Structural and Functional Alterations in the Cortex in a Rodent Model of First-Episode Psychosis

From the Department of Neurophysiology of the Ruhr University Bochum Chair: Prof. Dr. phil. habil. Denise Manahan-Vaughan

Inaugural Dissertation for the Attainment of a Doctor’s Degree in Medicine at the High Medical Faculty of the Ruhr University Bochum

presented by Thomas Grüter from Lippstadt 2014

Dean: Prof. Dr. med. A. Bufe Referee: Prof. Dr. phil. habil. D. Manahan-Vaughan 1st Co-referee: Prof. Dr. med. M. Brüne 2nd Co-referee: Prof. Dr. rer. nat. T. Gloveli

Date of oral examination: 17 November 2015

Abstract

Problem: Schizophrenia is a common mental disorder that is characterised by positive and negative symptoms, as well as cognitive and social deficits. To date, the origin and aetiology of schizophrenia are still obscure and sufficient treatment for the negative and cognitive symptoms is unavailable. However, several studies indicate that early treatment results in a better outcome.

Method: Dysfunctional glutamate receptors play a central role in schizophrenia pathology. This has led to the glutamate hypothesis of psychosis/ schizophrenia. In this study, an animal model of first-episode psychosis that implements a single high-dose treatment of the non-competitive N- methyl-D-aspartate receptor (NMDAR) antagonist MK801 in rats was used in order to examine resultant changes in neurotransmitter receptor expression. Due to the significance of glutamatergic neurotransmission for synaptic plasticity, plasticity-relevant receptors were in focus. Alter- ations in expression of the NMDAR subunits GluN1, GluN2A, and GluN2B, the γ-aminobutyric acid (GABA) receptors GABAA and GABAB, the dopamine D1 and D2 receptors, as well as the metabotropic glutamate (mGlu) receptors 1, 2/3, and 5 were analysed via immunohistochemical labelling 1 and 4 weeks after antagonist treatment. Particular attention was paid to the prefrontal cortex (PFC) and hippocampus, as these structures are known to be affected in psychosis. By means of in-situ hybridisation, experience-dependent cellular activity was assessed 1 and 4 weeks after MK801-treatment by evaluating hippocampal expression of the immediate early gene Arc at rest and following spatial learning.

Results: An increase of GABAA receptor expression and, in addition, a decrease of mGlu1 receptor expression were found 1 week after NMDAR antagonism, in both the PFC and hippocampus.

At that time-point, GABAB receptor expression was up-regulated in the hippocampus and

NMDAR subunit GluN2B was down-regulated in the PFC. Four weeks after treatment, GABAB and D1 receptors expressions were significantly increased in both PFC and hippocampus,

GABAA receptor expression was decreased in the PFC, and GluN2B subunit expression was increased in the hippocampus. GluN1, GluN2A, mGlu2/3, mGlu5 and D2 receptors were unaffected at both time-points. Furthermore, basal hippocampal Arc gene expression was increased in MK801-treated animals. In contrast, no difference was detected between Arc gene expression in vehicle and MK801-treated rats after novel spatial exploration either 1 or 4 weeks after treatment.

Conclusions: Long-term alterations in receptor distribution occur concurrently with enhanced basal cellular excitability in the hippocampus and PFC in an animal model of first-episode psychosis. These changes might underlie disturbances of synaptic plasticity, neuronal oscillations, and learning and memory that are known to occur in this animal model and provide novel insights into the dynamics of schizophrenia pathology.

Table of contents:

1. Introduction ...... 6 1.1. Schizophrenia ...... 6 1.2. Brain regions affected by schizophrenia ...... 8 1.2.1. Hippocampal formation ...... 8 1.2.2. Prefrontal cortex ...... 11 1.3. Hypotheses for the occurrence of psychotic symptoms ...... 13 1.4. Animal model of NMDA receptor antagonism ...... 15 1.5. Properties of significant receptors in schizophrenia...... 16 1.5.1. N-methyl-D-aspartate receptors ...... 17 1.5.2. γ-Aminobutyric acid receptors ...... 18 1.5.3. Dopamine receptors ...... 19 1.5.4. Metabotropic glutamate receptors ...... 20 1.6. Immediate early genes ...... 21

2. Objectives ...... 23

3. Materials and Methods ...... 24 3.1. Animals ...... 24 3.2. Drug treatment ...... 24 3.3. Immunohistochemical labelling ...... 24 3.4. In situ-hybridisation ...... 26 3.5. Brain slice assessment ...... 30 3.6. Analysis ...... 30

4. Results ...... 32 4.1. Immunohistochemistry ...... 32 4.1.1. Time-dependent alterations in the expression of GluN2B, but not GluN1 and GluN2A subunits in the hippocampus and prefrontal cortex after MK801-treatment ...... 32

4.1.2. Differences in the expression of GABAA and GABAB receptors in the hippocampus and prefrontal cortex after MK801-treatment ...... 34

4.1.3. Dopamine D1 but not dopamine D2 receptor expression is chronically elevated in the hippocampus and prefrontal cortex after MK801- treatment ...... 37 4.1.4. The expression of metabotropic glutamate receptor mGlu1, but not mGlu5 or mGlu2/3 is transiently reduced in the hippocampus and prefrontal cortex after MK801-treatment ...... 39 4.1.5. Synopsis of neurotransmitter receptor expression alterations after MK801-treatment ...... 41 4.2. In situ-hybridisation ...... 42 4.2.1. MK801-treatment alters basal Arc gene expression in the hippocampus and following spatial learning ...... 42 4.2.2. The exploratory behaviour of rats is not affected 1 and 4 weeks after MK801-treatment ...... 45

5. Discussion ...... 46 5.1. MK801 - a valid model in schizophrenia research? ...... 46 5.2. Is the rodent prefrontal cortex comparable to the human prefrontal cortex? ...... 48 5.3. Impact of changed receptor expression on the course of psychotic symptoms ...... 49 5.3.1. The role of altered GluN2 subunit composition in synaptic plasticity and learning and memory ...... 50 5.3.2. Changes in neuronal activity by altered GABA receptors expression have consequences for synaptic plasticity and cognitive function ...... 53 5.3.3. Contribution of changed dopamine receptor expression to synaptic plasticity and learning and memory ...... 58 5.3.4. Influence of changed metabotropic glutamate receptor expression on synaptic plasticity and learning and memory ...... 60 5.4. Enhanced neuronal activity in the animal model of psychosis ...... 62 5.4.1. Connection of Arc gene expression to neuronal activity, synaptic plasticity, and learning and memory ...... 62 5.4.2. Consequences of altered neuronal activity in the animal model and in schizophrenia patients ...... 64

5.5. The hippocampus and prefrontal cortex – key structures in the occurrence of schizophrenia? ...... 68 5.6. Prefrontal-hippocampal interplay ...... 69 5.7. The role of γ-oscillations in cognitive impairments ...... 70 5.8. New treatment strategies for schizophrenia ...... 71 5.9. Limitations and remaining questions ...... 73

6. Conclusion ...... 74

7. References ...... 75

8. Appendix ...... 97

List of Abbreviations:

AB Antibody AMPAR α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor ANOVA analysis of variance BDNF brain-derived neurotrophic factor CA cornu ammonis cAMP cyclic adenosine monophosphate dlPFC dorsolateral prefrontal cortex GABA γ-aminobutyric acid GABAR γ-aminobutyric acid receptor GAD67 67 kDa glutamic acid decarboxylase ICD International Classification of Diseases LTD long-term depression LTP long-term potentiation mGlu metabotropic glutamate mPFC medial prefrontal cortex MRI magnet resonance imaging mRNA messenger ribonucleic acid n-Goat normal goat serum NMDAR N-methyl-D-aspartate receptor OD optical density PBS phosphate buffered saline PCP phencyclidine PET positron emission tomography PFC prefrontal cortex SSC saline-sodium citrate buffer TBS tris-buffered saline Tx Triton-X-100

List of Figures:

Figure 1: The anatomy and function of the hippocampal formation ...... 9 Figure 2: Behavioural protocol for induction of spatial learning ...... 26 Figure 3: Spatial learning in the empty holeboard ...... 27 Figure 4: Nuclear, Arc, and GAD67 staining ...... 27 Figure 5: The analysed areas in the prefrontal cortex and hippocampus ...... 30 Figure 6: Time-dependent changes occur in NMDAR subunit GluN2B after MK801-treatment ...... 33

Figure 7: MK801-treatment affects GABAA receptor expression ...... 35

Figure 8: GABAB receptor expression is up-regulated after MK801-treatment ...... 36 Figure 9: The dopamine D1 receptor is chronically up-regulated after MK801- treatment ...... 38 Figure 10: MK801-treatment reduces transiently mGlu1 expression ...... 40 Figure 11: Summary of all alterations in neurotransmitter receptor expression after MK801-treatment ...... 41 Figure 12: Arc gene expression is affected at rest and following holeboard (HB) experience by MK801 1 week after application ...... 43 Figure 13: Arc gene expression is affected at rest and following holeboard (HB) experience by MK801 4 weeks after application ...... 44 Figure 14: MK801-treatment does not affect exploratory behaviour ...... 45 Figure 15: Anatomical separation of the human and rodent prefrontal cortex ...... 48 Figure 16: Interplay between pyramidal cells and interneurons ...... 56 Figure 17: Ceiling effect impairs increasing neuronal excitability ...... 67 Figure A1: GluN1 subunit expression has not changed after MK801-treatment...... 97 Figure A2: GluN2A subunit expression is unaffected after MK801-treatment ...... 98 Figure A3: MK801-treatment does not alter D2 receptor expression ...... 99 Figure A4: mGlu2/3 receptors expression has not changed after MK801- treatment ...... 100 Figure A5: mGlu5 receptor expression remains equal after MK801-treatment ...... 101

1. Introduction

The following introduction is divided into three parts:

1. A characterisation of schizophrenia focusing on relevant brain regions and possible hypotheses of pathophysiological mechanisms;

2. The introduction of the subsequent animal model of psychosis used in this study, that is based on N-methyl-D-aspartate receptor (NMDAR) antagonism;

3. A description of the receptors examined in this study, as well the immediate early gene Arc.

1.1. Schizophrenia

Schizophrenia is a common mental disorder. The prevalence of schizophrenia amounts to about 0.4%, and the lifetime morbid risk amounts to about 0.72% of the adult population. Men are affected earlier and show a higher incidence than women, at a ratio of 1.4:1. On average, the onset of this disease is between the ages of 15 and 35 (McGrath et al., 2008).

Schizophrenia is diagnosed by its clinical appearance. This disease is characterised by a distinctive pattern of mental disorders which significantly impact on patient quality of life of patients. Typically, negative symptoms, such as apathy or blunted emotional responses, occur at an early phase, whereas positive symptoms, such as delusions and hallucinations, appear later on in the disease’s progression. However, the order of symptoms is variable and is accompanied by cognitive impairments at each stage of development (Häfner et al., 1999). Cognitive symptoms are believed to be the best predictor of functional outcome, such as optimal adaptation to the outside world (Lesh et al., 2011). Two major diagnostic systems aim to standardise the clinical diagnosis: on the one hand, the Diagnostic and Statistical Manual of Mental Dis- orders published by the American Psychiatric Association and, on the other hand, the International Classification of Diseases (ICD) by the World Health Organization. Currently, the ICD-10 version is the one most widely used in Germany and it requires fulfilment of the following criteria for a diagnosis of schizophrenia to be made:

At least one of the following symptoms must be present for at least one month:  Thought echo, thought insertion/withdrawal/broadcast  Delusions of control, influence or passivity, delusional perception  Auditory hallucination, running commentary or in dialogue  Persistent bizarre delusions or two or more of the following must be present for at least one month:

 Persistent hallucinations in any modality

 Formal thought disorder

 Catatonic behaviour

 Negative symptoms

At the moment of diagnosis, other organic brain diseases, affective disorders or drug- related appearances must be excluded.

The treatment of schizophrenia is based on a psychotherapeutic and pharmacological approach. In antipsychotic treatment, first generation (also called conventional or typical) and second generation (also called atypical) antidopaminergics are used. Besides aripiprazole, both first and second generation antipsychotic drugs block the dopamine D2 receptor. Despite their relative effectiveness in treating the positive symptoms, first generation antipsychotics, such as haloperidol and chlorpromazine, treat negative and cognitive symptoms insufficiently (Chue and Lalonde, 2014) and exhibit a range of adverse side-effects including extrapyramidal movement disorders (Malhotra et al., 1993). Second generation antipsychotics, such as risperidon or amisulpride, are increasingly popular because of their relatively improved effectiveness in treatment of negative symptoms and reduced extrapyramidal side-effects. In contrast, second generation antipsychotics exhibit reduced antipsychotic power and induce weight gain (Jibson and Tandon, 1998). However, recent meta-analysis has demonstrated no major differences between first and second generation antipsychotics in symptom control (Ellenbroek, 2012). Numerous studies indicate an improved symptomatic outcome following early pharmacological and psychotherapeutic treatment. It is hypothesised that patients in a prodomal phase (“at-risk”-mental state) or that recently experienced first-episode schizophrenia respond significantly better to treatment regimes, leading to an increased chance of preventing chronification and

subsequent comorbidities, and better chances of remission (Marshall et al., 2005; Álvarez-Jiménez et al., 2012). This leads to the question which cellular and molecular changes occur in the early stages of schizophrenia, and to what extent these changes can be targeted in innovative treatment approaches.

1.2. Brain regions affected by schizophrenia

Imaging studies comparing schizophrenia patients with healthy controls have revealed areas that are believed to contribute to the development of the disease: in the centre of attention are the hippocampal formation and prefrontal cortex (PFC). In magnet resonance imaging (MRI) studies of schizophrenia patients, alterations in the volume of the hippocampal formation and PFC are evident (Shepherd et al., 2012). Furthermore, studies using functional MRI demonstrate alterations in prefrontal activity (He et al., 2013) and a correlation between the clinical success of pharmacological treatment and treatment-related prefrontal changes (Karch et al., 2012). Moreo- ver, animal models of hippocampal lesions replicate the schizophrenia phenotype, at least in part (Tseng et al., 2009). These observations support that the hippocampal formation and PFC contribute to schizophrenia-related impairments. The following description reviews the physiological, anatomical, and structural composition of the hippocampal formation and PFC, whereas an overview of the role of these structures in the pathophysiology of schizophrenia is presented in section 5.5.

1.2.1. Hippocampal formation

The following description of the hippocampal formation is primarily based on Am- aral and Lavenex (2007).

The hippocampal formation, located in the medial temporal lobe, includes the entorhinal cortex, the dentate gyrus, the hippocampus, and the subiculum. The hippocampus can be divided into frontal and dorsal portions and the cornu ammonis (CA) 1 to 4 by different histological structures and their functional inputs as specified later. The following refers to the dorsal part.

Figure 1: The anatomy and function of the hippocampal formation

The dentate gyrus, the hippocampus, and the subiculum are parts of the allocortex, a brain region in the cerebral cortex that is characterised by only three cell layers. The dentate gyrus consists of a superficial, relatively cell-free molecular layer and the granule cell layer that contains the soma of the principal neurons. The hilus is located between these layers. In the hippocampus, the soma of the principal cells is represented by the pyramidal cell layer that is tightly packed in CA1 and more loosely in CA3. The Stratum oriens, an externally located and relatively cell-free layer includes the basal dendrites of the pyramidal cells and interneurons, and is innervated by the CA3-CA3 associational connections and the CA3-CA1 Schaffer collateral connections. The Stratum lucidum, a narrow acellular zone containing mossy fibers from the DG to the CA3, exists only in the CA3 region directly above the pyramidal cell layer. The Stratum radiatum, located above the pyramidal cell layer in CA1 and CA2, and above the Stratum lucidum in CA3, includes mossy fibers, CA3-CA3 associational, and CA3-CA1 Schaffer collateral connections. The Stratum lacunosum- moleculare is the most superficially located layer, comprising the termination of fibers from the entorhinal cortex and other regions.

The hippocampal formation represents a complex neuronal circuit of signal transmission. The main afferent input into the hippocampal circuit arises from the entorhinal cortex. The entorhinal cortex has a tight bidirectional connection to several regions of the neocortex, for instance the associational, perirhinal, parahippocampal, and prefrontal cortices (Anderson et al., 2007). The information received from the entorhinal cortex is passed to the granule cells of the dentate gyrus and pyramidale cells of the CA regions via the perforant path, so-called of its characteristic of “perforat-

ing” the subiculum. From the dentate gyrus, mossy fibers project to the CA3 region. The CA3 pyramidal cells then project information to the CA1 region via Schaffer collateral axons. Finally, the CA1 region sends its afferents mostly to the subiculum. Even though most information is transmitted in a functional unidirectional unit containing a circuit projection from the entorhinal cortex to the dentate gyrus, the CA regions, the subiculum, and back to the entorhinal cortex, there are several additional, in part back-feeding projections (Witter et al., 2014). Efferent information is especially signalled from the CA1 region and the subiculum via the fornix to mammil- lary bodies and from the entorhinal cortex to higher cortical areas (Anderson et al., 2007).

