Faculty of Medicine, Dentistry and Health Sciences Department of Anatomy and Neuroscience

Brock and Furness Labs ~ Research Projects

Information for prospective honours and PhD students, 2010-11...


Our research themes:


On this page you will find:


GENERAL INFORMATION

Principal investigators

All postdoctoral fellows have full time research positions:

 

Associate Professor James Brock

Rm: E238
Ph: 8344 5811
Email: j.brock@unimelb.edu.au
Associate Professor James Brock is expert in vascular biology, electrophysiology and spinal cord injury research.


Professor John B Furness

Rm: E238
Ph: 8344 8859
Email: j.furness@unimelb.edu.au

Professor John Furness has substantial research experience in many facets of neuroscience, especially in immunohistochemistry and organ physiology.

 

Dr Daniel Poole

Rm: E212
Ph: 8344 9994
Email: d.poole@unimelb.edu.au

Dr Daniel Poole is expert in receptor trafficking and second messenger signaling.  He is expert in enteric neurobiology.

 

Dr Tony Frugier

Rm: E240
Ph: 8344 4782
Email: tony.frugier@unimelb.edu.au
Dr Tony Frugier has expertise in all aspects of molecular biology.  He has made significant studies of neuropathological changes in neuronal injury and ischemic damage.

 

Dr Romke Bron

Rm: E202
Ph: 8344 5806
Email: r.bron@unimelb.edu.au

Dr Romke Bron is a molecular biologist examining ghrelin and its roles and consequences of spinal cord injury.

 

Dr Trung Nguyen

Room: E212
Ph: 8344 9994
Email: tvnguyen@unimelb.edu.au

Dr Trung Nguyen is an expert in electrophysiology of enteric neurons and works on the characterisation of ion channels and their regulation in normal and disturbed tissues.

 

Dr Trent Reardon

Rm: E641
Ph: 8344 5797
Email: treardon@unimelb.edu.au
Dr Trent Reardon is a muscle physiologists working on neural control of vascular muscle.



Location

You are welcome to visit us in our lab on the Ground Floor of the East Wing of the Medical (Tri-radiate) Building.


Research program

Our research program combines structural, physiological, pharmacological and neurochemical studies of the enteric nervous system, and visceral afferent (sensory) neurons. Members of our laboratory have backgrounds in molecular biology, physiology, biochemistry, cell biology, pharmacology and physics. We are also involved in drug discovery research in these areas. We have a strong interest in ion channels and their regulation.

Methods

We use a wide variety of methods including immunohistochemistry, confocal microscopy, molecular biology, patch clamp recording, intracellular microelectrode recording, retrograde neuronal tracing, in vitro and in vivo reflex studies, biophysical analysis of neuron properties, whole animal physiology, behavioural testing, and pharmacological analysis in vivo and in vitro.

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Projects 1-3: Autonomic dysfunction in spinal cord injury

To most observers, the dominant impact of spinal cord injury (SCI) is impaired mobility.  However, it is impairment of autonomic nervous system (ANS) function that socially isolates, increases dependence, precipitates hospital re-admission and causes premature death.  We are applying a combination of unique clinical and preclinical approaches to develop novel approaches to treating ANS dysfunction in SCI.  The autonomic centres in the spinal cord are disconnected from central control.

Project 1: Changes in vascular function after spinal cord injury.

Associate Professor James Brock, Mr Hussain Al Dera, Dr Trent Reardon
Cardiovascular disease is the greatest cause of premature death after the initial trauma of injury is overcome.  Loss of blood pressure control means loss of a fundamental of human haemodynamics – maintaining brain blood flow when erect.  Therefore, SCI patients are hypertensive when supine and hypotensive to the point of losing consciousness when erect.  Their blood vessels are inadequately controlled and their physiological states are abnormal.  In this project we are investigating the changes in neural control of blood vessels that occurs after spinal cord injury or local denervation.  In this project you will record muscle tension in isolated vessels and you will conduct a pharmacological analysis of blood vessel responsiveness and nerve control.


Project 2: The effects of spinal cord injury on urinary bladder barrier integrity.

Dr Romke Bron, Dr Tony Frugier, Associate Professor James Brock, Prof John Furness
A mysterious change occurs in the bladder lining following spinal cord injury.  Although the injury is far from the bladder, the lining epithelium (the urothelium) loses its integrity and becomes highly permeable, allowing bacterial invasion of the bladder wall.  Loss of neural influence on the bladder lining and breakdown of barrier function causes elevated rates of urinary tract infections in SCI; this is a major cause of hospital re-admission and loss of functional independence.  In this project you will use techniques of molecular biology and immunohistochemistry to characterise the changes that occur in the bladder after spinal cord injury


Project 3: Regulation of digestive function after spinal injury.

