Differential effects of classical neuroleptics and a newer generation antipsychotics on the MK-801 induced behavioural primitivization in mouse

Progress in Neuro-Psychopharmacology & Biological Psychiatry 30 (2006) 521 – 530 www.elsevier.com/locate/pnpbp

Differential effects of classical neuroleptics and a newer generation antipsychotics on the MK-801 induced behavioural primitivization in mouse Marie Nilsson a,*, Katarina Ryde´n Markinhuhta b, Maria L. Carlsson a a

The Arvid Carlsson Institute for Neuroscience at the Institute of Clinical Neuroscience, The Sahlgrenska Academy at Go¨teborg University, Medicinaregatan 11, Box 432, SE-405 30 Go¨teborg, Sweden b Carlsson Research AB, Biotech-Center, Arvid Wallgrens Backe 20, SE-413 46 Go¨teborg, Sweden Accepted 23 November 2005 Available online 18 January 2006

Abstract Cognitive dysfunction plays an important role in mental disorders like schizophrenia and may involve inadequate glutamatergic signalling in different regions of the brain, mediated by e.g. glutamatergic N-methyl-d-aspartate (NMDA) receptors. In rodents, NMDA receptor antagonists often increase motor activity; in addition they induce a more primitive and undifferentiated behavioural pattern, which we believe may correspond to some of the cognitive defects seen in schizophrenia. In the present study, the movement pattern of mice treated with the uncompetitive NMDA receptor antagonist MK-801 in conjunction with six antipsychotic agents, some with reported clinical effects on cognition, was characterised and quantified. The classical neuroleptic drugs chlorpromazine and trifluoperazine, the atypical antipsychotic agents ziprasidone and olanzapine, the gamino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-receptor potentiator CX516 and the serotonin (5-HT)2A-antagonist M100907 were tested. In accordance with previous observations, MK-801 was found to induce a primitive and monotonous behavioural pattern dominated by forward locomotion; spatial movements, the number of switches between the states moving and stationary, and rearing frequency were reduced. All test substances counteracted MK-801-induced hyperactivity, but differed in their ability to improve behavioural quality. Chlorpromazine and trifluoperazine were unable to restore behavioural diversity while ziprasidone, olanzapine, CX516 and M100907 restored it to varying degrees. A striking similarity in movement pattern was seen between the hypoglutamatergic mice treated with the AMPA-receptor agonist CX516, and those receiving the 5HT2A-antagonist M100907. D 2005 Elsevier Inc. All rights reserved. Keywords: Cognition; Mice; MK-801; Schizophrenia; Video tracking

1. Introduction Cognitive dysfunction is present in a majority of patients suffering from schizophrenia and often precedes the onset of positive and negative symptoms (Cannon et al., 2002; Cornblatt and Keilp, 1994; Goldberg et al., 1990). The cognitive impairments include problems with attention, memory and executive functions (Hoff et al., 1992; Saykin et al., 1994). The importance of cognitive function as an outcome measure has been investigated, and it has been shown that

Abbreviations: AMPA, a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; CCD, charge-coupled device; 5-HT, serotonin; NMDA, N-methyl-daspartate; PLS, Partial Least Square; PLS-DA, Partial Least Square Discriminant Analysis. * Corresponding author. Tel.: +46 31 773 3328; fax: +46 31 773 3401. E-mail address: [email protected] (M. Nilsson). 0278-5846/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2005.11.010

cognitive dysfunction affects social and vocational abilities, and quality of life, to a greater extent than positive symptoms do (Green, 1996; Jaeger and Douglas, 1992). Therefore, in the evaluation of antipsychotic medications, the importance of assessing effects on cognition, along with effects on positive and negative symptoms, has received increasing attention. The causes underlying cognitive disturbances in schizophrenia are not known, but aberrant signalling in the medial temporal lobe and prefrontal cortical regions of the brain is often discussed in this context. Prefrontal cortical structures are involved in working memory (Goldman-Rakic and Selemon, 1997), and functional brain imaging studies have shown an inability of schizophrenic patients to activate frontal cortex in cognitive tasks (Catafau et al., 1994; Rubin et al., 1991; Volz et al., 1997). These are regions that have a high concentration of glutamatergic neurones and where neuroanatomical and biochemical abnormalities have been observed in schizophrenia,

