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Minocycline, a second-generation tetracycline, as a neuroprotective agent in an animal model of schizophrenia
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Minocycline, a second-generation tetracycline, as a neuroprotective agent in an animal model of schizophrenia Yechiel Levkovitz a,⁎, Uri Levi b , Yoram Braw a , Hagit Cohen b a
The Emotion-Cognition Research Center, The Shalvata Mental Health Care Center, P.O.B. 94. Hod-Hasharon, Israel Ministry of Health Beer-Sheva Mental Health Center, Anxiety and Stress Research Unit, Faculty of Health Sciences, Ben-Gurion University of the Negev, Israel b
A R T I C LE I N FO
AB S T R A C T
Article history:
Minocycline is a second-generation tetracycline with a distinct neuroprotective profile. The
Accepted 28 March 2007
current study assessed the effects of minocycline in an animal model of schizophrenia, the
Available online 1 April 2007
non-competitive NMDA antagonist (dizocilpine maleate; MK801). The effects of minocycline were compared to those of haloperidol, a dopamine antagonist used for the treatment of
Keywords:
schizophrenia. The study protocol involved daily intraperitoneal injections of minocycline
Minocycline
(35 mg/kg) for three consecutive days. On the fourth day, the rats were injected with MK801
Tetracycline
and assessed for visual–spatial memory (Morris water maze) and sensorimotor gating
NMDA
(acoustic startle response, ASR, and the prepulse inhibition of the ASR). The findings indicate
MK801
that MK801 caused cognitive visuo-spatial memory deficits and changes in sensorimotor
Schizophrenia
gating, similar to those evident in schizophrenia. Minocycline reversed these cognitive
MWM
effects of MK801 and this effect was similar to that of haloperidol. The results of this study
ASR
suggest that minocycline may have protective properties against the cognitive effects of the
PPI
MK801 animal model of schizophrenia. The discussion addresses potential mechanisms underlying the effects of minocycline and possible directions for future research. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
The glutamatergic system has been implicated in the etiology and pathophysiology of schizophrenia (SZ) (Carlsson, 2001; Coyle, 1996; Tamminga, 1998). Glutamate, acting on NMDA receptors, is the principal activation signal for the production of nitric oxide (NO) (Ignarro and Murad, 1995; Szabo, 1996). Activation of NMDA receptor leads to a toxic calcium influx that activates numerous enzymes, including neuronal NO synthase (NOS). NO is able to further increase the excitotoxicity by enhancing glutamate release from presynaptic neurons and inhibiting glial glutamate transporters (Meffert et al., 1994; Montague et al., 1994; Pogun et al., 1994; Trotti et al., 1996).
The current study investigated the potential use of minocycline, a NOS inhibitor, for the treatment of SZ. Minocycline is a second-generation tetracycline that exerts anti-inflammatory and antimicrobial effects, while having a distinct neuroprotective profile (Golub et al., 1998; Ryan and Ashley, 1998; Yrjanheikki et al., 1999). Minocycline has excellent brain tissue penetration, is clinically well tolerated and completely absorbed when taken orally (Aronson, 1980; Barza et al., 1975). Its neuroprotective abilities were related with its ability to inhibit NOS, leading to decreases in NO intercellular levels (Amin et al., 1996). Also related are its effects on P-38 MAPK, caspase 1 and caspase 3 expression and cytochrome C release (Chen et al., 2000; Du et al., 2001; Tikka et al.,
⁎ Corresponding author. Fax: +972 9 9798643. E-mail address: [email protected] (Y. Levkovitz). Abbreviations: SZ, schizophrenia; Mino, minocycline; Halo, haloperidol 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.03.080
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2001; Zhu et al., 2002). These properties of minocycline suggest that it may play a role in the treatment of disorders involving neuronal damage, such as SZ. This suggestion is strengthened by positive findings in studies using animal models of ischemic injury brain damage and degenerative diseases (Arvin et al., 2002; Chen et al., 2000; Wu et al., 2002; Yrjanheikki et al., 1998, 1999) and initial reports of human SZ patients treated with minocycline (Miyaoka et al., 2007). The current study investigates the potential use of minocycline in the treatment of SZ, utilizing an animal model (NMDA antagonist, dizocilpine maleate/MK801). NMDA antagonists are considered a highly valid model of SZ, used to determine neurochemical correlates of psychosis and as agents in the search for new antipsychotics (Corbett et al., 1995; Johansson et al., 1999a; Yang et al., 1991). Administration of NMDA antagonists to healthy humans produces positive symptoms, negative symptoms and cognitive deficits, as manifested in SZ (Coyle, 1996; Duncan et al., 1999; Javitt and Zukin, 1991; Krystal et al., 1994). These drugs also exacerbate SZ symptoms with some symptoms more responsive to atypical neuroleptics (Duncan et al., 1999; Lahti et al., 1995; Steinpreis, 1996). With regard to the NO system, NMDA antagonists decrease in neuronal NO levels by blocking Ca2+ influx (Alagarsamy et al., 1994; Noda et al., 1996; Osawa and Davila, 1993). This common physiological pathway of minocycline and NMDA antagonists (decrease in NO inter-cellular) suggests a synergetic effect of the two components. In line with this suggestion, NOS inhibitors (e.g., L-NAME) enhanced the behavioral effects NMDA antagonists, while a NO donor showed an opposite effect (Bujas-Bobanovic et al., 2000a,b; Noda et al., 1995). However, other studies indicate that NOS inhibitors may actually block NMDA antagonist-induced effects (Johansson et al., 1997, 1998, 1999b). The current study investigated minocycline effects on MK801-induced deficits, in light of these earlier contrasting findings. Behavioral analyses focused on cognitive domains deficient in human SZ patients, memory and sensorimotor gating (Heinrichs and Zakzanis, 1998; Nuechterlein et al., 2004). Moreover, these behavioral analyses were found to be sensitive to the effects of NMDA antagonists (Ahlander et al., 1999; D'Hooge and De Deyn, 2001; Johansson et al., 1997). At the first stage of the study we investigated the effects of an NMDA antagonist (dizocilpine maleate; MK801) on the performance of the rats, using the Morris Water Maze (MWM) and behavioral analysis of the acoustic startle response, ASR (base-line, habituation and prepulse inhibition of the ASR). Next, we investigated the ability of minocycline to reverse the behavioral effects of MK801. The effects of minocycline were compared to those of haloperidol, a potent typical antipsychotic that blocks D2 dopamine receptors.
2.
Results
2.1. Determining the minocycline dose for reversing MK801 effects The suitable minocycline dose was determined by a dose– response analysis (Fig. 1). A significant main-effect of ‘group’
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Fig. 1 – Escape latency (mean ± SEM) of the six treatment groups in the four MWM trials (averaged over the experimental trails). *p < 0.05; **p < 0.01; ***p < 0.001.
was found for escape latencies in the MWM [F(5,35) = 9.699, p < 0.001]; post hoc Scheffe tests indicated that MK801-treated rats had longer escape latencies (poorer performance) compared to the saline group (p < 0.001). Moreover, a qualitative analysis indicated that MK801-treated rats did not behave adaptively when encountering the hidden platform, quickly jumping off the platform and continuing swimming (similar to the effects of ketamine, another NMDA antagonist; Wesierska et al., 1990). Rats treated with 30 and 35 mg/kg minocycline showed similar escape latencies to those of saline-treated rats. In contrast, rats treated with lower doses of minocycline had longer escape latencies than the saline-treated rats (p < 0.01) and similar to those of MK801-treated rats. Next, an ANOVA was performed for each MWM trial (trails 1–4) in order to choose the most effective of the two minocycline doses (30 or 35 mg/kg). There were no significant differences in trial 1 [F(5,35) = 1.955, n.s.], but a significant ‘group’ main-effect was found in trials 2–4 [F(5,35) = 5.176, p < 0.001; F(5,35) = 6.751, p < 0.001; F(5,35) = 7.330, p < 0.001; respectively]. Scheffe tests indicated that the 35 mg/kg minocycline dose was more effective, evident in the shorter escape latencies of these rats (mino 35 mg/kg + MK801), compared to the MK801 group (trail 4; p < 0.05).