Ms Dorota Ferens, Dr Mark Habgood, Associate Professor James Brock, Professor John Furness
The daily need for assistance with defecation in spinal injured patients is unpleasant, time-consuming and expensive for patient and carer.  Moreover, it does not avert the single most socially crippling consequence of SCI - uncontrolled defecation.  In this project we are studying the mechanisms in the lower lumbar spinal regions communicating with the colon that could be manipulated to allow for controlled, timed and safely reproducible emptying of the bowel by patients.    We have discovered a novel pharmacological method to trigger colorectal emptying.  This project will test in animal models this and other therapies for treating injured patients.  You will learn how to instrument animals and record from them, in vivo.

 

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Projects 4-7: The roles of the hormone ghrelin in autonomic control and the biology of ghrelin receptors

We have discovered that the hormone ghrelin, best known for roles in growth hormone release and stimulation of appetite, has important functions in controlling autonomic organs from the spinal cord level and probably from the brain stem.  In linked projects, we are investigating the roles of ghrelin using in vivo recording from rats, and the distributions of sites of action using in situ hybridisation, reporter mice and activity-dependent labeling of neurons.


Project 4: The cardiovascular effects of ghrelin

Dr Daniela Sartor, Dr Trung Nguyen, Dr Brid Callaghan, Ms Dorota Ferens, Associate Professor James Brock, Prof John Furness
We have evidence that ghrelin affects the cardiovascular system at three levels:  in the brain stem, where ghrelin, through unknown mechanisms, lowers blood pressure, in the spinal cord, where ghrelin acts on sympathetic preganglionic neurons to increase blood pressure, and at peripheral sites  where it dilates blood vessels.  At each site, the pharmacology of interactions with ghrelin receptors differs.  In this project you will examine an aspect of the influence of ghrelin and ghrelin receptor agonists the cardiovascular system

 

Project 5: The molecular basis for ghrelin receptor heterogeneity

Dr Romke Bron, Ms Billie Hunne, Associate Professor James Brock, Prof John Furness

The receptor pharmacology of ghrelin in quite unusual.  Despite there being only one molecularly defined receptor, the effects of agonists at different sites vary considerably.  For example, at growth hormone secreting cells in the pituitary gland, ghrelin is a full agonist, whereas des-acyl ghrelin has no effect.  At some blood vessels, both ghrelin and des-acyl ghrelin are agonists, and one site has been discovered in the spinal cord at which ghrelin is an agonists and des-acyl ghrelin is an antagonist.  In this project you will use methods including laser microdissection to isolate regions of receptor variability and extract protein and mRNA.  The protein will be separated by 2-D gel electrophoresis and immunoreactive proteins will be identified.  If appropriate, these will be subjected to mass spectrophoretic analysis.  mRNA will be characterised by size and sequence.


Project 6: Novel compounds that act at ghrelin receptors: pharmacological and functional analysis

Dr Brid Callaghan, Prof John Furness, Dr Romke Bron, Dr Jonathan Baell, Ms Kung Ban, Ms Dorota Ferens
A range of ligands has been synthesized that act as ligands for the ghrelin receptor.  Many of these have been reported in the patent literature without their biological activities being revealed.  In this project you will collaborate with medicinal chemists to decide on strategies to prepare compounds.  You will test these by pharmacological analysis using transfected cells and isolated tissues.

 

Project 7: Ghrelin receptor distribution revealed in reporter mice

Ms Billie Hunne, Dr Romke Bron, Prof John Furness, Dr Alan Lomax
We have available mice that are engineered to express a fluorescent protein under the control of the promotor for the ghrelin receptor.  Using these mice, you will determine the pathways in which the ghrelin receptor is involved.  The reporter mice will be used to locate sights of electrophysiological recording to investigate the functions and roles of the ghrelin receptors.

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Projects 8-12: Enteric neuropathy:  ischemia-reperfusion injury

The changes caused by ischemia are recognised as important in causing gut dysfunction, but very little is known about effects at a cell level.  We have recently found that effects on neurons are substantial and type specific, for example the nitric oxide neurons are swollen to almost double their normal volume.  Moreover, there is a large, but generally unrecognised, inflammatory reaction associated with ischemia.  A substantial part of the damage appears to be exerted by free-radical formation, including the production of nitric oxide.  In these projects, we are investigating the mechanisms behind the damage, and are using transgenic animals and pharmacological interventions to investigate protection against ischemia-reperfusion injury.

