EVALUATION OF NOVEL DUAL-HIT MODELS OF ‘SCHIZOPHRENIA-LIKE’ SYMPTOMS IN THE RAT PHILIP LAURENCE ROY GASKIN, MPharmacol. Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy JULY 2014 Abstract Schizophrenia is a debilitating disorder comprising positive, negative and cognitive deficits with a poorly-defined neurobiological basis. Animal models with greater translational reliability and validity are essential to develop improved therapies and aid understanding of disease aetiology. This thesis utilised the well-established isolation rearing developmental disruption model of schizophrenia in the rat as the base for producing novel ‘dual-hit’ combination models of the disease, with the aim of improving disease validity and model robustness. Pharmacological insults were added to the isolation rearing model, first in the form of prenatal administration of the antimitotic agent methylazoxymethanol (MAM), and subsequently perinatal treatment with the N-methyl-D-aspartate receptor antagonist phencyclidine (PCP). The resulting ‘dual-hit’ models were assessed for behavioural and neurobiological validity to schizophrenia, and the incurred deficits challenged with the atypical antipsychotic risperidone and the putative adjunct therapy lamotrigine. Combination of isolation rearing and prenatal MAM on gestational day 17 did not produce more robust behavioural deficits than isolation rearing alone, but did cause marked reductions in hippocampal volume, akin to those observed in the clinic. Addition of perinatal PCP treatment on post-natal days seven, nine and eleven to the isolation rearing protocol produced more robust behavioural deficits, with limitations. Baseline hyperlocomotion in a novel arena in three cohorts was accompanied by an elevated locomotor response to acute PCP treatment, highlighting sensitization. Visual and spatial learning deficits were i observed in the novel object discrimination task, whilst fear-motivated conditioning was impaired in a conditioned emotional response paradigm. Preattentional processing was also somewhat deficient in combination-treated animals in the prepulse inhibition of acoustic startle paradigm. Inconsistent deficits in visuo-spatial learning and cognitive flexibility were observed in a Morris water maze task. Acute treatment with the atypical antipsychotic compound risperidone at 0.5mg/kg caused marked sedation. At lower doses, pretreatment 30 mins prior to behavioural testing elevated prepulse inhibition and reversed emotional conditioning deficits, and returned baseline locomotor activity to levels similar to control. There was no effect on visual reference memory deficits. Conversely, pretreatment with the sodium-channel blocker lamotrigine reversed a deficit in visual reference memory, but had no effect on sensorimotor gating or fear-motivated conditioning. These data suggest that the combination of isolation rearing and perinatal PCP treatment to rats produces a model of schizophrenia-like symptoms that possesses some validity to the human condition, but lacks the desired robustness of a preclinical model. Further validation and improvement may allow this model to become a useful tool in on-going preclinical research. ii Acknowledgements Firstly, I would like to thank my supervisors Prof Kevin Fone and Dr Steve Alexander for their constant support and advice throughout my study at the University of Nottingham. Alongside my colleagues and friends Allison McIntosh, Sinead Shortall, Dave Watson, Caitlin Jones, Maddy King, Amy Warner, Stacey Knapp and Ian Topham, the community I have been part of in the School of Biomedical Sciences has been an on-going source of encouragement and assistance that I couldn’t have done without. I would like to particularly acknowledge the contribution that Ian Topham and a plethora of undergraduate students have made to this thesis through assistance with in vivo data acquisition. Additional thanks go to Maria Toledo-Rodriguez for enthusiastically guiding me through the unfamiliar world of ex vivo biochemistry. Outside of the lab, thanks go to all the teammates, coaches and opponents I have taken the field with as part of the Nottingham Outlaws and Nottingham Caesars American Football teams. There are few better ways to clear the mind after a hard day in the lab than banging heads in the trenches. I would like to acknowledge my family. Huge thanks goes to my new wife Mims for putting up with me throughout my PhD study, and always putting a smile back on my face. I’d also like to thank my parents and sister for their unending support during my seemingly endless time at University. This thesis is for each of them, as without them it would not have existed. iii Table of Contents Chapter 1 – General Introduction…………………………………………………….1 1.1 Schizophrenia…………………………………….