2.2. Effects of minocycline (35 mg/kg) on MK801-induced spatial learning/memory deficits as demonstrated by the MWM There was a significant ‘group’ main-effect for escape latencies in the MWM [F(5,44) = 68.92, p < 0.001]; Scheffe tests indicated that the MK801-treated rats had the longest escape latencies (poorer performance) compared to all other groups (p < 0.001). Both minocycline and haloperidol treated rats (mino + MK801 and haloperidol + MK801 groups) had shorter escape latencies than rats treated with MK801 alone (with no significant differences between the two groups). However, both treatment groups showed poorer performance compared to rats treated with saline, haloperidol alone and minocycline alone (p < 0.001 for all comparisons). See Fig. 2.
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Fig. 2 – Escape latency (mean ± SEM) of the six treatment groups in the four MWM trials (averaged over the experimental trails). ***p < 0.001.
The ANOVA also indicated significant main-effects of ‘trial’ and ‘group’ × ‘trial’ interaction [F(3,132) = 238.84, p < 0.001; F(15,132) = 19.00, p < 0.001; respectively]. The ‘trail’ maineffect corresponds to a decrease in escape latencies with each trial (general visuo-spatial learning). As for the source of the interaction effect, significant group differences were found in trials 2–4 [F(5,44) = 1.710, n.s.; F(5,44) = 34.82, p < 0.001; F(5,44) = 46.538, p < 0.001; F(5,44) = 31.46, p < 0.001; for trails 1–4, respectively]. The MK801-treated rats showed longer escape latencies than the saline-treated rats in trials 2–4 (p < 0.001). As for the effects of minocycline and haloperidol, Scheffe tests indicated an increasing differentiation with each trial (see Fig. 3). Specifically, in trial 2, neither minocycline nor haloperidol had an effect. In trial 3 only minocycline had a significant effect indicated by faster escape latencies of minocycline-treated rats (compared to the MK801 treatment group; p < 0.001). In trial 4, both minocycline and haloperidol treatments affected the rats' performance as expressed by significantly shorter escape latencies compared to the MK801-treated rats (p < 0.001 for both comparisons).
Fig. 3 – Mean ± SEM of base-line habituation of the ASR, averaged over 4 stimulus presentations. **p < 0.01; ***p < 0.001.
Fig. 4 – Mean ± SEM of % of habituation of the ASR for pulses for the six treatment groups. ***p < 0.001.
2.3. Effects of minocycline on MK801-induced ASR changes The ANOVA for base-line ASR (see Fig. 3) revealed a significant ‘group’ main-effect [F(5,25) = 12.489, p < 0.001]; Scheffe tests indicated that MK801-treated rats had significantly larger ASR, compared to all other treatment groups (p < 0.01 for MK801 compared to minocycline alone treatment group; p < 0.001 for all other comparisons). Significant ‘group’ main-effect was also found in the repeated-measures ANOVA for the habituation of the ASR (% habituation) [F(5,25) = 22.96, p < 0.001]. Scheffe tests indicated that MK801-treated rats had significantly lower % habituation of the ASR compared to all treatment groups (p < 0.001; see Fig. 4). This finding points toward the effectiveness of minocycline in preventing the MK801 effects.
2.4. Effects of minocycline on MK801-induced changes in sensorimotor gating as evaluated by the PPI of the ASR The ANOVA for %PPI revealed a significant ‘group’ main-effect (Fig. 5) [F(5,25) = 11.74, p < 0.001]; Scheffe tests indicated that
Fig. 5 – Mean ± SEM of % of PPI for the six treatment groups to four stimulus intensities (72, 76, 80 and 82 dB). #p < 0.1, * p < 0.05, **p < 0.01; ***p < 0.001.
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the MK801-treated rats had lower %PPI compared to most treatment groups (p < 0.05 — saline; p < 0.001 — haloperidol + MK801, haloperidol alone), while the approaching significance with the minocycline treatment group (p = 0.06). Minocycline reversed these effects of MK801, as revealed by the significant difference in %PPI between minocycline + MK801 and MK801 (alone) treated rats (p < 0.01). There was also a significant ‘prepulse intensity’ maineffect with prepulse stimulus intensity (72, 76, 80 and 84 dB) related to %PPI [F(3,23) = 25.35, p < 0.001]. This main-effect, however, was qualified by a significant ‘group’ × ‘prepulse intensity’ interaction [F(15,64) = 8.82, p < 0.001]; follow-up ANOVAs for each treatment group indicated that larger prepulse stimulus intensities were related to higher %PPI (a larger inhibition of the ASR) in the saline, minocycline (alone), mino + MK801, haloperidol + MK801 and haloperidol (alone) groups [F(3,12) = 10.75, p < 0.01; F(3,12) = 6.15, p < 0.01; F(3,12) = 12.21, p < 0.001; F(3,15) = 56.58, p < 0.001; F(3,12) = 9.24, p < 0.01; respectively]. While the ANOVA for the MK801-treated rats also showed a significant main-effect [F(3,15) = 35.069, p < 0.001], rats belonging to this group did not show the linear increase in % PPI with prepulse stimulus intensities seen in all other treatment groups (see Fig. 5). In summary, MK801-treated rats showed poorer performance in the MWM (evident in trials 2–4 of the MWM). MK801 also effected the sensorimotor gating of the rats, as indicated by the ASR and PPI of the ASR. Minocycline reversed the effects of MK801, similarly to the control dopamine antagonist, haloperidol.
3.
