Epilepsy & Neuropharmacology
How do Anti-Epileptic Drugs Work?
Supervisor: Dr Chris French
Location: Department of Medicine (RMH/WH), Royal Melbourne Hospital
Contact: Dr Chris French T: 8344 3276 E: frenchc@unimelb.edu.au
Despite many years of use and research, it is still not clear how even some of the oldest forms of anti-epileptic drugs work. That which is known is generally based on the effects of these compounds on single neurons, rather than examining how activity of the whole inter-connected neural network of the mammalian CNS is modulated. This project involves studying the effects of AED’s at several levels of organization of the CNS – single channel (voltage-gated sodium, potassium and calcium channels), single neuron, principal neuron/interneuron dynamics, as well as glial cell effects. Patch clamp techniques are used for recording dissociated neuron and neurons in the intact brain slice, and these observations will be extended with high-speed calcium imaging with conventional microscopy as well as multiphoton techniques. This projects affords excellent opportunities for skill development in electrophysiology, pharmacology, advanced microscopy and computational neuroscience.
How do Antipsychotic Drugs Trigger Seizures?
Supervisor: Dr Chris French
Location: Department of Medicine (RMH/WH), Royal Melbourne Hospital
Contact: Dr Chris French T: 8344 3276 E: frenchc@unimelb.edu.au
The treatment of psychosis and schizophrenia has been greatly improved with the use of anti-psychotic drugs such as chlorpromazine, haloperidol and newer drugs such as clozapine. One significant side effect of these drugs is that they tend to lower the threshold for epileptic seizures to occur. The aim of this project is to quantify enhanced seizure activity with this type of drug using the in vitro brain slice technique. Seizure provocation threshold, synaptic transmission and single neuron properties will be assessed using rat hippocampal brain slices after acute application of these drugs.
This project will be a great introduction to basic in vitro synaptic electrophysiology, whole-cell patch clamping and basic pharmacological manipulations to assess dopaminergic activity.
Modelling Epilepsy and Epilepsy Drug Effects-Computational Neuroscience Project
Supervisor: Dr Chris French
Location: Department of Medicine (RMH/WH), Royal Melbourne Hospital
Contact: Dr Chris French T: 8344 3276 E: frenchc@unimelb.edu.au
It is unclear how large scale electrical oscillations in the CNS are produced with epileptic seizures. Simple hyper-excitability of individual ion channel types and abnormalities of synaptic transmission are undoubtedly important. However, at the network level, recurrent excitation and inhibition from interneurons must be crucial, and may explain why some anti epileptic drugs (AED's) produce paradoxical exacerbation of seizures. This project involves modelling small networks (initially just 2 neurons) to examine the dynamics of seizure production, as well as how certain anti-epileptic drugs suppress or occasionally exacerbate network oscillations. This modelling involves incorporating novel experimental data from this laboratory on normal and drug affected ion channel mechanisms, as well as the effect of glial (supporting cells) cell interactions. The program "Neuron" will be mainly used for the simulations. Some programming experience is necessary, but the modelling language is relatively simple. This project provides an opportunity to gain an in-depth understanding of ion channel kinetics and non-linear behaviour of individual neurons and networks, with a strong clinical relevance.
Multi-Electrode Recording in the Rat Brain
Supervisor: Dr Chris French
Location: Department of Medicine (RMH/WH), Royal Melbourne Hospital
Contact: Dr Chris French T: 8344 3276 E: frenchc@unimelb.edu.au
Although immense advances have occurred in recording electrical signals from the CNS, these observations tend to be of single cells in a matrix of many millions of neurons and hence give very limited information about how the whole highly interconnected network functions. One solution to this problem is to use banks of tetrodes, bundles of four 10-20 micron diameter electrodes to record many cells simultaneously, either from a single region or from different parts of the brain. Up to 32 electrodes can be implanted with our system, and sophisticated spike detection and analysis algorithms are available to organize the complex multiple signals recorded. This recording technique can also be easily adapted to exploring epileptiform discharges in models of both focal and generalised epilepsy (including drug effects), which will the main aim of this project. This project provides opportunity to learn cutting-edge electrophysiological and computing analysis techniques for assessment of function of the mammalian nervous system.
ADAM22 and LGI1: role in epilepsy and synapse development
Supervisor: Dr Giovanna D’Abaco, Dr Andrew Morokoff
Location: Deparment of Surgery, Royal Melbourne Hospital
Contact: Dr Giovanna D’Abaco (giovanna.dabaco@mh.org.au
Background: ADAM22 and its ligand LGI1, have been described to play diverse roles in the brain. For instance, ADAM22 knockout mice die early from seizures and ataxia and LGI1 mutations have been found in human subjects with temporal love epilepsy. LGI1 binding to ADAM22 takes place at the synaptic membrane and leads to hyper-excitability mediated by glutamate receptors. Both proteins are expressed highly during development of the hippocampus and cerebellum, however little is known about their exact role. This project aims to explore the effects of ADAM22 and LGI1 on brain an synaptic development using a number of approaches. ADAM22 -/- mice and +/- mice will be compared in panel of behavioural testing as well as histopathological brain assessment. The effect on seizures will be assessed by placing electrodes in the brain of the mice and EEG monitoring for up to 8 weeks. Furthermore, in vitro testing, eg using neuronal and synaptic differentiation assays, as well as stem cell differentiation assays will be performed in the laboratory.
Does a novel mutatin in the rat Cav3.2 T-type Ca2+ channel gene increase burst firing of neurons in vivo in a rat model of genetic absence epilepsy?
Supervisors: Dr. Kim Powell, Professor Terence O’Brien
Location: The Department of Medicine (RMH/WH), The Royal Melbourne Hospital, The University of Melbourne.
