed with Image Master PlatinumTM software (GE Health care). Protein spots with p < 0.05 and >1.3-fold change were considered significant. Spots of interest were picked from sliver-stained gels, followed by in-gel trypsin digestion and peptide extraction (Wang et al., 2007) for protein identification by tandem mass

spectrometry analysis using ABI 4800 MALDI TOF/TOFTM MS Analyzer (Applied Biosystems, Foster City, CA, USA). The combined peptide mass fingerprinting (PMF) and MS/MS peptide fragmentation data were submitted to the NCBInr database and SwissProt database using the software MASCOT version 2.2 (Matrix Science). For all significant protein identifications, both protein and total ion

Fig. 2. Open field and amphetamine test. Error bars refer to ±SEM. Distances moved in the open field and amphetamine test with 5-min time-bins.

X. Zhang et al. / Brain Research Bulletin 116 (2015) 16–24

19

Fig. 3. Gel image and PLS-DA analysis. (A) A representative two-dimensional gel shows the protein spots expressed in C57BL/6N mice and 129X1/SvJ mice. The molecular weight is indexed on the right side of the figure. Proteins with significant differences in expression are highlighted in green detected. (B) A multivariate analysis using partial least squares-discriminative analysis (PLS-DA) carried out using SIMCA-P + 12 software (Umetrics). This analysis indicated that C57BL/6N mice could be differentiated from 129X1/SvJ mice on the basis of the whole medial Prefrontal Cortex protein expression. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

scores were above or equal to C.I. 99%. The biological functions of differentially expressed protein sequences were explored using blast2go software (https://www.blast2go.com/). 2.4. Multivariate statistical analysis of full proteome profile Multivariate analysis using partial least squares-discriminant analysis (PLS-DA, SIMCA 12.0, Umetrics, Umea, Sweden) was performed to examine whether C57BL/6N mice were different from 129X1/SvJ mice on the basis of full proteome profile (Karp et al., 2005). 2.5. Western blot validation Prefrontal tissue harvested from mice used for behavioural testing was used for western blot confirmation of the 2D experiment. mPFC of male and female C57BL/6N and 129X1/SvJ mice was homogenised at 4 ◦ C in lysis buffer (1:5, wt/vol) containing 1 mM EDTA and 20 mM phenylmethylsulphonyl fluoride. Proteins from resultant supernatant were determined (Bio-Rad Protein Assay)

for sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Protein (20 ␮g/lane) was subjected to electrophoresis on a 10% (wt/vol) polyacrylamide gel in SDS, and gels subsequently processed for electroblotting to polyvinylidene difluoride (PVDF) membranes. The blotted PVDF membranes were saturated with 5% (wt/vol) of skimmed milk in Tris Buffer Saline, PH 7.4 and 0.1% (vol/vol) of Tween 20 for 1 h at room temperature. The membranes were sequentially incubated with primary antibodies to the following proteins: Superoxide dismutase (SOD2, rabbit polyclonal IgG antibody, 1:500 dilution, Cat# sc-30080, Santa Cruz Biotechnology) and glyceraldehyde-3phosphate dehydrogenase (GAPDH, rabbit polyclonal IgG antibody, 1:1000 dilution, Cat# ab9485, Abcam) overnight at 4 ◦ C following by a peroxidase-labelled anti-mouse/rabbit IgG (1:2000 dilution, Boehringer Mannheim, Germany) for 1 h at room temperature. After thorough washing, positive bands were revealed using ECL western blotting detection reagents and autoradiography film (Amersham, Biosciences, UK). The intensities of the bands were quantified using IMAGE QUANT software (Molecular Dynamics, USA).

20

X. Zhang et al. / Brain Research Bulletin 116 (2015) 16–24

Table 1 Differential protein expression in C57BL/6N mice relative to 129X1/SvJ mice. Classification

Spot no.