Discussion
The findings indicate that MK801 induced extensive deficits in spatial memory and learning, as evident in both quantitative and qualitative analyses of performance in the MWM (Morris et al., 1982a). MK801-treated rats also showed less habituation of the ASR and lower PPI of the ASR, changes in sensorimotor gating similar to those seen in SZ patients (Davis, 1980; Davis et al., 1993). These findings replicated the effects of other NMDA antagonists and correspond to cognitive deficits of human SZ patients (Heinrichs and Zakzanis, 1998; Mansbach, 1991; Nuechterlein et al., 2004; Wass et al., 2006). Next, we utilized the MWM and found a clear dose-dependent effect of minocycline, similar to the effects of minocycline in other models of CNS damage (e.g., Levkovitch-Verbin et al., 2006; Yrjanheikki et al., 1999). Three days of pre-treatment with minocycline completely reversed the effects of an acute treatment regimen of MK801, an NMDA antagonist. This effect is evident in the similar behavioral profile of minocycline and saline-treated rats. Minocycline reversed the effects of MK801 on base-line, habituation and PPI of the ASR. Minocycline was similarly effective in the MWM, mainly in the third and fourth experimental trials (pointing toward effects on learning processes, with limited effect on activity levels). Several putative mechanisms can account for these findings: first, the effects of minocycline may be related to its neuroprotective properties against neuronal damage, through inhibition of the proliferation and activation of microglia (Tikka et al.,
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2001). Second, minocycline may exert its effect through the NO system, associated with spatial learning and sensorimotor gating (Geyer et al., 2001; Liang et al., 1994; Wass et al., 2006). As detailed in the Introduction section, both MK801 and minocycline decrease inter-cellular NO levels (Alagarsamy et al., 1994; Amin et al., 1996; Noda et al., 1996; Osawa and Davila, 1993). These common physiological pathways suggest that minocycline may exacerbate the effects of NMDA receptor antagonists. In line with this possibility, NOS inhibitors (e.g., LNAME) enhanced NMDA antagonist-induced behaviors, while a NO donor showed an opposite effect (Bujas-Bobanovic et al., 2000a,b; Noda et al., 1995). However, other studies indicate that NOS inhibitors may actually block NMDA antagonistinduced effects. For example, several NOS inhibitors (e.g., ARR 17477, L-NAME) reversed the effects of NMDA antagonists (Johansson et al., 1997, 1998, 1999b). The current study is in agreement with the latter group of findings, suggesting that minocycline opposes MK80-induced deficits. They correspond to Johansson et al.'s (1997, 1998, 1999b) findings and add convergent validity by using a different NMDA antagonist (MK801) and NOS inhibitor (minocycline). It was suggested that Johansson et al.'s (1997, 1998, 1999b) findings may stem from over-dependence on measures of activity and stereotyped behavior (Bujas-Bobanovic et al., 2000a,b). The current study indicates that this may not be the case since the effects of the NOS inhibitor were found when we used additional well-validated behavioral paradigms. The effect of minocycline in countering the effects of MK801 may be related to glutamate compensatory mechanisms. NMDA antagonists facilitate glutamate release stemming from compensatory response to NMDA receptor blockade (Adams and Moghaddam, 2001; Krystal et al., 1999; Moghaddam, 1994; Moghaddam et al., 1997). In turn, the glutamate release affects non-NMDA (AMPA/kainate) receptors, leading to behavioral effects and neural damage (Breese et al., 2002; Olney et al., 1999). The dependence upon a compensation mechanism suggests that only a long duration of NMDA receptor blockade will lead to these effects (Olney et al., 1999). Different durations or dosages of NMDA antagonist treatments may explain the contrasting findings on the effects of NOS inhibitors on NMDA antagonist administration. Our current findings highlight the potential role of minocycline as a protective agent against MK801-induced deficits. They suggest that minocycline may serve as a potential therapeutic tool for disorders such as SZ. This putative suggestion should be accompanied by the recognition of the inherent limitations in the use of animal models to accurately reflect complex disorders. For example, the construct validity of these models is limited, at least until the biochemical nature of SZ is deciphered (Krystal, 1999; Lipska and Weinberger, 2000). Bearing these inherent limitations in mind, it is our hope that the current study findings will contribute to paving the way for future research that will clarify minocycline's potential. These future studies can help in the clinical validation of minocycline as neuroprotective agent by utilizing additional behavioral paradigms, animal species and antipsychotic comparison drugs. Such research also holds a promise to expand our knowledge of the pathophysiology of SZ.
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4.
Experimental procedures
4.1.
Animals
The study animals were adult male Sprague–Dawley rats (total N = 91; 41 for the minocycline dose determination and 50 for the main experimental procedure) weighing 150–200 g, from the ‘Animal Behavior Laboratory’ in Ben-Gurion University of the Negev. They were housed four per cage in a vivarium with stable temperature and a reversed 12-hour light/dark cycle, with unlimited access to food and water. The animals were habituated to housing conditions for at least 10 days during which they were pre-handled to minimize the stress that can be induced following needle injection (Ryabinin et al., 1999). The paradigm consisted of handling them once daily by picking them up with a gloved hand. Experimental procedures were performed during the dark phase using a dim light. All testing procedures were approved by the university's Animal Care Committee and carried out in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals.
4.2.
Materials
4.2.1.
Drug solutions
4.3.2. (PPI)
Acoustic startle response (ASR) and prepulse inhibition
Both experimental paradigms were performed in a room that was adjacent to the laboratory facilities and an animal stall that was not used for other experiments. The animals were tested in pairs, with startle chambers counterbalanced across the different experimental groups. Startle response and prepulse inhibition were measured using two ventilated startle chambers (SR-LAB system, San Diego Instruments, San Diego, CA). Each chamber consisted of a Plexiglas cylinder resting on a platform inside a sound-attenuated ventilated chamber. A high-frequency loudspeaker inside the chamber produced both a continuous broadband background noise of 70 dB and the selected acoustic stimuli. Movement inside the tube was detected by a piezoelectric accelerometer below the frame. The amplitude of a whole body startle to an acoustic pulse was defined as the average of 100 1-ms accelerometer readings collected from pulse onset. These signals were then digitized and stored by a computer. Sound levels within each test chamber were measured routinely by a sound level meter (Radio Shack) to ensure consistent presentation. The SR-LAB calibration unit was used routinely to ensure consistent stabilimeter sensitivity between test chambers and over time (Swerdlow and Geyer, 1998).
4.4.
Procedure
4.4.1.
General handling (preliminary) procedure
4.2.1.1. Dizocilpine maleate (MK801).
Fresh solutions of dizocilpine maleate MK801 (Sigma-Aldrich, Israel) were prepared in physiological saline (0.9% NaCl in sterile distilled water) for each group of rats. A dose of 0.1 mg/kg was used, as described in Kipnis et al. (2004).
4.2.1.2. Minocycline. Minocycline hydrochloride (Sigma, St. Louis, MO) was freshly dissolved in phosphate-buffered saline (KPBS, pH 7.2, 37 °C). A 35 mg/kg dose was chosen after a pretest using 20–40 mg/kg doses (see the Procedure section and Fig. 1). 4.2.1.3. Haloperidol. Haloperidol (Sigma-Aldrich) was dissolved with a minimal amount of glacial acetic acid (∼10 μl) and then diluted with lukewarm 5.5% D-glucose, with a final pH ∼ 6.0. 0.4 mg/kg dose was used for the MWM, 0.3 mg/kg for sensorimotor gating analyses. 4.2.1.4. Saline.
Saline was used for control injections.
4.3.
Apparatus
4.3.1.
Morris water maze (MWM)
The MWM consisted of a water pool (diameter = 1.8 m, depth = 0.6 m) containing water at 26 ± 1 °C. A hidden platform was located 1.5 cm below the water surface. Within the testing room, only distal visuo-spatial cues were available to the rats for location of the submerged platform. A video camera was placed above the center of the pool and a video tracking system with online digital output directly fed data into a computer. Data were analyzed by an Etho-Vision automated tracking system (Noldus Information Technology b.v., Wageninpen, The Netherlands).
Ten days before the beginning of the experiment, the rats underwent a handling procedure conducted once daily and aimed at reducing experimental stress (Ryabinin et al., 1999). The handling procedure consisted of picking the rat up with a gloved hand and stroking it.
4.4.2. Dose-dependent effect of intraperitoneal (IP) injections of minocycline compared to those of MK801 and saline The effective dose of minocycline to be used in the study was assessed in a trial procedure. Rats (N = 41) were injected IP with either minocycline in one of four doses (20, 25, 30 and 35 mg/kg) or with saline for three consecutive days before behavioral testing. We initiated minocycline treatment 3 days in order to achieve a sufficient drug level (based on; Levkovitch-Verbin et al., 2006), taking into consideration the fact that minocycline is more effective when given before CNS injury, compared to administration shortly after the onset of injury (Du et al., 2001; Yrjanheikki et al., 1998). Fifteen minutes before conducting the behavioral procedures (the fourth day), the rat was injected with IP MK801 0.1 mg/kg (as described in; Kipnis et al., 2004) or saline (for the rats that received saline earlier), for a total volume of 1 ml/kg body weight. Next, the rats underwent four consecutive trials in the MWM paradigm. The MWM was used to assess dose– response effects because of its high levels of reliability and validity (Morris et al., 1982b; Schimanski and Nguyen, 2004).