On the basis of this neuronal connection, the hippocampal formation functions in the consolidation of declarative memory (Squire, 1992). This first became eminently apparent in the patient H. M. after he underwent a bilateral resection of the medial temporal lobe to arrest pharmacoresistent epilepsy (Scoville, 1954). As a result, the patient suffered from anterograde amnesia for facts and events, but was able to re- trieve learned information earlier in childhood. Experiences made briefly before resection were lost. After hippocampectomy, the patient was only able to store information for a very short period of time, but surprisingly he was able to acquire visuo- motor skills (Milner, 1972), demonstrating the segregation of brain structures involved in declarative and procedural memory. In animal studies with hippocampal lesions and more recently in human studies with neuroimaging (positron emission tomography (PET) or functional MRI), the role of the hippocampal formation in generation of declarative (and spatial) memory, and its absence of contribution to forms of memory such as implicit memory was verified (Cohen et al., 1999). More particularly, it became apparent that the hippocampal formation plays a role in the consolidation of new information but has only a time-limited role in storage after completion (Squire et al., 2001).

Furthermore, the hippocampal formation appears to be involved in other functions: For instance, O’Keefe and Dostrovsky (1971) described place cells that are activated depending on the subject’s location within his or her environment and thereby contribute to cognitive maps that enable representations of spatial memory (Burgess et al., 2002).

Besides this more global view of the functions of the hippocampal formation, the different subunits seem to have a specific function in memory-formation. The dentate gyrus, together with the CA3 region, is believed to dissociate similar neuronal patterns and thereby prevent interference and enable novelty detection. This process is called pattern separation (Bakker et al., 2008). In contrast, the CA3 region is believed to enable episodic memory by acting as an autoassociative network that forms asso- ciations between new input and previously established memory traces, a process called pattern completion (Hoang and Kesner, 2008). Last but not least, the CA1 region appears to compare retrieved information from the CA3 region and the entorhinal cortex to provide a mismatch detector (Chen et al., 2011).

1.2.2. Prefrontal cortex

The human PFC is a heterogeneous formation responsible for multiple complex behavioural patterns (Seniów, 2012). Injuries caused by neoplasia, haemorrhage, or degenerative processes lead to frontal lobe disorders characterised by a disturbance in executive functions (Seniów, 2012). Depending on the location of the injury, the severity varies greatly. Symptoms range from cognitive impairments, such as a shortened attention span, to inappropriate behaviour and difficulty to inhibit emo- tions (Seniów, 2012). This diversity may rely on the heterogeneous composition of the PFC that is represented by its subdivision into an orbitofrontal, dorsolateral, and medial part (Euston et al., 2012).

The function of the prefrontal subunits is still a matter of debate (Kesner and Churchwell, 2011). On a functional basis, Wise et al. (1996) separated the subareas of the PFC according to their involvement in problem-solving strategies and rules that are differently complex. They claim that the orbitofrontal and medial PFC (mPFC) support only simple strategies to solve problems, whereas the dorsolateral PFC (dlPFC) is involved in more comlex situations. To prove that, they conducted lesions studies demonstrating a role of the orbitofrontal and medial PFC in simple object and place learning tasks, respectively, whereas the dlPFC was engaged in tasks depending on matching and association (Wise et al, 1996; Kesner and Church- well, 2011).

In addition, several functional attributes are assigned to single cortices: First, the orbitofrontal cortex seems to be important for control and appraisal of emotional impulses and impressions, and therefore has a considerable influence on social adjustment and decision-making (Barbas, 2007; Kim et al., 2007; Seniów, 2012). Sec- ondly, the dlPFC appears to enable higher cognitive performances and goal-directed behaviour by means of maintenance and manipulation of memory items and strategies, constant integration of context information, control of interference, motor planning and reward anticipation (Szameitat et al., 2006; Tsujimoto et al., 2007; Seamans et al., 2008; Seniów, 2012). Finally, the mPFC is supposed to select and evaluate responses in conflicting situations and to compare them to past experiences and possible alternative actions (Seamans et al., 2008). Thereby, schematic behavioural patterns of appropriate actions are formed and selected in conjunction with the amygdala and hippocampus (Euston et al., 2012).

All subunits of the PFC appear to be essential for working memory (Kesner and Churchwell, 2011). It has been proposed that the different subunits are involved in working memory for different modalities, for instance the dlPFC may process spatial information while the ventral PFC processes visual information (Goldman-Rakic, 1996). But, further analysis has shown that each subregion only tends to process pre- ferred modalities but that even single neurons can switch in modality (Rao et al., 1997; Kesner and Churchwell, 2011).

All in all, the functional subdivision of the PFC is still a matter of much debate. One reason might be that the subareas in the PFC are highly interconnected (Barbas and Pandya, 1989), hence, Wilson et al. (2010) proposed that the PFC operates only in toto and a complete functional separation might be impossible.

Similar to the primate PFC, the rodent PFC can be divided into a medial, ventral medial, lateral and ventral PFC (Kesner and Churchwell, 2011). The medial and ventral medial PFC were analysed in our MK801-animal model for reasons of comparability to the medial and dorsolateral PFC in human that play a central role in the pathology of schizophrenia (cf. section 5.2). Regarding topology, cytoarchitectonics, and connections, the rodent medial and ventral medial PFC can be separated into the precen- tral and anterior cingulate, the prelimbic and infralimbic, and the medial orbital cortices (Kesner and Churchwell, 2011). These areas interact with several brain structures, for instance the neocortex, thalamus, and several limbic structures (Kesner and

Churchwell, 2011). Since the ventral striatum projects to the midline thalamic nucleus via the substancia nigra and pallidum, the PFC is involved in so-called basal ganglia-thalamocortical circuits (Groenewegen et al., 1997).

1.3. Hypotheses for the occurrence of psychotic symptoms

In the pathogenesis of schizophrenia symptoms, several factors contribute to its overall clinical appearance. Until today, it is still obscure which aspects cause the positive and negative symptoms and which alterations merely consequences of prior alterations. Currently, hypotheses based on neurotransmitter alterations, genetic alterations, and early life events attempt to explain the genesis of schizophrenia.

The dopamine hypothesis was proposed to explain the occurrence of psychotic symptoms, because virtually all antipsychotic drugs feature a relatively specific antidopa- minergic effect. This assumption was supported by the demonstration of a positive correlation between the affinity of drugs to dopamine receptors and their antipsychotic potential (Seeman et al., 1975). Later, Laruelle et al. (1996) confirmed, using single-photon emission computed tomography, that patients suffering from schizophrenia exhibit enhanced striatal dopamine release after treatment with amphetamine, a drug that causes the complete release of dopamine stored in vesicles. Thus, they could prove that the degree of severity of positive symptoms in schizophrenia patients is positively correlated to the ability of their dopaminergic system to release dopamine in response to activating stimuli. However, even in the study by Laruelle et al. (1996), the amount of dopamine release did not correlate with the appearance of negative symptoms. Clinical observations indicate that most first generation antipsychotic drugs have only a negligible effect on negative symptoms thus casting doubt on the exclusiveness of the dopamine hypothesis (Tamminga et al., 1998).

Later, the glutamate hypothesis was proposed based on data from NMDAR antagonists, such as phencyclidine (PCP), ketamine, or MK801 (Olney and Farber, 1995). These agents generate psychotic symptoms in healthy humans and enhance psychotic symptoms of schizophrenia patients (Lahti et al., 2001). They are also able to elicit negative symptoms (Olney and Farber, 1995). The concrete molecular pathogenesis of NMDAR dysfunction in schizophrenia patients is still unclear: several different mechanisms were found, such as alterations in pre- and postsynaptic systems, chang-

es in gene regulation and expression, and impairments in receptor contribution and function (Javitt et al., 2012). In addition, polymorphisms of the GluN2B receptor gene GRIN2B (Allen et al., 2008) were found in schizophrenia patients.

A third hypothesis, based on deficient inhibition of γ-aminobutyric acid (GABA), was proposed (Squires and Saederup, 1991; Kalkman and Loetscher, 2003). Striking- ly, alterations in GABAergic neurons is one of the most consistent abnormalities in post-mortem analyses of the brains of schizophrenics (Akbarian et al., 1995; Guidotti et al., 2000; Volk et al., 2000; Hashimoto et al., 2005, 2008a, b; Duncan et al., 2010). Moreover, experiments with GABA receptor (GABAR) antagonists have demonstrated impairments in cognitive function similar to those observed in schizophrenia patients (Enomoto et al, 2011; Paine et al., 2011). Thus, reduced GABAergic influence is still believed to be a core feature in schizophrenia pathology.

In addition, serotonin might play an important role. Parallels in behaviour between subjects under the influence of serotonergic drugs, such as semisynthetic lysergic acid diethylamide, and schizophrenia patients have led to the consideration of serotonin in schizophrenia pathology. Besides comparable positive symptoms, both, patients and with serotonergic drugs treated rodents or humans exhibit equal deficits in startle habituation and prepulse inhibition postulated as an indicator for thought disorder (Geyer and Vollenweider, 2008). In addition, second generation antipsychotic drugs feature a further relatively potent blockade of 5-HT2 receptors that is believed to be partly responsible for their effectiveness on negative symptoms (Meltzer, 1999).

Furthermore, Chronister and DeFrance (1982) postulated a potential role of histamine in the origin of pathologically elevated dopamine states. Both, decreased histamine receptors and increased histamine degradation in hippocampal projections to the nucleus accumbens result in a relative increase of dopamine that mimics several psychotic symptoms and might play a part in the pathogenesis of schizophrenia (Chronister and DeFrance, 1982; Heleniak and O’Desky, 1999).

Even though alterations in neurotransmitter systems have successfully been connected to schizophrenia pathogenesis, the occurrence of schizophrenia symptoms seem to be multi-factorial and may be explained by the vulnerability-stress model (Nuechter- lein and Dawson, 1984). This model claims that psychotic symptoms only become

manifest if stressful life events occur against the background of an already pre- existing subclinical vulnerability. This predisposition may comprise genetic factors or early life events, such as delivery complications, whereas initiating stress factors are characterised by acute life events, such as moving to a foreign environment or substance abuse. The former might explain the relatively high incidence of psychotic symptoms among migrants (McGrath et al., 2008).

1.4. Animal model of NMDA receptor antagonism

A leading theory concerning the occurrence of schizophrenia is the glutamate hypothesis. Since psychotic symptoms occur in healthy humans after treatment with PCP (Javitt and Zukin, 1991) and ketamine (Krystal et al., 1994), these noncompetitive irreversible NMDAR antagonists have often been used to mimic positive, negative and cognitive symptoms in rodents.

In 1982, a promising new compound to emulate psychosis was produced: the un- competitive irreversible NMDAR antagonist MK801 ([+]-5-methyl-10,11-dihydro- 5H-dibenzo-[a,d]-cyclohepten-5,10-imine hydrogen maleate). MK801 displays unique properties: brain concentrations of MK801 peak thirty minutes after systemic application, whereas the elimination half-life is about two hours (Vezzani et al., 1989). In comparison to PCP and ketamine, MK801 features a higher binding affinity (Kornhuber and Weller, 1997) and selectivity (Wong et al., 1986) for the PCP binding site within the NMDAR channel pore, whereas the dissociation rate is significantly lower (MacDonald et al., 1991). Thus, MK801 has been described as a quite specific and virtually “irreversible” NMDAR antagonist (Rosenmund et al., 1993; Dzubay and Jahr, 1996; Talukder et al., 2010).

A systemic single treatment of MK801 in rats is used as a model for first-episode psychosis (Wöhrl et al., 2007; Manahan-Vaughan et al., 2008a, b; Wiescholleck and Manahan- Vaughan, 2012, 2013a, b). Such an application causes various alterations in both receptor expression and distribution, and electrophysiological properties (Ru- jescu et al., 2006). Immediately following injection, γ-power oscillations in CA1 area and dentate gyrus is increased (Kittelberger et al., 2012), that might affect neural coding and communication, especially across networks (Lisman, 2012). Furthermore, the ability to induce hippocampal long-term potentiation (LTP) that is believed to

comprise a cellular correlate of learning and memory is disrupted in vitro and in vivo after treatment (Coan et al., 1987; Abraham and Mason, 1988; Manahan-Vaughan et al., 2008a, b; Wiescholleck and Manahan-Vaughan, 2012, 2013a, b). In contrast, hippocampal long-term depression (LTD) is not altered after MK801-treatment (Manahan-Vaughan et al., 2008b), suggesting the elevations in neuronal excitability may play a role in the aetiology of the psychosis-like status in rodents.

MK801-treated animals modify their behaviour following systemic application. Im- mediately after injection, hyperlocomotion and stereotypy arise (Wöhrl et al., 2007; Manahan-Vaughan et al., 2008a), whereas prepulse inhibition (Manahan-Vaughan et al., 2008a) and abilities in social interactions are compromised (Rung et al., 2005). In contrast to behavioural impairments, hippocampal- and prefrontal-dependent memory are affected, and this deficits is long-lasting (Wiescholleck and Manahan- Vaughan, 2013a, b). Deficits in avoidance tasks (Benvenga and Spaulding, 1988), radial maze tasks (Manahan-Vaughan et al., 2008a, b), and spatial delayed alternation tasks (Verma and Moghaddam, 1996) have also been reported.

1.5. Properties of significant receptors in schizophrenia

Schizophrenia pathology manifests in structural and functional alterations, such as changed receptor expression and impaired neuronal information encoding. This can be expected to result in a mutual regulation: changes in receptor expression affect neuronal functioning and vice versa. Synaptic plasticity is a major mechanism for neuronal information encoding and subsequent learning and memory. As it has been shown that synaptic plasticity is impaired in both the MK801 animal model (Mana- han-Vaughan et al., 2008a, b; Wiescholleck and Manahan-Vaughan, 2012, 2013a, b) and schizophrenia patients (Hasan et al., 2011), it is vitally important to elucidate the molecular background of these deficits. Synaptic plasticity predominantly requires adequate NMDAR functioning (Lüscher and Malenka, 2012), but persistent forms of synaptic plasticity is not only affected by NMDAR expression, but also by the glutamatergic (Manahan-Vaughan, 1997; Naie and Manahan-Vaughan, 2004, 2005a, b; Altinbilek and Manahan-Vaughan, 2009), GABAergic (Coulter and Carlson, 2007), and dopaminergic (Kulla and Manahan-Vaughan, 2000; Manahan-Vaughan and Kulla, 2003; Lemon and Manahan-Vaughan, 2006, 2012) systems. Strikingly, altera-

tions in the NMDAR (Kristiansen et al., 2007; Gonzalez-Burgos and Lewis, 2012), GABA receptors (Benes et al., 1996a), dopamine receptors (Laruelle et al., 2003), and metabotropic glutamate receptor (Gupta et al., 2005; Volk et al., 2010) expressions have been demonstrated in studies of schizophrenia patients. In the following section, these receptors are introduced from a pharmacological point of view.

1.5.1. N-methyl-D-aspartate receptors

The NMDAR is an ionotropic glutamate receptor required for the induction of synaptic plasticity such as LTP and LTD (Lüscher and Malenka, 2012), and is able to induce excitotoxity (Zhou et al., 2013). Every NMDAR is assembled in a hetero- tetrameric manner by two obligatory GluN1 subunits and two GluN2A-D and/or GluN3A-B subunits arising from separated genes GRIN1, GRIN2A-D, and GRIN3A-B, respectively (Paoletti, 2011). Since six facultative subunits are known, there is a large variety of receptor compositions, whereas the most NMDARs are formed by a GluN1/GluN2 subunit mixture (Paoletti, 2011). The assembly of four subunits in one NMDAR serves as an integral membrane protein that exhibits a selective permeability for cations (especially Ca2+). Influx requires, on the one hand, activation of GluN1 and, where necessary, GluN3 subunits by glycine or D-serine and activation of the GluN2 subunit by glutamate. On the other hand, it also requires a postsynaptic depolarisation that uncouples a pore blocking Mg2+ ion. In comparison to other ionotropic receptors, such as α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid receptors (AMPAR) and kainate receptors, NMDARs are slow to activate and deactivate (Paoletti, 2011). Due to its significant role in synaptic plasticity and excitotoxity, NMDARs are subject of multiple allosteric modulations, for instance by hydrogen and zinc (Gladding and Raymond, 2011; Paoletti, 2011).