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suggesting an underlying cortical network dysfunction involving inadequate glutamatergic signalling mediated by e.g. Nmethyl-d-aspartate (NMDA) receptors. The most important evidence supporting deficient glutamatergic function in schizophrenia is provided by observations of the mental effects of NMDA antagonists. Phencyclidine and subanaesthetic doses of ketamine in healthy volunteers produce symptoms closely resembling those seen in persons with schizophrenia, including positive as well as negative and cognitive symptoms (Krystal et al., 1994; Malhotra et al., 1996). Moreover, subanaesthetic doses of ketamine given to schizophrenic patients produce psychotic symptoms that have many similarities to the individuals’ own specific positive symptoms (Lahti et al., 1995). Conventional neuroleptic drugs like haloperidol and chlorpromazine only weakly alleviate cognitive dysfunction at best and, in fact, certain cognitive abilities often deteriorate instead (Krausz et al., 2000; Medalia et al., 1988). In contrast, several studies suggest that the newer generation antipsychotic compounds improve some of the cognitive impairments in schizophrenic patients (Cuesta et al., 2001; Meltzer and McGurk, 1999; Sharma, 1999). There are reports of improvement in verbal fluency and attention with clozapine treatment, improvement in attention, memory and executive functioning with risperidone and improvement in verbal learning, verbal fluency and executive functioning with olanzapine, although the global performance does not seem to be restored to the level of healthy controls with these agents. However, there is disagreement between different studies and many findings have not been replicated. In a critical review of clinical trials concerning the neurocognitive effects of atypical antipsychotics (Keefe et al., 1999), the authors draw attention to the various limitations in the trial designs, such as 1) incomplete information about treatment and cognitive status prior to the initiation of new medication, 2) inappropriate dosage, 3) small sample size and 4) the scarcity of studies performed with double-blind design. Administration of NMDA antagonists to rats and mice is often used to create an experimental model of schizophrenia. In rodents, NMDA receptor antagonists cause hyperactivity, which may correspond to positive symptomatology of schizophrenia. In addition NMDA antagonists induce a primitive and undifferentiated behavioural pattern. In a previous study (Nilsson et al., 2001) the behavioural pattern of mice pretreated with the NMDA receptor antagonist MK-801 was characterised using a video tracking software. The MK-801 treated animals’ behaviour consisted of monotonous forward locomotion with a reduction of spatial movements, rearing frequency and number of voluntary stops. We believe that this loss of behavioural sophistication may correspond to some of the cognitive defects seen in schizophrenia. To further examine this possibility the present study was undertaken: the movement pattern of mice treated with the uncompetitive NMDA receptor antagonist MK-801 in conjunction with six established or purported antipsychotic agents with different pharmacological profiles, some with reported clinical effects on cognition, was characterised and quantified.

The classical neuroleptic drugs chlorpromazine and trifluoperazine, the newer generation antipsychotic agents ziprasidone and olanzapine, the AMPA receptor modulator CX516 and the selective 5-HT2A-antagonist M100907 were compared. 2. Material and methods 2.1. Animals Male NMRI mice (B&K Universal AB, Sollentuna) weighing 25 –30 g at the time of testing were used in all experiments. The mice were housed in groups of eight in ventilated, wood wool-enriched, Macrolon type II cages for at least one week before the experiments were carried out. (Temperature: 20– 22 -C; 12 h light/dark cycle with lights on at 6.00 AM; free access to water and food pellets.) The experimental design was approved by the Go¨teborg ethic committee for animal experimentation. 2.2. Drugs and drug administration (+)-MK-801 hydrogen maleate (Research Biochemical Inc, USA), chlorpromazine hydrochloride (AB Leo, sweden), trifluoperazine dihydrochloride (Pharma-Rodia) and CX516 (courtesy Cortex Pharmaceuticals Inc, USA) dissolved in physiological saline. Olanzapine (courtesy Eli Lilly and Company, USA) and M100907 (courtesy Hoechst Marion Roussell, USA) were dissolved in approximately 5 Al acetic acid and diluted with glucose (5.5%) to the desired concentration. Ziprasidone (courtesy Pfizer, USA) was dissolved in a cyclodextrine solution containing hydroxypropyl-h-cyclodextrine (80 mg/ml) and citric acid (4.2 mg/ml) dissolved in physiological saline. All drugs were given 30 min before the registration of behaviour started, with the exception of CX516 which was given 5 min before. All substances were given intraperitoneally, with the exception of olanzapine which was given subcutaneously. The injection volume was 10 ml/kg for all substances. 2.3. Experimental procedure On the day of the experiment the animals were marked, weighed and administered either test substance or vehicle. After appropriate pre-treatment, the animals were individually introduced into an unfamiliar, open field arena and videotaped for 30 min. Four arenas were simultaneously videotaped from above with one single CCD monochrome video camera. The camera was connected to an S-VHS videocassette recorder. The arenas were made of black plexiglass (l, w, h: 46  33  35 cm), rubbed with sandpaper and indirectly illuminated to avoid reflexes and shadows. The floors of the arenas were covered with grey gravel that had earlier been exposed to other mice. The videotapes were after completion of the experiment analysed with the video-tracking program Ethovision 1.95 from Noldus Information Technologies b.v. The program gives for each sampling occasion the position of the mouse, together with the animal’s body area seen from the overhead camera; the