4.5.
Main experimental procedure
Rats (N = 50) were injected with minocycline (35 mg/kg) for three consecutive days before the behavioral testing (in
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accordance with; Levkovitch-Verbin et al., 2006). On each day of behavioral testing, the rats were IP injected with either haloperidol (alone or in combination with MK801) or saline (alone or minocycline, saline, minocycline + MK801, and MK801 groups) 30 min before behavioral assessments, an effective time-frame for inducing behavioral effects in rodents (Svensson, 2000). Fifteen minutes before undergoing the behavioral procedures on the fourth day, the rat was injected IP with MK801 or saline, for a total volume of 1 ml/kg body weight. Afterwards the rats underwent the three behavioral procedures described below (Table 1).
4.6.
The Morris Water Maze (MWM) paradigm
The MWM (Morris, 1984) was used as a well-validated hippocampus dependent test of visual–spatial memory (D'Hooge and De Deyn, 2001; Eichenbaum et al., 1990; Logue et al., 1997; Schimanski and Nguyen, 2004). Further support of the ecological validity of the MWM for the purposes of the current study can be found in memory deficits exhibited by SZ patients, accompanied by hippocampus abnormalities (Green, 1996; Green et al., 2000; Halliday, 2001; Shenton et al., 2001). The MWM tested the ability of an animal to locate a hidden platform submerged under water by using extra-maze cues from the test environment was examined. The rats were trained in a pool 1.8 m in diameter and 0.6 m high, containing water at 26 ± 1 °C. A 10 cm square transparent platform was hidden in a constant position (1 cm below the water level) in the pool. Only distal visuo-spatial cues were available to the rats for location of the submerged platform. The rats were given trials to find the hidden platform (acquisition phase). The escape latency, i.e., the time required by the rat to find and climb onto the platform, was recorded for up to 120 s. Each rat was allowed to remain on the platform for 30 s, after which it was removed to its home cage. If the rat did not find the platform within 120 s, it was manually placed on it and returned to its home cage after 30 s.
4.7. Acoustic startle response (ASR) and prepulse inhibition (PPI) paradigms Assessment of the ASR and PPI was employed as ecologically valid paradigms for assessing sensorimotor reactivity and the inhibition of the startle reflex by a weak pre-stimulus (PPI) (Davis, 1980; Davis et al., 1993). ASR and PPI were measured using two startle chambers, with each chamber consisting of a
Plexiglas cylinder resting on a platform inside a soundattenuated, ventilated chamber (Materials section). The animals were tested in pairs, with startle chambers counterbalanced across the different experimental groups. At the beginning of the experiment, the animals were placed inside the tube and the startle session started with a 5-min acclimatization period during which a high-frequency loudspeaker inside the chamber produced a continuous broadband background noise of 68 dB. Following the acclimatization period, four startle pulses (120 dB for 30 ms) were presented. Movement inside the tube was detected by a piezoelectric accelerometer below the frame. The amplitude of the whole body startle to an acoustic pulse was defined as the average of 100 1-ms accelerometer readings collected from pulse onset. These signals were then digitized and stored in a computer. The SR-LAB calibration unit was used routinely to ensure consistent stabilimeter sensitivity between test chambers and over time. The paradigm allowed the measurement of base-line ASR (the transient force resulting from the movements of the platform evoked by the ASR to the first startle pulse), as well as the animal's ability to habituate to repetitive loud pulses of noise (habituation of the ASR). PPI of the ASR was used as a measure of sensorimotor gating, with the startle chambers used to assess the ability of prepulse stimuli to block the startle response. The prepulses were broadband noise bursts of either 72, 76, 80, or 84 dB and of 20 ms in duration. The interval between the prepulse and the startle pulse was 80 ms. Each session consisted of six blocks of 10 trials. Each block included four different trial types: two startle pulse-alone trials, four prepulses at different intensities followed by startle pulse, four prepulses alone at four intensities and one no-stimulus trial. The different trial types were presented pseudo-randomly with a variable inter-trial interval of 10–20 s.
4.8.
Data analysis
4.8.1.
Morris Water Maze (MWM)
Escape latency (defined as the time between placing the rat in the pool until it located the platform) was measured during behavioral testing. Selection of minocycline dose was conducted using an analysis of variance (ANOVA) with a betweensubject measure of ‘group’ (treatment group; MK801, saline and four minocycline doses (20, 25, 30, 35 mg/kg)]. Since the analysis showed similar effects of 30 and 35 mg/kg minocycline doses, we conducted follow-up ANOVA's for trails 1–4 in
Table 1 – Study design Experimental procedure
Days −10–0
Treatment MK801 Minocycline + MK801 Haloperidol + MK801 Minocycline Haloperidol Saline
Handling
Daily injections (days 1–3) Saline Minocycline Saline Minocycline Saline Saline
Pre-behavioral testing (day 4) 30 min 15 min Saline Saline Haloperidol Saline Haloperidol Saline
Notes: MWM, Morris Water Maze; ASR, acoustic startle response; PPI, prepulse inhibition.
MK801 MK801 MK801 Saline Saline Saline
Behavioral testing (day 4) MWM ASR PPI
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the MWM (see the Results section). Analysis of the main MWM experiment was conducted using a repeated-measures ANOVA using a between-subject measure of ‘group’ [treatment groups; MK801, MK801 + minocycline, minocycline, MK801 + haloperidol, haloperidol and saline] and a withinsubject measure of ‘trial’ (effects among groups in the four different trials). In order to elucidate the of significant interactions follow-up ANOVAs were conducted for each MWM trail. In all analysis, significant group differences were followed by Scheffe post hoc tests.
4.8.2.
Acoustic startle response (ASR)
The paradigm allowed the measurement of base-line ASR (the transient force resulting from the movements of the platform evoked by the ASR to the first startle pulse), as well as the animal's ability to habituate to repetitive loud pulses of noise (habituation of the ASR). Habituation of ASR was defined as a decrement in startle response to an initially novel stimulus when presented repeatedly; the decrease in ASR was calculated as the percent of amplitude decrease between the mean of the first five amplitude values and the mean of the last 25 amplitude values: % habituation = 100 × [(average startle amplitude in Block 1) − (average startle amplitude in Block 5 / (average startle amplitude in Block 1)]. Results were analyzed by two-way ANOVA with a betweensubject measure of ‘group’ (treatment group). Significant group differences in the ANOVA's were followed by Scheffe post hoc tests.
4.8.3.
Prepulse inhibition (PPI)
Startle magnitude is expressed in all the procedures as arbitrary units. Percent PPI (%PPI) was calculated as the mean startle magnitude to stimulus-alone minus the mean startle magnitude to prepulse stimulus trials, all divided by the mean stimulus-alone trials and multiplied by 100. The overall mean %PPI was calculated for the four prepulse intensities (72, 74, 80, 84 dB). Results were analyzed by twoway ANOVA with a between-subject measure of ‘group’ and ‘prepulse intensity’ (72, 76, 80, 84 dB). Follow-up repeated ANOVAs were conducted for each group in order to ascertain the source of significant interactions. In all ANOVAs significant group main-effects were followed by Scheffe post hoc tests.