Contact: Dr Kim Powell T: 8344 3273 E: kpowell@unimelb.edu.au ,
Professor Terence O’Brien T: 8344 5479 E: obrientj@unimelb.edu.au
Voltage-gated calcium (Ca2+) channels are believed to play a critical role the generation of the hypersynchronous oscillatory thalamocortical activity that underlies absence seizures. Mutations in the Cav3.2 T-type Ca2+ channel gene have been reported in patients with childhood absence epilepsy (CAE) patients. Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are widely used model of absence epilepsy. In this model, Cav3.2 mRNA expression and T-type Ca2+ currents3 are elevated in the reticular nucleus of the thalamus (nRT), and we have shown similar elevations in the cortex. An increasing body of evidence, including from our laboratory, indicates that the seizures in GAERS originate focally in the somatosensory cortex. It is also known that the thalamus plays a critical role in allowing the seizures to occur, the basis of which is pathological oscillatory thalamocortical activity.
Together this data implicates the Cav3.2 channel in the pathogenesis of this disease although whether functional abnormalities in the channel play a causative role in absence epilepsy is unknown. Linking an absence phenotype to a mutation in this channel would provide a priori case for a causative role. To this end we have identified that GAERS carry a homozygous single nucleotide missense mutation in a highly conserved region the III-IV linker domain of the Cav3.2 T-Type Ca2+ gene (R1584P).
Importantly, with our Canadian collaborators, we have shown that this mutation is dependent upon exonic splicing for its functional consequences to be expressed in-vitro (i.e. its requires the presence of exon 25 [Cav3.2 (+25)] to produce significantly faster recovery from channel inactivation and greater charge transference during high frequency bursts). This gain-of-function mutation, the first reported in the GAERS polygenic animal model, has a novel mechanism of action.
The current project will attempt to link this novel mutation with a cellular epileptic phenotype in-vivo. For these in vivo studies adult male F2 progeny of both NEC (non-epileptic control rats)xGAERS and GAERSxNEC double-cross matings who are homozygous (+/+) for the R1584P mutation will be compared to those who do not carry the mutation (-/-).Single-cell juxtacellular recordings of cortical neurons and extracellular field recordings will made in vivo, under neurolept anaesthesia, along with EEG recording of the related sensorimotor cortex. Neuronal firing patterns in the somatosensory cortex and reticular thalamus, between and during seizures, will be compared between animals with and without the mutation. Variables to be examined will include: the firing rate, the burst firing percentage, the number of action potentials per burst and the intraburst firing rate. The location of the recorded cells will be confirmed at the end of each experiment by juxtacellular labelling with neurobiotin.
Neuropsychiatric, neurocognitive, quality of life and bone health outcomes in patients with epilepsy treated with Levetiracetam (Keppra) versus older AEDs as substitution monotherapy (KONQUEST)
Supervisors: Dr Raju Yerra, Dr Marian Todaro, Prof Terence O’Brien
Location: The Comprehensive Epilepsy Program, Department of Neurology, The Royal Melbourne Hospital.
Contact Dr. Raju Yerra E: raju.yerra@mh.org.au ,
Dr Marian Todaro E: Marian.Todaro@mh.org.au ,
Professor Terence O’Brien T: 8344 5479 E: obrientj@unimelb.edu.au
This is an investigator initiated study that aims to compare neurocognitive, neuropsychiatric, Quality of Life (QOL) and bone health outcomes in a cohort of patients with focal epilepsy treated with Levetiracetam (LEV) as the substitution Anti Epileptic Drug (AED) monotherapy, with those in whom carbamazepine (CBZ) or sodium valproate (VPA) are started as first substitution drug. Seizure control and adverse drug effects will be compared as secondary outcome variables. This patient population was chosen as it represents a clinically important common group of patients who have not been adequately studied. Previous studies of LEV and other “new” AEDs have been performed in either medically refractory patients (who have taken multiple AEDs) or newly treated patients. If positive the results of this study will provide evidence for wider use of the new drugs as monotherapy, especially in early stages of epilepsy, and help improve the health outcomes of patients with epilepsy. With the bone health and body composition study we aim to study changes in bone health and body composition with anti epileptic drug therapy. There is increasing concern about the long-term increased risk for fracture and bone disease in patients taking long-term anti-epileptic drugs, and this study aims to determine if the newer drug (LEV) may have an advantage in this regard over the older drugs.
The study population is over 100 patients with epilepsy recruited from the Epilepsy, Neurology and First Seizure outpatient clinics of the Royal Melbourne Hospital who have failed treatment with first AED either, due to lack of efficacy or side effects. The subjects were randomised to treatment with LEV or with CBZ or VPA. If the initial AED was CBZ or PHT the subject will be randomised to LEV or VPA, and conversely if the initial AED was VPA the subject will be randomised to LEV or CBZ. Subjects are then followed for 12 months with assessment of their epilepsy, drug side-effects, mood, quality of life, cognition and bone health.
Evaluation of dynamin inhibitors as novel therapies for epilepsy
Supervisors: Prof. Terence J. O’Brien, Professor Phil Robinson and Dr. Nigel Jones.
Location: The Department of Medicine (RMH), Melbourne, and the Department of Physiology, Children’s Medical Research Institute, Sydney.
Contact: Prof Terence J. O’Brien T: 8344 5479 E : obrientj@unimelb.edu.au,
Professor Phil Robinson E: probinson@cmri.com.au ,
Dr. Nigel Jones T: 8344 6729 E: ncjones@unimelb.edu.au
Background: The group of Phil Robinson at the CMRI have discovered the principle that dynamin modulators can control synaptic transmission. Consequently, they have engineered the first generation of small molecule dynamin inhibitors and have preliminary evidence for their effectiveness as anticonvulsant drug candidates using in vivo models. The GTPase activity of the enzyme dynamin is a novel molecular target for epilepsy. Blocking dynamin produces inhibition of neuronal synaptic vesicle endocytosis (SVE) and reduced synaptic transmission. The common feature of all anti-epileptic drugs (AEDs) is a reduction in synaptic transmission. For most AEDs the mechanistic basis of this reduction is uncertain. In a 2006 publication in Nature Neuroscience Professor Robinson’s group showed that inhibition of SVE by blocking dynamin leads to an activity-dependent run-down in synaptic transmission. The unique aspect of this discovery is the lack of effect on acute or brief bursts of synaptic transmission - being inhibited only after high or prolonged stimulation. We propose that molecules based on SVE inhibition would reflect a new and better AED design, especially in those cases where sufferers fail to respond to or tolerate conventional treatments. SVE inhibition has the unique ability to block sustained neuronal burst firing, as occurs during an epileptic seizure, while allowing normal neuronal transmission to occur under most physiological situations. By targeting only neurons experiencing prolonged or unusually high frequency stimulation, such drugs may have fewer effects in the absence of a seizure thus reducing the risk of many of the side-effects associated with AED therapy.