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animals’ body areas are measured in pixels. The analysis results in a track record, which describes the animals’ behavioural pattern during the observation period. The first 10 min of recording were used for further analyses. The mice were decapitated immediately after completion of the video recording.

regarded as spontaneous movements as they are not influenced by the restrictions of the walls of the arena. Needless to say, this may not be true if the animal’s behaviour is highly stereotyped; to make an assessment of this, we always watch the videotapes and observe the gross appearance of the animals.

2.4. Behavioural measures

2.5. Statistical analyses

Table 1 gives a short description of the variables used in the analyses. The video tracking was performed at a sampling frequency of 12.5 Hz. In the subsequent analysis, down sampling steps 2, 5 and 10 were applied to some of the variables, i.e. these variables were calculated at four different sampling frequencies, 12.5, 6.25, 2.5 and 1.25 Hz (denoted as _1 –4 where 1 corresponds to 12.5 Hz). Others were filtered in such a way that the animals had to move a certain distance before the variables were calculated. The outcome of the variable calculations is determined both by the complexity of the animals’ behaviour and the resolution used to obtain the measures. Hence, the variables are analysed at different sampling frequencies as well as with different filtering. The variables were calculated on the entire arena as well as in a middle zone (m), defined as the central part located 6 cm or more away from the border of the arena. The reason for performing separate analyses on activities performed in the middle zone, is that the spatial variables, e.g. turning, sinuosity and meander, as well as the number of stops, can here be

The data were evaluated using both multivariate and univariate statistical methods. For multivariate evaluation, Partial Least Square Discriminant Analysis (PLS-DA) was applied (Sjo¨stro¨m et al., 1986; Wold et al., 1984, 1999). In Partial Least Square (PLS) analysis, the relationship between the measured variables is explored and the covariances are used to reduce the original set of variables to a smaller set, commonly referred to as latent variables or principal components. The principal components are not correlated to each other and they show the significant relation between the original variables. PLS-DA is an extension of PLS that accomplishes an optimal projection to latent variables that focuses on class separation. The objective with PLS-DA is to find a model that separates classes of observations on the basis of their X-variables. The X-matrix in this case consists of the measured behavioural variables shown in Table 1. In order to encode a class identity, we used a Y-data matrix of dummy variables describing the class membership of each observation. A dummy variable is an artificial variable that assumes a

Table 1 Variables used in analyses AM _1 – 4 AMm _1 – 4 AM0.5 AMm0.5 AM1 AMm1 sdAM0.5 sdAMm0.5 sdAM1 sdAMm1 DM _1 %IZ _1 R _1 – 4

Rm _1 – 4 S_1 – 4

Sm _1 – 4 ST_1 – 4 STm _1 – 4 T_1 – 4 Tm _1 – 4 sdA meanA

Mean of absolute meander, unsigned change in direction of movement between two consecutive samples relative to the distance moved (ranges from 0- to 180-/cm). Mean of absolute meander in middle zone. The same definition as AM, except that the change of direction is not calculated unless the distance moved between two consecutive samples exceeds a distance moved of 0.5 cm. AM0.5 in middle zone. Same as AM except for that the change of direction is not calculated unless the distance moved exceeds 1 cm. AM1 in middle zone. Standard deviation of absolute meander, otherwise like AM0.5. sdAM0.5 in middle Standard deviation of absolute meander, otherwise like AM1. sdAM0.5 in middle. Distance moveda Percent time spent in middle zone Number of rearings — detected as a reduction of the animal_s body image as seen by the overhead video camera. A rearing is defined when the proportional change in body area of a certain sample area and an interval of previous consecutive sample areas (calculated with a moving average of 5 samples) exceeds a threshold change of 15%. Rearing in middle zone. Sinuosity, change of direction associated with a given path length. The sample co-ordinates are recalculated (rediscretised) to an equidistant path length of 7 cm, and the changes in direction are calculated on the new sample co-ordinates. S is defined as the standard deviation of all recalculated turn angles divided by the square root of the rediscretisation step length. Sinuosity in the middle zone. Number of changes between the states moving/not moving (=number of stops). Animals are regarded as ‘‘not moving’’ when the velocity is below 5 cm/s, calculated with a moving average of 5 samples. Number of stops in the middle zone. Turning (same definition as for sinuosity but with a path length of 0.5 cm). Turning in the middle zone. Standard deviation of the digitised image of the animals body area, measured in pixels Mean of the digitised images of the animal_s body area, measured in pixels.

a Distance moved was not included in the multivariate evaluation of the data since this variable has such a strong discriminating importance for MK-801 treated animals. The analysis was instead focused on discrimination based on alterations in body posture and path shape.

