Original Paper Dev Neurosci 2014;36:18–28 DOI: 10.1159/000357495
Received: April 24, 2013 Accepted after revision: November 21, 2013 Published online: January 24, 2014
Late-Life Effects of Chronic Methamphetamine Exposure during Puberty on Behaviour and Corticostriatal Mono-Amines in Social Isolation-Reared Rats Laetitia Strauss a Christiaan B. Brink a Marisa Möller a Dan J. Stein c Brian H. Harvey b a
Division of Pharmacology, School of Pharmacy, and b Centre of Excellence for Pharmaceutical Sciences, Faculty of Health Sciences, North-West University, Potchefstroom, and c MRC Unit for Anxiety and Stress Disorders, Department of Psychiatry, University of Cape Town, Cape Town, South Africa
Abstract Chronic methamphetamine (MA) abuse results in an acute psychosis indistinguishable from paranoid schizophrenia. However, less is known of the interaction between MA use and environmental insults, and how this contributes to lateonset psychopathology. Using social isolation rearing (SIR), a neurodevelopmental animal model of schizophrenia, we investigated the association between changes in corticostriatal mono-amines and putative behaviours related to MA-induced psychosis in isolation and group-housed rats following chronic MA or saline exposure. Weaned male offspring of MA-naive female Wistar rats, either group- or isolation-housed from postnatal day (PND) +21, received saline (2 ml/kg s.c. b.i.d.) or an escalating dose of MA (0.2–6 mg/kg s.c. b.i.d.) for 16 days from PND +35 to +50. On PND +78, offspring were tested for deficits in social interactive behaviour (SIB) and prepulse inhibition (PPI) of startle, with frontal cortex and striatum harvested for the assessment of monoamine concentrations. SIR significantly reduced rearing
© 2014 S. Karger AG, Basel 0378–5866/14/0361–0018$39.50/0 E-Mail [email protected] www.karger.com/dne
time, staying together, approaching and anogenital sniffing (outward-directed SIB), but increased self-grooming and locomotor activity (self-directed SIB), and also induced profound deficits in PPI. Pubertal MA exposure in group-housed animals also induced similar alterations in outward- and selfdirected SIB and reduced PPI. Combined MA + SIR exposure evoked a similarly intense behavioural response as SIR or MA separately, with no exacerbation evident. Neither treatment separately nor together affected corticostriatal serotonin or noradrenaline levels, although frontal cortical dopamine (DA) levels were significantly increased in SIR and MA + group-housed animals. A trend towards further elevated frontal cortical DA was noted in the MA + SIR treatment group. Striatal DA was unaltered by all treatments. This study provides the first evidence that chronic pubertal MA exposure evokes postpubertal psychosis-like behaviours in rats of similar intensity to that induced by a neurodevelopmental animal model of schizophrenia (SIR). Moreover, the study is unique in that these behavioural changes occur together with associated changes in frontal cortical but not striatal DA, without affecting other mono-amines, and strongly implicates frontal cortical DA changes in the psychotogenic effects of early-life MA exposure or environmental insult. Although MA exposure in animals with a history of environmental insult (i.e. MA + SIR) has similar effects, combined
Brian H. Harvey, PhD Centre of Excellence for Pharmaceutical Sciences, School of Pharmacy Faculty of Health Sciences, North-West University Potchefstroom, 2520 (South Africa) E-Mail Brian.Harvey @ nwu.ac.za
Downloaded by: Stockholms Universitet 198.143.54.1 - 9/4/2015 8:38:04 AM
Key Words Postweaning exposure · Early-life adversity · Wistar rat · Late-life psychopathology · Frontal cortex · Striatum · Cumulative risk · Dual diagnosis · Stress
© 2014 S. Karger AG, Basel
Introduction
As a drug of abuse, methamphetamine (MA) is fast becoming a major global health problem [1–3]. MA is transported by dopamine (DA), serotonin (5-HT) and noradrenaline (NA) transporters to cause a reverse transport of mono-amines and a resulting increase in synaptic concentrations [1]. In fact MA is a potent releaser of DA, NA and 5-HT [1, 2], while it also inhibits the reuptake of these mono-amines [1]. Chronic MA use, however, tends to deplete DA levels [4, 5]. These pronounced effects on dopaminergic transmission are presumed to mediate the psychotogenic effects of MA as well as increased oxidative stress and neurotoxicity after long-term abuse of the drug [1]. The symptoms of MA-induced psychosis, such