Fold differences

p-Value

Protein name

NCBI protein ID

1.88 lower 8.77 lower

0.028 0.007

Gi|31980762 Gi|55153885

175

Undetectable in 129X1/SvJ

0.023

196 205

Undetectable in 129X1/SvJ Undetectable in 129X1/SvJ

0.001 0.003

224

Undetectable in 129X1/SvJ

0.019

235

Undetectable in 129X1/SvJ

4.82E−06

Superoxide dismutase Glyceraldehyde-3-phosphate dehydrogenase Proteasome subunit alpha type-6 Peroxiredoxin 6 Proteasome subunit alpha type-1 NADH dehydrogenase 1 alpha subcomplex submit 10 Alpha-enolase

12.7909 lower

2.01E−04

Growth factor receptor-bound protein 2

Gi|6680083

124

9.22 lower

0.017

14-3-3 protein zeta/delta

Gi|1841387

107 127 129 130 139 143 222 226 237

4.3778 lower Undetectable in C57BL/6N Undetectable in C57BL/6N Undetectable in C57BL/6N Undetectable in C57BL/6N Undetectable in C57BL/6N Undetectable in 129X1/SvJ Undetectable in 129X1/SvJ Undetectable in 129X1/SvJ

0.039 0.003 4.16E−05 0.006 1.10E−05 0.001 0.008 0.021 0.002

Gi|31981577 Gi|13384630 Gi|5729783 Gi|12408324 Gi|33416314 Gi|299522842 Gi|228480253 Gi|37604188 Gi|126521835

239

Undetectable in 129X1/SvJ

0.010

Prefoldin subunit 2 Stathmin-2 Complexin-2 Complexin-1 Tubulin beta-2A chain Dynactin subunit 2 Septin-2 Septin-5 T-complex protein 1 subunit beta Coronin-1

4.710 lower

0.006

Gi|18152793

Undetectable in 129X1/SvJ Undetectable in 129X1/SvJ

0.012 1.95E−06

Pyruvate dehydrogenase E1 component subunit beta, mitochondria 28S ribosomal protein S22 Ornithine aminotransferase

1 48 Energy metabolism

Cell communication/signal transduction

Microtubule and neurotransmitter transport

Mitochondrial matrix

16

43

217 232

3. Results 3.1. Behavioural tests 3.1.1. PPI There was no strain difference in baseline startle responses in both sexes, see Fig. 1A. There was a significant main effect of strain on PPI (F(1,36) = 10.727, p = 0.002), and the main effect of sex was also significant (F(1,36) = 10.618, p = 0.002). The dataset was therefore split by strain and sex for further exploration. Both sexes of 129X1/SvJ mice had significantly higher PPI than C57BL/6N mice [females (F(1,18) = 4.826, p = 0.041); males (F(1,18) = 6.184, p = 0.023)]; but C57BL/6N females had higher PPI than males (F(1,18) = 7.357, p = 0.014); see Fig. 1.

Gi|6755198 Gi|6671549 Gi|33563282 Gi|148708069 Gi|34784434

Gi|4895037

Gi|13384904 Gi|8393866

3.2.2. Individual protein differences Analysis of covariance (ANCOVA) revealed significant strain differences in the expression of 22 proteins. The identities of these protein spots, their fold differences in C57BL/6N relative to 129X1/SvJ, and the peptide sequences matched for each protein are shown in Table 1. The biological functions of differentially expressed protein are shown in Fig. 4. 3.3. Western blot validation Western blot analyses confirmed that the expression of superoxide dismutase (SOD2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was significantly lower in C57BL/5N mice relative to 129X1/SvJ mice (Fig. 5). 4. Discussion

3.1.2. Open field and amphetamine challenge C57BL/6N mice had higher baseline locomotor activity (distance moved in the open field test) than 129X1/SvJ (F(1,36) = 38.623, p < 0.001); and this activity did not significantly change following a saline injection. Baseline activity was therefore included as a covariate in the analysis of response to amphetamine; C57BL/6N mice were significantly more active in response to amphetamine than 129X1/SvJ (F(1,35) = 24.570, p < 0.001), see Fig. 2.

Deciding the appropriate in-bred strain of mouse to adopt in experimental models relevant to neuropsychiatric disorders is challenging given the different anatomical, biochemical, neuropathological or behavioural features of individual strains (He and Shippenberg, 2000; Kuzmin and Johansson, 2000; Simpson et al., 1997). In this study, we observed that young adult C57BL/6N mice and 129X1/SvJ had distinct behavioural and proteomic phenotypes. Relative to 129X1/SvJ mice, C57BL/6N mice had lower PPI and were more sensitive to amphetamine challenge.