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discrete numerical value for the class description, in this case denoting treatment group. The results of these analyses are presented as low dimensional projections, loading plots, where the relations between the X and Y variables are visualised. The loading plot shows how the measured variables are correlated and how they are influenced by the treatment. Variables close to each other have similar characteristics and a location far from origin indicates high discriminating importance. The significance of the components was evaluated by means of cross-validation (Stone, 1974; Wold, 1978) and only significant components are shown in the figures. The preprocessing of all data consisted of unit variance scaling and mean-centring (Jackson, 1991). The Simca-P+program, version 10.0.2.0, UMETRICS, was used for the calculations. Univariate comparisons between groups for individual variables were performed with the non-parametric Kruskal – Wallis test followed by Mann – Whitney U-test. An extended variant of the Mann – Whitney U-test that tests for trends among ordered groups (Cuzick, 1985) was used to evaluate dose – response relations. For the group comparisons, asterisks in the graphical presentations denote significance at the 95% level or above. A diagonal line above columns indicates significant trends in dose –response at the 95% level or above. Bar chart figures were presented as means and S.E.M. 3. Results 3.1. Behavioural measures The effects of chlorpromazine (0.3, 0.9, 1.2 mg/kg), trifluoperazine (0.33, 1, 3 mg/kg) olanzapine (0.083, 0.25, 0.75 mg/kg), ziprasidone (0.33, 1, 3 mg/kg), CX516 (45, 90, 120 mg/kg) and M100907 (0.001, 0.01, 0.1 mg/kg) on MK-801 (0.2 mg/kg) induced alterations of movement pattern in mice were univariately analysed with respect to distance moved, turning in the middle zone, standard deviation of meander in the middle zone, number of rearings, and number of stops in the middle zone, measured at the highest sampling frequency, 12.5 Hz. Fig. 1a shows the effects of the six antipsychotic compounds in MK-801-treated mice with respect to distance moved. All tested substances reduced dose-dependently MK801 induced hyperactivity. The effects on the spatial variable turning are shown in Fig. 1b: the MK-801-induced decrease in turning was dose-dependently counteracted by olanzapine, ziprasidone, CX516 and M100907 but not by chlorpromazine and trifluoperazine. In this graph, the column for the highest dose of ziprasidone was truncated at 200% (mean for this group was 260 and S.E.M 58). The effects of the tested compounds on standard deviation of meander (Fig. 1c) were similar to those observed on turning, except for a more moderate effect of the highest dose of ziprasidone. The MK801 induced decrease in standard deviation of meander was not counteracted by chlorpromazine, trifluoperazine or M100907. The MK-801 induced reduction in rearing frequency shown in Fig. 1d could not be reversed by any of the tested compounds. In fact, there was a significant trend that increasing dose of test

