Description of Facilities
Receptor autoradiography provides information about the localisation and density of specific receptors in the brain. In our centre, receptor autoradiography experiments are routinely performed on various neurotransmitter receptors. This involves the use of radiolabelled ligands to determine the tissue distributions of receptors. We typically do in vitro receptor autoradiography, where the radioligand is applied to tissue after sectioning. Alternatively, ligands can be administered into the circulation (with subsequent tissue removal and sectioning), termed in vivo receptor autoradiography. The ligands are generally labelled with 3H (tritium), 35S or 125I. After incubating tissue with the radioligand, sections are washed and dried. Autoradiographs (images) are then generated, either by exposing tissue to X-ray film or by capturing signals directly with a Beta Imager. Computer assisted analysis programs then allow us to quantify the amount of radioactivity, and hence, receptor density, in a region of interest. We use this technique to determine whether certain treatments or disease states alter the expression of receptors when compared to normal tissue.
G-protein assays give information about the functionality of receptors of interest. GTPgammaS (GTPγS, guanosine 5'-O-[gamma-thio]triphosphate) is the nonhydrolyzable G-protein-activating analogue of guanosine triphosphate (GTP). Its 35S labelled radioligand [35S]GTPgammaS is used in autoradiography and G-protein binding studies in our centre. Normally in G-protein-mediated signal transduction, binding of an activated G protein-coupled receptor (agonist-occupied) to a G-protein stimulates the dissociation of GDP from the a subunit of the G-protein, allowing GTP to bind in its place. The activated G-protein a subunit and the βγ subunits dissociate and interact with effectors such as adenylyl cyclase, phospholipase C and ion channels. The intrinsic GTPase activity of the α subunit hydrolyses GTP to GDP, ending the activation cycle. GTP-γS, however, is a nonhydrolysable analogue of GTP which produces persistent activation of α subunits. The functional coupling between receptors and Gi proteins can therefore be assayed by measuring [35S]GTPγS binding. Currently in our centre we have examined dopamine D2 and cannabinoid CB1 receptors using this assay.
Western Blot is used to detect certain proteins in a specific area of brain tissue by antibody-binding. The protein content of homogenised brain tissue is quantified by measuring the density of a specific antibody bound to the protein of interest. Firstly, tissue samples are homogenised with a buffer which prevents breakdown of the protein content. Each sample is then loaded into a gel and electrophoresis separates the proteins by size. The separated proteins are then transferred to a membrane and labelled with a primary antibody specific to the protein of interest. The primary antibody is then labelled with a secondary antibody, which has an attached enzyme that reacts with a chemiluminescent agent to produce detectable light. The protein content is measured by exposure to light sensitive film and densitometric analysis. In our facility we use this method to detect various disease-relevant proteins such as neuregulin1, ErbB4, NR2B, PSD-95.
ISH allows us to detect and localise a specific gene sequence and its expression in brain tissue. A radiolabelled probe is used to bind to the gene sequence and it is quantified by measuring the radiation intensity in clearly defined areas of the brain tissue sections. For hybridisation, tissue is cut into 14μm slides with a cryostat and mounted on slides. The tissue is fixed to keep the target RNA sequences in place. The radioactive probe hybridises to the target sequence and then the excess probe is washed away. Then, the radioactive probe, bound to the target RNA, gets localised and quantified in the tissue by exposure to radio-sensitive film and analysis is performed with the BioRad gene detection system. In our facility, we use 35S labelled probes to detect disease-relevant genes, including NMDA, trkB, BDNF, NPY.
Polymerase chain reaction (PCR) is a technique that allows us to identify and amplify a particular DNA sequence in the genome so that it can be selectively visualised. Primers (short DNA fragments) being complementary to the target region allow us to identify the sequence and the DNA polymerase serves to multiply the target region. We use PCR in our lab primarily to determine the genotype of our knockout animals, ie identifying the wildtype, Neuregulin-1 or GPCR12 gene or the respective mutated gene sequence.