as paranoia, hallucinations and delusions [3], are similar to acute paranoid schizophrenia, and both conditions share several similarities in positive and negative symptoms [6], as well as cognitive deficits [7]. Importantly, MA abusers are 11 times more likely to develop psychosis than the general population [3]. Individuals exposed to early-life stress or adversity might be more vulnerable to developing late-life behavioural abnormalities [8]. Schizophrenia is widely regarded as a neurodevelopmental disorder, being causally related to pre-, peri- and postnatal stress [9]. Whereas MA abuse is most often associated with acute psychosis [3, 10, 11], with up to 50% of cases resolving spontaneously [12], chronic MA can cause long-term psychosis [13, 14] that is more often seen with higher doses and prolonged use [14]. It has been suggested that MA abuse may trigger or unmask psychosis in susceptible individuals [14]. Whether altered dopamine can fully explain this association, or whether other confounds such as environmental insults are involved, requires clarification. With MA abuse often occurring during early adolescence, the question arises whether early-life MA abuse places a vulnerable individual at greater risk for developing psychosis later in life. Epidemiological studies would suggest that it does [3, 6, 15, 16], although this question has never been formerly studied either at the clinical or preclinical level. Neuro-Behavioral Effects of MA in SIR
Animal models of early-life insults can assist in delineating the mechanisms that underlie the interplay between MA abuse and vulnerability to develop MA psychosis [17]. Drug abuse and social isolation are two important nongenetic factors that contribute towards the development of schizophrenia [18], while postweaning social isolation rearing (SIR) of rodents is regarded as a valid neurodevelopmental model of schizophrenia [8]. Deficits in social behaviours, as well as in sensorimotor gating as determined by prepulse inhibition (PPI) of the startle response, are valid behavioural measures of deficits that are analogous to those observed in schizophrenia [8]. SIR induces profound deficits in these responses [19], whereas it also causes neurochemical abnormalities that closely parallel those observed in schizophrenia, such as mono-amine and glutamate changes [20, 21]. Finally, these biobehavioural changes respond to antipsychotic drugs [19, 22]. When considering a link with MA, SIR also evokes corticostriatal oxidative stress [19], a pathological process evident in both schizophrenia [23] and MA abuse [1, 2]. This study investigated the effect of SIR and chronic MA exposure during puberty, separately and combined, on sensorimotor gating and social interactive behaviour in rats. Since altered frontostriatal DA, 5-HT and NA pathways may play an important role in the psychosis of schizophrenia and MA abuse [24–26], we also studied the ensuing effects on these mono-amines. We hypothesize that SIR or MA would evoke typical schizophrenialike behaviours, as well as altered corticostriatal monoamines, akin to that of schizophrenia, and that these changes would be worsened in combined SIR-MA-exposed animals.
Materials and Methods Animals Male Wistar rats (250–300 g on the day of testing) were randomly separated at weaning (postnatal day 21, PND 21) into group(3–4 rats/cage) and isolation-reared (1 rat/cage) conditions for 8 weeks (until PND 77). Identical cages containing sawdust were used, under controlled conditions (22 ± 1 ° C; 50 ± 10% humidity; 12-hour light/dark cycle with lights on at 6.00 h; food and water ad libitum). SIR animals were maintained in an environment with no enrichment and minimal handling, with sawdust changed once a week. The study was undertaken in accordance with the guidelines for the care and use of laboratory animals in South Africa (NorthWest University ethics approval No.: NWU-000105-11-S5).
Drug Treatment MA was dissolved in saline before being administered according to body weight on that specific day. On PND 21 weaned male rat pups were divided into SIR and group-reared rats. Rats were
Dev Neurosci 2014;36:18–28 DOI: 10.1159/000357495
19
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exposure was not additive with regard to behavioural or neurochemical changes. We conclude that a ceiling effect or compensatory mechanisms prevent more pronounced neurobehavioural deficits occurring following MA + SIR treatment, at least under the current study conditions.