compound further reduced the rearing frequency. Fig. 1e shows that MK-801 reduced the number of stops in the middle zone in the trifluoperazine, olanzapine and M100907 experiment and tended to do so in the ziprasidone and CX516 experiments. Olanzapine increased the number of stops in the doses 0.25 and 0.75 mg/kg and there was a significant dose-dependent trend. Ziprasidone increased the number of stops significantly at 0.33 mg/kg and M100907 at the dose of 0.01 mg/kg. No effects on the number of stops were seen in the chlorpromazine experiment. 3.2. Multivariate evaluation A multivariate PLS-DA analysis was applied on the six different experiments with the purpose of assessing all measured variables and all antipsychotic treatments simultaneously. To adjust for different baseline levels, values were normalised to the level of the MK-801 treated group in the different experiments. From each experiment 4 –5 individuals treated with the same antipsychotic and with motor activity levels similar to controls were included in the analysis together with controls and MK-801-treated animals. The analysis was based on 52 observations, 51 behavioural variables (Table 1) and 8 dummy variables, one for each treatment group. The analysis resulted in a six-component model, explaining 40%, 23%, 11%, 5.8%, 5.7% and 1.9%, respectively, of the variance in the behavioural variables. The result is presented as loading plots showing how the measured variables are correlated and how they are influenced by the treatments. In Fig. 2a, a loading plot for the first two components shows that the different treatments are mainly separated along component 1. Treatment groups located at one side of the plot show relatively higher values of variables presented on that same side and lower values of variables presented on the opposite side of the plot. This means that MK + CX516, MK + M100907, MK + ziprasidone and MK + olanzapine to different extents have higher values in all measures of path shape, i.e. variables describing the horizontal spatial variability in the movement path, compared to the MK + chlorpromazine and MK + trifluoperazine treated groups as well as MK-801 alone. In component 1, olanzapine seems to be the treatment that most clearly increased horizontal spatial variability in MK-801 treated mice. The second component appears to mainly reflect the variation in body posture. Controls are characterised by a high level of rearing and variation in body area, while all other treatment groups are more characterised by high values of mean body area, indicating a more flattened body posture. The third component, shown in Fig. 2b, further discriminates between the first generation antipsychotics and the newer generation of compounds; it is shown here that mice receiving MK + chlorpromazine or MK + trifluoperazine have a different movement pattern with respect to the number of stops. These two treatment groups are in the third component characterised by the number of stops recorded at the lowest sampling frequencies 2.5 and 1.25 Hz (ST_3 –4), indicating that these mice spent longer time periods not in motion. This was most evident in the MK + trifluoperazine group.

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a

300 250

Distance moved

525

z=-2.75*

z=-2.69**

+

z=-3.45***

+

200 150

z=-3.35***

+

+

*

*

*

*

* *

*

*

+

+

*

100 50

z=-3.92***

z=-3.10**

*

*

0 chlorp

Turning in middle

b

triflu

olanz

zipr

CX516

z=3.81***

200

*

*

150

z=2.36*

*

*

*

+

+

+

50

z=2.58**

* *

z=0.57

100

+

260 (58)

z=3.65***

z=1.43

M100907

+

+

0 chlorp

triflu

olanz

zipr

CX516

M100907

c SD Meander in middle

200 z=2.84**

150 100 50

*

z=2.81** z=0.61

z=0.72

+

** +

+

+

z=2.43*

*

+

z=1.61

+

0 chlorp

Rearing

d

triflu

CX516

M100907

100

50

z=-2.53*

+

z=-4.43***

+

+

z=-3.85**

*

**

chlorp

triflu

z=-3.65***

z=-2.32*

z=0.63

+ zipr

CX516

z=-0.15

*

* M100907

z=1.82

*

* z=-0.55

z=-1.39

+

+

+

z=-2.97**

** *

100 50

+

*

200 150

z=-5.57***

+

*

olanz

300 250

Stops in middle

zipr

150

0

e

olanz

0 chlorp

ctr

triflu

MK-801

olanz

zipr

dose 1

CX516

dose 2

M100907

dose 3

Fig. 1. Effects of chlorpromazine (0.3, 0.9, 1.2 mg/kg), trifluoperazine (0.33, 1, 3 mg/kg), olanzapine (0.083, 0.25, 0.75 mg/kg), ziprasidone (0.33, 1, 3 mg/kg), CX516 (45, 90, 120 mg/kg) and M100907 (0.001, 0.01, 0.1 mg/kg) on MK-801 (0.2 mg/kg) induced hypoglutamatergia. a) Distance moved, b) turning in middle zone, c) standard deviation of meander in middle zone, d) rearing and e) number of stops in middle zone. Shown are group means and S.E.M for the first 10 min of recording. All values are expressed as percent of the vehicle treated group for easier visual comparison between the six experiments. ‘‘Dose 1’’ in the figure legend corresponds to the lowest dose of each antipsychotic compound. N = 4 – 5. *p < 0.05 compared to MK-801, +p < 0.05 compared to control (Mann – Whitney U-test). A line above bars indicates significant trend in the dose – response with Cuzick’s test. The sampling frequency was 12.5 Hz.