This project would test one or more of these candidate dynamin inhibitor treatments for anti-epileptic and anti-epileptogenic effects in “true” epilepsy models of generalized genetic (i.e. GAERS) and acquired focal epilepsy (post-status epilepticus and electrical amygdala kindling) to provide data predictive of efficacy for human epilepsies.
Skills: Small animal handling and neurosurgery (electrode implantations), rat electroencephalography recordings, brain perfusion and fixation, brain histological techniques, drug administration and neuropharmacological principles.
Investigating the role of Stargazin and AMPA receptors in contributing to the epileptic phenotype of GAERS
Supervisors: Dr Kim Powell, Dr Jeremy Kennard, Professor Terence O’Brien
Location: Department of Medicine (RMH/WH), Royal Melbourne Hospital
Contacts: Dr. Kim Powell T: 8344 3273 E: kpowell@unimelb.edu.au
Dr Jeremy Kennard T: 8344 3273 E: jkennard@unimelb.edu.au
Professor Terence O’Brien T: 8344 5479 E: obrientj@unimelb.edu.au.
Project Overview: Absence seizures, one of the most common seizure types in humans with idiopathic generalised epilepsy (IGE), are generalised non-convulsive events characterised by recurrent episodes of staring with unresponsiveness. Absence seizures most commonly affect children and adolescents who can experience hundreds of seizures per day and if left untreated can lead to disruptions in learning. Despite the important recent identification of genetic mutations in some rare families with IGEs showing a monogenic inheritance, in the common situation (>95% of sufferers) with complex inheritance patterns the genetic determinants of the absence seizures is still unknown. These epilepsies are presumed to be polygenic, with more than one genetic variation contributing to the phenotype, but the nature of these variations and how they interact to result in epilepsy remains to be determined. GAERS rats are a strain of rats which spontaneously develop generalized absence seizures.
AMPA receptors are ionotropic transmembrane receptors for the excitatory neurotransmitter, glutamate that mediates fast synaptic transmission in the central nervous system. Stargazin, a member of a new family of proteins called Transmembrane AMPA Receptor regulatory Proteins (TARPs), is critical for the trafficking and anchoring of AMPA receptors to synaptic membranes. Stargazin also influences electrophysiological properties of AMPA receptors including the slowing of deactivation and reducing desensitization rates. This newly identified TARP role for stargazin may have major functional implications on the homeostatic balance of neuronal excitation, and potentially for the pathophysiology of epilepsy. Indeed research from our laboratory using a genetic animal model has linked an increase in stargazin expression in the brain to absence epilepsy. Associated with an increase in stargazin expression is an increase in AMPA receptor expression only at the plasma membrane. This would be expected to enhance neuronal excitability and therefore be potentially pro-epileptic.
The specific aims of this project are
- To correlate thalamocortical expression of stargazin with seizure expression.
- To examine for differences in expression (membrane vs. cytosol) of AMPA receptor subunits in juvenile pre-epileptic and adult epileptic GAERS and for association with stargazin expression.
- To determine if stargazin associates with a specific AMPA receptor subunit and if there is a developmental switch in this preference associated with the onset of absence seizures.
- To determine if there is a genetic cause for the increase in stargazin expression.
Skills
The skills expected to be learnt from this project include: Small animal handling and neurosurgery (electrode implantations), EEG recordings and analysis, biochemical and molecular analysis (real time PCR, western blotting, multiplex ligation-dependent probe amplification analysis).
Post traumatic brain injury and epilepsy onset: Imaging the brain to investigate neural circuits and appropriate therapy interventions
Supervisor: Professor Terence O’Brien, Dr Damian Myers, Prof Rod Hicks, Dr Nigel Jones
Location: Department of Medicine, (RMH) and the Centre for Molecular Imaging, The Peter MacCallum Cancer Institute
Contact:
Dr Damian Myers T: 8344 6449/0401 766608, E: damianem@unimelb.edu.au;
Prof Terence J O’Brien T: 8344 5479 E: obrientj@unimelb.edu.au;
Dr Nigel Jones T: 8344 6729 E:: ncjones@unimelb.edu.au
Closed-head traumatic brain injury (TBI) is a common condition that has dramatic and often long-lasting impacts on the patient and their family. The annual incidence of significant TBI in developed countries has been estimated to be 1/1000
One of the dramatic and disabling long-term consequences of TBI is the development of post-traumatic epilepsy (PTE), which occurs in up to 25% of patients with moderate to severe injuries. With penetrating brain injuries the incidence is over 50%.
Epilepsy is defined as the occurrence of recurrent unprovoked seizures and is a prevalent neurological disorder as it affects up to 3% of the population in a lifetime and 0.5-1% at any one time. PTE often has severe morbidity and is difficult to treat as the seizures that develop are highly refractory complex partial seizures.
There is a lack of information about the mechanisms underlying the late epileptic, neurocognitive and neuropsychiatric changes occurring post-TBI. Neuronal plasticity occurring after TBI may explain the altered neuronal circuitry that, potentially, involves multiple cellular processes including neuronal death, axonal sprouting with formation of aberrant circuitry, neurogenesis and altered circuit connectivity caused by both axonal and dendritic plasticity.