The specific properties of the NMDAR are regulated by its subunit composition. The NMDAR subunits GluN2A and GluN2B are characterised by both a relatively large conductance, long opening time (3-5 ms) and high sensitivity to Mg2+ block. They display similarities in terms of several biophysical properties in comparison to GluN2C and GluN2D. However, there are differences between GluN2A and GluN2B: in comparison to GluN2B, GluN2A displays higher maximal open probability, peak current, and faster rise time, but it has a lower glycine and glutamate affinity, faster glutamate decay and deactivation. On the whole, a higher agonist con-

centration is necessary to activate GluN2A containing receptors in a fast, but strong manner. Nevertheless, this is not sufficient to reach the same charge transfer per activation comparable to GluN2B (Yashiro and Philpot, 2008; Paoletti, 2011).

Different receptor localisations, such as synaptic, perisynaptic (within 200-300 nm of the postsynaptic density), and extrasynaptic, appear to influence receptor function by differing in preferential subunit composition, open probability, and downstream pathways (Paoletti, 2011). Many studies have illustrated that GluN2A and GluN2B are preferentially located on synaptical and extrasynaptical sides, respectively (Glad- ding and Raymond, 2011). Each subcellular localisation has its unique protein expression and receives different amounts of glutamate release. Synaptic NMDARs receive a relatively high amount of glutamate release, whereas a glutamatergic spill- over from presynaptic or other sources for example from glia is necessary for activation of extrasynaptic NMDAR (Gladding and Raymond, 2011). However, even though GluN2A and GluN2B are found dominantly in synaptical and extrasynaptical localisation, respectively, both subunits have been found on the other side. Apart from inverse primary expression, lateral receptor diffusion has been described: NMDARs are able to change their localisation from extrasynaptic to synaptic and vice versa. Against this background, a perisynaptic NMDAR might be a receptor in transit (Gladding and Raymond, 2011). These possible changes in receptor distribution hinder a clear functional distribution to each subunit.

1.5.2. γ-Aminobutyric acid receptors

GABA, one of the major inhibitory neurotransmitters in the human brain, activates the ionotropic GABAA and the metabotropic GABAB receptors.

GABAA receptors are pentameric ion channels displaying two binding sites, one for

GABA and a second one for benzodiazepines. GABAA receptors generate two forms of inhibition: firstly, phasic inhibition is formed in the postsynaptic membrane and mediates an inhibitory postsynaptic potential by transmission of chloride in the course of milliseconds (<10 ms). By mediating a phasic inhibitory output, GABAer- gic interneurons prevent overexcitation of principal neurons and play a fundamental role in the generation of rhythmic activities in the complete neuronal network (Far- rant and Nusser, 2005). Secondly, tonic inhibition is created by extrasynaptically

located high-affinity GABAA receptors, providing long-lasting inhibitory effects. These receptors occur, for instance, in granule cells of dentate gyrus (Nusser and Mody, 2002), CA1 pyramidal cells (Bai et al., 2001) and certain inhibitory interneurons in the CA1 region of the hippocampus (Semyanov et al., 2003). Tonic inhibition seems to have a more modulating effect on single neurons by regulating the magni- tude and duration of the response to a given excitatory influence (Farrant and Nusser, 2005).

GABAB receptors are G-protein coupled receptors and regulated by their principal and auxiliary subunits. The principal subunit controls the surface expression and receptor localisation, whereas the auxiliary subunit influences the kinetics and response to ligand binding. In general, GABAB receptors produce slow inhibitory synaptic conductance (seconds to minutes) after strong simultaneous stimulation of several interneurons both pre- and postsynaptically (Gassmann and Bettler, 2012). Postsyn- aptic localised GABAB receptors stimulate G protein-activated inwardly rectifying potassium channels (Gähwiler and Brown, 1985) resulting in hyperpolarisation of the postsynaptic membrane. Presynaptic GABAB receptors mediate a negative feedback to calcium gating ion channels (Mintz and Bean, 1993) at inhibitory and excitatory terminals by released GABA from the same neuron or by spill-over from neighbour- ing synapses, respectively. Furthermore, GABAB receptors reduce cyclic adenosine monophosphate (cAMP) levels via inhibition of adenylate cyclase, subsequently decrease protein kinase A activity (Xu and Wojcik, 1986) and thereby reduce vesicle priming (Sakaba and Neher, 2003). Likewise, the GABAB βγ-subunit is able to reduce vesicle fusion to the presynaptic terminals by coupling with SNARE proteins (Jarvis et al., 2000; Blackmer et al., 2001). All of these presynaptic functions have the consequence of reduced neurotransmitter release.

1.5.3. Dopamine receptors

There are five known dopamine receptors, D1 to D5. These five dopamine receptors can be divided into two groups according to their transduction pathway and genetic homology: D1-like receptors (D1 and D5) and D2-like receptors (D2, D3, and D4) (Beaulieu and Gainetdinov, 2011). Both, dopamine D1 and D5 receptors have been found in pyramidal cells and in cells in the strata oriens and radiatum. Furthermore,

the D1-like messenger ribonucleic acid (mRNA) can be detected in granule cells in the dentate gyrus (Hansen and Manahan-Vaughan, 2014).

The dopamine receptor D1 is characterised by a strict postsynaptic localisation and a

Gs-coupled signal transduction. After activation, the D1 pathway results in activation of the adenylate cyclase and production of cAMP, and thereby multiple stimulating processes are triggered, such as locomotor activity (Beaulieu and Gainetdinov, 2011).

In contrast to D1, the dopamine receptor D2 is found both pre- and postsynaptically and acts via Gi signal transduction. Gi proteins have the opposite effect of Gs proteins by inhibiting the synthesis of cAMP. Presynaptically localised receptors are known to suppress the neurotransmitter release in the sense of fine-tuning of neuronal output (Beaulieu and Gainetdinov, 2011). Whereas presynaptic D2 receptors reduce locomotor activity, postsynaptic D2 receptors stimulate it (Beaulieu and Gainetdinov, 2011). Interestingly, pre- and postsynaptic D2 receptors are activated at different levels of dopamine release. Presynaptic D2 receptors are stimulated at low doses of agonists, whereas postsynaptic D2 receptors are only activated at higher doses. This mechanism provides a dose-dependent bidirectional effect (Beaulieu and Gainetdi- nov, 2011).

1.5.4. Metabotropic glutamate receptors

Metabotropic glutamate (mGlu) receptors are G-protein-coupled receptors. G- protein-coupled receptors are activated by extracellular ligands that trigger the re- placement of guanosine 5-diphosphate with guanosine 5-triphosphate. The affiliated guanosine 5-triphosphate causes the dissociation of the α- and βγ-subunit and powers the interaction with various molecules until guanosine 5-triphosphate is hydrolysed to guanosine 5-diphosphate (Mukherjee and Manahan-Vaughan, 2013).

The eight different mGlu receptors are divided into three groups according to their genetic similarity, transduction mechanisms and pharmacological properties (Mukherjee and Manahan-Vaughan, 2013): group I consists of mGlu1 and mGlu5, group II consists of mGlu2 and 3, and group III consists of mGlu4, 6, 7, and 8. Due to the heterogenous nature of group III mGlu receptors and the focus of the present study, the following description is restricted to group I and II mGlu receptors.

Group I mGlu receptors (mGlu1 and mGlu5) are mainly coupled to Gq proteins and provoke, via phospholipase Cβ, the hydrolysis of phosphatidylinositol 4,5- disphosphate into inositol 1,4,5-trisphosphate and diacylglycerol. Thereby, intracel- lular Ca2+ release occurs and protein kinase C becomes activated mostly resulting in cell depolarisation and increases in cell excitability (Ferraguti et al., 2008).

Group I mGlu receptors are not only located in neuronal cells, but also in glial cells. They are distributed in the whole central nervous system predominantly at postsynaptic localisation (Ferraguti et al., 2008). A differentiated localisation of group I mGlu receptors is regulated by homer proteins. Furthermore, homer proteins are important for regulating molecules that determine receptor activity and ligand response (Niswender and Conn, 2010).

Group II mGlu receptors, comprising mGlu2 and 3, are predominantly coupled to pertussis toxin sensitive G-proteins (Gi/o protein). After activation, Gi proteins inhibit the adenylate cyclase activity, resulting in decreased production of cAMP (Mukher- jee and Manahan-Vaughan, 2013).

In the brain, both receptors were found on neurons, whereas mGlu3 is also expressed in glia (Mukherjee and Manahan-Vaughan, 2013). As a result of their presynaptical localisation, their reduction of the second messenger cAMP causes decreased neurotransmitter release (Mukherjee and Manahan-Vaughan, 2013).

1.6. Immediate early genes

Immediate early genes are characterised by rapid and transient expression after receptor or cell activation. This heterogeneous group mainly expresses transcription factors, for instance c-fos and zif268, but some encode for structural proteins, such as homer1a and Arc. All of these are able to modify synaptic function (Lanahan and Worley, 1998).

The immediate early gene Arc, also called activity-regulated cytoskeleton-associated protein or Arg3.1, precipitates in neuronal dendrites where synapses were recently activated (Link et al., 1995; Lyford et al., 1995). Arc gene translation is almost only located in the postsynaptic density of non-GABAergic calcium-calmodulin kinase II- positive glutamatergic neurons in the hippocampus and neocortex (Vazdarjanova et al., 2006). Gene transcription, mRNA transportation, docking and translation is ex-

tensively influenced by the extracellular-signal-regulated kinase (Bramham et al., 2010; Korb and Finkbeiner, 2011). It is a key booster at each step and is, for instance, positively regulated by brain-derived neurotrophic factor (BDNF) TrkB receptor, group I mGlu receptor, and NMDAR (Bramham et al., 2010; Korb and Fink- beiner, 2011). Furthermore, Arc gene expression is negatively influenced by AM- PAR activity (Rao et al., 2006). After transcription, most of the expressed Arc mRNA is transported via microtubules by means of messenger ribonucleoprotein particles. Arc docks with F-actin at regions of enhanced activity, in order to facilitate and maintain synaptic and structural plasticity (Bramham et al., 2010; Korb and Finkbeiner, 2011). Arc has several functions and is linked to learning and behaviour, NMDAR-LTP, mGlu-LTD and AMPAR endocytosis, homeostatic scaling, structural plasticity, and an as yet unknown nuclear function (Korb and Finkbeiner, 2011).

2. Objectives

Schizophrenia is a very severe disease that is characterised by positive, negative, and cognitive symptoms. However, to date adequate treatment is still unavailable, especially for the negative and cognitive symptoms. The initiating triggers and cause- determining mechanisms are still obscure, not least because schizophrenia is difficult to examine in humans due to comorbidity and drug treatment. The first-episode of psychosis appears to have a central role in treatment since recent studies have shown that early intervention might define and even halt the development of this disease. It is believed that the molecular basis of schizophrenia is founded in a disturbance in expression and function of glutamatergic, GABAergic, and dopaminergic systems.

Against this background, the structural and functional changes associated with schizophrenia were simulated using an animal model of first-episode psychosis that in- volves acute treatment of the NMDAR antagonist, MK801. To describe time- dependent shifts in receptor expression in central pathogenic structures, the surface expressions of the NMDAR, mGlu receptors 1, 2/3, and 5, GABAA and GABAB receptors, and dopamine D1 and 2 receptors in the PFC and hippocampus were analysed 1 and 4 weeks after MK801-treatment. Furthermore, the expression of the immediate early gene Arc in the hippocampus in naïve animals and after spatial learning was examined 1 and 4 weeks after MK801-treatment, in order to detect alterations in the activity levels of hippocampal neurons.

3. Materials and Methods 3.1. Animals

The present study was carried out in accordance with the European Communities Council Directive of September 22nd, 2010 (2010/63/EU) for care of laboratory animals and after approval of the local government ethics committee (Bezirksregierung, Arnsberg). Male Wistar rats (7-8 weeks, Charles River, Germany) were housed indi- vidually in a temperature- and humidity-controlled vivarium with a constant 12-hour light-dark cycle (lights on from 6 a.m. to 6 p.m.) with ad libitum food and water ac- cess. All surgical procedures and experiments were conducted during the day.

3.2. Drug treatment

The NMDAR antagonist [+]-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]-cyclohepten- 5,10-imine hydrogen maleate (MK801, Tocris, Germany) was dissolved in 0.9% physiological saline. MK801 (5 mg ⁄ kg) or vehicle (10 ml ⁄ kg) were injected intra- peritoneally 7 days before commencement of first experiments. The concentration of MK801 was chosen in accordance with previous studies conducted by the group of Prof. Dr. Manahan-Vaughan in which the same dose proved to be effective in inducing long-lasting effects (Wöhrl et al., 2007; Manahan-Vaughan et al., 2008a, b; Wie- scholleck and Manahan-Vaughan, 2012, 2013b). A single high-dose treatment, as opposed to chronic low-dose treatment, has been picked in order to model exclusively the very first acute psychosis-related experience. Directly after injection, acute transient psychosis-like behaviours (locomotion, ataxia, and stereotypy) were observed as described elsewhere (Wöhrl et al., 2007) in order to evaluate the effectiveness of the treatment.

3.3. Immunohistochemical labelling

For the immunohistochemical experiments, MK801 injected animals were compared to vehicle injected animals. Furthermore, the labelling was conducted in one group 1 week after treatment and in a second group 4 weeks after treatment. All animals were handled for two days before experiments. Prior to brain removal, animals were anaesthetised with sodium pentobarbital (50 mg/kg intraperitoneal, Narcoren, Merial

GmbH, Hallbergmoos, Germany). Deeply anaesthetised rats were, at the time-point of exitus, initially perfused through the left ventricle into the aorta with 100 ml Ring- er’s solution containing 1000 IU Heparin (Liquemin N 25000, Roche, Basel, Swit- zerland) until no blood was effluent anymore, followed by approx. 500 ml 4% paraformaldehyde in phosphate buffered saline (PBS) pH 7.4. The extracted brains were stored in paraformaldehyde over night und cryoprotected in sucrose (30%) diluted in PBS for 5 days at 4°C. Serial coronal sections (30 µm thick) were collected via a freezing microtome (Leitz Kryomat 1703).

In order to reduce unspecific background staining in the slices, endogenous peroxidase was blocked by 0.3% hydrogen peroxide (H2O2) in PBS for 20 min. Then, endogenous biotin and electrostatic loadings of proteins were reduced by 20% avidin (Avidin-biotin blocking kit, Vector Laboratories, Burlingame, USA) in dilution medium for 90 min. Afterwards, the primary antibodies (AB) were applied in 20% biotin (Avidin-biotin blocking kit) containing dilution medium overnight in their corresponding concentration: GluN1 (1:400, monoclonal mouse AB, 556308, Becton, Dickinson and Company, Franklin Lakes, USA), GluN2A (1:750, polyclonal rabbit AB, sc-9056, Santa Cruz Biotechnology, Santa Cruz, USA), GluN2B (1:200, polyclonal goat AB, sc-1469, Santa Cruz), GABAA (1:400, monoclonal mouse AB,

MAB341, Merck Millipore, Billerica, USA), GABAB (1:500, monoclonal mouse

AB, ab55051, Abcam, Cambridge, UK), D1 (1:100, polyclonal goat AB, sc-1434, Santa Cruz), D2 (1:1000, polyclonal rabbit AB, AB1558, Merck Millipore), mGlu1 (1:400, polyclonal rabbit AB, ab82211, Abcam), mGlu2/3 (1:200, polyclonal rabbit AB, ab1553, Merck Millipore), and mGlu5 (1:200, polyclonal rabbit AB, ab5675, Merck Millipore). The corresponding secondary AB, rat-absorbed biotinylated horse- anti-mouse (BA-2001), goat-anti-rat (BA-1000), or horse-anti-goat (BA-9500) AB, all from Vector Laboratories, were added 1:500 in dilution medium for 90 min. After applying the avidin-biotin-peroxidase complex detection system (ABE-Elite, Vector Laboratories) 1:1000 in dilution medium for 90 min, the reaction was visualised by incubation in 3,3’-Diaminobenzidine (Sigma-Aldrich, St. Louis, USA) dissolved in

0.01% H2O2 containing PBS 1:2 for 10 min.

For staining of GABAA, GABAB, GluN2B, D1, D2, mGlu1, and mGlu5, the dilution medium was composed of 10% normal serum (Vector Laboratories) diluted in PBS containing 0.2% Triton-X-100 (Tx, Sigma-Aldrich) for the primary AB and the avi-

din-biotin blocking kit, and PBS containing 0.1% Tx for the secondary AB and ABC-Elite. For staining of GluN1, GluN2A, and mGlu2/3, 1% bovine serum albumin (BSA, Sigma-Aldrich) in tris-buffered saline (TBS) containing idem Tx was used as the dilution medium.