Acknowledgment Esther Eshkol is thanked for editorial assistance.
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Minocycline, a second-generation tetracycline, as a neuroprotective agent in an animal model of schizophrenia Yechiel Levkovitz a,⁎, Uri Levi b , Yoram Braw a , Hagit Cohen b a
The Emotion-Cognition Research Center, The Shalvata Mental Health Care Center, P.O.B. 94. Hod-Hasharon, Israel Ministry of Health Beer-Sheva Mental Health Center, Anxiety and Stress Research Unit, Faculty of Health Sciences, Ben-Gurion University of the Negev, Israel b
A R T I C LE I N FO
AB S T R A C T
Article history:
Minocycline is a second-generation tetracycline with a distinct neuroprotective profile. The
Accepted 28 March 2007
current study assessed the effects of minocycline in an animal model of schizophrenia, the
Available online 1 April 2007
non-competitive NMDA antagonist (dizocilpine maleate; MK801). The effects of minocycline were compared to those of haloperidol, a dopamine antagonist used for the treatment of
Keywords:
schizophrenia. The study protocol involved daily intraperitoneal injections of minocycline
Minocycline
(35 mg/kg) for three consecutive days. On the fourth day, the rats were injected with MK801
Tetracycline
and assessed for visual–spatial memory (Morris water maze) and sensorimotor gating
NMDA
(acoustic startle response, ASR, and the prepulse inhibition of the ASR). The findings indicate
MK801
that MK801 caused cognitive visuo-spatial memory deficits and changes in sensorimotor
Schizophrenia
gating, similar to those evident in schizophrenia. Minocycline reversed these cognitive
MWM
effects of MK801 and this effect was similar to that of haloperidol. The results of this study
ASR
suggest that minocycline may have protective properties against the cognitive effects of the
PPI
MK801 animal model of schizophrenia. The discussion addresses potential mechanisms underlying the effects of minocycline and possible directions for future research. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
The glutamatergic system has been implicated in the etiology and pathophysiology of schizophrenia (SZ) (Carlsson, 2001; Coyle, 1996; Tamminga, 1998). Glutamate, acting on NMDA receptors, is the principal activation signal for the production of nitric oxide (NO) (Ignarro and Murad, 1995; Szabo, 1996). Activation of NMDA receptor leads to a toxic calcium influx that activates numerous enzymes, including neuronal NO synthase (NOS). NO is able to further increase the excitotoxicity by enhancing glutamate release from presynaptic neurons and inhibiting glial glutamate transporters (Meffert et al., 1994; Montague et al., 1994; Pogun et al., 1994; Trotti et al., 1996).
The current study investigated the potential use of minocycline, a NOS inhibitor, for the treatment of SZ. Minocycline is a second-generation tetracycline that exerts anti-inflammatory and antimicrobial effects, while having a distinct neuroprotective profile (Golub et al., 1998; Ryan and Ashley, 1998; Yrjanheikki et al., 1999). Minocycline has excellent brain tissue penetration, is clinically well tolerated and completely absorbed when taken orally (Aronson, 1980; Barza et al., 1975). Its neuroprotective abilities were related with its ability to inhibit NOS, leading to decreases in NO intercellular levels (Amin et al., 1996). Also related are its effects on P-38 MAPK, caspase 1 and caspase 3 expression and cytochrome C release (Chen et al., 2000; Du et al., 2001; Tikka et al.,
⁎ Corresponding author. Fax: +972 9 9798643. E-mail address: [email protected] (Y. Levkovitz). Abbreviations: SZ, schizophrenia; Mino, minocycline; Halo, haloperidol 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.03.080
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2001; Zhu et al., 2002). These properties of minocycline suggest that it may play a role in the treatment of disorders involving neuronal damage, such as SZ. This suggestion is strengthened by positive findings in studies using animal models of ischemic injury brain damage and degenerative diseases (Arvin et al., 2002; Chen et al., 2000; Wu et al., 2002; Yrjanheikki et al., 1998, 1999) and initial reports of human SZ patients treated with minocycline (Miyaoka et al., 2007). The current study investigates the potential use of minocycline in the treatment of SZ, utilizing an animal model (NMDA antagonist, dizocilpine maleate/MK801). NMDA antagonists are considered a highly valid model of SZ, used to determine neurochemical correlates of psychosis and as agents in the search for new antipsychotics (Corbett et al., 1995; Johansson et al., 1999a; Yang et al., 1991). Administration of NMDA antagonists to healthy humans produces positive symptoms, negative symptoms and cognitive deficits, as manifested in SZ (Coyle, 1996; Duncan et al., 1999; Javitt and Zukin, 1991; Krystal et al., 1994). These drugs also exacerbate SZ symptoms with some symptoms more responsive to atypical neuroleptics (Duncan et al., 1999; Lahti et al., 1995; Steinpreis, 1996). With regard to the NO system, NMDA antagonists decrease in neuronal NO levels by blocking Ca2+ influx (Alagarsamy et al., 1994; Noda et al., 1996; Osawa and Davila, 1993). This common physiological pathway of minocycline and NMDA antagonists (decrease in NO inter-cellular) suggests a synergetic effect of the two components. In line with this suggestion, NOS inhibitors (e.g., L-NAME) enhanced the behavioral effects NMDA antagonists, while a NO donor showed an opposite effect (Bujas-Bobanovic et al., 2000a,b; Noda et al., 1995). However, other studies indicate that NOS inhibitors may actually block NMDA antagonist-induced effects (Johansson et al., 1997, 1998, 1999b). The current study investigated minocycline effects on MK801-induced deficits, in light of these earlier contrasting findings. Behavioral analyses focused on cognitive domains deficient in human SZ patients, memory and sensorimotor gating (Heinrichs and Zakzanis, 1998; Nuechterlein et al., 2004). Moreover, these behavioral analyses were found to be sensitive to the effects of NMDA antagonists (Ahlander et al., 1999; D'Hooge and De Deyn, 2001; Johansson et al., 1997). At the first stage of the study we investigated the effects of an NMDA antagonist (dizocilpine maleate; MK801) on the performance of the rats, using the Morris Water Maze (MWM) and behavioral analysis of the acoustic startle response, ASR (base-line, habituation and prepulse inhibition of the ASR). Next, we investigated the ability of minocycline to reverse the behavioral effects of MK801. The effects of minocycline were compared to those of haloperidol, a potent typical antipsychotic that blocks D2 dopamine receptors.
2.
Results
2.1. Determining the minocycline dose for reversing MK801 effects The suitable minocycline dose was determined by a dose– response analysis (Fig. 1). A significant main-effect of ‘group’
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Fig. 1 – Escape latency (mean ± SEM) of the six treatment groups in the four MWM trials (averaged over the experimental trails). *p < 0.05; **p < 0.01; ***p < 0.001.
was found for escape latencies in the MWM [F(5,35) = 9.699, p < 0.001]; post hoc Scheffe tests indicated that MK801-treated rats had longer escape latencies (poorer performance) compared to the saline group (p < 0.001). Moreover, a qualitative analysis indicated that MK801-treated rats did not behave adaptively when encountering the hidden platform, quickly jumping off the platform and continuing swimming (similar to the effects of ketamine, another NMDA antagonist; Wesierska et al., 1990). Rats treated with 30 and 35 mg/kg minocycline showed similar escape latencies to those of saline-treated rats. In contrast, rats treated with lower doses of minocycline had longer escape latencies than the saline-treated rats (p < 0.01) and similar to those of MK801-treated rats. Next, an ANOVA was performed for each MWM trial (trails 1–4) in order to choose the most effective of the two minocycline doses (30 or 35 mg/kg). There were no significant differences in trial 1 [F(5,35) = 1.955, n.s.], but a significant ‘group’ main-effect was found in trials 2–4 [F(5,35) = 5.176, p < 0.001; F(5,35) = 6.751, p < 0.001; F(5,35) = 7.330, p < 0.001; respectively]. Scheffe tests indicated that the 35 mg/kg minocycline dose was more effective, evident in the shorter escape latencies of these rats (mino 35 mg/kg + MK801), compared to the MK801 group (trail 4; p < 0.05).