The neural changes that occur during the onset and development of PTE are poorly understood so this project has been designed to investigate structural and functional changes that occur in the cortex and hippocampus, key structures of the brain neural network circuitry.
Several projects are available that include techniques such as small animal MRI and positron emission tomography (PET), video-EEG monitoring and histological techniques to investigate neural network changes associated with seizure onset after head trauma; another project area involves the study of neurocognitive and neurobehavioural testing to study the consequences of traumatic brain injury; advanced confocal microscopy and fluorescence imaging techniques.
The following projects have been designed to investigate the progressive neurological changes that occur post-traumatic head injury. The long-term aim is to investigate potential therapies that may protect the neural circuitry immediately after injury. To date, no effective neuroprotective strategies that have significant, long-term, benefits have been developed to treat PTE.
Project 1: A study of the neurocognitive and neurobehvioural changes that occur after closed-head traumatic brain injury;
Project 2: Structural and functional changes in the brain monitored by FDG-PET and MRI after closed-head traumatic brain injury;
Project 3: Post-traumatic brain injury and neurogenesis: Tracking neurological changes in post-traumatic brain injury using advanced fluorescence imaging techniques
These projects will be conducted through the Department of Medicine at the Royal Melbourne Hospital and imaging will be performed at both the Howard Florey Institute and the Centre for Molecular Imaging at the Peter MacCallum Cancer Institute.
Investigations into the role of neuropeptide y in a genetic rat model of absence epilepsy
Supervisor: Prof Margaret Morris and Prof Terence J O’Brien.
Location: Department of Medicine (RMH) and Department of Pharmacology, University of New South Wales.
Contact : Prof Terence J O’Brien T: 8344 5479 E : obrientj@unimelb.edu.au
Professor Margaret Morris E: m.morris@unsw.edu.au
- Absence epilepsy is one of the most common idiopathic generalised epilepsy syndromes. The underlying neurophysiological correlate of absence epilepsy is a pathological activation of rhythmic thalamocortical activity. However, the underlying aetiology for this disorder is still unknown.
- There is increasing evidence that neuropeptide Y has a role in modulating seizures in acquired focal epilepsies, however there has been little investigation of its possible role in generalised epilepsy syndromes.
- This study will investigate the effect of intracerberbal microinfusions of neuropeptide Y into selected intracerebral thalamocortical brain regions on the number and total duration of absence seizure in the Genetic Absence Epilepsy Rats of Strasbourg (GAERS) model. Absence seizures will be quantified on the basis of the SWDs recorded on EEG for 90 minutes following the infusion. The effect of infusion antagonists and agonists of various neuropeptide Y receptors will also be evaluated.
- The second stage of the project will investigate the effect of enhancing NPY expression focally in selected thalamocortical using an recombinant adenovirus viral vector.
Skills: Small animal handling and neurosurgery (electrode implantations, microinjection catheter implantations), rat electroencephalography recordings, brain perfusion, fixation and histological preparation, immunohistochemistry.
A model of functional disconnections to study the pathophysiology of psychosis and epilepsy
Supervisor: Dr Nigel Jones and Prof Terence J O’Brien.
Location: Department of Medicine (RMH)
Contact: Dr Nigel Jones T: 8344 6729 E: ncjones@unimelb.edu.au
Functional disconnections in cortico-thalamo-cortical (CTC) systems, the neuronal circuits of attention, cognition and perception, are thought to underlie dysfunctions of conscious integration such as those seen in schizophrenia. More than 80% of the neurons that make up the CTC systems are glutamatergic. There is considerable evidence to suggest that NMDA-type glutamate receptors are implicated in the pathophysiology of schizophrenia. Non-competitive NMDA receptor antagonists (PCP, ketamine, MK-801), at subanaesthetic doses, induce cognition impairment, schizophreniform psychosis, hallucinations, and exacerbate both positive and negative symptoms in schizophrenic patients. In rodents, ketamine produces a wide spectrum of abnormal behaviour relevant to schizophrenia. The neuronal mechanisms underlying transient disruption in NMDA receptor function remain to be determined. CTC circuits generate coherent synchronized gamma frequency (30-80 Hz) oscillations during conscious brain operations. Disruption of cognition-related coherences of gamma oscillations between cortical areas is a major functional abnormality in schizophrenic patients.
It has been shown that patients with generalised epilepsy have increased baseline (i.e. between seizures) gamma activity on the EEG compared to non-epileptic control subjects. Work in our laboratory in the Department of Medicine has demonstrated that the Genetic Absence Epilepsy Rats from Strasbourg (GAERS), a well validated animal model of genetic generalized epilepsy, display a range of behavioural and emotional abnormalities that are consistent with those seen in models of schizophrenia-like psychosis. These rats, and their non-epileptic counterparts (NEC rats), have been respectively selectively breed for the presence or absence of the epileptic phenotype. The co-segregation of the psychiatric behavioral and epileptic phenotypes over more than 60 generations suggests an aetiological link between the two. This project will also explore the hypothesis that GAERS have an abnormal response of cortical gamma activity to the administration of NMDA antagonists. If true, this would provide a neurophysiological correlate for the link between the epilepsy and schizophrenic like phenotypes in GAERS.
Note: this project is also listed under Neuropsychiatry and Stress Biology
Antiepileptic drugs and effects on bone health
Supervisor: Dr Damian Myers, Dr Andrew Stevenson, Professor John Wark, and Professor Terence O’Brien.
Location: Department of Medicine (RMH)
Contacts: Dr Damian Myers T: 8344 6449/0401766608 E: damianem@unimelb.edu.au; Dr Andrew Stevenson E: Andrew.stevenson@csiro.au; Professor John Wark T: 9342 7109 E: jdwark@unimelb.edu.au
Recent clinical studies have confirmed that long-term administration of antiepileptic drug (AED) theapies affect bone mineral density (BMD) and incrase risk of bone fracture. Epilepsy is a common neurological disorder typically requiring life-long treatment with neuroactive drugs such as carbamazepine and valproate. The problem of AED-associated bone disease must be addressed. Our research group has developed a model to study AED-induced changes in bone and the emphasis of this project will involve the use of bone protective therapies to overcome the AED-induced bone loss.