Human post-mortem tissue is a valuable resource that enables us to examine protein, mRNA and morphological status in disease states compared to controls. Specifically, we use human post-mortem brain tissue from schizophrenia subjects and compare this to controls matched for age, gender etc. We receive our brain tissue from the NSW Tissue Resource Centre and The Stanley Foundation. We implement some of our molecular techniques such as receptor autoradiography, in situ hybridization, western blot and immunohistochemistry to determine the chemical changes that occur in the schizophrenia brain. This allows us to identify targets for further investigations into symptom pathology as well as treatment targets that we can study in our animal models and ultimately in the clinical setting.
Cell cultures are essential in understanding cellular pathways of disease-relevant proteins and their interactions. Immortalised human cell lines are representative of particular human cell types. Primary cell cultures are cultured directly from a brain structure from an animal model (transgenic mouse…) and reflect the health/disease state of this model on a cellular level. Cells of both human cell lines and primary cell cultures are grown and maintained at an appropriate temperature and gas mixture (typically, 37°C, 5% CO2 for mammalian cells) in a cell incubator. After chemical treatment of interest, cells can be fixed and stained with specific markers (fluorescent antibodies for example).
Animal models are essential in preclinical research as they allow us to mimic symptoms of a disease that are similar to the human condition. Animal models can be created in various ways. In our lab, we have genetic, pharmacological, metabolic and environmental rat and mouse models of disease.
Genetic animal models in our lab include Neuregulin-1 and GPCR12 knockout mice. Neuregulin-1 is a major candidate gene for the vulnerability to schizophrenia. Neuregulin-1 knockout mice have been shown before to exhibit a vulnerability to develop schizophrenia-like symptoms, including deficits in cognition and prepulse inhibition similar to what is observed in schizophrenia patients. We examine these mice for neurochemical parameters that could underlie the vulnerability to develop disease symptoms and explore the potential of novel drugs. GPCR12 knockout mice have been shown as an animal model for obesity. Knockout animals show an increased weight gain and hormonal states similar to what is observed in obese patients. We explore neurochemical parameters that could underlie these changes and will further validate this animal model as a potential model for psychiatric diseases.
Our pharmacological animal models comprise treatment with (1) Phencyclidine (PCP), MK801 and Ro-631908 to induce schizophrenia-like symptoms and (2) treatment with AOM to induce colon cancer. Treatment with NMDA receptor antagonists like PCP and MK801 is well established as an animal model for schizophrenia. In our lab, we use both perinatal animal treatment to mimic the development of schizophrenia in humans and chronic treatment of adult animals to explore neurochemical changes underlying schizophrenia as well as a basis to test novel drugs. By treating animals early during brain development with these drugs, we hope to discover possible mechanisms whereby alterations in key brain pathways can lead to disorders such as schizophrenia. We have already found that this treatment alters neurotransmitter expression and behaviour in later life, which has relevance to schizophrenia pathology. To further elucidate the role of NMDA receptors in schizophrenia, we also study the effects of related compounds such as Ro 63-1908 (antagonist of the NR2B subtype of the NMDA receptor) treatment on neurochemistry and behaviour.
Metabolic animal models are created by exposing animals to diets that are known to induce obesity in humans. These are well-established procedures in our lab using high-fat, high-carbohydrate or cafeteria-style diets to investigate the underlying neurochemical mechanisms of obesity development in humans and nutrition-based interventions to reduce weight gain.
Environmental animal models are based on the exposure to environmental stimuli that are known to induce or improve disease states in humans. In our lab, we use enriched environment and social isolation housing of vulnerable animals in early adolescence to mimic developmental factors that influence the genesis of schizophrenia in humans.
Animal models to reveal mechanisms of antipsychotic drugs. These animal modes are created by treatment of rats and mice with various typical (such as haloperidol) and atypical (e.g. olanzapine and aripiprazole) antipsychotics to investigate the neuropharmacological mechanisms underlying their therapeutic efficacy and side-effects. We have established (1) rodent models to study antipsychotic modulation of neural transmissions after short-term and chronic treatments, and (2) a rat model (with olanzapine treatment) to mimic human antipsychotic-induced obesity.
Microdialysis allows determination of chemical factors that serve neuronal communication or its modification in the extracellular space between neurons. To measure neurotransmission in vivo, a microdialysis probe is implanted in the brain area of interest. A semi-permeable membrane allows the sampling of neurochemical factors in the perfusion fluid. Samples gained from the freely moving and behaving animals are analysed via HPLC. In our lab, we focus on the analysis of glutamate, GABA and dopamine.