 

Project 8: Consequences of ischemia-reperfusion injury in the human intestine

Dr Tony Frugier, Mr Gene Venables, Dr Daniel Poole, Ms Michelle Thacker, Ms Louise Pontell, Prof John Furness, Dr Mehrdad Nikfarjam, Professor Robert Jones.
This project utilises human small intestine that has been subjected to ischemia and reperfusion.  This project is part of an ongoing research program aimed at understanding the numerous biochemical and molecular changes following ischemia and reperfusion in humans. The availability of human small intestine tissue makes this project unique.  Real-time quantitative PCR, ELISA, Western-blot and immunohistochemical methods are some of the techniques the prospective student will be trained for and use for this project.


Project 9: The reactions of enteric neurons and glia to ischemia-reperfusion injury

Ms Michelle Thacker, Dr Tony Frugier, Mr Gene Venables, Ms Billie Hunne, Ms Louise Pontell, Prof John Furness.
This project uses immunohistochemical methods to investigate the changes that occur in the enteric nervous system when blood flow is interrupted and restored.  This includes investigation of changes in neuronal and glial morphology and chemistry, including cytoskeletal disruption, the occurrence of cell death (using TUNEL staining) and the nitrosylation of proteins.  You will learn how to conduct experimental surgery, methods of multiple labelling immunohistochemistry and confocal analysis.


Project 10: Calcium imaging of enteric glia

Ms Michelle Thacker, Dr Trung Nguyen, Prof John Furness
Enteric glia have important roles in controlling the environments of neurons and restricting the changes in neuronal function that can be caused by accumulation of metabolites or by damage to tissues.  Glial cells react to various factors, including cytokines and inflammatory mediators.  Many of these signal through changes in cytoplasmic calcium.  Enteric glia exhibit morphological changes in responses to stress, but how the stress signals are transduced is unknown.  In this project you will learn how to record calcium signals in living cells and will use this technology to investigate the responses of glial cells to inflammatory mediators and the stress of ischemia/ reperfusion.

 

Project 11: Investigation of the role of nitric oxide in ischemia/ reperfusion injury using knock out mice

Dr Tony Frugier, Mr Gene Venables, Ms Leni Rivera, Ms Michelle Thacker, Prof John Furness
We have good evidence that nitric oxide (NO), that is produced by the enzyme nitric oxide synthase (NOS), has a role in the enteric neuronal damage that is caused by ischemia-reperfusion.  The main evidence is that NOS neurons are selectively affected and there is nitrosylation of proteins in the enteric nervous system.  To test this theory, we will cause ischemia-reperfusion injury to mice in which neuronal NOS has been knocked out and to wild-type mice.  Effects on the swelling of NOS neurons, on the nitrosylation of protein and on cell death will be measured.


Project 12: Intestinal ischemia/ reperfusion injury – protective strategies

Dr Tony Frugier, Mr Gene Venables, Dr Daniel Poole, Prof John Furness
A major problem arising during intestinal transplantation surgery is ischemia/reperfusion (I/R) injury to enteric neurons that results in disordered gastrointestinal function.  This problem could possibly be avoided or reduced by neuroprotective treatment before or at the time of surgery if the nature of the events was better understood and appropriate strategies are developed.  Recent investigations have revealed that nitric oxide (NO) produced by damaged cells may have a key role in causing cell injury.  In this project we will test the protection that results from inhibition of the NO-producing enzyme: nitric oxide synthase (NOS).

 

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Project 13-16: The enteric nervous system and the control of digestive function

Project 13: Changes in digestive function caused by pharmacological targeting of ion channels and receptors

Ms Dorota Ferens, Prof John Furness
The enteric nervous system, which controls digestive tract movements, can generate a range of behaviours of the intestine, from strong propulsive movements to mixing activity.  It would be useful, therapeutically, to be able to switch between movement patterns.  This project investigates the ways in which drugs that target ion channels of enteric neurons can modify behaviour of the organ.  In this project you will learn about recording physiological parameters, gastrointestinal motility, blood pressure and heart rate in an anesthetized rat model.

 

Project 14: Receptor Trafficking in Enteric Neurons of the Inflamed Intestine

Dr Daniel Poole
Many G-protein coupled receptors (GPCRs) are internalized from the cell surface into endosomes in response to agonist binding. As this effectively controls the number of receptors available for subsequent agonist binding, endocytosis and recycling of GPCRs are key regulators of cellular responsiveness to agonists.  During and following periods of intestinal inflammation there are significant changes in intestinal motility and secretion and this is partly attributable to the hyperexcitability of enteric neurons.  Changes in trafficking of receptors expressed by these neurons have yet to be examined. In this project you will examine the distribution and trafficking of receptors in enteric neurons during and after periods of intestinal inflammation and stress, with an emphasis on the neurokinin 1, somatostatin receptor 2A and mu opioid receptors.  Changes in the expression of key regulators of endocytosis will be examined by PCR and Western blot and changes in receptor signaling will be examined by Ca2+ imaging (NK1R) and ERK assays.

Techniques: immunofluorescence, quantitative microscopy, Western blot, PCR, Ca2+ imaging.