The common aim of the projects listed below is to determine whether the loss of bone associated with anti-epilepsy therapies can be prevented by the administration of bone protective therapies. The two protective agents to be tested are bisphosphonate and parathyroid hormone (PTH).
Project 1: AED–induced changes in bone macrostructure, microstructure and bone
strength: AIM: To image and quantify, in in vivo longitudinal studies, the effects of anti-epilepsy drugs on bone using peripheral quantitative computed tomography (pQCT) (for changes in bone macrostructure & strength) and phase-contrast X-ray imaging (PCI tomography to assess bone microarchitecture at high resolution). The two interventions, bisphosphonate and PTH will be assessed on bone parameters; images will be acquired at 8, 16 and 24 weeks.
Project 2: AED-induced changes in measures of bone turnover: AIM: To measure biochemical markers of bone turnover and key metabolic factors in the serum (vitamin D, PTH, osteocalcin, calcium) in our model of AED-induced bone loss and to determine whether the interventions, bisphosphonate or PTH, affect the biochemical outcomes
Project 3: AED-induced changes in macro- and micro-architectural features of bone: AIM: To assess whether the bone-protective agents, bisphosphonate or PTH, inhibit bone remodelling after treatment with the AED. Microarchitectural changes to bone will be imaged using phase-contrast X-ray (PCX) imaging and tomography. These techniques provide high resolution images (in micron range) using X-ray projection-based techniques. These projects involve collaborations with other institutes.
This work will be conducted in the Department of Medicine at the Royal Melbourne Hospital and advanced imaging techniques will be performed in collaboration with the CSIRO Materials Science and Engineering division in Clayton.
Neurodegenerative diseases: Investigation of neuronal circuit activity using fluorescence imaging combined with electrophysiology
Supervisor: Dr Damian Myers, Dr Gareth Moorhead, Dr Chris French and Dr Chris Ryan, Department of Medicine (RMH/WH), University of Melbourne, The Royal Melbourne Hospital, CSIRO Materials Science and Engineering.
Location: Department of Medicine (RMH/WH), Royal Melbourne Hospital and the CSIRO Materials Science and Engineering Division in Clayton
Contact:
Dr Damian Myers T: 8344 6449/0401 766608 E: damianem@unimelb.edu.au
Neurodegenerative disorders such as Alzheimer’s disease, stroke, brain tumors, epilepsy and traumatic brain injury have long-lasting impacts on patients and their families. Dysregulation of neuronal networks caused by such disease processes underlies the neurocognitive, neurobehavioural and neuropsychiatric changes that occur and typically involves altered neuronal circuit activity in the hippocampus and/or the thalamocortical circuits. The capacity of neuronal circuits to change and the subsequent altered function of neurons is termed neuronal plasticity. In the laboratory, this can be assessed using fluorescence-based microscopy combined with electrophysiology.
The aim of this project is to design and implement an electrode array for investigation of the perforant pathway in the hippocampus. This novel approach to study neuronal changes will be used in future studies to define hippocampal neuronal network activity in normal and disease states. A key component of this project will be validation of the electrode array measures using a traditional field recording approach. Electrophysiological recordings and neuronal activity will be combined to assess temporal changes in electrophysiological output with cell activity based on calcium ion transients measured using calcium ion-sensitive fluorescent probes.
Techniques to be used in this project include in vitro slicing of brain for the study of neuronal circuit activity and monitoring of neuronal activity using Ca2+-sensitive fluorescent probes with fast sensitive CCD cameras. An electrode array will be developed with validation and preliminary experiments performed using field recordings as described previously.
This project will be conducted through the Department of Medicine at the Royal Melbourne Hospital and the CSIRO Materials Science and Engineering Division in Clayton.
Investigation of the role of Y receptors in the seizure suppression effect of valproate in a rat model of genetic genealised epilepsy
Supervisors: Prof. Terence O’Brien and Prof. Margaret Morris
Location: The Department of Medicine, The Royal Melbourne Hospital and The Department of Pharmacology, The University of New South Wales.
Contacts: Professor Terence O’Brien T: 8344 5479 E: obrientj@unimelb.edu.au ,
Prof. Margaret Morris: E: m.morris@unsw.edu.au
Description: Valproate is the drug of choice for treatment of primary generalised epilepsy, but its mechanisms of action is still uncertain. There is a delayed onset of maximal effect following commencement of valproate treatment, suggesting that upregulation of a secondary messenger may be involved in its anti-epileptic action. Recent work has demonstrated that chronic valpraote administration in rats results in upregulation of expression of neuropeptide Y (NPY) in brain regions critical to the generation of generalisied seizures. We have evidence that NPY has powerful seizure suppression effects in the genetic absence epilepsy rats from Strasbourg (GAERS), a genetic rat model of absence epilepsy, predominantly via effects on the Y2 receptor subtype. This project will investigate if the anti-seizure effects of NPY are mediated through NPY related mechanisms, and if so identify the receptors mediating this effect. A positive outcome of the study may lead to new drugs that more specifically target the epilepsy reducing some of the common undesirable side effects of valproate.
Skills: Small animal handling and neurosurgery (electrode/cannula implantations), rat electroencephalography recordings, drug administration, brain perfusion and fixation, brain histology, immunohistochemistry, stereological neuronal cell counting and analysis techniques.