Day
9.00 h
15.00 h
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
0.2 0.6 1.0 1.4 1.8 2.2 2.6 3.0 3.4 3.8 4.2 4.6 5.0 5.4 5.8 6.0
0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2 3.6 4.0 4.4 4.8 5.2 5.6 6.0 6.0
injected twice daily and subcutaneously with either saline or an escalating dose of MA, starting with 0.2 mg/kg on PND 35 and ending with 6 mg/kg on PND 50 (table 1) [27–29]. Dosing with MA or saline took place at 9.00 and 15.00 h every day for 16 consecutive days. The study consisted of 4 treatment groups (10 rats/group) designated as follows: group-housed + saline; group-housed + MA; SIR + saline; SIR + MA. After behavioural testing from PND 78, all animals were sacrificed 24 h later by decapitation without any prior use of an anaesthetic agent. Thereafter frontal cortices and striata were dissected for use in the neurochemical analyses. Behavioural Tests Following vehicle/drug treatments, the animals were housed under prescribed SIR or group housing conditions until PND 78, at which time the behavioural tests were performed, as described below. All behavioural tests were performed sequentially between 1 and 4 h shortly after the start of the dark cycle (i.e. 19.00–22.00 h), with social behaviour testing performed first (least stressful test) followed by the PPI test on days 77 and 78, respectively [22]. Social Interaction Test Social interactive behaviour (SIB) in an open field was monitored to determine spontaneous social interactive (outwardly directed) and self (inwardly)-directed behaviours [34, 35]. SIB is also performed as a measurement of spontaneous locomotor activity in rodents [35, 36]. The arena comprised a black square floor (70 × 70 cm) with opaque walls (40 cm high), with the floor divided into 35 × 35 cm equal squares [37], and illuminated with a red light (70 lx). A digital video camera mounted above the arena recorded all spontaneous SIB. Two rats were placed in the centre of the floor and allowed to adapt for 30 s. One of the two rats was tested for SIB with an unknown test partner that did not differ by more than 10 g in weight. Both test rats received the same treatment and prior experience. Time spent in active SIB (rearing, approaching and staying togeth-
20
Dev Neurosci 2014;36:18–28 DOI: 10.1159/000357495
er, anogenital sniffing), as well as self-directed behaviour (selfgrooming, squares crossed) [36] were scored for a period of 10 min using video analyses by an independent investigator blind to housing and treatment condition. During this period the evaluator remained outside the testing room. The arena was cleaned with 10% ethanol solution between testing [35]. Testing was performed between 19.00 and 22.00 h. PPI Test PPI was assessed in two illuminated and ventilated soundattenuated startle chambers (SR-Lab, San Diego Instruments, San Diego, Calif., USA). A stabilimeter system consisting of a transparent Plexiglas cylinder (diameter 8.2 cm, length 20 cm) mounted on a Plexiglas frame is present in each startle chamber. The acoustic noise bursts are provided by a speaker mounted 24 cm above the cylinder. A piezo-electric accelerometer mounted below the frame detects and transduces the startle response to a Hewlett-Packard personal computer that collates the data using SR-Lab software. Startle amplitudes are defined as the average of one hundred 1-ms stabilimeter readings collected from the stimulus onset. The stabilimeter was calibrated before each test. A protocol was set up according to previously described methods [19]. The startle session began with a 5-min acclimatization period, with a continuous 68-dB(A) background noise applied throughout the test session, followed by 4 blocks of trials with either pulse, no-pulse or prepulse trials, with a total of 100 trials. Percent PPI for 4 prepulse intensities – 72, 76, 80 and 84 dB(A) – was used and calculated according to the formula: percent PPI = [(pulse-alone amplitude – prepulse + pulse amplitude)/pulsealone amplitude] × 100 [30, 31]. The last 10 trials consisted of single 40-ms 120-dB(A) pulse-alone startle stimuli. The total of 100 trials was delivered with an average interval of 25 s. The first and last 10 pulse-alone stimuli (block 1 and block 4, respectively) and the 20 pulse-alone stimuli that were included in the PPI block itself (block 2 and block 3) were used to obtain a measure of mean startle amplitude suggestive of habituation to repeated delivery of the startling stimuli [19, 32, 33]. Neurochemical Analysis Preparation of Brain Tissue To avoid the effects of behavioural testing on the neurochemical analyses, rats were sacrificed by decapitation 24 h after the final behavioural test. Fresh brain tissue was used for the macrodissection on ice of the frontal cortex and striatum. These brain regions were first identified according to stereological coordinates [38] and subsequently fixed in relation to anatomical landmarks as previously described [20]. The frontal cortex was dissected after first removing the olfactory bulb from the cortex and then cutting around the anterior tip of the corpus callosum. When dissecting the striatum, the dorsal side of the brain was placed side up. The two cerebral hemispheres were split and the striatum was dissected with the corpus callosum as external limits and the external walls of the lateral ventricles as internal limits. The striatum and frontal cortex were then snap frozen in liquid nitrogen and stored at –80 ° C until the day of mono-amine analysis.