2.2. Effects of minocycline (35 mg/kg) on MK801-induced spatial learning/memory deficits as demonstrated by the MWM There was a significant ‘group’ main-effect for escape latencies in the MWM [F(5,44) = 68.92, p < 0.001]; Scheffe tests indicated that the MK801-treated rats had the longest escape latencies (poorer performance) compared to all other groups (p < 0.001). Both minocycline and haloperidol treated rats (mino + MK801 and haloperidol + MK801 groups) had shorter escape latencies than rats treated with MK801 alone (with no significant differences between the two groups). However, both treatment groups showed poorer performance compared to rats treated with saline, haloperidol alone and minocycline alone (p < 0.001 for all comparisons). See Fig. 2.
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Fig. 2 – Escape latency (mean ± SEM) of the six treatment groups in the four MWM trials (averaged over the experimental trails). ***p < 0.001.
The ANOVA also indicated significant main-effects of ‘trial’ and ‘group’ × ‘trial’ interaction [F(3,132) = 238.84, p < 0.001; F(15,132) = 19.00, p < 0.001; respectively]. The ‘trail’ maineffect corresponds to a decrease in escape latencies with each trial (general visuo-spatial learning). As for the source of the interaction effect, significant group differences were found in trials 2–4 [F(5,44) = 1.710, n.s.; F(5,44) = 34.82, p < 0.001; F(5,44) = 46.538, p < 0.001; F(5,44) = 31.46, p < 0.001; for trails 1–4, respectively]. The MK801-treated rats showed longer escape latencies than the saline-treated rats in trials 2–4 (p < 0.001). As for the effects of minocycline and haloperidol, Scheffe tests indicated an increasing differentiation with each trial (see Fig. 3). Specifically, in trial 2, neither minocycline nor haloperidol had an effect. In trial 3 only minocycline had a significant effect indicated by faster escape latencies of minocycline-treated rats (compared to the MK801 treatment group; p < 0.001). In trial 4, both minocycline and haloperidol treatments affected the rats' performance as expressed by significantly shorter escape latencies compared to the MK801-treated rats (p < 0.001 for both comparisons).
Fig. 3 – Mean ± SEM of base-line habituation of the ASR, averaged over 4 stimulus presentations. **p < 0.01; ***p < 0.001.
Fig. 4 – Mean ± SEM of % of habituation of the ASR for pulses for the six treatment groups. ***p < 0.001.
2.3. Effects of minocycline on MK801-induced ASR changes The ANOVA for base-line ASR (see Fig. 3) revealed a significant ‘group’ main-effect [F(5,25) = 12.489, p < 0.001]; Scheffe tests indicated that MK801-treated rats had significantly larger ASR, compared to all other treatment groups (p < 0.01 for MK801 compared to minocycline alone treatment group; p < 0.001 for all other comparisons). Significant ‘group’ main-effect was also found in the repeated-measures ANOVA for the habituation of the ASR (% habituation) [F(5,25) = 22.96, p < 0.001]. Scheffe tests indicated that MK801-treated rats had significantly lower % habituation of the ASR compared to all treatment groups (p < 0.001; see Fig. 4). This finding points toward the effectiveness of minocycline in preventing the MK801 effects.
2.4. Effects of minocycline on MK801-induced changes in sensorimotor gating as evaluated by the PPI of the ASR The ANOVA for %PPI revealed a significant ‘group’ main-effect (Fig. 5) [F(5,25) = 11.74, p < 0.001]; Scheffe tests indicated that
Fig. 5 – Mean ± SEM of % of PPI for the six treatment groups to four stimulus intensities (72, 76, 80 and 82 dB). #p < 0.1, * p < 0.05, **p < 0.01; ***p < 0.001.
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the MK801-treated rats had lower %PPI compared to most treatment groups (p < 0.05 — saline; p < 0.001 — haloperidol + MK801, haloperidol alone), while the approaching significance with the minocycline treatment group (p = 0.06). Minocycline reversed these effects of MK801, as revealed by the significant difference in %PPI between minocycline + MK801 and MK801 (alone) treated rats (p < 0.01). There was also a significant ‘prepulse intensity’ maineffect with prepulse stimulus intensity (72, 76, 80 and 84 dB) related to %PPI [F(3,23) = 25.35, p < 0.001]. This main-effect, however, was qualified by a significant ‘group’ × ‘prepulse intensity’ interaction [F(15,64) = 8.82, p < 0.001]; follow-up ANOVAs for each treatment group indicated that larger prepulse stimulus intensities were related to higher %PPI (a larger inhibition of the ASR) in the saline, minocycline (alone), mino + MK801, haloperidol + MK801 and haloperidol (alone) groups [F(3,12) = 10.75, p < 0.01; F(3,12) = 6.15, p < 0.01; F(3,12) = 12.21, p < 0.001; F(3,15) = 56.58, p < 0.001; F(3,12) = 9.24, p < 0.01; respectively]. While the ANOVA for the MK801-treated rats also showed a significant main-effect [F(3,15) = 35.069, p < 0.001], rats belonging to this group did not show the linear increase in % PPI with prepulse stimulus intensities seen in all other treatment groups (see Fig. 5). In summary, MK801-treated rats showed poorer performance in the MWM (evident in trials 2–4 of the MWM). MK801 also effected the sensorimotor gating of the rats, as indicated by the ASR and PPI of the ASR. Minocycline reversed the effects of MK801, similarly to the control dopamine antagonist, haloperidol.
3.