Sodium Channels in Epilepsy
Supervisors: Dr Chris French, Prof Terence O’Brien
Location: Department of Medicine (RMH/WH), Royal Melbourne Hospital
Contact: Dr Chris French T: 8344 3260 E: frenchc@unimelb.edu.au
Laboratory Overview. The O’Brien Laboratory in the Department of Medicine, University of Melbourne, has a wide range of research activities related to the neurological disorder epilepsy. Projects include molecular biological studies, in vivo and in vitro electrophysiology, advanced imaging techniques, animal behaviour models, pharmacogenomics as well as comprehensive clinical
Project Overview. The project will be to study voltage-gated sodium channels, membrane proteins that are the basis of almost all electrical signaling in the nervous system, and so of the greatest significance in normal function, as well as disease states including epilepsy. Properties of normal channels in rat brain cells and cloned channels in tissue culture will be studied, as well as the effects of common anti-epileptic drugs (AED’s). We are particularly interested in examining how minor genetic variations impact on AED action. Opportunities for mathematical modeling and computational simulations of nerve cell activity are also available.
The project thus offers a very wide range of possibilities for advanced skill acquisition, including molecular biological techniques, patch-clamping and computational neuroscience. Several publications are anticipated. Additionally, a very high priority is placed on basic research skill acquisition, including experimental design and analysis, statistical techniques, familiarity with common molecular biological methods, as well as public presentation of research findings.
The role of Grainyhead-like genes in neural tube deficits induced by valproate
Supervisors: Professor Stephen Jane, Bone Marrow Research Laboratories; Professor Terence O’Brien, Epilepsy and Neuropharmacology Group; Professor Frank Vajda, Epilepsy and Neuropharmacology Group
Location: The Department of Medicine (RMH/WH), The Royal Melbourne Hospital
Contacts: Professor Stephen Jane T: 9342 8641 E: jane@wehi.edu.au
Professor Terence O’Brien T: 8344 5479 E: obrientj@unimelb.edu.au
Professor Frank Vajda E: vajda@netspace.net.au
Valproate is one of the most common medications prescribed for patients with epilepsy, and is the drug of choice for primary generalised epilepsy. Additionally it is commonly prescribed for a number of non-epileptic conditions, such as bipolar disorder and chronic pain. Women chronically taking valproate for these conditions not uncommonly become pregnant despite the fact that it is established to increase the risk of major birth defects, including neural tube defects (NTD). The risk of NTD in a foetus exposed to valproate during the first trimester is greater than that for other anti-epileptic drugs. However, the mechanism by which valproate causes NTD is currently unknown. Research in the BMRL has demonstrated that a highly conserved family of mammalian genes, the Grainyhead family, play essential roles in a range of developmental events including neural tube closure (Nature Medicine 9: 1513-1519, 2003; Science 308: 411-413, 2005). The Grainyhead genes encode transcription factors which act through target genes to mediate their effects. This project aims to determine if these genes play a role in valproate induced neural tube deficit. If successful this model can be used to address a number of clinically important questions, including to design and test interventions (e.g. folate supplementation) or valproate-analogues which may reduce the occurrence of this serious drug-induced teratogenicity. The study methodology will involve feeding breeding pairs of transgenic or control mice expressing different levels grainyhead-like proteins to one of two different treatments: (i) 4g/kg of chow feed – a dose established to produce clinical relevant blood levels in mice – prior to and during pregnancy; (ii) normal chow feed. Two groups of control wild type mice will be feed each of the diets respectively. The litters will be examined for the incidence and nature of neural tube defects in the pups.
Epigenetic regulation of gene expression in epilepsy
Supervisors: Dr Nigel Jones, Professor Terence O’Brien, Dr Kim Powell
Location: Department of Medicine (RMH/WH), Clinical Sciences Building, Royal Melbourne Hospital, University of Melbourne.
Contact: Dr. Nigel Jones T: 8344 6729 E : ncjones@unimelb.edu.au
Prof. Terence O’Brien T: 8344 5490 E: obrientj@unimelb.edu.au
Dr. Kim Powell T: 8344 3273 E: kpowell@unimelb.edu.au
Background: Epigenetics describes the way chromatin/DNA structure can influence the gene expression. This relatively new field of molecular biology is well-advanced in cancer research, but has received little to no attention with respect to neurological conditions such as epilepsy. Changes in gene expression are heavily implicated in the disease process of epilepsy (referred to as epileptogenesis) which turns a normal healthy brain into an epileptic brain. Epigenetic alterations are a strong candidate to mediate such gene expression changes. This program seeks to investigate epigenetic changes associated with epilepsy to determine whether such modifications in chromatin structure contribute to epileptogenesis. We are currently focussing on two genes.
Research project 1: Brain-Derived Neurotrophic factor (BDNF).
BDNF is heavily implicated in both epilepsy and neuronal plasticity (a form of neuronal reorganisation thought to be crucial in the development of disease). Previous research has shown also that expression of this gene can be epigenetically regulated to influence learning, and also may be a mechanism by which the ketogenic diet successfully treats epilepsy. This project will examine the chromatin structural alterations (DNA methylation and histone acetylation) at the promoter regions of BDNF and determine their influence on BDNF gene expression in epilepsy, and also explore whether pharmacological modification of these sites can impede/reverse the process of epileptogenesis.
Research project 2: Reelin.
Reelin is a guidance molecule implicated in brain development. It is also implicated in epilepsy, with reelin down-regulation thought to be responsible for pathological hallmarks of the disease, such as dentate granule cell dispersion. This project will examine Reelin expression and DNA methylation at the Reelin promoter region, and relate these changes to alterations in behaviour and seizure frequency in epileptic animals compared with controls.
Skills: Small animal handling; behavioural testing for anxiety/depression related behaviours and cognitive function; animal models of epilepsy; small animal surgery and EEG recording; extensive molecular biology techniques, including real-time qPCR, Western blotting, and techniques specific for epigenetic analysis (Methylation Specific PCR, Bisulfite Specific PCR etc).
Imaging neurogenesis using Magnetic Resonance Spectroscopy
Supervisors: Dr Nigel Jones, Dr Dennis Velakoukis, Professor Gary Egan
Location: Department of Medicine (RMH/WH), Clinical Sciences Building, Royal Melbourne Hospital, University of Melbourne.