Regional Brain Mono-Amine Analysis DA, 5-HT and NA levels were quantified by high-performance liquid chromatography (HPLC) with electrochemical detection [39]. Samples were weighed and thawed followed by the
Strauss/Brink/Möller/Stein/Harvey
Downloaded by: Stockholms Universitet 198.143.54.1 - 9/4/2015 8:38:04 AM
Table 1. Escalating dosage regimen (mg/kg) for MA from PND 35
to 50
addition of 1 ml homogenization buffer (0.5 mM sodium metabisulphite; 0.3 mM Na 2-EDTA; 0.1 M perchloric acid, 60%). The tissue was ruptured by sonication (2 × 12 s, amplitude of 14 μm) and allowed to stand on ice for 20 min to precipitate proteins and facilitate extraction of mono-amines. Thereafter samples were centrifuged at 4 ° C in an ultracentrifuge for 20 min at 16,000 rpm (24,000 g). The pH of the samples was then adjusted to pH 5 with 10 M potassium acetate (1 drop/ml). A 200-μl aliquot was removed and placed into another tube, whereupon 20 μl of the internal standard, isoprenaline, was added. The final sample was vortexed and centrifuged, with 10 μl injected onto the HPLC column. Mono-amine concentrations were determined by comparing the area under the peak to that of the mono-amine standard (isoprenaline; range 5–50 ng/ml; Chromeleon® Chromatography Management System version 6.8 data acquisition and analysis software). Linear standard curves (regression coefficient greater than 0.99) were found in this range. Mono-amine concentrations were expressed as nanograms per milligram wet weight of tissue.
Group-reared saline SIR saline
40
Results
Behavioural Studies: Percent PPI The mean startle amplitudes of the group-reared controls and the isolated rats during 4 consecutive blocks of ten 120-dB stimuli, as well as the group- or SIR-housed animals receiving saline or MA, showed no significant overall differences in mean startle amplitude (data not shown). Furthermore, no significant habituation following repeated stimulation between block 1 and block 4 of startle was observed (data not shown). Figure 1a depicts the percentage of PPI for the salinetreated group versus SIR animals at the various prepulse intensities tested. Two-way ANOVA revealed a significant percent PPI · housing condition interaction [F(3, 54) = 3.75, p = 0.02; fig. 1a]. A Tukey post hoc test indicated that percent PPI was significantly reduced in the SIR rats compared to their socially housed controls, with a significant overall effect evident at each prepulse intensity: 72 dB (p < 0.01), 76 dB (p < 0.01), 80 dB (p < 0.05), 84 dB (p < 0.01).
Neuro-Behavioral Effects of MA in SIR
30
** **
10 0
**
*
20
72
76
a
80
84
Prepulse intensity (dB) 50 40
Group-reared saline Group-reared MA SIR saline SIR MA
30 * * * *
20
* *
** * 10 0
**
*** * **
72
76
b
80
84
Prepulse intensity (dB)
Fig. 1. Sensorimotor gating at the different prepulse intensities, as indicated, in the saline-treatment cohort (a; n = 10/group) and in group-reared and SIR rats in the drug treatment cohort (b; n = 10/ group), as determined by percentage of PPI. Two-way ANOVA with Tukey post hoc test was performed to compare the data of the two treatment groups in the saline treatment cohort with repeated measures for the PPI intensities (a). In the treatment cohort a 3-way ANOVA followed by a Tukey post hoc test with repeated measures was performed to compare the different drug treatment groups with their respective control (b). a * p < 0.05, ** p < 0.01 versus groupreared saline (2-way ANOVA, Tukey test with repeated measures), at 72, 76, 80 and 84 dB(A), respectively. b * p < 0.05, ** p < 0.01, *** p < 0.001 versus group-reared saline (3-way ANOVA, Tukey test with repeated measures), at 72, 76, 80 and 84 dB(A), respectively.