3.2. 2D gel electrophoresis

4.1. PPI

3.2.1. Whole proteome analysis A total of 364 individual spots were visualised across the group of 12 mPFC gels (Fig. 3A). All these spots were included in the partial least squares-discriminant analysis (PLS-DA). The PLS-DA indicated a clear separation between two strains (Fig. 3B).

C57BL/6N mice had a relative impairment in PPI, and female C57BL/6N mice had higher levels of PPI than males of this strain (Fig. 1B). These inter-strain differences in PPI occurred in the absence of significant differences in baseline startle reactivity. This is important because 129X1/SvJ mice have been reported to

X. Zhang et al. / Brain Research Bulletin 116 (2015) 16–24

21

Fig. 4. The biological functions of selected protein identified using Blast2go (https://www.blast2go.com/).

suffer progressive hearing loss in adulthood (JAX® Mice Database). Hearing loss would be expected to impair PPI in 129X1/SvJ, but the opposite was true here, suggesting hearing was intact in our cohort. The low levels of PPI in C57BL/6N male mice observed here have been reported previously (Matsuo et al., 2010). However, PPI in female C57B6N mice has not previously been examined and we

found that females had higher PPI than male mice. Lower levels of PPI are consistently reported in male patients with schizophrenia (Braff et al., 2001; Cadenhead et al., 1993); but female patients with schizophrenia have higher PPI than men with the disorder (Kumari et al., 2004). Thus, the relatively low PPI in the C57BL/6N strain in male mice especially, is reminiscent of the pattern of PPI impairment in schizophrenia.

Fig. 5. Western blot analysis of differentially expressed target proteins in C57BL/6N and 129X1/SvJ mice. (A) Gel image of protein products in C57BL/6N and 129X1/SvJ mice. (B) The relative expression of the target proteins/␤-actin. Columns show mean ±SEM (n = 20 for C57BL/6N mice. n = 14 for 129X1/SvJ mice). *p < 0.05.

22

X. Zhang et al. / Brain Research Bulletin 116 (2015) 16–24

4.2. Amphetamine sensitivity We found that C57BL/6N mice were more sensitive to amphetamine than 129X1/SvJ mice (Miner, 1997), even when baseline activity differences were controlled for. This result is consistent with findings previously reported in C57BL/6J mice (Chen et al., 2007). Specifically, the enhanced response to amphetamine in C57BL/6J mice is thought to be caused by a greater amphetamineinduced dopamine efflux in C57BL/6J mice than in 129S2/SvHsd mice (Chen et al., 2007). A similar mechanism may underscore the difference in amphetamine response in our ‘related’ C57 and 129 strains. Amphetamine-induced locomotion is thought to model a core pathophysiology of schizophrenia (Tenn et al., 2003), suggesting that the C57BL/6 sub-strains may usefully mirror this aspect of the condition. Taken together, relatively low levels of PPI, sex differences in PPI, and amphetamine sensitivity in C57BL/6N mice compared to 129X1/SvJ mimic the differences observed in people with neurodevelopmental conditions such as schizophrenia compared to typically developing peers (Braff and Geyer, 1990; McAlonan et al., 2002; Meltzer, 1980). This has implications for the choice of strain in studies of schizophrenia. For example, C57BL/6N mice may be a good choice to examine new antipsychotic medications (Geyer et al., 1990; Swerdlow et al., 1994). In contrast, when the aim is to investigate null mutations predicted to impair PPI or enhance amphetamine sensitivity, 129X1/SvJ mice may be more appropriate.