Project 15: Effects of Milk-Derived Opioid Agonists on Enteric Neurons

Dr Daniel Poole
The digestion of milk and milk products gives rise to casein-derived peptides (casomorphins), which can act as agonists at opioid receptors, leading to alteration in intestinal motility and secretion.  These peptides may partly underlie milk intolerance in infants and ‘cheese addiction’.  In this project you will examine the effects of casomorphins on receptor endocytosis, using mu, delta and kappa opioid receptors heterologously expressed in cell lines.  You will also examine the effects of casomorphins on endogenous opioid receptors expressed by enteric and central neurons and detect specific uptake using fluorescently-labeled peptides.  Activation of cells by casomorphins will be examined using assays for cAMP production and ERK phosphorylation.  The pharmacological effects of casomorphins on gut motility and secretion will be examined in vitro and in vivo and related to data from the microscopy studies.

Techniques: immunofluorescence, quantitative confocal microscopy, neuronal culture, cell culture, live imaging, peptide labelling, plate-based assays for cAMP and ERK activation, Western blotting, pharmacological assays, gastric emptying and bead propulsion

 

Project 16: Developing Effective Transfection Methods for Enteric Neurons

Dr Daniel Poole
The effective use of standard cell biology techniques in the field of enteric neurobiology has been greatly restricted by the relative inefficiency in which enteric neurons can be transfected.  This has limited the use of e.g. siRNA knockdown of target proteins or the expression of tagged proteins. In this project you will optimize methods for transfection (electroporation, Ca2+ phosphate) of cultured myenteric neurons using eGFP as a marker of effective transfection and pan-neuronal markers to quantify relative efficiency.  Neuronal viability following transfection will be assayed by Ca2+ imaging and effective knockdown of proteins will be examined by Western blotting. If time allows, you will then use these techniques to examine receptor trafficking in the soma and neurites of myenteric neurons using real-time imaging of fluorescently-tagged receptors and FRAP.

Techniques used: Neuronal culture, electroporation, Ca2+ imaging, Western blotting, live imaging, FRAP.

 

 

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Project 17: Vascular neurobiology

Project 17: Changes in neurovascular transmission in diabetes

Ms Niloufer Johansen, Dr Trent Reardon, Associate Professor James Brock
In both type 1 and type 2 diabetes there can be changes in the neural control of blood vessels leading to impaired regulation of blood pressure and tissue perfusion.  In this project, we are concentrating on the early effects of diabetes on nerve-mediated contractions of small arteries supplying skin.  Loss of neural control of these vessels has been suggested to be an early change that leads to microvascular disease in skin of diabetic patients, which impairs wound healing and increases the risk of gangrene particularly in the extremities (feet).  In this project you will use blood vessel myography and smooth muscle electrophysiology to monitor changes in artery function and combine this with immunohistochemistry to assess changes in arterial innervation.


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Project 18: Pain and sensory mechanisms

Project 18: The initiation of action potentials in corneal afferent endings

Associate Professor James Brock, Dr Jason Ivanusic
The mechanisms whereby sensory stimuli are transduced into action potentials in the sensory nerve endings of nociceptors are poorly understood.  This project uses an electrophysiological technique that allows electrically activity to be recorded directly from fine unmyelinated sensory nerve terminals.  Using this technique we are investigating the location within the nerve terminal axons at which action potentials are initiated.  This knowledge is fundamental to understanding the function of sensory nerve terminals.  This project would be particularly suited those engineering skills.

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Projects 19-20: Techniques in Histopathological Analysis

Project 19: Histopathology in mutant mice

Ms Tina Cardamone, Ms Louise Pontell, Mr Steve Pouniotis, Prof John Furness
One of the most important skills needed for the analysis of mutant mice, where the mutation is not known, or the consequences of mutation are unexpected, is histopathological analysis.  The skills to do this type of study are much in demand.  In this project, you will be set the task of conducting histopathological analysis of three different mutants.  You will learn how to prepare tissues, how to conduct a systematic analysis, how to use sophisticated imaging hardware and software and how to report histopathological results.

 

Project 20: Histological Investigation of Enteric Neuropathy and tissue damage

Ms Louise Pontell, Prof John Furness, Ms Tina Cardamone
Damage to enteric neurons, and to other cell types in the intestinal wall, can cause death, and, if not death, results in considerable morbidity.  We are using animal models of the intestinal damage that occurs in inflammatory bowel disease and in ischemia/ reperfusion injury to the intestine.  We are also investigating changes in human intestine.  This project aims at characterising the tissue damage that occurs and to relate the tissue damage to functional disorders.  You will learn methods of histopathology and how tissues are analysed.  You will also learn how to take electronic images and prepare histopathology reports.

 

 

 

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