Figure 1b depicts the percent PPI in the drug treatment study. Using 3-way ANOVA, the following significant interactions in SIR and socially housed animals was observed: percent PPI · treatment [F(3, 144) = 7.679, p < 0.0001], percent PPI · housing condition [F(3, 144) = 17.88, p < 0.0001] and treatment · housing condition [F(3, 144) = 0.179, p < 0.0001]. Tukey post hoc analysis
Dev Neurosci 2014;36:18–28 DOI: 10.1159/000357495
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Statistical Analysis PPl was analysed by a 2-way factorial ANOVA with PPI as within-subject factor and housing as between-subject factor, followed by Tukey post hoc tests. ANOVA with repeated measures was used for the startle amplitude and different PPI intensities as within-subject factors. For the treatment cohort, 3-way ANOVA with Tukey post hoc tests with repeated measures were used for the percent PPI measurements. The various aspects of SIB, as well as regional brain neurochemistry, were analysed with respect to treatment (saline, MA) and housing condition (social, SIR) using 3-way ANOVA and the Tukey post hoc test. In all cases, data are expressed as the mean ± standard error of the mean, with a p value of
Received: April 24, 2013 Accepted after revision: November 21, 2013 Published online: January 24, 2014
Late-Life Effects of Chronic Methamphetamine Exposure during Puberty on Behaviour and Corticostriatal Mono-Amines in Social Isolation-Reared Rats Laetitia Strauss a Christiaan B. Brink a Marisa Möller a Dan J. Stein c Brian H. Harvey b a
Division of Pharmacology, School of Pharmacy, and b Centre of Excellence for Pharmaceutical Sciences, Faculty of Health Sciences, North-West University, Potchefstroom, and c MRC Unit for Anxiety and Stress Disorders, Department of Psychiatry, University of Cape Town, Cape Town, South Africa
Abstract Chronic methamphetamine (MA) abuse results in an acute psychosis indistinguishable from paranoid schizophrenia. However, less is known of the interaction between MA use and environmental insults, and how this contributes to lateonset psychopathology. Using social isolation rearing (SIR), a neurodevelopmental animal model of schizophrenia, we investigated the association between changes in corticostriatal mono-amines and putative behaviours related to MA-induced psychosis in isolation and group-housed rats following chronic MA or saline exposure. Weaned male offspring of MA-naive female Wistar rats, either group- or isolation-housed from postnatal day (PND) +21, received saline (2 ml/kg s.c. b.i.d.) or an escalating dose of MA (0.2–6 mg/kg s.c. b.i.d.) for 16 days from PND +35 to +50. On PND +78, offspring were tested for deficits in social interactive behaviour (SIB) and prepulse inhibition (PPI) of startle, with frontal cortex and striatum harvested for the assessment of monoamine concentrations. SIR significantly reduced rearing
© 2014 S. Karger AG, Basel 0378–5866/14/0361–0018$39.50/0 E-Mail [email protected] www.karger.com/dne
time, staying together, approaching and anogenital sniffing (outward-directed SIB), but increased self-grooming and locomotor activity (self-directed SIB), and also induced profound deficits in PPI. Pubertal MA exposure in group-housed animals also induced similar alterations in outward- and selfdirected SIB and reduced PPI. Combined MA + SIR exposure evoked a similarly intense behavioural response as SIR or MA separately, with no exacerbation evident. Neither treatment separately nor together affected corticostriatal serotonin or noradrenaline levels, although frontal cortical dopamine (DA) levels were significantly increased in SIR and MA + group-housed animals. A trend towards further elevated frontal cortical DA was noted in the MA + SIR treatment group. Striatal DA was unaltered by all treatments. This study provides the first evidence that chronic pubertal MA exposure evokes postpubertal psychosis-like behaviours in rats of similar intensity to that induced by a neurodevelopmental animal model of schizophrenia (SIR). Moreover, the study is unique in that these behavioural changes occur together with associated changes in frontal cortical but not striatal DA, without affecting other mono-amines, and strongly implicates frontal cortical DA changes in the psychotogenic effects of early-life MA exposure or environmental insult. Although MA exposure in animals with a history of environmental insult (i.e. MA + SIR) has similar effects, combined
Brian H. Harvey, PhD Centre of Excellence for Pharmaceutical Sciences, School of Pharmacy Faculty of Health Sciences, North-West University Potchefstroom, 2520 (South Africa) E-Mail Brian.Harvey @ nwu.ac.za
Downloaded by: Stockholms Universitet 198.143.54.1 - 9/4/2015 8:38:04 AM
Key Words Postweaning exposure · Early-life adversity · Wistar rat · Late-life psychopathology · Frontal cortex · Striatum · Cumulative risk · Dual diagnosis · Stress
© 2014 S. Karger AG, Basel
Introduction