4.3. Proteomics The pattern of protein expression in the prefrontal cortex of C57BL6N mice was clearly distinct from 129X1/SvJ mice. Blast2go analysis showed that the proteins differentially expressed were those associated with oxidative metabolism, receptor protein signalling, cell communication and signal transduction and energy pathways (Table 1, Fig. 4). These protein differences may have functional consequences. For example, relatively lower SOD, pyruvate dehydrogenase (PDH) and GAPDH in C57BL/6N mice suggests a vulnerability to oxidative stress in that strain (Brown, 1992; Gardner et al., 1994; Maier and Chan, 2002); and oxidative stress has been linked to weak PPI and sensitivity to amphetamine (Cabiscol et al., 2000; Hakak et al., 2001; Jiang et al., 2013). Strain differences in the expression of structural proteins tubulin beta, prefoldin, stathmin and dynactin imply differences in microtubule dynamics (Tkachev et al., 2003), which is essential for synaptic transmission (Feng, 2006; Ikegami et al., 2007; Ly and Verstreken, 2006; Shumyatsky et al., 2005). Synaptic transmission may also be altered by other proteins differentially expressed, such as the presynaptic proteins Complexin I and II (Brose, 2008; Strenzke et al., 2009; Südhof and Rothman, 2009). In particular, differences in the excitatory/inhibitory balance in these strains may result from their distinct protein profile. For example, expression of Sept2 regulates glial glutamate uptake through interaction with the astrocyte glutamate transporter (GLAST) (Kinoshita et al., 2004) and was higher in C57BL/6N tissue. In contrast, lower levels of 14-3-3 protein zeta/delta in C57BL/6N mice may have ‘knock-on’ disinhibitory effects on protein kinase C (PKC) which regulates the activities of other glutamate transporters, GLT-1 and EAAC1, Finally, glutamate can be synthesised from ␣-ketogutarate by ornithine aminotransferase (OAT) (Greenamyre, 2001) and greater levels of OAT in C57BL/6N mice relative to 129X1/SvJ mice may facilitate this excitatory pathway in C57BL/6N mice. We cautiously suggest a relative excitatory/inhibitory shift towards glutamate in C57BL6N mice. This would be expected to disrupt PPI and enhance sensitivity to

amphetamine in these mice (Pycock and Horton, 1979; Takahashi et al., 2007). 4.4. Limitations We acknowledge a number of limitations in our study. Only two mouse strains were compared; therefore it is unknown whether results hold across comparisons of either strain to other in-bred strains or even closely ‘related’ strains. For example, it has been reported that genetic manipulations that result in an enhanced resistance to depression-like pathologies may be more difficult to ascertain in mice with C57BL/6J background, but the opposite might be true for C57BL/6N animals (Sturm et al., 2015). We elected to study the mPFC proteome only. Nevertheless, many interconnected brain regions are involved in the expression of the complex behavioural phenotypes examined. The proteomics approach adopted here was exploratory in nature. It is not the most sensitive omics method and does not directly sample genetic differences. The standard global scale proteomic analysis applied does address potential protein differences in subcellular fractions such as mitochondria, nucleus or plasma membrane. Thus, false negative results are possible and future studies examining subcellular compartments, though challenging, would be useful to reduce false negative rates (Vercauteren et al., 2007). However, a strength of our study was the inclusion of female animals. Exclusion of female animals in experiments and inadequate analysis of data by sex has been suggested to contribute to the troubling rise of irreproducibility in preclinical biomedical research (Clayton and Collins, 2014; Landis et al., 2012). We have therefore tried to conform to calls from the US National Institutes of Health (NIH) and Office of Research on Women’s Health for a balance of male and female animals in preclinical studies unless single sex inclusion is specifically warranted (May 2014). 5. Conclusions Modelling of human neurodevelopmental disorders in animals is extremely challenging given the subjective nature of many symptoms, the lack of biomarkers and objective diagnostic tests, and the early state of the relevant neurobiology and genetics. Background strain characteristics have considerable relevance for interpreting results from studies of genetically altered animals or those exposed to environmental insults or drug treatment (Gershenfeld and Paul, 1998). C57BL/6N and 129X1/SvJ strains are widely used in the field of translational neuroscience and differences observed here highlight the importance of strain selection. We suggest that the C57BL/6N mouse may usefully proxy features of the neurodevelopmental disorders and could have application in pre-translational screening of new therapeutic approaches. The 129X1/SvJ strain in contrast, might be better suited to experimental studies of causal risk factors expected to lower PPI and increase amphetamine sensitivity. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements The authors would like to thank the staff of the Laboratory animal Unit in The University of Hong Kong for their expert assistance with breeding and husbandry. This study was supported by research funding from the Hong Kong Universities General Research Fund Award to Dr Grainne McAlonan (HKU 774710).