For enhancing the staining of GluN1, GluN2A, and mGlu2/3, the sections were left in the primary AB solutions for 5 days at 4°C. Following the secondary AB, the sections were enhanced with biotinylated tyramine as described by Adams (1992).

Except after application of avidin, upon all steps the slices were washed 3 times each 5 min with dilution medium and the entire procedure was done at room temperature if not other specified. After mounting on gelatinised slides, the slides were dehydrat- ed through an ascending alcohol series and finally treated with xylene. Finally, they were cover-slipped with DePeX (Serva, Heidelberg, Germany).

3.4. In situ-hybridisation

For the in situ-hybridisation, MK801 injected animals were compared to vehicle injected animals. Furthermore, the hybridisation was conducted in one group 1 week after treatment and in a second group 4 weeks after treatment. To evaluate neuronal activity after spatial learning, all in situ-hybridisation experiments were conducted in naïve animals and animals that performed the following behavioural protocol:

Figure 2: Behavioural protocol for induction of spatial learning

All animals were handled for two days before experiments. One day before experi- ment, the rats were habituated for 1 h to a recording chamber (40 x 40 cm and 50 cm in height). On the experimental day, they were acclimatised to the same chamber again for at least 60 min. Following this, the empty holeboard, a gray board with empty holes (5.5 cm diameter and 4.5 cm deep) in each corner was inserted. It has

been shown that exploration of a novel empty holeboard facilitated LTP in the dentate gyrus and CA1 region (Kemp and Manahan-Vaughan, 2004, 2008). After 6 min of exploration time, starting after the first direct hole exploration, brains were re- moved. The exploration time of each hole was counted separately.

A B

Figure 3: Spatial learning in the empty holeboard A: Schematic illustration of the empty holeboard B: Picture of a rat performing the empty holeboard experience

For Arc labelling, the following in situ-hybridisation protocol was conducted to all animals: After extraction, the brains were quick-frozen in isopentane over dry ice and stored at -80°C. The unfixed brains were cut coronally 20 µm thick on a Cryostat (Leica CM3050 S) at approximately -20°C and mounted directly on superfrost plus slides (Gerhard Menzel GmbH, Braunschweig, Germany) and stored at -80°C until further processing.

A B C

Figure 4: Nuclear, Arc, and GAD67 staining A: Nuclei are labelled with DAPI (blue) B: Arc gene expression is labelled with Cy5 (red) C: Interneurons are identified with GAD67-Cy2 labelling (green)

The fluorescence in-situ-hybridisation was conducted by adapting the procedure used by Guzowski and Worley (2001). The Arc complementary deoxyribonucleic acid was produced by Entelechon (Bad Abbach, Germany) according to the sequence of Lyford et al. (1995). Using the linearised deoxyribonucleic acid, the antisense RNA probe labelled with fluorescein at the uridine-5'-triphosphate site was created using the in vitro transcription kit Ambion MaxiScript Kit (Invitrogen, Carlsberg, USA) by Dr. Verena Aliane (Dept. of Neurophysiology, RUB). The length of the RNA probe was monitored by gel electrophoresis by Mrs Anke Galhoff (Dept. of Cell Physiolo- gy, RUB).

After drying the slides for at least 1 h, they were fixed with 4% paraformaldehyde, rinsed in 2x-saline-sodium citrate buffer (2x-SSC) for 2 min and then deposited in an acetic anhydride solution containing 0.5% acetic anhydride (Acros Organics, New Jersey, USA), 1.48% triethanolamine, and 0.92% sodium chloride dissolved in dieth- ylpyrocarbonate water (DEPC-water, C. Roth, Karlsruhe, Germany). Upon washing the slides 5 times in 2x-SSC for 1 min, they were deposited in an acetone/methanol (1:1) solution for 5 min and then again washed in 2x-SSC for 5 min. Afterwards, the slides were set in a humid chamber that was prepared with a 1:1 2x-SSC/deionised formamide solution (Sigma-Aldrich). After covering with 1x-prehybridisation buffer (Sigma-Aldrich), they were incubated for 30 min.

For the hybridisation step, 1 ng mRNA probe was solved in 1 µg 1x-hybridisation buffer (Sigma-Aldrich) per glass slide, heated at 90°C for 5 min and placed directly on ice. After rinsing in 2x-SSC, the slices were incubated with the Arc mRNA in the humid chamber at 56°C over night.

The next day, the slides were placed in a rack and washed 3 times for 5 min with 2x- SSC. Then, 10µg/ml ribonuclease A (from bovine pancreas, Sigma-Aldrich) in 2x- SSC was added for 15 min at 37°C. Then, the slides were washed first with 2x-SSC for 10 min, then with 0.5x-SSC 10 minutes at room temperature, 30 min at 56°C, and finally 10 min again at room temperature. To block endogenous peroxidase, the slices were pretreated with 3% H2O2 in 1x-SSC for 15 min, followed by 3 times washing with 1x-SSC for 5 min and once 5 min with TBS. To inhibit unspecific binding of proteins, a solution of 10% normal goat serum (n-Goat) in TBS was added for 60 min in the humid chamber. Anti-fluorescein-peroxidase (polyclonal sheep Fab- fragment, 11426346910, Roche) was applied in a dilution of 1:400 in TBS and 1% n-

Goat for 2 h. After washing 3 times 5 min with TBS-Tween 20 (Polysorbate 20), a solution of biotinylated tyramine, H2O2 (0.02%), and TBS was applied (1:1:100) for 20 min to enhance the fluorescence signal. The slices were washed again 4 times for 2 min with TBS and then incubated in a dilution of StrepAvidin Cy5 (Dianova, Hamburg, Germany) 1:3000 with 1% bovine serum albumin in TBS-Tween for 30 min. Afterwards, the slides were cleared first in TBS-Tween, then in PBS each 5 min. In order to label the nuclei of the cells, 4',6-diamidino-2-phenylindole (DAPI, Invitrogen) was added in a concentration of 1:10000 in PBS, followed by 3 times washing with PBS for each 5 min.

For 67 kDa glutamic acid decarboxylase (GAD67) staining, the slices were incubated in 10% n-Goat and PBS-0.2% Tx for 90 min at the next day. Afterwards, they were covered with the AB for GAD67 (1:100, monoclonal mouse AB, MAB-5406, Merck Millipore) in a dilution of PBS-0.2% Tx and 1% n-Goat overnight. Next day, the slices were rinsed in PBS 3 times for 5 min and additionally incubated with Dylight- 488 (1:250, Cy2-labelled goat-anti-mouse AB, 115-485-166, Dianova) in 1% n-Goat in PBS-0.2% Tx. After washing 3 times for 5 min with PBS and drying over night, the slides were cover slipped with Vectorshield Hard Set mounting medium for fluorescence (Vector Laboratories).

3.5. Brain slice assessment

A B

C D

Figure 5: The analysed areas in the prefrontal cortex and hippocampus

Representation of the analysed areas in the PFC (A and B) and hippocampus (C and D) in Nissl stain (A and C) and as schematic illustration (B and D). Fig. 5B is based on Paxinos and Watson (1986).

For both the in situ-hybridisation and immunohistochemistry, hippocampal brain areas were assessed at approximately -3.3 mm and -4.3 mm relative to Bregma. Ad- ditionally, for the immunohistochemistry, prefrontal areas at approximately 3.2 mm and 2.2 mm were used. For both techniques, Nissl stains using 1% toluidine blue were made for surveillance of quality and spatial orientation. In addition, negative controls were prepared for supervision of specificity. For this purpose each first AB and each secondary AB was used alone following the staining protocols. No staining could be observed in these negative controls indicating that the observed staining in the immunohistochemistry and the in situ-hybridisation experiments was specific.

3.6. Analysis

Immunohistochemical data were collected using a Leica microscope and analysing software Neurolucida (MBF Bioscience). The dorsal hippocampus and the mPFC

were measured. The hippocampus was divided into the dentate gyrus, the CA1, CA4, and a common CA2/3 region. The optical density (OD) of the strata oriens, pyramidale, and radiatum were measured in the CA and the stratum granulosum and the hilus in the dentate gyrus for all markers except of GABAB. For examination of this metabotropic GABA-receptor, positive cells were counted in the same areas. In the mPFC, a global analysis of all layers of the anterior cingulated, prelimbic, and infralimbic cortices were performed. For GABAB, receptor-containing cells of the lamina ganglionaris (layer V) were counted. Data were acquired from both hemispheres of two slides per animal and background staining was measured in the cingulum and subtracted from results.

In the in situ-hybridisation experiments, both MK801 and vehicle injected groups were compared for each time-point. Per animal, three consecutive slices were labelled and z-stacks were obtained from the CA1, CA3 and dentate gyrus region of one hemisphere of each slice using a Zeiss ApoTome (63x magnification). Only cells whose entire nucleus was visible in the z-stack were included and the percentage of intranuclear Arc-positive cells within the hippocampal regions was analysed. Fur- thermore, all GAD67-positive interneurons and glial cells were excluded from the analysis. To control for bias, measurements were carried out masked and spot- checked by two additional scientists.

In all immunohistochemical experiments, a multifactorial one-way analysis of variance (ANOVA) with the between-groups factor TREATMENT (MK801, vehicle) and REGION (PFC, DG, CA4, CA2/3, CA1) was used separately for each receptor

(GluN1, GluN2A, GluN2B, GABAA, GABAB, D1, D2, mGlu1, mGlu2/3, mGlu5) and time point (1 week, 4 weeks) to evaluate differences in receptor distribution. For the analysis of in situ-hybridisation, a multifactorial one-way ANOVA with the between-groups factor TREATMENT (MK801, vehicle) and REGION (DG, CA3, CA1) was used separately for each behavioural protocol (with or without empty holeboard exploration) and time point (1 week, 4 weeks) to evaluate differences in Arc gene expression. In all ANOVAs, Duncan’s post hoc test was used. To compare the exploration behaviour of MK801 and vehicle treated rats on the empty holeboard, an unpaired t-test was performed. Significance levels were set at the 5% level. All data were shown as mean ± standard error of mean.

4. Results 4.1. Immunohistochemistry 4.1.1. Time-dependent alterations in the expression of GluN2B, but not GluN1 and GluN2A subunits in the hippocampus and prefrontal cortex after MK801-treatment

Changes in NMDAR expression have been described both in animal models of psychosis and in the post-mortem brains of psychotic patients (Wilson et al., 1998; Kris- tiansen et al., 2007; Gonzalez-Burgos and Lewis, 2012). Here, it was examined whether changes occur in subunit expression of the NMDAR in the hippocampus and PFC. The investigated receptor subunits are GluN1, GluN2A and GluN2B.

In MK801-treated animals, GluN1 and GluN2A subunit expression was not altered either 1 or 4 weeks after drug administration compared to controls.

In contrast, the multifactorial ANOVA showed an effect for the factor TREAT- MENT (F(1,40)=16.98, p<0.001) and REGION (F(4,40)=5.02, p=0.002) for the GluN2B expression after 1 week. The post-hoc test revealed that 1 week post- injection, the GluN2B subunit expression was down-regulated in the PFC (vehicle 29.69±3.44 OD, MK801 19.70±0.91 OD, p=0.040). Four weeks after injection, an effect for the factor TREATMENT (F(1,40)=14.26, p<0.001) and REGION (F(4,40)=13.55, p<0.001) was evident. At that time point, GluN2B expression was only up-regulated in the dentate gyrus (vehicle 28.60±2.11 OD, MK801 38.13±2.6 OD, p=0.004). These alterations suggest that adaptive modifications in the expression of GluN2B occur.

1 week

Vehicle MK801

90 80 70

Vehicle 60 50 40 *

30 optical density optical 20 10

MK801 0

4 weeks

Vehicle MK801 90 80

Vehicle 70 60 50 * 40 * 30

optical density optical 20 10

MK801 0

Figure 6: Time-dependent changes occur in NMDAR subunit GluN2B after MK801-treatment

4.1.2. Differences in the expression of GABAA and GABAB receptors in the hippocampus and prefrontal cortex after MK801-treatment

GABA is critical for the regulation of excitability in the hippocampus (Coulter and

Carlson, 2007). Therefore, possible changes in the expression of GABAA and GAB-

AB subtypes of the GABAR were explored.

For the GABAA expression after 1 week, the multifactorial ANOVA showed an effect for the factor TREATMENT (F(1,40)=30.21, p<0.001) and REGION (F(4,40)=41.26, p<0.001). After 4 weeks, the ANOVA showed an effect for the factor TREATMENT (F(1,40)=14.92, p<0.001) and REGION (F(4,40)=80.48, p<0.001). The post-hoc test revealed that GABAA expression initially increased 1 week after MK801 administration in the hippocampus (CA1 vehicle 48.21±3.36 OD, MK801 60.32±4.11 OD, p=0.036; CA4 vehicle 57.92±3.70 OD, MK801 vehicle 71.96±4.58 OD, p=0.014), but by 4 weeks after treatment, expression had declined to levels equivalent to that seen in controls. In the PFC, the GABAA expression increased 1 week after treatment (vehicle 63.36±3.12 OD, MK801 81.68±5.13 OD, p=0.001), but reversed into a reduction 4 weeks after treatment compared to controls (vehicle 72.50±1.47 OD, MK801 61.07±3.32 OD, p=0.003).

For the GABAB expression after 1 week, the ANOVA showed an effect for the factor TREATMENT (F(1,40)=4.34, p=0.044) and REGION (F(4,40)=1226.76, p<0.001). Four weeks after injection, the ANOVA showed an effect for the factor TREAT- MENT (F(1,40)=42.59, p<0.001) and REGION (F(4,40)=555.14, p<0.001). In the hippocampus, GABAB expression was specifically increased in the dentate gyrus both 1 and 4 weeks after MK801-treatment (1 week vehicle 115.49±12.20 cells/0.1mm2, MK801 160.40±11.22 cells/0.1mm2, p=0.002; 4 weeks vehicle 73.11±3.49 cells/0.1mm2, MK801 118.37±9.99 cells/0.1mm2, p=0.006). In the PFC, the up-regulation was only significant 4 weeks after administration (vehicle 375.25±14.60 cells/0.1mm2, MK801 542.50±28.79 cells/0.1mm2, p<0.001).

Changes in GABAA expression in the hippocampus and PFC were transient, whereas increases in GABAB expression in the hippocampus were sustained throughout the 4 week assessment. The latter effects were localised to the dentate gyrus. Increases in expression in the PFC had become significant 4 weeks after treatment.

1 week

Vehicle MK801

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optical density optical 20 10

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Figure 7: MK801-treatment affects GABAA receptor expression

1 week

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700 )

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) * 2 600 *

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Figure 8: GABAB receptor expression is up-regulated after MK801-treatment

4.1.3. Dopamine D1 but not dopamine D2 receptor expression is chronically elevated in the hippocampus and prefrontal cortex after MK801-treatment

Dopamine D1 and D2 receptors are not only important for persistent forms of synaptic plasticity and learning in the hippocampus (Kulla and Manahan-Vaughan, 2000; Manahan-Vaughan and Kulla, 2003; Lemon and Manahan-Vaughan, 2006, 2012), they are also chronically dysfunctional in psychotic patients (Laruelle et al., 2003). Therefore, the expression of both receptors in the hippocampus and PFC of vehicle- and MK801-treated animals was examined.

For the dopamine D1 receptor expression after 1 week, the multifactorial ANOVA showed an effect for the factor TREATMENT (F(1,40)=7.45, p=0.009) and RE- GION (F(4,40)=26.26, p<0.001). Four weeks after injection, the multifactorial ANOVA showed an effect for the factor TREATMENT (F(1,40)=17.10, p<0.001) and REGION (F(4,40)=32.65, p<0.001). The post-hoc test revealed that dopamine D1 receptor expression in both the hippocampus as well as the PFC increased only 4 weeks after injection. At that time-point, a strong increase was measurable in the dentate gyrus (vehicle 4.54±0.76 OD, MK801 9.36±0.89 OD, p=0.004) and PFC (vehicle 12.65±0.82 OD, MK801 16.90±1.18 OD, p=0.007).

Dopamine D2 receptor expression after MK801-administration was not altered compared to controls at either of the time-points studied. This suggests that D1 is chronically over-expressed in the animal model of psychosis.