As a drug of abuse, methamphetamine (MA) is fast becoming a major global health problem [1–3]. MA is transported by dopamine (DA), serotonin (5-HT) and noradrenaline (NA) transporters to cause a reverse transport of mono-amines and a resulting increase in synaptic concentrations [1]. In fact MA is a potent releaser of DA, NA and 5-HT [1, 2], while it also inhibits the reuptake of these mono-amines [1]. Chronic MA use, however, tends to deplete DA levels [4, 5]. These pronounced effects on dopaminergic transmission are presumed to mediate the psychotogenic effects of MA as well as increased oxidative stress and neurotoxicity after long-term abuse of the drug [1]. The symptoms of MA-induced psychosis, such as paranoia, hallucinations and delusions [3], are similar to acute paranoid schizophrenia, and both conditions share several similarities in positive and negative symptoms [6], as well as cognitive deficits [7]. Importantly, MA abusers are 11 times more likely to develop psychosis than the general population [3]. Individuals exposed to early-life stress or adversity might be more vulnerable to developing late-life behavioural abnormalities [8]. Schizophrenia is widely regarded as a neurodevelopmental disorder, being causally related to pre-, peri- and postnatal stress [9]. Whereas MA abuse is most often associated with acute psychosis [3, 10, 11], with up to 50% of cases resolving spontaneously [12], chronic MA can cause long-term psychosis [13, 14] that is more often seen with higher doses and prolonged use [14]. It has been suggested that MA abuse may trigger or unmask psychosis in susceptible individuals [14]. Whether altered dopamine can fully explain this association, or whether other confounds such as environmental insults are involved, requires clarification. With MA abuse often occurring during early adolescence, the question arises whether early-life MA abuse places a vulnerable individual at greater risk for developing psychosis later in life. Epidemiological studies would suggest that it does [3, 6, 15, 16], although this question has never been formerly studied either at the clinical or preclinical level. Neuro-Behavioral Effects of MA in SIR
Animal models of early-life insults can assist in delineating the mechanisms that underlie the interplay between MA abuse and vulnerability to develop MA psychosis [17]. Drug abuse and social isolation are two important nongenetic factors that contribute towards the development of schizophrenia [18], while postweaning social isolation rearing (SIR) of rodents is regarded as a valid neurodevelopmental model of schizophrenia [8]. Deficits in social behaviours, as well as in sensorimotor gating as determined by prepulse inhibition (PPI) of the startle response, are valid behavioural measures of deficits that are analogous to those observed in schizophrenia [8]. SIR induces profound deficits in these responses [19], whereas it also causes neurochemical abnormalities that closely parallel those observed in schizophrenia, such as mono-amine and glutamate changes [20, 21]. Finally, these biobehavioural changes respond to antipsychotic drugs [19, 22]. When considering a link with MA, SIR also evokes corticostriatal oxidative stress [19], a pathological process evident in both schizophrenia [23] and MA abuse [1, 2]. This study investigated the effect of SIR and chronic MA exposure during puberty, separately and combined, on sensorimotor gating and social interactive behaviour in rats. Since altered frontostriatal DA, 5-HT and NA pathways may play an important role in the psychosis of schizophrenia and MA abuse [24–26], we also studied the ensuing effects on these mono-amines. We hypothesize that SIR or MA would evoke typical schizophrenialike behaviours, as well as altered corticostriatal monoamines, akin to that of schizophrenia, and that these changes would be worsened in combined SIR-MA-exposed animals.
Materials and Methods Animals Male Wistar rats (250–300 g on the day of testing) were randomly separated at weaning (postnatal day 21, PND 21) into group(3–4 rats/cage) and isolation-reared (1 rat/cage) conditions for 8 weeks (until PND 77). Identical cages containing sawdust were used, under controlled conditions (22 ± 1 ° C; 50 ± 10% humidity; 12-hour light/dark cycle with lights on at 6.00 h; food and water ad libitum). SIR animals were maintained in an environment with no enrichment and minimal handling, with sawdust changed once a week. The study was undertaken in accordance with the guidelines for the care and use of laboratory animals in South Africa (NorthWest University ethics approval No.: NWU-000105-11-S5).
Drug Treatment MA was dissolved in saline before being administered according to body weight on that specific day. On PND 21 weaned male rat pups were divided into SIR and group-reared rats. Rats were
Dev Neurosci 2014;36:18–28 DOI: 10.1159/000357495
19
Downloaded by: Stockholms Universitet 198.143.54.1 - 9/4/2015 8:38:04 AM
exposure was not additive with regard to behavioural or neurochemical changes. We conclude that a ceiling effect or compensatory mechanisms prevent more pronounced neurobehavioural deficits occurring following MA + SIR treatment, at least under the current study conditions.