X. Zhang et al. / Brain Research Bulletin 116 (2015) 16–24

References Belknap, J.K., Crabbe, J.C., Young, E., 1993. Voluntary consumption of ethanol in 15 inbred mouse strains. Psychopharmacology (Berl) 112, 503–510, http://dx.doi. org/10.1007/bf02244901 Bogerts, B., Meertz, E., Schönfeldt-Bausch, R., 1985. Basal ganglia and limbic system pathology in schizophrenia: a morphometric study of brain volume and shrinkage. Arch. Gen. Psychiatry 42, 784–791, http://dx.doi.org/10.1001/archpsyc. 1985.01790310046006 Braff, D.L., Geyer, M.A., 1990. Sensorimotor gating and schizophrenia: human and animal model studies. Arch. Gen. Psychiatry 47, 181–188, http://dx.doi.org/10. 1001/archpsyc.1990.01810140081011 Braff, D.L., Geyer, M.A., Swerdlow, N.R., 2001. Human studies of prepulse inhibition of startle: normal subjects, patient groups, and pharmacological studies. Psychopharmacology (Berl) 156, 234–258, http://dx.doi.org/10.1007/ s002130100810 Brose, N., 2008. For better or for worse: complexins regulate SNARE function and vesicle fusion. Traffic 9, 1403–1413, http://dx.doi.org/10.1111/j.1600-0854. 2008.00758.x Brown, G.K., 1992. Pyruvate dehydrogenase E1 alpha deficiency. J. Inherit. Metab. Dis. 15, 625–633, http://dx.doi.org/10.1007/bf01799619 Bryant, C.D., Zhang, N.N., Sokoloff, G., Fanselow, M.S., Ennes, H.S., Palmer, A.A., McRoberts, J.A., 2008. Behavioral differences among C57BL/6 substrains: implications for transgenic and knockout studies. J. Neurogenet. 22, 315–331, http:// dx.doi.org/10.1080/01677060802357388 Cabiscol, E., Piulats, E., Echave, P., Herrero, E., Ros, J., 2000. Oxidative stress promotes specific protein damage in Saccharomyces cerevisiae. J. Biol. Chem. 275, 27393–27398, http://dx.doi.org/10.1074/jbc.m003140200 Cadenhead, K.S., Geyer, M.A., Braff, D.L., 1993. Impaired startle prepulse inhibition and habituation. Am. J. Psychiatry 150, 1862–1867, http://dx.doi.org/10.1176/ ajp.150.12.1862 Chen, R., Zhang, M., Park, S., Gnegy, M.E., 2007. C57BL/6J mice show greater amphetamine-induced locomotor activation and dopamine efflux in the striatum than 129S2/SvHsd mice. Pharmacol. Biochem. Behav. 87, 158–163, http:// dx.doi.org/10.1016/j.pbb.2007.04.012 Clayton, J.A., Collins, F.S., 2014. NIH to balance sex in cell and animal studies. Nature 509, 282–283, http://dx.doi.org/10.1038/509282a Crawley, J.N., 1996. Unusual behavioral phenotypes of inbred mouse strains. Trends Neurosci. 19, 181–182, http://dx.doi.org/10.1016/s0166-2236(96)20021-9 Feng, J., 2006. Microtubule: a common target for parkin and Parkinson’s disease toxins. Neuroscientist 12, 469–476, http://dx.doi.org/10.1177/1073858406293853 Flores, G., Wood, G.K., Liang, J.-J., Quirion, R., Srivastava, L.K., 1996. Enhanced amphetamine sensitivity and increased expression of dopamine D2 receptors in postpubertal rats after neonatal excitotoxic lesions of the medial prefrontal cortex. J. Neurosci. 16, 7366–7375. Gardner, P.R., Nguyen, D., White, C.W., 1994. Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and in rat lungs. Proc. Natl. Acad. Sci. U. S. A. 91, 12248–12252, http://dx.doi.org/10.1073/pnas.91.25. 12248 Gerlai, R., 1996. Gene-targeting studies of mammalian behavior: is it the mutation or the background genotype? Trends Neurosci. 19, 177–181, http://dx.doi.org/ 10.1016/s0166-2236(96)20020-7 Gershenfeld, H., Paul, S., 1998. Towards a genetics of anxious temperament: from mice to men. Acta Psychiatr. Scand. 98, 56–65, http://dx.doi.org/10.1111/j.16000447.1998.tb05968.x Geyer, M.A., Swerdlow, N.R., Mansbach, R.S., Braff, D.L., 1990. Startle response models of sensorimotor gating and habituation deficits in schizophrenia. Brain Res. Bull. 25, 485–498. Greenamyre, J.T., 2001. Glutamatergic influences on the basal ganglia. Clin. Neuropharmacol. 24, 65–70, http://dx.doi.org/10.1016/0361-9230(90)90241-q Hakak, Y., Walker, J.R., Li, C., Wong, W.H., Davis, K.L., Buxbaum, J.D., Haroutunian, V., Fienberg, A.A., 2001. Genome-wide expression analysis reveals dysregulation of myelination-related genes in chronic schizophrenia. Proc. Natl. Acad. Sci. U. S. A. 98, 4746–4751, http://dx.doi.org/10.1073/pnas.081071198 He, M., Shippenberg, T.S., 2000. Strain differences in basal and cocaine-evoked dopamine dynamics in mouse striatum. J. Pharmacol. Exp. Ther. 293, 121–127. Hefner, K., Whittle, N., Juhasz, J., Norcross, M., Karlsson, R.-M., Saksida, L.M., Bussey, T.J., Singewald, N., Holmes, A., 2008. Impaired fear extinction learning and cortico-amygdala circuit abnormalities in a common genetic mouse strain. J. Neurosci. 28, 8074–8085, http://dx.doi.org/10.1523/jneurosci.4904-07.2008 Ikegami, K., Heier, R.L., Taruishi, M., Takagi, H., Mukai, M., Shimma, S., Taira, S., Hatanaka, K., Morone, N., Yao, I., 2007. Loss of ␣-tubulin polyglutamylation in ROSA22 mice is associated with abnormal targeting of KIF1A and modulated synaptic function. Proc. Natl. Acad. Sci. U. S. A. 104, 3213–3218, http://dx.doi. org/10.1073/pnas.0611547104 Jablensky, A., 2000. Epidemiology of schizophrenia: the global burden of disease and disability. Eur. Arch. Psychiatry Clin. Neurosci. 250, 274–285, http://dx.doi.org/ 10.1007/s004060070002 Jiang, Z., Rompala, G.R., Zhang, S., Cowell, R.M., Nakazawa, K., 2013. Social isolation exacerbates schizophrenia-like phenotypes via oxidative stress in cortical interneurons. Biol. Psychiatry 73, 1024–1034, http://dx.doi.org/10.1016/j. biopsych.2012.12.004 Karp, N.A., Griffin, J.L., Lilley, K.S., 2005. Application of partial least squares discriminant analysis to two-dimensional difference gel studies in expression proteomics. Proteomics 5, 81–90, http://dx.doi.org/10.1002/pmic.200400881