1 week

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70 Vehicle 60 50 40 30 20 density optical 10

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Figure 9: The dopamine D1 receptor is chronically up-regulated after MK801-treatment

4.1.4. The expression of metabotropic glutamate receptor mGlu1, but not mGlu5 or mGlu2/3 is transiently reduced in the hippocampus and prefrontal cortex after MK801-treatment

Group I and II metabotropic glutamate (mGlu) receptors are very important for hippocampal synaptic plasticity and spatial learning (Manahan-Vaughan, 1997; Naie and Manahan-Vaughan, 2004, 2005a, b; Altinbilek and Manahan-Vaughan, 2009). It was therefore examined whether changes in these receptors occur in MK801-treated animals.

For the mGlu1 expression after 1 week, the multifactorial ANOVA showed an effect for the factor TREATMENT (F(1,40)=57.33, p<0.001) and REGION (F(4,40)=5.66, p=0.001). Four weeks after injection, the multifactorial ANOVA showed an effect for the factor REGION (F(4,40)=13.18, p<0.001). In MK801-treated animals, mGlu1 was down-regulated both in the hippocampus (dentate gyrus vehicle 39.86±1.51 OD, MK801 30.79±2.05 OD, p=0.001; CA1 vehicle 44.10±2.17 OD, MK801 37.85±1.14 OD, p=0.025; CA2/3 vehicle 39.34±1.81 OD, MK801 31.77±1.84 OD, p=0.006; CA4 vehicle 40.01±1.60 OD, MK801 30.70±1.79 OD, p=0.001) and the PFC (vehicle 44.84±1.79 OD, MK801 35.88±1.19 OD, p=0.002) 1 week, but not 4 weeks after injection. By contrast, expression of mGlu5 and mGlu2/3 receptors was unaltered compared to controls.

1 week

Vehicle MK801 90

70 Vehicle 60 * * * * * * * * * 50 40 30

20 optical density optical 10

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90 80

Vehicle 70 60 50 40 30

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Figure 10: MK801-treatment reduces transiently mGlu1 expression

4.1.5. Synopsis of neurotransmitter receptor expression alterations after MK801-treatment

Figure 11: Summary of all alterations in neurotransmitter receptor expression after MK801-treatment Summary of all analysed receptors in the PFC (A) and hippocampus (B). A ratio >1 indicates an up-regulation in MK801-treated rats relative to vehicle treated rats.

All in all, there was an increase of GABAA receptor expression and, in addition, a decrease of mGlu1 receptor expression was found 1 week after NMDAR antagonism, in both the PFC and hippocampus. At that time-point, GABAB receptor expression was up-regulated in the hippocampus and NMDAR subunit GluN2B was down- regulated in the PFC. Four weeks after treatment, GABAB and D1 receptors expressions were significantly increased in both PFC and hippocampus, GABAA receptor

expression was decreased in the PFC, and GluN2B subunit expression was increased in the hippocampus. GluN1, GluN2A, mGlu2/3, mGlu5 and D2 receptors were unaffected at both time-points.

4.2. In situ-hybridisation 4.2.1. MK801-treatment alters basal Arc gene expression in the hippocampus and following spatial learning

To analyse changes in neuronal activity in the hippocampus 1 and 4 weeks after the treatment with MK801 in rats, Arc gene expression was compared to the expression in vehicle injected controls. The multifactorial ANOVA showed for the group without holeboard exploration after 1 week post-injection an effect for the factor TREATMENT (F(1,24)=18.06, p<0.001). The post-hoc test showed that 1 week after injection vehicle-treated animals had a significantly lower Arc gene expression in CA1 (vehicle 12.67±2.08 %, MK801 22.29±2.49 %, p=0.0099) and CA3 (vehicle 11.44±1.98 %, MK801 19.59±2.53 %, p=0.027) regions compared to MK801-treated animals, both without novel spatial learning experience. Four weeks after injection, a second cohort of animals was assessed. The multifactorial ANOVA showed for the group without holeboard exploration after 4 weeks post-injection an effect for the factor TREATMENT (F(1,24)=20.61, p<0.001) and REGION (F(2,24)=4.69, p=0.019). At this time-point, higher Arc gene expression was evident in CA1 (vehicle 16.85±0.63 %, MK801 29.25±4.04 %, p=0.009), CA3 (vehicle 15.86±2.41 %, MK801 26.08±4.28 %, p=0.028) and dentate gyrus (vehicle 9.95±2.51 %, MK801 19.27±1.55 %, p=0.045) in the MK801-treated animals compared to vehicle injected animals, both without learning paradigm. In contrast, no difference was detected between Arc gene expression in vehicle and MK801-treated rats after novel spatial exploration either 1 or 4 weeks after treatment. These data support that MK801- treatment results in chronic and persistent elevations of Arc gene expression, even under circumstances where learning has not occurred.

CA1 no HB HB

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Figure 12: Arc gene expression is affected at rest and following holeboard (HB) experience by MK801 1 week after application

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Figure 13: Arc gene expression is affected at rest and following holeboard (HB) experience by MK801 4 weeks after application

4.2.2. The exploratory behaviour of rats is not affected 1 and 4 weeks after MK801-treatment

20 18 16 14 12 10 8 6

exploration time (s) time exploration 4 2 0 Vehicle 1 MK801 1 Vehicle 4 MK801 4 week week weeks weeks

Figure 14: MK801-treatment does not affect exploratory behaviour

To rule out that the differences in basal Arc gene expression results from changes in the behaviour of the rats, activity during exploration of the empty holeboard was assessed. The time that each rat spent exploring the different holes, i.e. dipping their heads into one of the holes, within a 6 min period was measured. The average exploration time of all four groups was 12.5±1,04s in 6 minutes (1 week vehicle 14.64±2.80s, 1 week MK801 12.46±1.75s, 4 weeks vehicle 10.57±1.92s, 4 weeks MK801 13.21±1.91s). The groups did not differ in the exploration time whether they were treated with MK801 or vehicle, nor between the incubation times of the drugs.

5. Discussion

In this study, it was found that long-term changes in neurotransmitter receptor expression occur in an animal model of psychosis. In particular, NMDAR GluN2B subunit, GABAA, GABAB, D1, and mGlu1 are affected in a subregion- and time- dependent manner. An increase of GABAA receptor expression and, in addition, a decrease of mGlu1 receptor expression were found 1 week after NMDAR antagonism, in both the PFC and hippocampus. At that time-point, GABAB receptor expression was up-regulated in the hippocampus and NMDAR subunit GluN2B was down- regulated in the PFC. Four weeks after treatment, GABAB and D1 receptors expressions were significantly increased in both PFC and hippocampus, GABAA receptor expression was decreased in the PFC, and GluN2B subunit expression was increased in the hippocampus. These changes appear to alter neuronal excitability: basal hippocampal Arc gene expression is increased in MK801-treated animals and, strikingly, no elevation in Arc gene expression was elicited in MK801-treated rats after performing spatial learning paradigm.

5.1. MK801 - a valid model in schizophrenia research?

To understand the development in pathogenesis of schizophrenia and generate new therapeutic strategies, animal models appear imperative since it is very rare that analysis can be conducted on the brains of patients that have not undergone pro- longed pharmacological treatment. Patients present several comorbidities which might bias post-mortem findings. Furthermore, in vivo studies in humans are gener- ally non-ethical. To evaluate the validity of the animal model using the NMDAR antagonist, MK801, the criteria by Willner (1986) for animal models in psychiatric research were adopted. Accordingly, three dimensions of validity are applicable: construction validity (comparability of the theoretical basis of the model to pathogenesis in human), face validity (comparability of the symptoms in the model to human), and predictive validity (prediction of human responsiveness in drug treatment). The following text is based on an interpretation of the MK801 model in light of these criteria that was first described by Wiescholleck and Manahan-Vaughan (2013a).

For high construction validity, the model must feature the same theoretical rational compared with pathogenesis in humans. Although the entire pathogenesis of schizophrenia is still obscure, several observations indicate that MK801 might mimic a key

mechanism in schizophrenia occurrence. After administration of NMDAR antagonists, healthy subjects display positive as well as negative and cognitive symptoms comparable to those observed in schizophrenia, and patients with schizophrenia suf- fer exacerbation of their symptoms (Jentsch and Roth, 1999; Lahti et al., 2001). Fur- thermore, several neuroimaging and post-mortem studies illustrate a multitude of alterations in the glutamatergic system, especially in NMDAR expression, thus confirming this system as a key element in schizophrenia (Jentsch and Roth, 1999). Even though a pharmacological model is not able to reproduce others pathologies such as disease-causing genetic changes, it serves as a model for the dynamic of schizophrenia development.

Regarding face validity, MK801-treated rats exhibit positive, negative, and cognitive impairments that are in part comparable with impairments seen in human patients. Immediately after single high dose treatment, rats display increased locomotor activity and stereotypy that is thought to reflect positive symptoms in schizophrenia (Wöhrl et al., 2007; Manahan-Vaughan et al., 2008a; Wiescholleck and Manahan- Vaughan, 2013a). As an indicator for negative symptoms, several studies have shown social withdrawal acutely after NMDAR antagonism in rats (Zou et al., 2008; Wiescholleck and Manahan-Vaughan, 2013a). Cognitive deficits are visible acutely and chronically after MK801-treatment, illustrated as impairments in learning and memory, executive function, attention span, and sensorimotor gating (Wiescholleck and Manahan-Vaughan, 2013a). For the latter, prepulse inhibition of the acoustic startle response is effective for measurements of sensorimotor gating across species (Braff and Geyer, 1990). In both MK801-treated rats and schizophrenia patients, disruption of prepulse inhibition has been shown immediately after treatment (Mana- han-Vaughan et al., 2008a) and during acute psychotic symptoms, respectively (Braff et al., 1999). LTP is believed to be a cellular correlation of learning and memory, and is chronically deficient in MK801-treated animals (Manahan-Vaughan et al., 2008a, b; Wiescholleck and Manahan-Vaughan, 2012, 2013a, b) comparable to impairments in LTP-like plasticity after transcranial stimulation in schizophrenia patients (Frantseva et al., 2008; Hasan et al., 2011). Finally, deficits in executive functions and attention have been seen in both human patients (Tyson et al., 2004) and MK801-treated rodents (Paine et al., 2011).

The third criteria of validity is the predictive validity, and addresses the question as to what extent human performance can be predicted by this animal model of schizophrenia/psychosis. One very important criterion for animal models of human disease is the responsiveness to drug treatment. Second generation antipsychotics, that have been shown to be effective in humans, also elicit a positive effect on deficits induced by NMDAR antagonism in rodents (Abdul-Monim et al., 2006). Furthermore, the dopamine D1 receptor agonist, and antagonists of several 5-HT receptor subtypes, SKF 38393, improve deficits in the MK801 animal model of psychosis (McLean et al., 2009) and in human patients (Davidson et al., 1990).

All in all, the MK801 animal model of psychosis seems to have a relatively high validity against the background of the difficulties of modelling psychiatric disorders in rodents.

5.2. Is the rodent prefrontal cortex comparable to the human prefrontal cortex?

The question of equivalence of human brain areas to animal brain areas is very difficult to answer, especially with regard to brain areas strongly connected to social behaviour and higher cognitive functions.

Figure 15: Anatomical separation of the human and rodent prefrontal cortex Modified from Wise (2008).

The rodent PFC is different in structure compared to the human PFC. For instance, the human PFC can be divided by the content of a homotypical, or rather, dysgranu- lar layer 4 into a granular dorsolateral and an agranular PFC (Wise, 2008). By con-

trast, the rodent PFC is completely agranular (Wise, 2008). Nevertheless, there are comparable agranular areas in both species: the infralimbic, prelimbic and anterior cingulate cortices that all belong to human mPFC (Brodmans’ areas 24, 25, and 32) and that are the areas used in this analysis (Seamans et al., 2008; Wise, 2008).

The agranular areas of both species are highly comparable in localisation and inter- connection. In human and rodents, the agranular PFCs have a similar localisation at the border of the allocortex (Wise, 2008). Moreover, both agranular PFCs share similar interactions with other brain areas such as premotor and somatosensory cortices, sensory cortices and limbic areas (Seamans et al., 2008). Additionally, the mPFCs of rats and primates have unique direct cortical projections to cholinergic basal forebrain and brainstem nuclei, noradrenergic locus coeruleus and to serotonergic dorsal and median raphe nuclei. The mPFC in both species gain dopaminergic inputs from the ventral tegmental area (Uylings et al., 2003; Seamans et al., 2008).

However, results from behavioural analyses blur this distinction. On the one hand, it is possible to separate several behaviours to orbitofrontal, dorsolateral, and medial areas across species (Uylings et al., 2003). On the other hand, studies have shown that the rodent mPFC is involved in motor planning and reward anticipation: charac- teristics that are also typical for primate granular dlPFC (Seamans et al., 2008). Yet, this finding is not surprising when it is taken into consideration that the human granular cortex probably evolved from the agranular cortex as higher cognitive functions was required (Preuss and Kaas, 1999).

In general, the rodent PFC serves as a suitable model for simplified mechanisms in preclinical studies and the extrapolation of common mechanisms (Brown and Bow- man, 2002; Seamans et al., 2008; Wise, 2008; Kesner and Churchwell, 2011). The rodent PFC is relatively comparable to the human agranular PFC. Nevertheless, some functional aspects are also comparable to human granular dlPFC.

5.3. Impact of changed receptor expression on the course of psychotic symptoms

Schizophrenia pathology is believed to emerge through genetic alterations and complications during maturation and manifests inter alia in receptor alterations. In this animal model of psychosis, receptor alterations were examined after putative first-

episode psychosis. The focus was on the glutamatergic, GABAergic, and dopaminergic systems.

5.3.1. The role of altered GluN2 subunit composition in synaptic plasticity and learning and memory

Each NMDAR is composed of an obligatory subunit, GluN1, and variable subunits, such as GluN2 or GluN3 (Paoletti, 2011). In this study, changes in expression of the subunits GluN1, GluN2A, and GluN2B were assessed in the MK801 animal model of psychosis. Time-dependent alterations were observed in the expression of GluN2B, but not GluN1 and GluN2A. Shortly after treatment, GluN2B was down- regulated in the PFC, whereas GluN2B was chronically up-regulated in the hippocampus.

Analyses of the post-mortem human brain tissue of psychotic patients have demonstrated very mixed results concerning GluN2 subunit transcription and surface expression (Kristiansen et al., 2007; Gonzalez-Burgos and Lewis, 2012). However, a direct comparison is confounded by the fact that psychotic patients undergo long- term pharmacological treatment and are affected by comorbidity. The possibility cannot be excluded that the MK801-model emulates certain, but not all, aspects of the pathophysiology of psychosis. In line with the results of the present study, the gene encoding the GluN2B subunit, GRIN2B, seems to be a risk gene as revealed by a recent meta-analysis (Allen et al., 2008). Furthermore, up-regulation of GluN2B subunit has been previously demonstrated in several animal models of NMDAR antagonism: in the first study, neonatal rats were treated with MK801 (1-2 mg/kg intraperitoneal for 2-4 h), which resulted in an increased GluN2B, GluN1, and GluN2A subunit expression in the hippocampus (Wilson et al., 1998). In the second study, increased GluN2B and GluN2A subunit expression occurs in rodent PFC 12 weeks after treatment with PCP (10 mg/kg subcutaneously) on postnatal days 7, 9, and 11 (Owczarek et al., 2011). However, differences in these studies with regard to age and treatment regimens also confound direct comparison. None of the animal studies to date has examined the long-term effect on NMDAR subunit expression of the insti- gation of first-episode psychosis in an animal model, as was the case in the present study.

Since only the functional GluN2 and not the obligatory GluN1 subunit differs in expression following MK801-treatment, a dysregulation in NMDAR function is more likely rather than a reduced amount of NMDAR expression in schizophrenia. Several studies in schizophrenia patients support this idea by demonstrating alterations in NMDAR interacting proteins and signalling molecules (Beneyto and Meador- Woodruff, 2008). Although specific alterations were restricted to GluN2B expression, MK801-treatment also caused changes in the GluN2A/GluN2B ratio.