Contact: Dr. Nigel Jones T: 8344 6729 E : ncjones@unimelb.edu.au
Dr Dennis Velakoukis E: Dennis.Velakoulis@mh.org.au
Professor Gary Egan E: gary.egan@florey.edu.au
Background: The realisation that the mammalian brain is capable of producing new neurons (a process termed ‘neurogenesis’) stimulated world-wide interest in many scientific disciplines, both with regards to normal brain function, and also a range of disease states. We now know that seizures, the hallmark symptom of epilepsy, stimulate a burst of neurogenesis in both animal models and in human patients. Intense speculation now surrounds the involvement of these newly born cells in the disease process of epilepsy. However, the limits of current technology allow us only to visualize these new cells in post-mortem tissue, making clinical translation of this research difficult. Through the use of advanced in vivo imaging (Magnetic Resonance Spectroscopy - MRS), this project aims to develop and characterize a method of visualizing newly born neurons in the functioning epileptic brain. Parallel studies are also being performed in human epilepsy patients.
Research plan: Seizures are induced in rats using a chemoconvulsant called Kainic acid, an insult known to induce neurogenesis in the brain. One week after the seizure, animals undergo a series of MRI and MRS scans at the Howard Florey Institute small animal imaging facility. The animals are then euthanized, and the brains processed for histological assessment of the extent of neurogenesis in seizure animals and controls. The MRI/MRS signals are processed for the presence of a biomarker using established protocols of our collaborators (Manganas et al, Science, 318:980-5, 2007), and correlated with the histological data.
Skills: Small animal handling; drug injections and the induction of status epilepticus; cardiac perfusions; immunohistochemistry; immunofluorescence; confocal microscopy; Magnetic Resonance Imaging and Magnetic Resonance Spectroscopy.
The impact of over-expression and under-expression of tissue Plasminogen Activator on epilepsy progression in mice
Supervisors: Dr Nigel Jones, Professor John Hamilton, Professor Terence O’Brien
Location: Department of Medicine (RMH/WH), Clinical Sciences Building, Royal Melbourne Hospital, The University of Melbourne
Contacts: Dr. Nigel Jones T: 8344 6729 E : ncjones@unimelb.edu.au
Professor John Hamilton T: 8344 5480 E: jahami@unimelb.edu.au
Professor Terence O’Brien T: 8344 5490 E: obrientj@unimelb.edu.au
Background.
The processes governing the development of limbic epilepsy are not well understood, but a growing body of literature supports the role of inflammatory mediators in this disease process. One such molecule is tissue Plasminogen Activator (tPA), a clinically used clot-busting enzyme which also has profound effects on cellular physiology in brain regions relevant to temporal lobe epilepsy. These effects, including modulation of cognitive processes, and influencing synaptic connectivity, provide strong rationale to promote tPA as a enzyme which may be involved in development of epilepsy.
Research Plan
The current proposal will investigate the role of tPA signalling in a mouse model of temporal lobe epilepsy. Using genetically engineered mice which are bred to either express an abundance of tPA, or a complete lack of tPA, we will determine the direct role of tPA on epilepsy progression. These experiments will incorporate the amygdala kindling model of limbic epilepsy in mice bred in the laboratories of our collaborators. The second aspect of the project will attempt to ascertain the mechanisms by which tPA might influence the progression of disease using immunocytochemical techniques.
Acquired skills will include small animal handling, neurosurgery, amygdala kindling, post-mortem processing, and immunocytochemistry.
Neuronal networks - wired differently in epilepsy?
Supervisors: Dr Verena Wimmer and Dr Steven Petrou
Location: Howard florey Institute, Royal Parade, The University of Melbourne
Contacts: Dr Verena Wimmer T: 8344 1847 E: verena.wimmer@florey.edu.au
Dr Steven Petrou T: 8344 1957 E: steven.petrou@florey.edu.au
GABA receptors play an important role in mediating inhibitory transmission in the brain. One less well know aspect of GABA receptor signalling is their function in the migration of “young” neurons during embryonic development, when GABA receptors guide the neurons to their appropriate position and allow them to make the correct connections with other cells. Our group has generated a mouse model of human epilepsy which is characterized by a mutation in the GABA receptor subunit gamma2. Our mice show the same epilepsy phenotype human patients have, absence seizures. This project aims at revealing changes in the “wiring” of neuronal networks in the epileptic mouse brain due to GABA receptor dysfunction in development. We will use state-of-the-art laser scanning imaging to visualize synaptic connections and find out how the epilepsy mutation has changed their number, type and specificity. These data will be important for our understanding of how genetic mutations affect different aspects of brain function.
Investigating the therapeutic potential of Cav3.2 Ca2+ channel blocking drugs in suppressing absence seizures in a polygenic rat model of idiopathic generalised epilepsySupervisors: Dr Kim Powell, Professor Terence O’Brien
Location: Department of Medicine (RMH/WH), Clinical Sciences Building, Royal Melbourne Hospital, University of Melbourne
Contacts: Dr. Kim Powell T: 8344 3273 E: kpowell@unimelb.edu.au
Professor Terence O’Brien T: 8344 5490 E: obrientj@unimelb.edu.au.
Project Overview
Absence seizures are one of the most common seizure types in humans with idiopathic generalised epilepsies (IGE). Aside from a few genes discovered in rare families where the epilepsy has a monogenic inheritance, the underlying genetic causes of the common IGEs are still largely unknown, but presumed to be polygenic. In an important, well characterised model of IGE with absence seizures, the Genetic Absence Epilepsy Rats from Strasbourg (GAERS), our group has discovered a single nucleotide missense mutation in the highly conserved III-IV linker region of the Cav3.2 T-type Ca2+ gene (R1584P, gcm) which correlates with seizure expression in GAERS double-crossed with NEC rats (F2 generation).