23

Kinoshita, N., Kimura, K., Matsumoto, N., Watanabe, M., Fukaya, M., Ide, C., 2004. Mammalian septin Sept2 modulates the activity of GLAST, a glutamate transporter in astrocytes. Genes Cells 9, 1–14, http://dx.doi.org/10.1111/j.1356-9597. 2004.00696.x Kumari, V., Aasen, I., Sharma, T., 2004. Sex differences in prepulse inhibition deficits in chronic schizophrenia. Schizophr. Res. 69, 219–235, http://dx.doi.org/10. 1016/j.schres.2003.09.010 Kuzmin, A., Johansson, B., 2000. Reinforcing and neurochemical effects of cocaine: differences among C57, DBA, and 129 mice. Pharmacol. Biochem. Behav. 65, 399–406, http://dx.doi.org/10.1016/s0091-3057(99)00211-7 Landis, S.C., Amara, S.G., Asadullah, K., Austin, C.P., Blumenstein, R., Bradley, E.W., Crystal, R.G., Darnell, R.B., Ferrante, R.J., Fillit, H., 2012. A call for transparent reporting to optimize the predictive value of preclinical research. Nature 490, 187–191, http://dx.doi.org/10.1038/nature11556 Li, Q., Cheung, C., Wei, R., Hui, E.S., Feldon, J., Meyer, U., Chung, S., Chua, S.E., Sham, P.C., Wu, E.X., 2009. Prenatal immune challenge is an environmental risk factor for brain and behavior change relevant to schizophrenia: evidence from MRI in a mouse model. PLoS ONE 4, e6354, http://dx.doi.org/10.1371/journal.pone. 0006354 Ly, C.V., Verstreken, P., 2006. Mitochondria at the synapse. Neuroscientist 12, 291–299, http://dx.doi.org/10.1177/1073858406287661 Maier, C.M., Chan, P.H., 2002. Book review: role of superoxide dismutases in oxidative damage and neurodegenerative disorders. Neuroscientist 8, 323–334, http://dx.doi.org/10.1177/107385840200800408 Matsuo, N., Takao, K., Nakanishi, K., Yamasaki, N., Tanda, K., Miyakawa, T., 2010. Behavioral profiles of three C57BL/6 substrains. Front. Behav. Neurosci., 4, http:// dx.doi.org/10.3389/fnbeh.2010.00029 McAlonan, G.M., Daly, E., Kumari, V., Critchley, H.D., van Amelsvoort, T., Suckling, J., Simmons, A., Sigmundsson, T., Greenwood, K., Russell, A., 2002. Brain anatomy and sensorimotor gating in Asperger’s syndrome. Brain 125, 1594–1606, http:// dx.doi.org/10.1093/brain/awf150 McGhie, A., Chapman, J., 1961. Disorders of attention and perception in early schizophrenia. Br. J. Med. Psychol. 34, 103–116, http://dx.doi.org/10.1111/j. 2044-8341.1961.tb00936.x Meltzer, H.Y., 1980. Relevance of dopamine autoreceptors for psychiatry. Schizophr. Bull. 6, 456–475, http://dx.doi.org/10.1093/schbul/6.3.456 Meyer, U., Feldon, J., Schedlowski, M., Yee, B.K., 2005. Towards an immunoprecipitated neurodevelopmental animal model of schizophrenia. Neurosci. Biobehav. Rev. 29, 913–947, http://dx.doi.org/10.1016/j.neubiorev.2004.10.012 Miner, L.L., 1997. Cocaine reward and locomotor activity in C57BL/6J and 129/SvJ inbred mice and their F1 cross. Pharmacol. Biochem. Behav. 