Discussion
The findings indicate that MK801 induced extensive deficits in spatial memory and learning, as evident in both quantitative and qualitative analyses of performance in the MWM (Morris et al., 1982a). MK801-treated rats also showed less habituation of the ASR and lower PPI of the ASR, changes in sensorimotor gating similar to those seen in SZ patients (Davis, 1980; Davis et al., 1993). These findings replicated the effects of other NMDA antagonists and correspond to cognitive deficits of human SZ patients (Heinrichs and Zakzanis, 1998; Mansbach, 1991; Nuechterlein et al., 2004; Wass et al., 2006). Next, we utilized the MWM and found a clear dose-dependent effect of minocycline, similar to the effects of minocycline in other models of CNS damage (e.g., Levkovitch-Verbin et al., 2006; Yrjanheikki et al., 1999). Three days of pre-treatment with minocycline completely reversed the effects of an acute treatment regimen of MK801, an NMDA antagonist. This effect is evident in the similar behavioral profile of minocycline and saline-treated rats. Minocycline reversed the effects of MK801 on base-line, habituation and PPI of the ASR. Minocycline was similarly effective in the MWM, mainly in the third and fourth experimental trials (pointing toward effects on learning processes, with limited effect on activity levels). Several putative mechanisms can account for these findings: first, the effects of minocycline may be related to its neuroprotective properties against neuronal damage, through inhibition of the proliferation and activation of microglia (Tikka et al.,
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2001). Second, minocycline may exert its effect through the NO system, associated with spatial learning and sensorimotor gating (Geyer et al., 2001; Liang et al., 1994; Wass et al., 2006). As detailed in the Introduction section, both MK801 and minocycline decrease inter-cellular NO levels (Alagarsamy et al., 1994; Amin et al., 1996; Noda et al., 1996; Osawa and Davila, 1993). These common physiological pathways suggest that minocycline may exacerbate the effects of NMDA receptor antagonists. In line with this possibility, NOS inhibitors (e.g., LNAME) enhanced NMDA antagonist-induced behaviors, while a NO donor showed an opposite effect (Bujas-Bobanovic et al., 2000a,b; Noda et al., 1995). However, other studies indicate that NOS inhibitors may actually block NMDA antagonistinduced effects. For example, several NOS inhibitors (e.g., ARR 17477, L-NAME) reversed the effects of NMDA antagonists (Johansson et al., 1997, 1998, 1999b). The current study is in agreement with the latter group of findings, suggesting that minocycline opposes MK80-induced deficits. They correspond to Johansson et al.'s (1997, 1998, 1999b) findings and add convergent validity by using a different NMDA antagonist (MK801) and NOS inhibitor (minocycline). It was suggested that Johansson et al.'s (1997, 1998, 1999b) findings may stem from over-dependence on measures of activity and stereotyped behavior (Bujas-Bobanovic et al., 2000a,b). The current study indicates that this may not be the case since the effects of the NOS inhibitor were found when we used additional well-validated behavioral paradigms. The effect of minocycline in countering the effects of MK801 may be related to glutamate compensatory mechanisms. NMDA antagonists facilitate glutamate release stemming from compensatory response to NMDA receptor blockade (Adams and Moghaddam, 2001; Krystal et al., 1999; Moghaddam, 1994; Moghaddam et al., 1997). In turn, the glutamate release affects non-NMDA (AMPA/kainate) receptors, leading to behavioral effects and neural damage (Breese et al., 2002; Olney et al., 1999). The dependence upon a compensation mechanism suggests that only a long duration of NMDA receptor blockade will lead to these effects (Olney et al., 1999). Different durations or dosages of NMDA antagonist treatments may explain the contrasting findings on the effects of NOS inhibitors on NMDA antagonist administration. Our current findings highlight the potential role of minocycline as a protective agent against MK801-induced deficits. They suggest that minocycline may serve as a potential therapeutic tool for disorders such as SZ. This putative suggestion should be accompanied by the recognition of the inherent limitations in the use of animal models to accurately reflect complex disorders. For example, the construct validity of these models is limited, at least until the biochemical nature of SZ is deciphered (Krystal, 1999; Lipska and Weinberger, 2000). Bearing these inherent limitations in mind, it is our hope that the current study findings will contribute to paving the way for future research that will clarify minocycline's potential. These future studies can help in the clinical validation of minocycline as neuroprotective agent by utilizing additional behavioral paradigms, animal species and antipsychotic comparison drugs. Such research also holds a promise to expand our knowledge of the pathophysiology of SZ.
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4.
Experimental procedures
4.1.
Animals
The study animals were adult male Sprague–Dawley rats (total N = 91; 41 for the minocycline dose determination and 50 for the main experimental procedure) weighing 150–200 g, from the ‘Animal Behavior Laboratory’ in Ben-Gurion University of the Negev. They were housed four per cage in a vivarium with stable temperature and a reversed 12-hour light/dark cycle, with unlimited access to food and water. The animals were habituated to housing conditions for at least 10 days during which they were pre-handled to minimize the stress that can be induced following needle injection (Ryabinin et al., 1999). The paradigm consisted of handling them once daily by picking them up with a gloved hand. Experimental procedures were performed during the dark phase using a dim light. All testing procedures were approved by the university's Animal Care Committee and carried out in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals.
4.2.
Materials
4.2.1.
Drug solutions
4.3.2. (PPI)
Acoustic startle response (ASR) and prepulse inhibition
Both experimental paradigms were performed in a room that was adjacent to the laboratory facilities and an animal stall that was not used for other experiments. The animals were tested in pairs, with startle chambers counterbalanced across the different experimental groups. Startle response and prepulse inhibition were measured using two ventilated startle chambers (SR-LAB system, San Diego Instruments, San Diego, CA). Each chamber consisted of a Plexiglas cylinder resting on a platform inside a sound-attenuated ventilated chamber. A high-frequency loudspeaker inside the chamber produced both a continuous broadband background noise of 70 dB and the selected acoustic stimuli. Movement inside the tube was detected by a piezoelectric accelerometer below the frame. The amplitude of a whole body startle to an acoustic pulse was defined as the average of 100 1-ms accelerometer readings collected from pulse onset. These signals were then digitized and stored by a computer. Sound levels within each test chamber were measured routinely by a sound level meter (Radio Shack) to ensure consistent presentation. The SR-LAB calibration unit was used routinely to ensure consistent stabilimeter sensitivity between test chambers and over time (Swerdlow and Geyer, 1998).
4.4.
Procedure
4.4.1.
General handling (preliminary) procedure
4.2.1.1. Dizocilpine maleate (MK801).
Fresh solutions of dizocilpine maleate MK801 (Sigma-Aldrich, Israel) were prepared in physiological saline (0.9% NaCl in sterile distilled water) for each group of rats. A dose of 0.1 mg/kg was used, as described in Kipnis et al. (2004).
4.2.1.2. Minocycline. Minocycline hydrochloride (Sigma, St. Louis, MO) was freshly dissolved in phosphate-buffered saline (KPBS, pH 7.2, 37 °C). A 35 mg/kg dose was chosen after a pretest using 20–40 mg/kg doses (see the Procedure section and Fig. 1). 4.2.1.3. Haloperidol. Haloperidol (Sigma-Aldrich) was dissolved with a minimal amount of glacial acetic acid (∼10 μl) and then diluted with lukewarm 5.5% D-glucose, with a final pH ∼ 6.0. 0.4 mg/kg dose was used for the MWM, 0.3 mg/kg for sensorimotor gating analyses. 4.2.1.4. Saline.
Saline was used for control injections.
4.3.
Apparatus
4.3.1.
Morris water maze (MWM)
The MWM consisted of a water pool (diameter = 1.8 m, depth = 0.6 m) containing water at 26 ± 1 °C. A hidden platform was located 1.5 cm below the water surface. Within the testing room, only distal visuo-spatial cues were available to the rats for location of the submerged platform. A video camera was placed above the center of the pool and a video tracking system with online digital output directly fed data into a computer. Data were analyzed by an Etho-Vision automated tracking system (Noldus Information Technology b.v., Wageninpen, The Netherlands).
Ten days before the beginning of the experiment, the rats underwent a handling procedure conducted once daily and aimed at reducing experimental stress (Ryabinin et al., 1999). The handling procedure consisted of picking the rat up with a gloved hand and stroking it.
4.4.2. Dose-dependent effect of intraperitoneal (IP) injections of minocycline compared to those of MK801 and saline The effective dose of minocycline to be used in the study was assessed in a trial procedure. Rats (N = 41) were injected IP with either minocycline in one of four doses (20, 25, 30 and 35 mg/kg) or with saline for three consecutive days before behavioral testing. We initiated minocycline treatment 3 days in order to achieve a sufficient drug level (based on; Levkovitch-Verbin et al., 2006), taking into consideration the fact that minocycline is more effective when given before CNS injury, compared to administration shortly after the onset of injury (Du et al., 2001; Yrjanheikki et al., 1998). Fifteen minutes before conducting the behavioral procedures (the fourth day), the rat was injected with IP MK801 0.1 mg/kg (as described in; Kipnis et al., 2004) or saline (for the rats that received saline earlier), for a total volume of 1 ml/kg body weight. Next, the rats underwent four consecutive trials in the MWM paradigm. The MWM was used to assess dose– response effects because of its high levels of reliability and validity (Morris et al., 1982b; Schimanski and Nguyen, 2004).