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a meanA S_1 S_4 S_2 Sm_1 S_3 Sm_2 Sm_4 %IZ_1 Sm_3 STm_1 olz Tm_4 1STm_2 AMm1 STm_3

M100

comp 2

MK-801

zipr

CX

ST_1

chlp ST_2 trif ST_3ST_4

AM1 sdAMm1

STm_4

sdAM1 AM_4 AM_3

T_4 Tm_2 Tm_3 Tm_1 AMm_1 AMm_4 T_3 AMm0.5 AMm_2 1T_2 1T_1 AM0.5 AMm_3 sdAMm0.5 AM_1 sdAM0.5 AM_2

Rm_3 Rm_4 Rm_1 Rm_2

ctr

sdA

R_4R_1 R_3 R_2

Comp 1

b ST_1

ST_2

MK-801

sdAM1 AM1 Rm_4 sdA 1Rm_3 R_3 R_4 R_2 Rm_2 R_1 AMm1 M100 Rm_1 ctrsdAMm1

comp 3

CX

zipr

chlp meanA

olz

T_2 Sm_3 Sm_4 T_1 Sm_2 SS_3 _2Sm_1 T_3 S_1 S_4 %IZ_1 AMm0.5 sdAMm0.5 Tm_1 Tm_2 Tm_3 1T_4 Tm_4 AM0.5 AMm_4 AMm_2 AMm_1 AMm_3 STm_1

STm_2 STm_3 ST_3

STm_4 AM_2

sdAM0.5

AM_1

AM_3

trif

AM_4 ST_4

comp 1 Fig. 2. PLS-DA of movement pattern in mice pretreated with MK-801 alone (0.2 mg/kg) or in combination with chlorpromazine, trifluoperazine, olanzapine, ziprasidone, CX516 or M10090. 4 – 5 individuals per group (receiving MK-801 + test substance) with motor activity levels similar to controls were included in the analysis, together with controls and MK-801 treated mice. Included doses were for chlorpromazine 0.3, 0.9, and 1.2 mg/kg, for trifluoperazine 1 and 3 mg/kg, for olanzapine 0.25 and 0.75 mg/kg, for ziprasidone 0.33 and 1 mg/kg, for CX516 90 and 120 mg/kg and for M100907 0.01 and 0.1 mg/kg. The analysis was based on 52 observations, 51 behavioural variables and 8 dummy variables representing treatment. The analysis resulted in a six component model explaining 40%, 23%, 11%, 5.8%, 5.7% and 1.9%, respectively, of the variance in the behavioural variables. a) Loading plot for component 1 and 2. b) Loading plot for component 1 and 3. Abbreviations; see Table 1.

3.3. Movement pattern In Fig. 3, representative tracks of the animals’ movement patterns are shown. Tracks from hypoglutamatergic mice treated with antipsychotic agents were chosen from individuals having motor activity levels similar to controls, to enable relevant comparisons. The first 5 min of video recording are shown. In accordance with previous observations, MK-801 induced monotonous forward locomotion, with only rare switches to other behavioural programs. Neither chlorpromazine nor trifluoperazine did to any greater extent restore the

complex behavioural movement pattern seen in the tracks from controls. Olanzapine, ziprasidone, CX516 and M100907, on the other hand, induced movement patterns of higher complexity at the horizontal level, albeit with different characteristics. For instance, CX516 and M100907 induced a special type of wide turns that was not seen with the other treatments. 4. Discussion In this study, the movement patterns of hypoglutamatergic mice pre-treated with established or presumed antipsychotic

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a) control

b) MK-801

c) MK+ chlorpromazine

d) MK+ trifluoperazine

e) MK+olanzapine

f) MK+ziprasidone

g) MK+CX516

h) MK+M100907

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Fig. 3. Representative tracks of the movement patterns in mice treated with MK-801 in combination with established or purported antipsychotic agents. The first 5 min of recording are shown. a) Vehicle, b) MK-801 (0.2 mg/kg), c) MK-801 + chlorpromazine (1.2 mg/kg), d) MK-801 + trifluoperazine (3 mg/kg), e) MK801 + olanzapine (0.25 mg/kg), f) MK-801 + ziprasidone (0.33 mg/kg) g) MK-801 + CX516 (90 mg/kg) and h) MK-801 + M100907 (0.1 mg/kg). Tracks from mice treated with the antipsychotic compounds were chosen from mice having motor activity levels similar to controls. Distance moved for the different mice were from a – h; 47, 26, 74, 54, 25, 31, 35, 40, 31, 25, 34, 30, 32, 33, 36 and 29 m. The sampling frequency was 12.5 Hz.

