From the Department of Neurophysiology of the Ruhr University Bochum Chair: Prof. Dr. phil. habil. Denise Manahan-Vaughan Structural and Functional Alterations in the Cortex in a Rodent Model of First-Episode Psychosis 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 nega- tive symptoms, as well as cognitive and social deficits. To date, the origin and aetiology of schiz- ophrenia 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 glutama- tergic 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 recep- tor 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 unaf- fected 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 psy- chosis. 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 in- sights 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 1 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 2 5.5. The hippocampus and prefrontal cortex – key structures in the occurrence