58, 25–30, http:// dx.doi.org/10.1016/s0091-3057(96)00465-0 Nestler, E.J., Hyman, S.E., 2010. Animal models of neuropsychiatric disorders. Nat. Neurosci. 13, 1161–1169, http://dx.doi.org/10.1038/nn.2647 Owen, E., Logue, S., Rasmussen, D., Wehner, J., 1997. 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 80, 1087–1099, http://dx.doi.org/10.1016/ s0306-4522(97)00165-6 Perry, W., Minassian, A., Lopez, B., Maron, L., Lincoln, A., 2007. Sensorimotor gating deficits in adults with autism. Biol. Psychiatry 61, 482–486, http://dx.doi.org/10. 1016/j.biopsych.2005.09.025 Pycock, C., Horton, R., 1979. Dopamine-dependent hyperactivity in the rat following manipulation of GABA mechanisms in the region of the nucleus accumbens. J. Neural Transm. 45, 17–33, http://dx.doi.org/10.1007/bf01243878 Ralph, R.J., Paulus, M.P., Geyer, M.A., 2001. Strain-specific effects of amphetamine on prepulse inhibition and patterns of locomotor behavior in mice. J. Pharmacol. Exp. Ther. 298, 148–155. Rutter, M., 1978. Diagnosis and definition of childhood autism. J. Autism Child. Schizophr. 8, 139–161, http://dx.doi.org/10.1007/BF01537863 Shumyatsky, G.P., Malleret, G., Shin, R.-M., Takizawa, S., Tully, K., Tsvetkov, E., Zakharenko, S.S., Joseph, J., Vronskaya, S., Yin, D., 2005. Stathmin, a gene enriched in the amygdala, controls both learned and innate fear. Cell 123, 697–709, http:// dx.doi.org/10.1016/j.cell.2005.08.038 Simpson, E.M., Linder, C.C., Sargent, E.E., Davisson, M.T., Mobraaten, L.E., Sharp, J.J., 1997. Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice. Nat. Genet. 16, 19–27, http://dx.doi.org/10.1038/ng059719 Strenzke, N., Chanda, S., Kopp-Scheinpflug, C., Khimich, D., Reim, K., Bulankina, A.V., Neef, A., Wolf, F., Brose, N., Xu-Friedman, M.A., 2009. Complexin-I is required for high-fidelity transmission at the endbulb of held auditory synapse. J. Neurosci. 29, 7991–8004, http://dx.doi.org/10.1523/jneurosci.0632-09.2009 Sturm, M., Becker, A., Schroeder, A., Bilkei-Gorzo, A., Zimmer, A., 2015. Effect of chronic corticosterone application on depression-like behavior in C57BL/6N and C57BL/6J mice. Genes Brain Behav. 14, 292–300, http://dx.doi.org/10.1111/gbb. 12208 Südhof, T.C., Rothman, J.E., 2009. Membrane fusion: grappling with SNARE and SM proteins. Science 323, 474–477, http://dx.doi.org/10.1126/science.1161748 Swerdlow, N.R., Braff, D.L., Taaid, N., Geyer, M.A., 1994. Assessing the validity of an animal model of deficient sensorimotor gating in schizophrenic patients. Arch. Gen. Psychiatry 51, 139–154, http://dx.doi.org/10.1001/archpsyc.1994. 03950020063007 Takahashi, K., Nagai, T., Kamei, H., Maeda, K., Matsuya, T., Arai, S., Mizoguchi, H., Yoneda, Y., Nabeshima, T., Takuma, K., 2007. Neural circuits containing pallidotegmental GABAergic neurons are involved in the prepulse inhibition of the

