Inhibitory regulation of the prefrontal cortex in an animal model of methamphetamine behavioural sensitization: implications for the maintenance of chronic psychoses

Methamphetamine is a potent psychostimulant that can induce psychosis among recreational and chronic users, with some users developing a persistent psychotic syndrome that is indistinguishable from schizophrenia with respect to positive, negative and cognitive symptomatology. Given that methamphetamine psychosis and schizophrenia are characterised by a persistent vulnerability to psychotic relapse, previous studies have placed these similar symptoms in the context of behavioural sensitization, a phenomenon whereby repeat exposure to a stimulus results in a progressively increased behavioural response to that stimulus following a period of abstinence. As such, examination of the neurobiological changes that mediate sensitization to methamphetamine could enable further understanding of the mechanisms responsible for different aspects of psychotic disease states. Inhibitory γ-aminobutyric acid (GABA)-mediated neurotransmission plays an important role in regulating the prefrontal cortex (PFC), with dysfunctional inhibitory control of the PFC believed to underlie certain symptoms reported across psychotic disorders. While GABAergic dysfunction in schizophrenia has been well described, research surrounding PFC GABA-mediated neurotransmission following methamphetamine sensitization has received considerably less attention. The current thesis therefore contributes to this existing body of empirical work by providing a multi-method evaluation of the proteomic, genomic and cellular changes associated with the GABAergic system across global and localised regions of the PFC following sensitization to methamphetamine. The aim of Chapter 2 was to investigate changes to protein expression in the PFCfollowing behavioural sensitization to methamphetamine using label free proteomics withmass spectrometry. Proteomic analysis revealed 96 proteins that were differentially expressedin the PFC of methamphetamine rats, with 20% of these proteins previously implicated in the neurobiology of schizophrenia. Specifically, methamphetamine sensitization downregulatedthe expression of GAD₆₇, parvalbumin and neuroligin2, and upregulated the expression ofgephyrin, key proteins in the regulation of inhibitory neurotransmission, placing suchbiological changes as potential mediators in the maintenance of vulnerability to psychosis. In light of the results from Chapter 2, the aim of Chapter 3 was to determine whethermethamphetamine sensitization altered GABAergic mRNA expression of the PFC. Usingquantitative polymerase chain reaction (qPCR), the mRNA expression of GABA transporters(GAT₁ and GAT₃), GABAA receptor subunits (α3 and β1), together with the GABAB1receptor, were upregulated in the PFC of sensitized rats compared with saline controls. Thesefindings indicate that GABAergic mRNA expression is significantly altered at the pre andpostsynaptic level following sensitization to methamphetamine in the PFC, which could havesignificant consequences on GABA-mediated neurotransmission. Given that the PFC is a heterogeneous structure, with anatomically and functionallydistinct subregions, the aim of chapter 4 was to extend the results of Chapter 3 and examineGABAergic mRNA expression across subregions of the PFC following methamphetaminesensitization, specifically the prelimbic (PRL) and orbitofrontal (OFC) cortices. GAD₆₇,GAD₆₅, GAT₁, GAT₃, VGAT, GABAT, GABAAβ2, GABAB1 mRNA expressions wereupregulated in the PRL while ionotropic GABAA receptor subunits α1, α3, α5 and β2 togetherwith GABAB2 were specifically upregulated in the OFC. These findings suggest thatalterations to GABAergic mRNA expression following sensitization to methamphetamine arebiologically dissociated between the OFC and the PRL, with GABAergic gene expressiondifferentially expressed in a brain-region and GABA-specific manner. Finally, in Chapter 5, the effect of methamphetamine sensitization on GABAergicinterneuronal cell types across the PRL and OFC, as examined through calcium bindingproteins and neuropeptides, was assessed through the use of qPCR. Results indicated thatcalbindin, calretnin, somatostatin, cholecystokinin, and vasoactive intestinal peptide mRNAexpressions were upregulated in the PRL while parvalbumin, somatostatin, cholecystokininand vasoactive intestinal peptide mRNA expressions were specifically upregulated in theOFC. These findings suggest that the OFC and PRL are associated with distinct inhibitorycellular profiles following sensitization to methamphetamine, which could have significantconsequences on inhibitory neurotransmission and neuronal synchronisation. In order todetermine whether the previously identified GABAergic changes co-localized within specificcell types, the last analysis investigated whether GABAergic mRNA expression correlatedwith interneuronal mRNA expression. Altered GABA neurotransmission within parvalbumincontainingneurons was indicated by the correlation of parvalbumin mRNA expression withthe expression of GAD₆₇ and GAT₁. The mRNA expressions of GAD₆₇, GAD₆₅ and GAT₁ also correlated with the expression of cholecystokinin. While the expression of GABAAα1and GABAAα5 correlated with the expression of somatostatin in the OFC, no othercorrelations between GABAA receptor mRNA and interneuronal markers were identified inthe OFC. Therefore, altered GABA neurotransmission mediated by ionotropic andmetabotropic receptors in the OFC may be localized to glutamatergic pyramidal cells. Overall, the results of this thesis indicate that GABAergic neurotransmission plays anadaptive role in the PFC following sensitization to methamphetamine, with multipleinhibitory proteins, genes and cellular markers altered following sensitization tomethamphetamine. However, a number of unexpected findings were revealed with respect tothe GABAergic changes typically observed in schizophrenia. As such, these findings providemolecular evidence that schizophrenia and methamphetamine psychosis, at least with respectto the GABAergic system using the METH sensitization paradigm, may be associated withdistinct inhibitory neuropathologies in the PFC. The functional, methodological and clinicalimplications of these results are also discussed throughout the thesis.