Day
9.00 h
15.00 h
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
0.2 0.6 1.0 1.4 1.8 2.2 2.6 3.0 3.4 3.8 4.2 4.6 5.0 5.4 5.8 6.0
0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2 3.6 4.0 4.4 4.8 5.2 5.6 6.0 6.0
injected twice daily and subcutaneously with either saline or an escalating dose of MA, starting with 0.2 mg/kg on PND 35 and ending with 6 mg/kg on PND 50 (table 1) [27–29]. Dosing with MA or saline took place at 9.00 and 15.00 h every day for 16 consecutive days. The study consisted of 4 treatment groups (10 rats/group) designated as follows: group-housed + saline; group-housed + MA; SIR + saline; SIR + MA. After behavioural testing from PND 78, all animals were sacrificed 24 h later by decapitation without any prior use of an anaesthetic agent. Thereafter frontal cortices and striata were dissected for use in the neurochemical analyses. Behavioural Tests Following vehicle/drug treatments, the animals were housed under prescribed SIR or group housing conditions until PND 78, at which time the behavioural tests were performed, as described below. All behavioural tests were performed sequentially between 1 and 4 h shortly after the start of the dark cycle (i.e. 19.00–22.00 h), with social behaviour testing performed first (least stressful test) followed by the PPI test on days 77 and 78, respectively [22]. Social Interaction Test Social interactive behaviour (SIB) in an open field was monitored to determine spontaneous social interactive (outwardly directed) and self (inwardly)-directed behaviours [34, 35]. SIB is also performed as a measurement of spontaneous locomotor activity in rodents [35, 36]. The arena comprised a black square floor (70 × 70 cm) with opaque walls (40 cm high), with the floor divided into 35 × 35 cm equal squares [37], and illuminated with a red light (70 lx). A digital video camera mounted above the arena recorded all spontaneous SIB. Two rats were placed in the centre of the floor and allowed to adapt for 30 s. One of the two rats was tested for SIB with an unknown test partner that did not differ by more than 10 g in weight. Both test rats received the same treatment and prior experience. Time spent in active SIB (rearing, approaching and staying togeth-
20
Dev Neurosci 2014;36:18–28 DOI: 10.1159/000357495
er, anogenital sniffing), as well as self-directed behaviour (selfgrooming, squares crossed) [36] were scored for a period of 10 min using video analyses by an independent investigator blind to housing and treatment condition. During this period the evaluator remained outside the testing room. The arena was cleaned with 10% ethanol solution between testing [35]. Testing was performed between 19.00 and 22.00 h. PPI Test PPI was assessed in two illuminated and ventilated soundattenuated startle chambers (SR-Lab, San Diego Instruments, San Diego, Calif., USA). A stabilimeter system consisting of a transparent Plexiglas cylinder (diameter 8.2 cm, length 20 cm) mounted on a Plexiglas frame is present in each startle chamber. The acoustic noise bursts are provided by a speaker mounted 24 cm above the cylinder. A piezo-electric accelerometer mounted below the frame detects and transduces the startle response to a Hewlett-Packard personal computer that collates the data using SR-Lab software. Startle amplitudes are defined as the average of one hundred 1-ms stabilimeter readings collected from the stimulus onset. The stabilimeter was calibrated before each test. A protocol was set up according to previously described methods [19]. The startle session began with a 5-min acclimatization period, with a continuous 68-dB(A) background noise applied throughout the test session, followed by 4 blocks of trials with either pulse, no-pulse or prepulse trials, with a total of 100 trials. Percent PPI for 4 prepulse intensities – 72, 76, 80 and 84 dB(A) – was used and calculated according to the formula: percent PPI = [(pulse-alone amplitude – prepulse + pulse amplitude)/pulsealone amplitude] × 100 [30, 31]. The last 10 trials consisted of single 40-ms 120-dB(A) pulse-alone startle stimuli. The total of 100 trials was delivered with an average interval of 25 s. The first and last 10 pulse-alone stimuli (block 1 and block 4, respectively) and the 20 pulse-alone stimuli that were included in the PPI block itself (block 2 and block 3) were used to obtain a measure of mean startle amplitude suggestive of habituation to repeated delivery of the startling stimuli [19, 32, 33]. Neurochemical Analysis Preparation of Brain Tissue To avoid the effects of behavioural testing on the neurochemical analyses, rats were sacrificed by decapitation 24 h after the final behavioural test. Fresh brain tissue was used for the macrodissection on ice of the frontal cortex and striatum. These brain regions were first identified according to stereological coordinates [38] and subsequently fixed in relation to anatomical landmarks as previously described [20]. The frontal cortex was dissected after first removing the olfactory bulb from the cortex and then cutting around the anterior tip of the corpus callosum. When dissecting the striatum, the dorsal side of the brain was placed side up. The two cerebral hemispheres were split and the striatum was dissected with the corpus callosum as external limits and the external walls of the lateral ventricles as internal limits. The striatum and frontal cortex were then snap frozen in liquid nitrogen and stored at –80 ° C until the day of mono-amine analysis.