24

X. Zhang et al. / Brain Research Bulletin 116 (2015) 16–24

startle reflex in mice. Biol. Psychiatry 62, 148–157, http://dx.doi.org/10.1016/j. biopsych.2006.06.035 Tenn, C.C., Fletcher, P.J., Kapur, S., 2003. Amphetamine-sensitized animals show a sensorimotor gating and neurochemical abnormality similar to that of schizophrenia. Schizophr. Res. 64, 103–114, http://dx.doi.org/10.1016/s09209964(03)00009-4 Tkachev, D., Mimmack, M.L., Ryan, M.M., Wayland, M., Freeman, T., Jones, P.B., Starkey, M., Webster, M.J., Yolken, R.H., Bahn, S., 2003. Oligodendrocyte dysfunction in schizophrenia and bipolar disorder. Lancet 362, 798–805, http://dx. doi.org/10.1016/s0140-6736(03)14289-4

Vercauteren, F.G., Flores, G., Ma, W., Chabot, J.G., Geenen, L., Clerens, S., Fazel, A., Bergeron, J.J., Srivastava, L.K., Arckens, L., 2007. An organelle proteomic method to study neurotransmission-related proteins, applied to a neurodevelopmental model of schizophrenia. Proteomics 7, 3569–3579, http://dx.doi.org/10.1002/ pmic.200700379 Wang, Y., Lam, K.S., Lam, J.B., Lam, M.C., Leung, P.T., Zhou, M., Xu, A., 2007. Overexpression of angiopoietin-like protein 4 alters mitochondria activities and modulates methionine metabolic cycle in the liver tissues of db/db diabetic mice. Mol. Endocrinol. 21, 972–986, http://dx.doi.org/10.1210/me.20060249.