Even though both subunits bear a striking resemblance in terms of their gene sequence and biophysical features, their differences seem to have a great impact on the development and processing of sensory experiences. Early development may be un- derstood as brain adjustment to new sensory inputs. In newborn rodents, GluN2B subunit expression dominates compared to GluN2A and peaks around postnatal day 7-10 (Cull-Candy et al., 2001). During ageing, the GluN2A/GluN2B ratio increases steadily until GluN2A is expressed in the entire brain (Cull-Candy et al., 2001). In- terestingly, whereas GluN2A subunit expression is completely absent immediately after birth, GluN2A does not totally replace GluN2B in the adult brain except in cerebellar granule cells (Cull-Candy et al., 2001). Further experiments with dark-reared animals have demonstrated that brain areas lacking sensory information exhibit a reduced switch in GluN2A/B ratio (Philpot et al., 2001). Likewise, early stress, for instance maternal deprivation, is sufficient to impair the GluN2A/B switch (Rodenas- Ruano et al., 2012). Importantly, only 2 h of light exposure are necessary to induce the subunit shift (Philpot et al., 2001). These observations indicate that neuronal activity may be one of the key factors in the control of NMDAR subunit surface expression. Moreover, synaptic GluN2A has been linked to neuroprotection by mobili- sation of the transcription factor cyclic-AMP response element binding protein (Har- dingham and Bading, 2010). In conclusion, the enhancement of the GluN2A/B ratio in the PFC in the MK801 animal model 1 week after treatment might be the consequence of exacerbated neuronal activity. Alternitavely, increased sensory input during first-episode psychosis might be a cell-survival response to reverse exacerbated neuronal activity and increased sensory input during first-episode psychosis, as indi- cated by the results of the Arc gene experiments.

Furthermore, alterations in the dopaminergic system are believed to be responsible for NMDAR distribution (Hallett et al., 2006). Dopamine D1 receptors lead to en-

hanced co-clustering of GluN2B subunits with the postsynaptic density protein 95 and thereby increase surface expression of GluN2B (Hallett et al., 2006). Thus, enhanced D1 expression might affect NMDAR subunit constellations, as shown by contemporaneously enhanced D1 and GluN2B rates in the MK801 animal model 4 weeks after treatment.

Altered NMDAR subunit expression is believed to elicit enormous effects on synaptic plasticity (Yashiro and Philpot, 2008): NMDARs are intrinsically involved in the induction of most forms of hippocampal LTP and LTD (Yashiro and Philpot, 2008), but the role of the NMDAR subunits is still controversial (Collingridge et al., 2004; Yashiro and Philpot, 2008; Fetterolf and Foster, 2011). Several studies emphasize the role of GluN2A and GluN2B in LTP and LTD, respectively. As outlined previously (cf. section 1.5.1), in comparison to GluN2A, GluN2B are slow gating ion channels with low peak currents and slow glutamate decay that, on the whole, have the feasi- bility for a higher calcium transfer (Yashiro and Philpot, 2008; Paoletti, 2011). How- ever, it has been suggested that their pharmacological properties of calcium transmission might be dependent on the induction protocol. During high frequency stimulation, the charge transfer by GluN2A exceeds that of GluN2B, whereas the opposite is the case under low frequency stimulation (Erreger et al., 2005), resulting in LTP and LTD, respectively. Furthermore, it has been demonstrated in hippocampal slice prep- arations that GluN2B is effective in some LTP protocols that combine pre- with postsynaptical stimulation, for instance, a pairing of low frequency stimulation with postsynaptic depolarisation (Berberich et al., 2007) or a pairing of single presynaptic stimuli with postsynaptic action potentials (Zhang et al., 2008). In addition, this assignment is strongly affected by several parameters. For instance, the receptor localisation seems to influence whether GluN2A or GluN2B, or both, induce LTP or LTD. As outlined previously (cf. section 1.5.1), GluN2B-containing NMDARs are preferentially located extrasynaptically (Gladding and Raymond, 2011). Massey et al. (2004) have shown in slices of the perirhinal cortex that LTD is triggered primarily by extrasynaptic NMDAR, but, conversely, synaptic NMDAR are also able to induce LTD (Köhr, 2006). This ambivalent role of the GluN2 subunits appears to be mediated by different downstream molecules for each direction of synaptic plasticity: GluN2B interacts with the Ras molecule resulting in AMPAR endocytosis as a mechanism for LTD induction (Kim et al., 2005), whereas GluN2B might facilitate

LTP via interaction with CaMKII (Barria and Malinow, 2005). Notably, in the intact brain of freely moving rats (Ge et al., 2010; Dong et al., 2012, 2013), only a role of GluN2A and GluN2B for LTP and LTD have been shown, respectively. In conclusion, even if GluN2A and GluN2B might be particularly involve in LTP and LTD, respectively (Ge et al., 2010), both subunits seem to be able to induce both forms of synaptic plasticity (Weitlauf et al., 2005; Berberich et al., 2005, 2007; Philpot et al., 2007; Müller et al., 2009).

The chronically increased expression of GluN2B in the MK801 animal model of psychosis, as reported in the present study, may explain why LTP is impaired and LTD is unaffected (Manahan-Vaughan et al., 2008b). Nevertheless, even though the threshold for LTP induction increases in this animal model of psychosis, it could be supposed that once LTP is triggered, it might be quite robust due to a high calcium charge transfer by GluN2B activation. To date, no study has yet provided evidence of a more robust LTP in the MK801 animal model of psychosis; this is probably due to alterations in other receptors than the NMDAR. In the light of alterations in the GA- BAergic system (cf. section 5.3.2) and elevations in neuronal excitability (cf. section 5.4.2) in this study, increased excitability can mean that only little space remains for potentiation of synapses. Thus, although induction is enabled, sustainment could be impaired (Moser and Moser, 1999). This can relate to concurrent deficits in certain forms of learning and memory (Manahan-Vaughan et al., 2008a, b; Wiescholleck and Manahan-Vaughan, 2012, 2013a, b).

5.3.2. Changes in neuronal activity by altered GABA receptors expression have consequences for synaptic plasticity and cognitive function

A time-dependent shift in GABAR distribution in the MK801-animal model of psychosis was observed. Thus, GABAA expression was transiently up-regulated, but chronically down-regulated, whereas long-lasting GABAB elevation occurred in both the PFC and hippocampus. Investigations in humans revealed mixed results: on the one hand, PET studies reported that the high affinity binding to the non- benzodiazepine side of GABAA is increased in the PFC (Benes et al., 1996a), in pyramidal cells of hippocampal CA1 region and in non-pyramidal cells of the CA3 re-

gion in schizophrenia patients (Benes et al., 1996b). On the other hand, further studies using single-photon emission computed tomography (Ball et al., 1998; Verhoeff et al., 1999) and PET (Squires et al., 1993) did not demonstrate alterations in GA-

BAR distribution or show decreases in GABAA expression in the cortex. This diver- gence might be explained by an allosteric uncoupling (Benes and Berretta, 2001) in receptor regulation, but further investigation is necessary.

Initial up-regulation in GABAA expression in the MK801 animal model may occur to compensate for GABAA dysfunction in psychosis (Montpied et al., 1991; Volk and Lewis, 2002). In rats that undergo chronic treatment with MK801, mRNA of the GABA membrane transporter 3 is up-regulated and mRNA of the enzyme responsible for GABA synthesis, GAD67, is down-regulated (Paulson et al., 2003; Behrens et al., 2007). Similarly, reduced mRNA and protein levels of GAD67 are consistently found in the hippocampus and PFC in human post-mortem studies (Akbarian et al., 1995; Guidotti et al., 2000; Volk et al., 2000; Hashimoto et al., 2005, 2008a, b; Dun- can et al., 2010). These changes are likely to provoke reduced GABA synthesis and enhanced clearance of GABA from the synaptic cleft resulting in compensatory enhancement of postsynaptic GABAR expression. Strikingly, the first direct measurements of GABA release via proton nuclear magnetic resonance spectroscopy revealed a trend for reduced GABA release in the anterior cingulate regions in older schizophrenia patients compared to older control subjects in correlation to poor attention performance (Rowland et al., 2012). Further pharmacological studies support this hypothesis by demonstrating that GABAA antagonism, aimed at mimicking insufficient activation of GABAR by GABA, emulates psychotic symptoms (Thomas- es et al., 2013).

Chronically, prefrontal GABAA expression turned into reduced surface expression and, in addition, a long-lasting GABAB up-regulation was found. Both changes might augment the abnormal neuronal interaction. Whereas GABAA receptors are postsynaptically located, GABAB receptors act as presynaptical autoreceptors mediating a negative feedback to calcium gating ion channels of its own neurons and thereby reducing neurotransmitter release (Mintz and Bean, 1993). Because these autoreceptors are activated by release of endogenous GABA, reduced release of GABA, as proposed in the MK801 animal model, might cause a compensatory up- regulation of these GABAB receptors. This interplay might result in a constant reduc-

tion of GABA release and slow increases of GABAB surface expression, as seen in our study. Moreover, a long-lasting decrease in postsynaptic GABAA expression might further reduce the residual GABAergic influence.

Since GABA synthesis and release is mainly mediated by GABAergic interneurons that modulate principal cells, altered GABA release is likely to impair interneuron function and that in turn might result in changes in excitability of principal neurons. In consequence, an altered GABAergic inhibitory output onto principal cells would result in a disinhibited and indeed up-regulated state of basal pyramidal activation (Homayoun and Moghaddam 2007; Gordon, 2010; Thomases et al., 2013).

Figure 16: Interplay between pyramidal cells and interneurons A: Intact excitatory and inhibitory balance B: Interneuronal NMDAR dysfunction disinhibits pyramidal output

The vital role of the GABAergic system in disinhibition of the pyramidal activity in the MK801 animal model of schizophrenia and schizophrenia patients is supported by several lines of evidence:

Firstly, separate measurements of the firing rates of interneurons and principal cells revealed that MK801 preferentially targets interneuronal NMDAR. After systemic, single, in vivo treatment of MK801 in awake rats, the firing rates of putative fast- spiking interneurons initially decreased, whereas the activity of surrounding pyramidal neurons increased with a delay of a few minutes (Homayoun and Moghaddam, 2007). Since most interneurons modulate neuronal activity via a GABAergic influence, reduced interneuronal activation by NMDAR should result in disinhibition of the postsynaptic cell (Breier et al., 1997; Miyamoto et al., 2000). This hypothesis was supported by a transgenic mice model of degraded NMDAR of cortical and hippocampal GABAergic interneurons demonstrating a profound disinhibition of principal cells (Belforte et al., 2010; Nakazawa et al., 2012).

Secondly, a disruption in GAD67 expression was reported in post-mortem studies (Akbarian et al., 1995; Guidotti et al., 2000; Volk et al., 2000; Hashimoto et al., 2005, 2008a, b; Duncan et al., 2010). Disrupted GAD67 expression is able to reduce miniature inhibitory postsynaptic current amplitude in pyramidal cells (Lau and Murthy, 2012). Since the GABAergic pathway exerts a major inhibitory control on excitatory output, a dysfunction might lead to disturbance in the balance of activity control. Confirming this hypothesis, optogenetic studies demonstrated that elevated prefrontal excitation leads to impaired cognition and social behaviour (Yizhar et al., 2011). Furthermore, increasing inhibition during elevated excitation reduces these impairments by normalizing the excitatory- inhibitory balance.

Thirdly, paired-pulse transcranial magnetic stimulation (TMS) in humans revealed differences in schizophrenia patients (Nakazawa et al., 2012). Paired-pulse TMS includes subsequent stimulation with lower- and higher-intensity pulses at intervals of a few milliseconds. At certain inter-stimulation-intervals, the first stimulus reduces the evoked potential that the second higher pulse would evoke if given alone. Since GABAR agonists are able to enhance cortical inhibition in paired-pulse TMS, the inhibition between the two pulses is considered to be a function of GABAergic inhibition. Reduced inhibition in paired-pulse TMS was demonstrated in schizophrenia

patients, which is likely to be due to dysfunctional GABAergic interneurons and neuronal suppression (Nakazawa et al., 2012).

5.3.3. Contribution of changed dopamine receptor expression to synaptic plasticity and learning and memory

In the present study, a chronic enhancement of D1 expression in both the dentate gyrus and PFC was found in the MK801 animal model of first-episode psychosis. Enhanced D1 expression in the PFC has also been found in in vivo studies in schizophrenia patients (Abi-Dargham et al., 2002, 2012). Using the D1 specific PET ligand [11C]NNC 112, Abi-Dargham et al. (2002) demonstrated enhanced binding potentials in the dlPFC of unmedicated schizophrenia patients. Furthermore, this up-regulated binding potential was correlated with poor achievement in the n-back task, a working memory assignment (Abi-Dargham et al., 2002). In addition to up-regulated receptor expression, studies with schizophrenia patients reveal a connection of low liquor concentration of the dopamine metabolite homovanillic acid, reduced regional cerebral blood flow in the dlPFC and poor working memory performance indicating that prefrontal function is dependent on prefrontal dopaminergic metabolism (Weinberger et al., 1988; Kahn et al., 1994). Further evidence of decreased immunoreactivity for both the dopamine synthetic enzyme, tyrosine hydroxylase, and the dopamine transporter in post-mortem studies (Akil et al., 1999) suggests that an up-regulation of D1 expression may be due to reduced dopamine release in the dlPFC in schizophrenia (Heinz et al., 2003). These connections support the hypothesis that deficient dopaminergic input at D1 receptors in the PFC might result in cognitive impairments and negative symptoms of schizophrenia (Davis et al., 1991), but direct evidence for this is currently lacking.

A different hypothesis postulates gene-driven disturbances in spine homeostasis as the origin of negative symptoms and dopaminergic dysregulation (Sato, 2012). Re- duced spine density and altered dendritic morphology of principal cells is a very prominent finding in post-mortem tissue of schizophrenia patients (Garey et al., 1998). Since it is known that spine homeostasis is closely controlled by D1 activity (Scott et al., 2002, 2006; Arnsten, 2011; Penzes et al., 2011), genetic changes for structural important genes, for instance neuregulin, dysbindin, and DISC1 (Harrison

and Weinberger, 2005), and subsequent disruption in spine homeostasis might result in an up-regulation of D1 surface expression and enhanced D2 expression.

Glutamatergic and dopaminergic inputs coincide on the dendrites of the same pyramidal neurons (Sesack et al., 2003) and since D1 receptors stabilise glutamate mediated synaptic transmission (Levine et al., 1996), it can be hypothesised that deficits in dopaminergic innervations and in LTP, as seen in both the animal model (Manahan- Vaughan et al., 2008a, b; Wiescholleck and Manahan-Vaughan, 2012, 2013a, b) and in patients (Hasan et al., 2011), might be in part compensated by an up-regulation of D1 expression. In line with this, D1 interacts with the NMDAR by increasing its activation and promoting cell survival mechanisms (Flores-Hernández et al., 2002). Thereby, D1 promotes NMDAR surface expression and vice versa (Fiorentini et al., 2003; Pei et al., 2004). In contrast, D1 receptor internalisation is induced by the postsynaptic density protein 95 (Zhang et al., 2007) so that the competing interplay of NMDAR and postsynaptic density protein 95 regulates D1 surface expression. Interestingly, the postsynaptic density protein 95 is down-regulated in rodent models of psychosis using psychostimulation or dopamine supersensitivity (Yao et al., 2004).

D1 interacts not only with NMDAR on pyramidal cells, but is also found on interneurons, whereby reduced cortical dopamine can result in impaired interneuron activity (Inan et al., 2013). Interneuronal D1 activation promotes GABAergic inhibition of pyramidal cells (Seamans et al., 2001; Gorelova et al., 2002). Thereby, D1 activation is capable of potentiating stimulated reactions and suppressing spontaneous activity in order to enhance the signal-to-noise ratio (Seamans et al., 2001; Gorelova et al., 2002). Thus, the proper functioning of a complete cell cluster is dependent on the influence of D1 on both GABAergic interneurons and pyramidal cells. Accordingly, clinical trials with D1 antagonists have not shown sufficient antipsychotic effects, but can even aggravate positive symptoms (de Beaurepaire et al., 1995; Karlsson et al., 1995).

The finding that enhanced D1 expression occurred in the dentate gyrus in the present study was surprising since the role of hippocampal D1 in schizophrenia is still obscure. One analysis of D1 mRNA expression in the hippocampus of schizophrenia patients revealed a decrease in the CA3 region in a specific subgroup of cells (Panta- zopoulos et al., 2004), but further results are lacking. The hippocampus receives do-

paminergic innervations mainly by the ventral tegmental area, but also by the sub- stantia nigra and retrorubral area A8 (Hansen and Manahan-Vaughan, 2014). How- ever, studies in rodents have shown that the dorsal hippocampus has insufficient dopaminergic innervation due to a lack of dopaminergic fibers from the ventral tegmental area. Instead, the dorsal hippocampus receives intense noradrenergic modulation by both hippocampal noradrenergic neurons and innervations by the locus coeruleus resulting in direct release of dopamine (Hansen and Manahan-Vaughan, 2014). Do- pamine release can be detected minutes after the incoming of novel information in the hippocampus (Ihalainen et al., 1999). Alterations in dopamine receptor distribution and associated dopamine innervation are believed to have an enormous impact on synaptic plasticity and learning and memory (Hansen and Manahan-Vaughan, 2014), since D1 receptors regulate bidirectional synaptic plasticity in vivo (Lemon and Manahan-Vaughan 2006, 2012; Hansen and Manahan-Vaughan, 2014). D1 receptors are able to facilitate both LTP and LTD in the PFC (Huang et al., 2004) and in hippocampus (Lemon and Manahan-Vaughan, 2006, 2012). In particular, pharmacological and genetic studies have demonstrated that D1-like receptor activation is able to facilitate early and late phases of LTP and LTD in the CA1 region, whereas in the dentate gyrus only enhancement of late-phase LTP is evident (Hansen and Mana- han-Vaughan, 2014). Results concerning the D1 modulation of LTD in the dentate gyrus are lacking. Enhanced D1 expression in the dentate gyrus, as seen in the MK801 model of psychosis, might compensate for deficits in synaptic transmission similar to those seen in the PFC.