4.5.
Main experimental procedure
Rats (N = 50) were injected with minocycline (35 mg/kg) for three consecutive days before the behavioral testing (in
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accordance with; Levkovitch-Verbin et al., 2006). On each day of behavioral testing, the rats were IP injected with either haloperidol (alone or in combination with MK801) or saline (alone or minocycline, saline, minocycline + MK801, and MK801 groups) 30 min before behavioral assessments, an effective time-frame for inducing behavioral effects in rodents (Svensson, 2000). Fifteen minutes before undergoing the behavioral procedures on the fourth day, the rat was injected IP with MK801 or saline, for a total volume of 1 ml/kg body weight. Afterwards the rats underwent the three behavioral procedures described below (Table 1).
4.6.
The Morris Water Maze (MWM) paradigm
The MWM (Morris, 1984) was used as a well-validated hippocampus dependent test of visual–spatial memory (D'Hooge and De Deyn, 2001; Eichenbaum et al., 1990; Logue et al., 1997; Schimanski and Nguyen, 2004). Further support of the ecological validity of the MWM for the purposes of the current study can be found in memory deficits exhibited by SZ patients, accompanied by hippocampus abnormalities (Green, 1996; Green et al., 2000; Halliday, 2001; Shenton et al., 2001). The MWM tested the ability of an animal to locate a hidden platform submerged under water by using extra-maze cues from the test environment was examined. The rats were trained in a pool 1.8 m in diameter and 0.6 m high, containing water at 26 ± 1 °C. A 10 cm square transparent platform was hidden in a constant position (1 cm below the water level) in the pool. Only distal visuo-spatial cues were available to the rats for location of the submerged platform. The rats were given trials to find the hidden platform (acquisition phase). The escape latency, i.e., the time required by the rat to find and climb onto the platform, was recorded for up to 120 s. Each rat was allowed to remain on the platform for 30 s, after which it was removed to its home cage. If the rat did not find the platform within 120 s, it was manually placed on it and returned to its home cage after 30 s.
4.7. Acoustic startle response (ASR) and prepulse inhibition (PPI) paradigms Assessment of the ASR and PPI was employed as ecologically valid paradigms for assessing sensorimotor reactivity and the inhibition of the startle reflex by a weak pre-stimulus (PPI) (Davis, 1980; Davis et al., 1993). ASR and PPI were measured using two startle chambers, with each chamber consisting of a
Plexiglas cylinder resting on a platform inside a soundattenuated, ventilated chamber (Materials section). The animals were tested in pairs, with startle chambers counterbalanced across the different experimental groups. At the beginning of the experiment, the animals were placed inside the tube and the startle session started with a 5-min acclimatization period during which a high-frequency loudspeaker inside the chamber produced a continuous broadband background noise of 68 dB. Following the acclimatization period, four startle pulses (120 dB for 30 ms) were presented. Movement inside the tube was detected by a piezoelectric accelerometer below the frame. The amplitude of the whole body startle to an acoustic pulse was defined as the average of 100 1-ms accelerometer readings collected from pulse onset. These signals were then digitized and stored in a computer. The SR-LAB calibration unit was used routinely to ensure consistent stabilimeter sensitivity between test chambers and over time. The paradigm allowed the measurement of base-line ASR (the transient force resulting from the movements of the platform evoked by the ASR to the first startle pulse), as well as the animal's ability to habituate to repetitive loud pulses of noise (habituation of the ASR). PPI of the ASR was used as a measure of sensorimotor gating, with the startle chambers used to assess the ability of prepulse stimuli to block the startle response. The prepulses were broadband noise bursts of either 72, 76, 80, or 84 dB and of 20 ms in duration. The interval between the prepulse and the startle pulse was 80 ms. Each session consisted of six blocks of 10 trials. Each block included four different trial types: two startle pulse-alone trials, four prepulses at different intensities followed by startle pulse, four prepulses alone at four intensities and one no-stimulus trial. The different trial types were presented pseudo-randomly with a variable inter-trial interval of 10–20 s.
4.8.
Data analysis
4.8.1.
Morris Water Maze (MWM)
Escape latency (defined as the time between placing the rat in the pool until it located the platform) was measured during behavioral testing. Selection of minocycline dose was conducted using an analysis of variance (ANOVA) with a betweensubject measure of ‘group’ (treatment group; MK801, saline and four minocycline doses (20, 25, 30, 35 mg/kg)]. Since the analysis showed similar effects of 30 and 35 mg/kg minocycline doses, we conducted follow-up ANOVA's for trails 1–4 in
Table 1 – Study design Experimental procedure
Days −10–0
Treatment MK801 Minocycline + MK801 Haloperidol + MK801 Minocycline Haloperidol Saline
Handling
Daily injections (days 1–3) Saline Minocycline Saline Minocycline Saline Saline
Pre-behavioral testing (day 4) 30 min 15 min Saline Saline Haloperidol Saline Haloperidol Saline
Notes: MWM, Morris Water Maze; ASR, acoustic startle response; PPI, prepulse inhibition.
MK801 MK801 MK801 Saline Saline Saline
Behavioral testing (day 4) MWM ASR PPI
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the MWM (see the Results section). Analysis of the main MWM experiment was conducted using a repeated-measures ANOVA using a between-subject measure of ‘group’ [treatment groups; MK801, MK801 + minocycline, minocycline, MK801 + haloperidol, haloperidol and saline] and a withinsubject measure of ‘trial’ (effects among groups in the four different trials). In order to elucidate the of significant interactions follow-up ANOVAs were conducted for each MWM trail. In all analysis, significant group differences were followed by Scheffe post hoc tests.
4.8.2.
Acoustic startle response (ASR)
The paradigm allowed the measurement of base-line ASR (the transient force resulting from the movements of the platform evoked by the ASR to the first startle pulse), as well as the animal's ability to habituate to repetitive loud pulses of noise (habituation of the ASR). Habituation of ASR was defined as a decrement in startle response to an initially novel stimulus when presented repeatedly; the decrease in ASR was calculated as the percent of amplitude decrease between the mean of the first five amplitude values and the mean of the last 25 amplitude values: % habituation = 100 × [(average startle amplitude in Block 1) − (average startle amplitude in Block 5 / (average startle amplitude in Block 1)]. Results were analyzed by two-way ANOVA with a betweensubject measure of ‘group’ (treatment group). Significant group differences in the ANOVA's were followed by Scheffe post hoc tests.
4.8.3.
Prepulse inhibition (PPI)
Startle magnitude is expressed in all the procedures as arbitrary units. Percent PPI (%PPI) was calculated as the mean startle magnitude to stimulus-alone minus the mean startle magnitude to prepulse stimulus trials, all divided by the mean stimulus-alone trials and multiplied by 100. The overall mean %PPI was calculated for the four prepulse intensities (72, 74, 80, 84 dB). Results were analyzed by twoway ANOVA with a between-subject measure of ‘group’ and ‘prepulse intensity’ (72, 76, 80, 84 dB). Follow-up repeated ANOVAs were conducted for each group in order to ascertain the source of significant interactions. In all ANOVAs significant group main-effects were followed by Scheffe post hoc tests.
Acknowledgment Esther Eshkol is thanked for editorial assistance.
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