Ethosuximide, a first line drug to treat patients with absence epilepsy, is commonly believed to act via effects on T-type Ca2+ channels. However side effects such as drowsiness, ataxia and blurred vision are common and some patients (20%) are refractory to its effects. Importantly there is some controversy as to whether it truly acts to suppress absence seizures specifically via effects on T-type Ca2+ channels. Our collaborators from Neuromed Pharmaceuticals (Vancouver, Canada) have developed novel selective T-type Ca2+channel antagonists. Two selective Cav3.2 channel blockers were highly effective at suppressing seizures in GAERS compared to vehicle treatment (DMSO) and standard doses of the two drug most commonly used to treat absence seizures in clinical practice, ethosuximide and valproate. Recently it has been shown that a genetic polymorphism in the sodium channel, SCN1A, has an effect on the proportion of two splice variants as well as an effect on anti-epileptic drug dosing.
Therefore the specific aims of this project are:
- To investigate whether the gcm affects the seizure suppression ability of selective Cav3.2 channel blocking drugs in double crossed F2 animals.
- To investigate whether T-type Ca2+ channel antagonists are effective at suppressing seizures when administered intra-cortically or intra-nRT in GAERS and F2 animals, and whether this is influenced by the gcm genotype.
Skills
The skills expected to be learnt from this project include: Small animal handling and neurosurgery (electrode implantations cannula placement, drug administration), EEG recordings and analysis.
Investigating genetic determinants of absence epilepsy in a polygenic rat model of idiopathic generalized epilepsy
Supervisors: Dr Kim Powell, Professor Terence O’Brien
Location: Department of Medicine (RMH/WH), Clinical Sciences Building, Royal Melbourne Hospital, University of Melbourne
Contacts: Dr. Kim Powell T: 8344 3273 E: kpowell@unimelb.edu.au
Professor Terence O’Brien T: 8344 5490 E: obrientj@unimelb.edu.au.
Project Overview
Absence seizures, one of the most common seizure types in humans with idiopathic generalised epilepsy (IGE), are generalised non-convulsive events characterised by recurrent episodes of staring with unresponsiveness. Absence seizures most commonly affect children and adolescents who can experience hundreds of seizures per day and if left untreated can lead to disruptions in learning. Despite the important recent identification of genetic mutations in some rare families with IGEs showing a monogenic inheritance, in the common situation (>95% of sufferers) with complex inheritance patterns the genetic determinants of the absence seizures is still unknown. These epilepsies are presumed to be polygenic, with more than one genetic variation contributing to the phenotype, but the nature of these variations and how they interact to result in epilepsy remains to be determined. GAERS rats are a strain of rats which spontaneously develop generalized absence seizures.
Recent evidence implicates the Cav3.2 T-type Ca2+ channel in the pathogenesis of genetic absence epilepsy, although whether functional abnormalities in this channel play a causative role is unknown. We have previously reported that GAERS (a genetic rat model of absence epilepsy) carry a homozygous single nucleotide missense mutation in the highly conserved III-IV linker region of the Cav3.2 T-type Ca2+ gene (R1584P, gcm) which correlates with seizure expression in GAERS double-crossed with NEC rats (F2 generation). Our collaborative group have also identified two Cav3.2 splice variants in rat thalamus (± exon 25) located only 13 residues downstream from the gcm site and demonstrated that channels containing the +exon 25 splice variant and the gcm are faster to recover from inactivation and have greater charge transference during high-frequency burst firing (as is seen during absence seizures.
The specific aims of this project are:
Skills
The skills expected to be learnt from this project include: Small animal handling and neurosurgery (electrode implantations), EEG recordings and analysis, biochemical and molecular analysis (real time PCR, in situ hybridization, immunohistochemistry).
Using a new mouse model of severe epilepsy to discover new antiepileptic drugs
Supervisors: Dr Chris Reid & Dr Steve Petrou
Location: Florey neuroscience Institutes (Howard Florey Building)
Contact: Dr Chris Reid T: 8344 1954 E: careid@unimelb.edu.au
Dr Steven Petrou T : 8344 1957 E : spetrou@unimelb.edu.au
Dravet syndrome is a severe form of epilepsy that is very difficult to treat and often results in death (http://www.ninds.nih.gov/disorders/dravet_syndrome/dravet_syndrome.htm). Our group has developed a new mouse model of the disease that is based on a human mutation. The mouse has all the major symptoms seen in patients with the disease. Some antiepileptic drugs reduce seizures in patient while others make the disease worse. We want to test these antiepileptic drugs on the mouse to see if they have the same ‘pharmaco-therapeutic’ profile as humans with the disease. This will validate the model potentially making it a powerful tool with which to test new and hopefully more effective antiepileptic treatments for Dravet syndrome.
Stopping Epilepsy before it starts
Supervisors: Dr Chris Reid & Dr Steve Petrou
Location: Florey neuroscience Institutes (Howard Florey Building)
Contact: Dr Chris Reid T: 8344 1954 E: careid@unimelb.edu.au
Dr Steven Petrou T : 8344 1957 E : spetrou@unimelb.edu.au
Idiopathic generalised epilepsy is a common form of epilepsy with a strong genetic component. Advances in gene discovery suggests that genetic profiling will allow us to predict what chance an individual has of getting epilepsy. In an exciting recent discovery our group has shown that the impact of an epilepsy mutation in early brain development can increase the chance of adults having seizures (Chui et al Annals of Neurology 2008). Therefore, if we can stop the impact of the epilepsy mutation in early development we may be able to stop epilepsy from ever occurring. This project has two parts. First, to administer antiepileptic drugs in the early part of brain development and see if we can reverse the impact of an epilepsy mutation. Second, to record early brain activity in a mouse model of idiopathic generalised epilepsy that is based on a human epilepsy mutation. This will determine what may be going wrong with the brain in the early developmental time window. Together, projects outlined here will help devise new therapeutic strategies that may allow us to stop epilepsy from ever occurring in susceptible patients.