…………………………..2 1.1.1 Background…..…………………………………….………………………….2 1.1.2 Schizophrenia aetiology……………………………………………………..2 1.1.2.1 Genetics……………………………………………………………………….2 1.1.2.2 Environment…………………………………………………………………..4 1.1.2.3 Genetics x Environment……………………………………………………..7 1.1.3 Neurobiological Basis of Schizophrenia……………………………………...8 1.1.3.1 The Dopamine Hypothesis…………………………………………………..8 1.1.3.2 The Glutamate Hypothesis……………………………………………………9 1.1.4 Treatment of Schizophrenia………………………………………………….14 1.2 Preclinical Research in Schizophrenia…………………………....………16 1.2.1 Locomotor Activity………………………………………………………….17 1.2.2 Locomotor Response to Acute NMDA receptor antagonist…………………18 1.2.3 Novel Object Discrimination………………………………………………...19 1.2.4 Prepulse Inhibition of the Acoustic Startle Response……………………….20 1.2.5 Conditioned Emotional Response…………………………………………...23 1.2.6 Morris Water Maze………………………………………………………….25 1.3 Preclinical Models of Schizophrenia ……….…………………………….27 1.3.1 Isolation Rearing…………………………………………………………….29 1.3.2 Methylazoxymethanol (MAM)……………………………………………...33 1.3.3 Phencyclidine (PCP)…………………………………………………………37 1.3.4 Dual-Hit Models Models…………………………………………………….43 Chapter 2 – Combined Gestational MAM Treatment and Post-Weaning Social Isolation as a Model of ‘Schizophrenia-like’ Symptoms in the Rat………………..46 2.1 Introduction…………………………………………………………….......47 2.1.1 Hypothesis…………………………………………………………………...49 2.2 Materials and Methods…………………………………………………….50 2.2.1 Animals………………………………………………………………………50 2.2.2 Locomotor Activity………………………………………………………….53 2.2.3 Novel Object Discrimination………………………………………………...53 2.2.4 Prepulse Inhibition of Acoustic Startle………………………………………55 2.2.5 Conditioned Emotional Response…………………………………………...56 2.2.6 Morris Water Maze…………………………………………………………..57 2.2.7 Neuroanatomical Analysis…………………………………………………...60 2.2.8 HPLC Analysis………………………………………………………………61 2.2.9 Statistical Analysis…………………………………………………………..64 2.3 Results……………………………………………………………………….65 2.3.1 Locomotor Activity………………………………………………………….65 2.3.2 Novel Object Discrimination………………………………………………...68 2.3.3 Prepulse Inhibition of Acoustic Startle………………………………………72 2.3.4 Conditioned Emotional Response…………………………………………...75 2.3.5 Morris Water Maze…………………………………………………………..78 2.3.6 Hippocampal Size……………………………………………………………81 2.3.7 Hippocampus and Frontal Cortex Neurochemistry………………………...85 2.4 Discussion………………………………………………………………….89 2.4.1 Conclusion………………………………………………………………….104 iv Chapter 3 – Combined Rearing in Social Isolation and Perinatal Phencyclidine Treatment as a Model of ‘Schizophrenia-like’ Symptoms in the Rat……………..105 3.1 Introduction……………………………………………………………….106 3.1.1 Hypothesis………………………………………………………………….107 3.2 Materials and Methods…………………………………………………...108 3.2.1 Animals…………………………………………………………………….108 3.2.2 Morris Water Maze Modifications…………………………………………110 3.2.3 Hyperactivity to Acute PCP Treatment in an Open Field………………….112 3.3 Results……………………………………………………………………...113 3.3.1 Locomotor Activity………………………………………………………...113 3.3.2 Novel Object Discrimination……………………………………………….117 3.3.3 Prepulse Inhibition of Acoustic Startle……………………………………..121 3.3.4 Conditioned Emotional Response………………………………………….124 3.3.5 Morris Water Maze…………………………………………………………126 3.3.6 Locomotor Response to Acute PCP………………………………………..141 3.4 Discussion………………………………………………………………….144 3.4.1 Conclusion………………………………………………………………….153 Chapter 4 – Evaluating the Effect of Risperidone in the Iso-PCP Dual-Hit Model of ‘Schizophrenia-like’ Symptoms in the Rat………………………………………...154 4.1 Introduction……………………………………………………………….155 4.1.1 Hypothesis………………………………………………………………….156 4.2 Materials and Methods…………………………………………………...157 4.2.1 Animals……………………………………………………………………..157 4.2.2 Drug Administration………………………………………………………..157 4.2.3 Behavioural Testing and Statistical Analysis………………………………159 4.3 Results……………………………………………………………………...161 4.3.1 Locomotor Activity………………………………………………………...161 4.3.2 Novel Object Discrimination……………………………………………….165 4.3.3 Prepulse Inhibition of Acoustic Startle……………………………………..169 4.3.4 Conditioned Emotional Response………………………………………….173 4.3.5 Morris Water Maze…………………………………………………………180 4.4 Discussion………………………………………………………………….187 4.4.1 Conclusion…………………………………………………………………193 Chapter 5 – Evaluating the Effect of Lamotrigine in the Iso-PCP Dual-Hit Model of ‘Schizophrenia-like’ Symptoms in the Rat………………………………………...194 5.1 Introduction……………………………………………………………….195 5.1.1 Hypothesis………………………………………………………………….196 5.2 Materials and Methods…………………………………………………...197 5.2.1 Animals……………………………………………………………………..197 5.2.2 Drug Administration………………………………………………………..197 5.2.3 Behavioural Testing and Statistical Analysis………………………………198 5.3 Results……………………………………………………………………...202 5.3.1