5.3.4. Influence of changed metabotropic glutamate receptor expression on synaptic plasticity and learning and memory

In the MK801 animal model of first-episode psychosis, a transient reduction in mGlu1, but not mGlu5 or group II mGlu receptor expression in the PFC and hippocampus was observed in the present study. These findings do not equate with the results of human studies, however (Gupta et al., 2005; Volk et al., 2010). This might be due to pharmacological therapy or to the relative age of the subjects, as an age- dependent change in mGlu1 receptor expression has been described (Simonyi et al., 2005).

MGlu1 interacts with various proteins and receptors, such as NMDAR (Mukherjee and Manahan-Vaughan, 2013). For instance, mGlu1 increases the NMDAR mediated currents by potentiating NMDAR exocytosis and subsequent surface expression (Lan et al., 2001) or by facilitation of NMDAR activation (Heidinger et al., 2002). The latter is mediated through phosphorylation of GluN2 subunits via a physical linkage of both receptors (Heidinger et al., 2002). Acute loss of mGlu1 after MK801- treatment may therefore be instrumental in changing the GluN2B expression that subsequently occurred in the MK801 animal model. Interestingly, activation of group I mGlu receptors facilitates not only NMDAR currents on pyramidale cells, but also on GABAergic interneurons, illustrated by agonism of group I mGlu receptors resulting in enhanced GABA-mediated spontaneous inhibitory postsynaptic currents (Chu and Hablitz, 1998). Acute failure of mGlu1 mediated influence might therefore enhance deficits in GABAergic inhibition as a core feature in schizophrenia.

The strong connection of mGlu1 to NMDAR function suggests that alterations in mGlu1 expression provoke changes in synaptic plasticity, as seen in the animal model of psychosis (Manahan-Vaughan et al., 2008a, b; Wiescholleck and Manahan- Vaughan, 2012, 2013a, b). Group I mGlu receptors play a critical role, in particular in the late-phase of synaptic plasticity (Manahan-Vaughan, 1997; Naie and Mana- han-Vaughan, 2004, 2005a, b; Bikbaev et al., 2008; Mukherjee and Manahan- Vaughan, 2013). In several pharmacological as well as in in vitro and in vivo studies in transgenic rodents, it has been shown that group I mGlu receptors are involved in protein synthesis and frequency-dependent induction and maintenance of both, LTP and LTD (Manahan-Vaughan, 1997; Naie and Manahan-Vaughan, 2004, 2005a, b; Bikbaev et al., 2008; Mukherjee and Manahan-Vaughan, 2013). The afferent frequency, the thereby activated amount of mGlu receptor in comparison to NMDAR (Hsu et al., 2011), and also the localisation of the mGlu receptors might be instrumental in determining whether LTP or LTD occur. While postsynaptic group I mGlu receptors lead to cell depolarization (Coutinho and Knöpfel, 2002), presynaptically located receptors alter glutamate release (Manzoni and Bockaert, 1995). The particularly important role of group I mGlu receptors in LTP might explain why changes in mGlu1 expression occur after MK801-treatment.

Moreover, MK801-treated animals are similar to mGlu1 knockouts in behavioral aspects: both, MK801-treated animals and mGlu1 knockouts exhibit impairments in

hippocampus-dependent learning in the Morris water maze (Conquet et al., 1994; Lobellova et al., 2013). Furthermore, mGlu1 knockout mice (Brody et al., 2003), MK801-treated rodents (Manahan-Vaughan et al., 2008a), and schizophrenia patients (Braff et al., 1999) exhibit deficits in prepulse inhibition of acoustic startle.

5.4. Enhanced neuronal activity in the animal model of psychosis

In both the MK801 animal model (Manahan-Vaughan et al., 2008a, b; Wiescholleck and Manahan-Vaughan, 2012, 2013a, b) and schizophrenia patients (Häfner et al., 1999; Hasan et al., 2011), alterations in learning and memory and synaptic plasticity have been shown. These impairments are crurial for the daily life of schizophrenia patients, since correct neuronal encoding of information is instrumental for proper cognitive function, a strong predictor for adequate adaptation to the outside world (Lesh et al., 2011). In order to evaluate changes in neuronal activity, Arc gene expression was analysed in the dentate gyrus and hippocampus.

5.4.1. Connection of Arc gene expression to neuronal activity, synaptic plasticity, and learning and memory

Arc, as an immediate early gene, is transcribed at a low level under basal (naïve) conditions and can be induced by several ionotropic and metabotropic receptors that are known to be activated by neuronal activity and new inputs (Guzowski et al., 1999; Korb and Finkbeiner 2011). In line with this, Arc gene expression is relatively high in cultured neurons with increased basic levels of activity. Arc gene expression recedes after application of tetrodotoxin, a blocker of voltage-gated, fast sodium channels, mimicking neuronal inactivity. Conversely, Arc gene expression rises after application of bicuculline, a GABAA blocker (Shepherd et al., 2006). On this basis, Arc gene expression is used as an indicator for neuronal activity (Bramham et al., 2010; Korb and Finkbeiner 2011).

Arc might be correlated with LTP since it has been demonstrated that LTP mediated by convulsive seizures and high frequency stimulation in the perforant path results in increased Arc gene expression in the dentate gyrus (Link et al., 1995). Conversely, in a pharmacological animal model of disrupted Arc gene expression, the maintenance

phase of LTP is impaired without affecting its induction leading to compromised long-term memory without affecting task acquisition and short-term memory (Guzowski et al., 2000; Guzowski, 2002). These results were validated in an Arc gene knockout mouse model (Plath at al., 2006). Moreover, there is evidence of the connection of Arc and LTP from studies of BDNF, a key element in synaptic transmission and learning and memory (Binder and Scharfman, 2004). Protocols that are able to generate late-phase LTP also induce the release of BDNF (Lu et al., 2008) and, conversely, induction of LTP is affected in BDNF knockdown mice (Lu et al., 2008). In a rat model of impaired Arc gene expression using antisense oligodesox- ynucleotides, it was shown that both BDNF-mediated LTP and memory consolidation are Arc-dependent (Messaoudi et al., 2007).

Furthermore, Arc seems to be correlated with LTD, since it has been shown that application of group I mGlu receptor agonist (R,S)-3,5-dihydroxyphenylglycine that is able to produce robust LTD is capable of inducing Arc synthesis (Brackmann et al., 2004). Thereby, Arc mediates endocytosis of Glu1 containing AMPAR, resulting in a reduction of spontaneous miniature excitatory synaptic currents (Shepherd et al., 2006). Conversely, acute inhibition of Arc synthesis causes enhanced AMPAR surface expression rates and subsequent increased amplitude of spontaneously evoked miniature excitatory synaptic currents (Shepherd et al., 2006), and prevents induction of mGlu-mediated LTD (Park et al., 2008; Waung et al., 2008). In contrast, induction of NMDAR dependent LTD neither by low frequent stimulation (Steward and Wor- ley, 2001) nor by application of NMDA (Waung et al., 2008) requires Arc synthesis; moreover, decreases in surface AMPARs caused by NMDAR dependent LTD does not rely on Arc-induced prolong changes in AMPAR endocytosis rates (Waung et al., 2008). However, NMDAR dependent LTD mediated through low frequency stimulation is impaired in Arc knockout (Plath et al., 2006) and overexpressing (Rial Verde et al., 2006) mice. A possible explanation might be that more dramatic changes in Arc levels (knockout or overexpression) affect both NMDAR and mGlu dependent LTD, whereas group I mGlu dependent LTD is sensitive to smaller activity-evoked changes (Bramham et al., 2010).

It has been demonstrated that shortly after accomplishing a learning task, corresponding brain regions show increased expression of Arc within the nuclei (Guzowski et al., 1999). Arc protein expression is necessary for forming long-lasting

memory, but not short-term memory (Guzowski et al., 2000). Even a single brief experience in learning tasks increases Arc gene expression (Montag-Sallaz and Mon- tag, 2003; Miyashita et al., 2009). In contrast to other immediate early genes, Arc gene expression seems to be most strongly induced by hippocampus-dependent spatial learning (Guzowski et al., 2001). It has been shown that Arc gene expression in the hippocampus and anterior cingulate cortex is required for memory consolidation of inhibitory avoidance training (McIntyre et al., 2005) and in the hippocampus and lateral amygdala for contextual and Pavlovian fear conditioning, respectively (Ploski et al., 2008; Czerniawski et al., 2011). By a variation of hippocampus-dependent and -independent memory tasks, the importance of the immediate early gene Arc as a cellular marker for activity-dependent elevations in neuronal excitability has been shown.

In summary, Arc gene expression is highly regulated by experience. It is thus a valu- able tool for the assessment of neuronal activity resulting from learning and of changes in neuronal excitability in disease states.

5.4.2. Consequences of altered neuronal activity in the animal model and in schizophrenia patients

The finding that GABAR, GluN2B, D1, and mGlu1 expressions are altered in the MK801 model of psychosis suggests that excitability levels may increase in the hippocampus following MK801-treatment. Thus, spatial learning-dependent neuronal activity was assessed following MK801 application. Strikingly, it was observed that basal hippocampal Arc gene expression is increased in MK801-treated animals. In contrast, novel spatial exploration conducted either 1 or 4 weeks after treatment failed to elicit further enhancement in Arc gene expression. In other words, neuronal activation is elevated in MK801-treated animals even in the absence of hippocampus-dependent learning.

Although Thomsen et al. (2010) have demonstrated that the response of Arc gene expression to treatment with the NMDAR antagonist PCP is age-dependent, several other studies show enhanced Arc gene expression after application of NMDAR antagonists similar to the results of the present study (Nakahara et al., 2000; Gotoh et al., 2002; Thomsen et al., 2009, 2010). However, none of these examined changes in

Arc gene expression in light of behavioural tasks in an animal model of first-episode psychosis, as was the case in the present study.

In line with the finding of receptor alterations in the MK801 animal model that might increase neuronal excitability and lead to deficits in interneuronal inhibition (cf. section 5.3.2), these increases in Arc gene expression in the MK801 psychosis model could reflect excessive neuronal activity that disrupts hippocampal information processing. The striking finding that Arc gene expression and therefore basal activity in the MK801 animal model is enhanced and unchanged after spatial learning, is paral- leled by findings from other studies that suggest that increased neuronal excitability accompany schizophrenia:

Firstly, enhanced neuronal excitability was demonstrated in the maternal immune activation (Savanthrapadian et al., 2013) and the antimitotic agent methylazoxymeth- anol acetate (Sanderson et al., 2012) animal models of psychosis.

Secondly, genetic analysis of schizophrenia patients revealed that single nucleotide polymorphisms in the gene set of voltage-gated cation channel are correlated to poor working memory performance (Heck et al., 2014), signifying a linkage between ion channels that control neuronal excitability and schizophrenia symptoms.

Thirdly, human studies demonstrated an elevated blood flow in the hippocampus of schizophrenia patients at rest (Medoff et al., 2001) as an indicator of higher neuronal activity. Strikingly, it was able to restore the blood flow by sufficient antipsychotic drug treatment.

And fourthly, functional MRI studies have provided further evidence (Whitfield- Gabrieli et al., 2009). In these studies, the default mode network, a network that is normally activated at rest and suppressed during brain activity, is examined in schizophrenia patients and healthy controls at rest and during performance of working memory tasks. Healthy subjects exhibit a significantly higher suppression of the default mode network while performing tasks in comparison to schizophrenia patients. This observation is correlated to better working memory performance. A lack in normal suppression might indicate impaired cortical inhibition and enhanced neuronal excitability in schizophrenia.

The results of Arc gene expression in the present study have not only indicate that elevated basal neuronal activity, but also deficits in further recruitment during spatial

learning occur. Strikingly, LTP that is believed to comprise a cellular correlate of learning and memory is disrupted in this animal model (Manahan-Vaughan et al., 2008a, b; Wiescholleck and Manahan-Vaughan, 2012, 2013a, b). It could be argued that irreversible NMDAR antagonism results in impaired LTP even if the number of NMDAR does not change. In contrast to this, the GluN1 subunit has a half-life of only 34 h and the GluN2 subunits even less (Huh and Wenthold, 1999). Since the impairments persist for up to several weeks following MK801-treatment (Manahan- Vaughan et al., 2008a, b; Wiescholleck and Manahan-Vaughan, 2012, 2013a, b), the NMDAR antagnonism on its own is not sufficient to explain these effects. Further- more, LTP in the dentate gyrus is not exclusively NMDAR-dependent (Manahan- Vaughan et al., 1998), thus NMDAR antagonism may not necessarily result in a complete failure of LTP. Therefore, it is more likely that NMDAR antagonism pro- vokes changes in NMDAR subunit expression that may contribute to a change in plasticity-related receptor homeostasis. In turn, this may enhance hippocampal activity and neuronal excitability and result in a saturation of synaptic plasticity (Moser and Moser, 1999). Moser and Moser (1999) have shown that hippocampus- dependent learning can be blocked by saturation of hippocampus LTP prior to training. Since an increase in LTP probably mirrors further recruitment of synapses (Pe- tersen et al., 1998; Moser and Moser, 1999), ongoing increases in LTP could be limited by the availability of naïve synapses. However, it is supposed that not every single synapse must be maximally potentiated until a ceiling effect might appear, since the effect of saturation of population LTP is likely to follow a sigmoidal function (Barnes et al., 1994), so that already an enhancement of excitation at about 30% might be sufficient to block further population LTP (Moser and Moser, 1999). In other words, even if excitability is elevated, there might be only little space left for potentiation of synaptic strength. Strikingly, negative deflections, in the sense of LTD, are inducible (Manahan-Vaughan et al., 2008b) or presumably facilitated, because of the resulting larger range from the elevated basal state and enhancements in GluN2B expression.

A B

Figure 17: Ceiling effect impairs increasing neuronal excitability A: Physiological baseline levels of neuronal excitability allow LTP sustainment. B: Increased baseline level of neuronal excitability impairs sustainment of LTP.

Strikingly, deficits in synaptic strengthening and plasticity have been observed in both the MK801 animal model and schizophrenia patients. In the animal model, the ability to induce hippocampal LTP is disrupted in vitro and in vivo in short- and long-term following MK801-treatment (Manahan-Vaughan et al., 2008a, b; Wiescholleck and Manahan-Vaughan, 2012, 2013a, b). Studies with anodal transcranial direct current stimulation have shown that LTP-like plasticity is impaired in schizophrenia patients according to the length and severity of disease (Hasan et al., 2011). This impaired synaptic plasticity in schizophrenia is validated in studies with transcranial magnetic stimulation indicating an inability to modulate excitability and a loss of synaptic adaptiveness and plasticity (Voineskos et al., 2013).

This in turn would explain why not only LTP but also hippocampus-dependent learning is impaired in the MK801-model (Manahan-Vaughan et al., 2008a, b; Wiescholleck and Manahan-Vaughan, 2012, 2013a, b) and why cognition is impaired in schizophrenia patients (Keefe et al., 1997; Nuechterlein et al., 2004; Ranganath et al., 2008; Ragland et al., 2009). Likewise, PET studies of schizophrenia patients have demonstrated an inability of further hippocampal recruitment while performing learning tasks (Heckers, et al., 1998; Weiss et al., 2003). Similarly, recent optogenet- ics studies have shown that elevated excitation, but not elevated inhibition in the PFC, led to impaired cognition and social behaviour (Yizhar et al., 2011).