Regional Brain Mono-Amine Analysis DA, 5-HT and NA levels were quantified by high-performance liquid chromatography (HPLC) with electrochemical detection [39]. Samples were weighed and thawed followed by the
Strauss/Brink/Möller/Stein/Harvey
Downloaded by: Stockholms Universitet 198.143.54.1 - 9/4/2015 8:38:04 AM
Table 1. Escalating dosage regimen (mg/kg) for MA from PND 35
to 50
addition of 1 ml homogenization buffer (0.5 mM sodium metabisulphite; 0.3 mM Na 2-EDTA; 0.1 M perchloric acid, 60%). The tissue was ruptured by sonication (2 × 12 s, amplitude of 14 μm) and allowed to stand on ice for 20 min to precipitate proteins and facilitate extraction of mono-amines. Thereafter samples were centrifuged at 4 ° C in an ultracentrifuge for 20 min at 16,000 rpm (24,000 g). The pH of the samples was then adjusted to pH 5 with 10 M potassium acetate (1 drop/ml). A 200-μl aliquot was removed and placed into another tube, whereupon 20 μl of the internal standard, isoprenaline, was added. The final sample was vortexed and centrifuged, with 10 μl injected onto the HPLC column. Mono-amine concentrations were determined by comparing the area under the peak to that of the mono-amine standard (isoprenaline; range 5–50 ng/ml; Chromeleon® Chromatography Management System version 6.8 data acquisition and analysis software). Linear standard curves (regression coefficient greater than 0.99) were found in this range. Mono-amine concentrations were expressed as nanograms per milligram wet weight of tissue.
Group-reared saline SIR saline
40
Results
Behavioural Studies: Percent PPI The mean startle amplitudes of the group-reared controls and the isolated rats during 4 consecutive blocks of ten 120-dB stimuli, as well as the group- or SIR-housed animals receiving saline or MA, showed no significant overall differences in mean startle amplitude (data not shown). Furthermore, no significant habituation following repeated stimulation between block 1 and block 4 of startle was observed (data not shown). Figure 1a depicts the percentage of PPI for the salinetreated group versus SIR animals at the various prepulse intensities tested. Two-way ANOVA revealed a significant percent PPI · housing condition interaction [F(3, 54) = 3.75, p = 0.02; fig. 1a]. A Tukey post hoc test indicated that percent PPI was significantly reduced in the SIR rats compared to their socially housed controls, with a significant overall effect evident at each prepulse intensity: 72 dB (p < 0.01), 76 dB (p < 0.01), 80 dB (p < 0.05), 84 dB (p < 0.01).
Neuro-Behavioral Effects of MA in SIR
30
** **
10 0
**
*
20
72
76
a
80
84
Prepulse intensity (dB) 50 40
Group-reared saline Group-reared MA SIR saline SIR MA
30 * * * *
20
* *
** * 10 0
**
*** * **
72
76
b
80
84
Prepulse intensity (dB)
Fig. 1. Sensorimotor gating at the different prepulse intensities, as indicated, in the saline-treatment cohort (a; n = 10/group) and in group-reared and SIR rats in the drug treatment cohort (b; n = 10/ group), as determined by percentage of PPI. Two-way ANOVA with Tukey post hoc test was performed to compare the data of the two treatment groups in the saline treatment cohort with repeated measures for the PPI intensities (a). In the treatment cohort a 3-way ANOVA followed by a Tukey post hoc test with repeated measures was performed to compare the different drug treatment groups with their respective control (b). a * p < 0.05, ** p < 0.01 versus groupreared saline (2-way ANOVA, Tukey test with repeated measures), at 72, 76, 80 and 84 dB(A), respectively. b * p < 0.05, ** p < 0.01, *** p < 0.001 versus group-reared saline (3-way ANOVA, Tukey test with repeated measures), at 72, 76, 80 and 84 dB(A), respectively.
Figure 1b depicts the percent PPI in the drug treatment study. Using 3-way ANOVA, the following significant interactions in SIR and socially housed animals was observed: percent PPI · treatment [F(3, 144) = 7.679, p < 0.0001], percent PPI · housing condition [F(3, 144) = 17.88, p < 0.0001] and treatment · housing condition [F(3, 144) = 0.179, p < 0.0001]. Tukey post hoc analysis
Dev Neurosci 2014;36:18–28 DOI: 10.1159/000357495
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Statistical Analysis PPl was analysed by a 2-way factorial ANOVA with PPI as within-subject factor and housing as between-subject factor, followed by Tukey post hoc tests. ANOVA with repeated measures was used for the startle amplitude and different PPI intensities as within-subject factors. For the treatment cohort, 3-way ANOVA with Tukey post hoc tests with repeated measures were used for the percent PPI measurements. The various aspects of SIB, as well as regional brain neurochemistry, were analysed with respect to treatment (saline, MA) and housing condition (social, SIR) using 3-way ANOVA and the Tukey post hoc test. In all cases, data are expressed as the mean ± standard error of the mean, with a p value of