agents, some with reported clinical effects on cognition were examined with the purpose of capturing features that might predict cognition-enhancing properties of novel antipsychotic agents. Untreated mice when placed in a novel environment present a varied behaviour with many different kinds of activities. In contrast, MK-801-treated mice display a monotonous movement pattern with a meagre behavioural repertoire; we believe that this loss of behavioural sophistication may correspond to cognitive deficits of schizophrenia. To trace the animals’ paths, a video tracking software enabling objective quantitative analyses was used. 4.1. Effects on behaviour of antipsychotic agents The agents tested in this study influenced the movement pattern of the hypoglutamatergic mice in different ways. All compounds were effective in reducing the MK-801-induced hyperactivity, suggesting an ameliorating effect on positive symptoms of schizophrenia. All compounds caused unique movement pattern alterations in hypoglutamatergic mice, with olanzapine, ziprasidone, CX516 and M100907 to varying

degrees reinstating some features of normal behaviour. An increased number of small tangles, characteristic for normal mouse behaviour, was seen and showed up mainly in the variables turning and standard deviation of meander in the middle zone. This effect was most evident in the olanzapine and ziprasidone groups. None of the tested compounds was able to counteract all aspects of the MK-801-induced movement alterations. For example exploratory rearing, which is considerably reduced by MK-801-treatment, was further impaired by all tested compounds. The classical neuroleptics chlorpromazine and trifluoperazine did not to any significant degree restore any component of normal behaviour in the hypoglutamatergic mice. In a previous study on hypoglutamatergic mice (Nilsson et al., 2001) where we compared the movement patterns produced by the atypical clozapine, the dopamine D2/5-HT2A antagonist risperidone and the selective 5-HT2A antagonist M100907 with the conventional neuroleptic haloperidol, a similar result was obtained: risperidone, clozapine and M100907 restored behavioural diversity to a certain extent, while haloperidol only reversed the MK-801-induced hyperactivity without improving

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behavioural quality. Taken together, it can be concluded from these results that conventional and newer agents with antipsychotic properties have different effects on MK-801induced movement alterations in mice. The newer agents are to varying degrees capable of bringing back some behavioural features that are lost in the hypoglutamatergic mice, although a complete restoration is not achieved. Assuming that the level of behavioural diversity in the present model reflects cognitive status, these results are in accordance with the clinical observations that the older generation of antipsychotics have at best only weakly beneficial effects on cognitive dysfunction, and may in fact often worsen it, while some of the newer antipsychotic agents seem to improve certain aspects of cognition in schizophrenia. The AMPA receptor potentiator CX516 improved behavioural quality in the hypoglutamatergic mice, and indeed in both preclinical (Hampson et al., 1998) and clinical studies, CX516 has been observed to improve certain cognitive functions. For instance, in schizophrenic patients CX516 improved attention and memory when added to clozapine (Goff et al., 2001). However, a recently performed monotherapy study with CX615 (Marenco et al., 2002) in schizophrenic patients revealed no clear improvement in cognition. The study was performed with patients partially refractory to treatment with neuroleptics, which might have influenced the outcome, or perhaps involvement of additional receptor systems is needed for cognitive enhancing effects in schizophrenic patients. In the present study CX516 and M100907 reintroduced some spatial variability in the movement path, particularly with respect to wide turns, but on the whole not to the same extent as olanzapine and ziprasidone. Interestingly, there was a considerable similarity in the behavioural pattern produced by CX516 and the 5-HT2A receptor antagonist M100907. This similarity was evident both in the tracks and in the multivariate analysis, where the loading plots showed positions of these agents in close vicinity to one another. Both in the present and in our previous study we could see that the older generation of antipsychotics induced a special movement pattern with respect to the number of stops calculated at the lowest sampling frequencies. The multivariate analysis showed that these treatment groups were characterised by relatively more stops measured at the sampling frequencies 2.5 and 1.25 Hz (ST_3 –4). These variables most likely capture a special behavioural feature that can also be observed on the video recordings: these animals have extended time periods of immobility, a phenomenon that might correspond to the parkinsonian side effects in patients. 4.2. Possible mechanism behind cognition enhancing effects of newer antipsychotic compounds The mechanism behind the putative cognition enhancing effects is at present not clear, but the newer generation of antipsychotic compounds do in general show lower dopamine D2-receptor affinity and higher affinity for 5-HT2A-receptors compared to conventional neuroleptics. Further, the newer antipsychotics have a more distinct selectivity for cortical/limbic