5.5. The hippocampus and prefrontal cortex – key structures in the occurrence of schizophrenia?

After schizophrenia was first described as dementia praecox, cognitive deficits were focused on as a main characteristic of this disease (Kraepelin, 1919). It became apparent that memory impairments, deficits in executive function and inattention have a very great impact on daily life and do not sufficiently respond to treatment (Green, 1996). Patients suffering from schizophrenia demonstrate impairments, especially in declarative memory (Ranganath et al., 2008; Ragland et al., 2009), but also in working memory (Keefe et al., 1997) and processing speed (Nuechterlein et al., 2004). Even subjects with at ultra-high risk of psychosis that have a genetic risk plus a decrease in mental state/functioning, but not acute psychosis (Brewer et al., 2005), and unaffected relatives of schizophrenia patients (Whyte et al., 2005) show poorer memory performance. Against the background of these cognitive deficits in declarative and working memory and executive functions, the PFC and hippocampus became the centre of interest.

By means of several different techniques, alterations in morphology have been shown to occur more frequently in the hippocampus of schizophrenics than in any other brain region (Steen et al., 2006). These alterations are even evident in patients after first-episode psychosis and ultra-high risk subjects (Velakoulis et al., 2006), and progress in disease (Chakos et al., 2005). Furthermore, PET studies revealed a correlation between altered metabolic activity in the hippocampus and schizophrenia symptoms (Molina et al., 2003). Similarly, high-resolution studies illustrated an increased basal perfusion in the hippocampus of schizophrenics correlated to the mag- nitude of psychosis (Lahti et al., 2006) and, strikingly, antipsychotic treatment reduces hippocampal perfusion parallel to schizophrenia symptoms (Medoff et al., 2001). These functional alterations in schizophrenic subjects are consistent with the evidence of post-mortem studies which show genetic mutations and alterations in protein expression (Harrison, 2004).

In this study, chronic alterations in the GABAB and D1 receptors and the NMDAR subunit GluN2B were observed in the dentate gyrus. These changes in receptor expression should enhance neuronal excitability and affect synaptic plasticity. The dentate gyrus provides pattern separation and thereby a separation of important and un- important information (Bakker et al., 2008). Thus, enhanced excitability in the den-

tate gyrus might diminish its filter function, allow amplified passing of low- frequency encoded information, and favour pattern completion. Furthermore, Arc gene expression was enhanced in the dentate gyrus at rest, but not after performing the learning paradigm. Thus, these impairments in the dentate gyrus seem to lead to an over-encoding of irrelevant information that in turn might result in impairments in learning and memory and provoke delusional mood (Tamminga et al., 2010).

In addition to hippocampal changes, the PFC often exhibits alterations in schizophrenia patients. MRI and post-mortem dissections revealed reduced grey matter volume in the PFC of patients compared to healthy subjects (Fornito et al., 2009). Furthermore, functional imaging illustrated a reduced ability to mobilise prefrontal regions in relevant cognitive tests (Knable and Weinberger, 1997). Nuclear magnetic resonance spectroscopy studies have demonstrated metabolic alterations in the PFC even in subjects with clinical at-risk syndromes including positive and negative symptoms as well as impairment of various cognitive domains, but no acute psychosis (Jessen et al., 2006). The reason for these functional changes might be based on genetic alterations, as well as on changes in several excitatory and inhibitory neuro- chemical systems that in turn might be responsible for dendritic spine pathologies (Bennett, 2011).

Similar to the results obtained in the dentate gyrus, a chronic increase of GABAB and D1 receptors occurs in the PFC in the MK801 animal model. Alterations in both receptors are believed to affect neuronal excitability. Studies have shown that attention is dependent on the activation level of the PFC (Pezze et al., 2014). Both, an increase and decrease in prefrontal activation can impair attention. Therefore, these alterations in prefrontal receptor distribution are likely to lead to deficits in executive function.

5.6. Prefrontal-hippocampal interplay

The brain is a complex organ that is highly interconnected within its numerous areas. Since schizophrenia is characterised by positive, negative, and cognitive symptoms, it is believed to be mediated not by one single structure, but by the interplay of different regions. As discussed previously (cf. section 5.5), the hippocampus and PFC seem to be central in schizophrenia pathology. It is well known that the hippocampus is connected with the PFC via multiple channels (Goldman-Rakic et al., 1984), an

interaction that is postulated to adjust memory processes in order to obtain remote goals (Churchwell and Kesner, 2011).

For the purpose of elucidating the role of this connection in psychosis, it has been shown that acute systemic MK801-treatment in rodents is sufficient to induce long- term enhancement of hippocampal-prefrontal synaptic response (Blot et al., 2013). In addition, ventral hippocampal stimulation at 20 Hz and 40 Hz is able to generate an enduring disinhibited prefrontal local field potential in animals treated with MK801 in periadolescence (Thomases et al., 2013). A tight balance in the excitatory hippocampal drive to prefrontal neuronal circuits appears to be essential for proper functioning since enhancements as, for example, in NMDAR antagonism animal models (Blot et al., 2013) as well as reductions as, for example, in animal models of neonatal ventral hippocampus lesion (Tseng et al., 2009) are effective in enhancing prefrontal response by postsynaptic structural and molecular rearrangements. Furthermore, Sig- urdsson et al. (2010) have shown in a genetic mouse model of psychosis that these mice show reduced hippocampal-prefrontal synchrony while performing a learning task measured via phase-locking of prefrontal cells to hippocampal theta oscillations and by coherence of prefrontal and hippocampal local field potentials.

Since it has been shown that direct infusion of MK801 into the hippocampus, but not PFC, is effective to increase corresponding pyramidal cell activity (Suzuki et al., 2002; Jodo et al., 2005), alterations in hippocampal activity might be responsible for changes in prefrontal receptor distribution. Hippocampal hyperexcitability, as shown in the present study, might result in increased output to the PFC that possibly pro- vokes adaptive changes in the PFC and thereby compounds deficits in hippocampus- dependent long-term memory.

5.7. The role of γ-oscillations in cognitive impairments

The results of the present study indicate that alterations in interneuronal NMDAR function may occur that produce insufficient inhibitory modulation of pyramidal cells and this in turn might result in enhanced basal pyramidal excitatory output and an inability of further recruitment during learning. Besides deficits in synaptic plasticity, enhanced basal activity might result in impaired functional performance by desynchronisation of the principal neurons’ activity (Nakazawa et al., 2012). Syn-

chronisation of neuronal activity at γ-band frequency is believed to be essential for proper cognitive function, thus alterations in cortical γ-oscillations might cause cognitive deficits in schizophrenia (Lesh et al., 2011). Cortical circuits of fast-spiking parvalbumin-positive GABAergic interneurons with pyramidal cells are believed to be a neuronal substrate of γ-oscillations (Gonzalez-Burgos and Lewis, 2012). Fur- thermore, schizophrenia patients exhibit enhanced γ-power at baseline, but reduced γ-stimulus-to-baseline ratios in tasks engaging the frontal cortex (Gandal et al., 2012). Further evidence of the significant role of fast-spiking interneurons in disturbances in γ-oscillations comes from conventional (Belforte et al., 2010) and optogenetic (Carlén et al., 2012) knockdown studies of interneuronal NMDAR: both have shown significant enhancement of baseline γ-oscillations and deficits in various behavioural paradigms. Strikingly, further induction of γ-oscillations by optogenetic stimulation was impaired in these animal models indicating a ceiling effect (McNally et al., 2013) comparable to the findings of no further recruitment of Arc gene expression during spatial learning. Interestingly, since enhanced γ-band electroencephalog- raphy recordings have been detected in schizophrenia patients while suffering from psychosis and auditory hallucinations, it has been proposed that reduced basal γ- oscillations might contribute to impaired cognitive function by deficits in communication between brain regions, but abnormally elevated γ-oscillations might cause positive symptoms by an overinterpretation of misleading signals (McNally et al., 2013).

All in all, there is substantial evidence of an altered function of parvalbumin-positive interneurons resulting in pyramidal cell hyperactivity. These changes in neuronal excitability are speculated, on the one hand, to enhance basal γ-oscillations, but, on the other hand, to impair evoked γ-oscillations and LTP by a possible ceiling effect. Taken together, this hypothesis could explain the observed deficits of synaptic plasticity and learning and memory in the MK801 animal model.

5.8. New treatment strategies for schizophrenia

Schizophrenia treatment is mainly based on pharmacological and psychotherapeuti- cal/ psychoeducational strategies. As outlined above (cf. section 1.1), both first and second generation antipsychotic drugs that block the D2 receptor are quite insuffi-

cient in the treatment of negative and cognitive symptoms and exhibit enormous risk for side-effects (Ellenbroek, 2012). Currently, several new therapeutic strategies are in development that are in accordance with the results of the present study.

Firstly, pharmacological approaches are implemented that focus on receptor modulation that is comparable to the present study: On the one hand, a blockage of the glycine transporter GlyT1, in order to prevent the removal of the NMDAR co-agonist glycine, might be promising, as it has been effective in the rescue of deficits in synaptic plasticity and learning and memory in MK801-treated rodents (Manahan- Vaughan et al., 2008b). On the other hand, initial studies with the D1 receptor agonist dihydrexidine (DAR-0100, phase II) have proven tolerability and shown significant enhancement in prefrontal perfusion in schizophrenia patients (Mu et al., 2007). Moreover, the compound stepholidine might be a potential drug in schizophrenia treatment because it exhibits dopamine D1/5 agonist as well as D2 antagonist charac- teristics as an extension of current pharmacological profiles (Jin et al., 2002).

Secondly, approaches have been developed that are aimed to target receptor expression. Similar to GABAergic alterations in the present study, it has been proposed that in schizophrenia promoter hypermethylation in GABAergic interneurons might cause aberrant gene expression of GAD67, GABA transporter 1 and NMDAR subunits (Costa et al., 2006). Strikingly, studies have demonstrated that valproic acid can reverse GAD1 promotor hypermethylation in rats and restore GABAergic function (Dong et al., 2007).

Thirdly, new non-invasive treatment options have been developed that focus on neuronal excitability. Underlining a deficit in synaptic plasticity as a core pathology in schizophrenia, tDCS seems to be potent to reduce certain positive (Brunelin et al., 2012; Shiozawa et al., 2013), negative (Palm et al., 2013) and even cognitive symptoms (Göder et al., 2013). Furthermore, there are initial reports that repetitive TMS applied to the dlPFC of schizophrenia patients can improve working memory by generation of γ-band oscillations (Barr et al., 2013).

On the whole, much effort has to be put into further development of new treatment strategies, since available drugs at present are insufficient in restoring cognitive function. Recently, several new strategies have been implemented, but have not yet brought the desired clinical success.

5.9. Limitations and remaining questions

The current study provides novel insights into how alterations in receptor expression might change activity levels in an animal model of first-episode psychosis. These results might explain deficits in synaptic plasticity and the emulation of positive, negative, and cognitive deficits.

However, this study has some limitations. First of all, in the experiments, rodents were used in order to model a psychiatric disorder. Against the background of limitations in human post-mortem and in vivo studies, animal models seem to be a significant complementary approach. However, they are not able to reproduce the entire schizophrenia pathology. Although there is compelling evidence that the MK801 animal model is valid for schizophrenia research, it must be borne in mind that acute antagonism emulates the first-episode of psychosis and not the entire pathogenesis. Recent studies have revealed that in schizophrenia pathogenesis, the periadolescent period is sensitive for functional maturation of prefrontal inhibitory circuits (O’Donnell, 2012). Even though schizophrenia seems to be a neurodevelopmental disease, the animal model used here emulates at least the first-episode of psychosis and subsequently enables examination of mechanistic changes. Thus, the present study has posed new questions as to possible alterations in other brain regions such as the ventral hippocampus, striatum and ventral tegmental area. Further studies could address the early development of schizophrenia, especially in the context of the different roles of neuronal subpopulations.

6. Conclusion

In this study, long-term changes in neurotransmitter receptor expression were found to accompany changes in neuronal excitability in an animal model of first-episode psychosis. In particular, NMDAR GluN2B subunits, GABAA, GABAB, D1, and mGlu1 are affected in a subregion- and time-dependent manner. In parallel, hippocampal basal Arc gene expression is persistently increased, and spatial learning elic- its no further Arc gene expression in MK801 rats, suggesting that chronic changes in hippocampal excitability occur. This may disturb synaptic information encoding and explain why cognition is disturbed in schizophrenia.

The results of the present study suggest that NMDAR antagonism contributes to changes in plasticity-related receptor homeostasis that might affect GABAergic interneurons’ function and result in disinhibition of principal cell function. The delicate interplay of glutamatergic, GABAergic, and dopaminergic receptors required for regular hippocampal function is clearly altered in this animal model of schizophrenia and suggests that effective strategies to treat this condition should aim to modulate the effective functioning of all three receptor groups.

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8. Appendix

1 week Vehicle MK801 90

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Figure A1: GluN1 subunit expression has not changed after MK801-treatment

1 week Vehicle MK801 90

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Figure A2: GluN2A subunit expression is unaffected after MK801-treatment

1 week Vehicle MK801 90 80

70 60

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Figure A3: MK801-treatment does not alter D2 receptor expression

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Figure A4: mGlu2/3 receptors expression has not changed after MK801-treatment

100

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Figure A5: mGlu5 receptor expression remains equal after MK801-treatment

101 Acknowledgments

I am most grateful to my supervisor Prof. Dr. Denise Manahan-Vaughan for guiding me during the last few years. She believed in me and gave me the opportunity to present my work to an international audience. I am thankful for her great support and the frequent lively exchange.

I would like to express my thanks to my second supervisor Prof. Dr. Martin Brüne for his willingness to advise me on my work.

Many thanks also to the Faculty of Medicine for the scholarship which was awarded to me during the research for this thesis. Furthermore, I would like to thank the Inter- national Graduate School of Neuroscience and the Collaborative Research Center 874 for admitting me in the Integrated Research Training Group.

I am indebted to my colleges in the Department of Neurophysiology. This thesis would not have been possible without their support. I particularly extend my thanks to Dr. Verena Aliane and Ute Neubacher who supported my work on the thesis and helped me wherever necessary. I am grateful for their patience and enthusiasm.

There are many other people I need to thank: I owe my gratitude to Juliane Böge, Dimitrula Winkler, Dr. Valentina Wiescholleck, and Jens Klausnitzer for their support and assistance. I would like to thank Hannah Twarkowski for her scientific and personal feedback. Moreover, I would like to thank Nadine Kollosch for animal care and Anke Galhoff for performing gel electrophoresis.

My gratitude to my family and especially Melanie cannot be expressed in words. Their sincere care and love lightened the work on my thesis and beyond.

Curriculum Vitae

THOMAS GRÜTER

PERSONAL DETAILS

Date of birth 18 September 1987 Nationality German

Place of Birth Lippstadt Email [email protected]

EDUCATION

10/2008 – 10/2014 Ruhr University Bochum, School of Medicine

1998 – 2007 Ratsgymnasium Rheda-Wiedenbrück

DISSERTATION

10/2011 – present MD-Project in the Department of Neurophysiology of the Ruhr University Bochum under the supervision of Prof. Dr. Manahan-Vaughan and Prof. Dr. Brüne (LWL University Hospital Bochum)

10/2011 – present Associated membership of the International Graduate School of Neuroscience (IGSN) and membership of the Collabora- tive Research Center 874 “Integration and Representation of Sensory Processes”

10/2011 – present Membership of the Research School (RURS)

PUBLICATION

Grüter, T., Wiescholleck, V., Aliane, A., Manahan-Vaughan, D. Altered neuronal excitability underlies impaired hippocampal synaptic plasticity and learning in an animal model of psychosis. in preparation.

POSTER PRESENTATIONS

Grüter, T., Wiescholleck, V., Aliane, A., Dubovyk, V., Manahan-Vaughan, D. (July 2014). Alterations in neuronal excitability and neurotransmitter receptor expression accompany deficits in hippocampal synaptic plasticity in an animal model of psychosis. in Federation of European Neuroscience Societies, 9th FENS Forum, Milan, Italy

Grüter, T., Manahan-Vaughan, D. Structural alterations in MK 801 treated rats: new insights into the genesis of schizophrenia. (October 2012). in NeuroNRW, Neuro- Visionen 8, RWTH Aachen, Germany

STIPENDS AND PRIZES

10/2013 Awarded the Faculty Prize of the RUB Medical Faculty as best student in clinical studies

10/2012 – 09/2013 Acquisition of a ,,Deutschlandstipendium" from the RUB and the Prosper Hospital Recklinghausen

10/2011 – 09/2012 Acquisition of a scholarship for MD research work from the RUB Medial Faculty

WORK EXPERIENCE

07/2011 – 06/2013 Student assistant in the emergency admission of the St Jo- hannes Hospital Dortmund

11 – 12/2010 Conducting of tutorial sessions on medical negotiation for the Department of Medical Psychology and Medical Sociology

SOCIAL COMMITMENT

2008 – 2012 Working in a voluntary capacity with “ICJA voluntary exchange worldwide”

2007 – 2008 Voluntary Year of Social Service in Brazil organized by ICJA