vs striatal areas, which is supposed to have significance for improvement in negative and cognitive symptoms but especially for reducing the risk for extrapyramidal side effects. The older neuroleptics block cortical/limbic and striatal D2-receptors equally well. The newer generation of antipsychotic compounds induce a larger increase in dopamine release in the prefrontal cortex compared to the striatum and nucleus accumbens (Kuroki et al., 1999). 5-HT1A receptor stimulation has also been proposed to be involved in the therapeutic effects of the newer antipsychotics, since stimulation of 5-HT1A receptors leads to an increased dopamine release in the prefrontal cortex (Gobert et al., 1998; Kuroki et al., 1999; Rollema et al., 2000). Indirect modulation of glutamatergic neurotransmission may play an important role in the mechanism of action of the atypical antipsychotic compounds. Antipsychotic agents may modulate glutamatergic transmission at both the cortical and the subcortical level. The regulation of glutamate receptor expression in response to treatment with different antipsychotics has been examined by several authors, and it appears that the ionotropic NMDA and a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunits are regulated differently by conventional vs newer antipsychotics (Healy and MeadorWoodruff, 1997; Riva et al., 1997; Tascedda et al., 1999, 2001). Another strategy for alleviating cognitive disturbances in schizophrenia involves direct modulation of glutamatergic transmission. For instance, attempts have been made to strengthen NMDA receptor-mediated transmission by stimulating the associated strychnine insensitive glycine receptor (Wood, 1995). Tsai et al. (1998) reported that the glycine agonist d-serine added to conventional neuroleptics and/or risperidone, had beneficial effects on positive, negative and cognitive symptoms in schizophrenic patients. Glutamatergic transmission can also be reinforced by manipulating another subtype of glutamate receptor, i.e. the AMPA receptor. This can be achieved by treatment with a so called AMPAKINE\, which acts as a positive modulator of the AMPA receptor. The AMPAKINE CX516 has been reported to improve some aspects of memory in healthy men (Ingvar et al., 1997), and there is one study describing the effects of CX516 added to clozapine with improvements in measures of attention and memory in schizophrenic patients (Goff et al., 2001). 4.3. Comparison with other behavioural models There is an abundance of animal models dealing with different aspects of cognition. A common feature of currently used models is that both a period of training and some kind of reward are required. A commonly used model is Morris’ water maze, modelling spatial learning. In this model where the animal is taught to find a hidden platform, clozapine has been shown to counteract the phencyclidine-induced prolongation of swimming latency to find the platform (Okuyama et al., 1997); haloperidol was in the same test unable to alter the prolonged swimming latency (Ogawa et al., 1994; Okuyama et al., 1997). Furthermore, on acquisition of delayed alternation in a T-maze, a test for spatial working memory, clozapine improved the MK801-induced delayed alternation impairment (Hauber, 1993). In

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a study where an elevated plus-maze was used to evaluate spatial long-term memory, the latency to reach one of the enclosed arms was decreased for control mice, while the latency for MK-801-treated mice was unaltered, upon re-testing the day that followed the first test occasion. At both first occasion and retesting, each mouse was placed at the end of one of the open arms facing away from the central part of the maze. In this test paradigm, low doses (0.063 and 0.125 mg/kg) of olanzapine decreased the latency for MK-801-treated mice to reach one of the enclosed arms (Ninan and Kulkarni, 1999). In a reversal learning paradigm, a test for attention and aspects of executive functioning, food deprived animals are trained to press levers according to a defined rule. On the test day the reward paradigm is reversed and they are required to change strategy in order to get a reward in form of a food pellet. In this test ziprasidone was shown to reverse the impairment induced by PCP treatment, whereas haloperidol, in contrast, further impaired the animals’ performance (Abdul-Monim et al., 2003). These findings in various experimental models of cognition seem to be in line with our results on classical and new antipsychotic agents, which in conjunction with the good agreement with clinical findings support the validity of our movement pattern analysis model for evaluating potential cognition enhancing effects of antipsychotic compounds. A major advantage with our model, which is based on spontaneous exploratory behaviour, is that it is free from confounding factors involving motivation and reward. Moreover, our model is quick and simple as it does not require training of the animals. 5. Conclusion It can be concluded from these result that conventional and newer agents with antipsychotic effect have different effect on MK-801-induced movement alterations in mice. The never agents are to varying degrees capable of bringing back some features that are lost in the MK-801 treated mice. Assuming that the level of behavioural diversity in MK-801 treated mice reflects cognitive status, these results are in concordance with the antipsychotic compounds’ clinical effects on cognition and support the validity of using movement pattern analysis in the evaluation of potential cognition enhancing effect of antipsychotic compounds. Acknowledgements The present study was supported by grants from the Swedish Medical Research Council (9067), Stiftelsen Psykiatriska Forskningsfonden, Sahlgrenska Universitetssjukhuset, Go¨teborg, Sweden, Wilhelm och Martina Lundgrens Vetenskapsfond and Svenska Lundbeckstiftelsen. References Abdul-Monim Z, Reynolds GP, Neill JC. The atypical antipsychotic ziprasidone, but not haloperidol, improves phencyclidine-induced cognitive deficits in a reversal learning task in the rat. J Psychopharmacol 2003;17: 57 – 65.

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