Blood Vessel Laboratory | Brain Modelling Laboratory | Cerebral Cortex Laboratory | Developmental Neurobiology Laboratory | Learning and Emotions Laboratory | Movement and Memory Laboratory | Neuronal Signalling Laboratory | Synapse and Hearing Laboratory

Groups in the Division of Neuroscience are studying the fundamental cellular and molecular mechanisms underlying vision, hearing, movement, memory, emotion, higher order cortical processing, development and cardiovascular control. It is through such basic understanding that disorders of the nervous system will be properly understood and cures found. Professor Bruce Walmsley

This past year the Division of Neuroscience entered the NHMRC grants scheme, with an outstanding level of success. The NHMRC-funded research projects are aimed at understanding fundamental mechanisms in the central nervous system. Also this year, Professor Trevor Lamb, Cambridge University, UK, was awarded a prestigious Federation Fellowship to pursue his research in the Division. Professor Lamb is an international authority on the mechanisms of phototransduction in the retina, and will be joining the School in January 2003. Dr Christian Stricker (Switzerland) has been appointed to the teaching staff of the new ANU medical school, and will establish a research laboratory in the Division of Neuroscience, with Dr Anna Cowan, in early 2003. The establishment of these new laboratories will add significantly to the strength of the Division in its investigations of basic neurophysiological processes, which are outlined in the following sections.

Professor Bruce Walmsley, Head of Division

Blood Vessel Laboratory

Professor Caryl Hill

Coordination of vascular responses occurs through effective signaling between adjacent cells in the walls of blood vessels. Homocellular coupling between endothelial cells or between smooth muscle cells, as well as heterocellular coupling between smooth muscle and endothelium, occurs through anatomically identifiable (e.g. with electron microscopy) gap junctions. These gap junctions are comprised of members of the connexin (Cx) family of proteins which oligomerize to form a channel to link the cytoplasm of adjacent cells, permitting electrical coupling and the exchange of ions and small molecules. For example, when perivascular nerves are activated, neurotransmitter impacts on the outside layer of smooth muscle cells and cell-to-cell coupling is important in recruiting the innermost smooth muscle cells for an effective vasomotor response. Numerous other stimuli initiate responses from the endothelial surface of blood vessels, through the release from endothelial cells of factors which subsequently act on the surrounding smooth muscle cells. These factors include vasoconstrictors such as endothelin, and potent vasodilators, such as nitric oxide, prostaglandins and endothelium-derived hyperpolarizing factor (EDHF).

Our current research program is focused on investigating the expression of gap junctions and their constitutive connexin isoforms amongst different blood vessels in order to determine their role in responses such as those illustrated above. We have found four Cx isoforms in rat arteries, Cxs 37, 40, 43 and 45. In addition, our data show that considerable heterogeneity exists in the pattern of connexin expression between conduit (elastic) arteries and resistance (muscular) arteries. In muscular arteries, in comparison with large elastic arteries, Cx37 may be more important than Cx43 for cell coupling within the smooth muscle layers. Cx45 was also expressed in the media of both vessels, but to a lesser degree. In the endothelium, on the other hand, Cxs 37, 40 and 43 were expressed in both types of artery, although the density of Cx40 plaques was significantly greater in the muscular artery. These differences in Cx isoform distribution are expected to result in differences in the conductance properties of the gap junctions within the different cellular layers of the vessels.

Changes in the incidence of gap junctions may also have considerable impact on vascular function. For example, we have shown that the role of EDHF as a relaxing factor is dependent on the presence of myoendothelial gap junctions, which connect the endothelium with the underlying smooth muscle cells. Furthermore, as artery size and the number of smooth muscle cell layers is reduced, EDHF activity and myoendothelial gap junction incidence is increased. These studies show that EDHF is more important in smaller resistance sized vessels than in larger vessels and that the relaxation most likely results from the electrical transfer of hyperpolarization between the two cellular layers.

During hypertension, the balance between vasoconstriction and vasodilation is perturbed and frequently the ability of the endothelium to release relaxing factors is compromised. In vessels isolated from hypertensive rats, we have also found significant reductions in the density of Cx40 expression, as well as reductions in endothelial cell size. Dr Shaun Sandow

Such changes are expected to result in alterations in the ability of the endothelium to conduct vasodilatory responses.

Vessels from hypertensive rats are more prone to exhibiting vasomotion or spontaneous changes in vessel diameter. This vasomotion may contribute to or result from the increased blood pressure. Our data in normotensive rats indicates that cell coupling via gap junctions is important in this activity although the precise mechanisms may vary between different arteries. In some arteries, these mechanisms involve the activation of intracellular second messenger pathways and the release of intracellular calcium, while in others, a complex interaction between intracellular and extracellular calcium results in specific membrane potential changes due to the activation of particular ion channels. Identification of the mechanisms underlying vasomotion in hypertensive animals may permit a more detailed determination of its function in this pathological condition.

Brain Modelling Laboratory

Dr John Clements

Information moving through the brain is encoded as bursts of electrical impulses, and waves of chemicals. A major goal of neuroscience research is decoding this information, and learning how it is processed. My lab is to helping 'break the code', by studying the dynamic properties of neurons as they are bombarded by transient bursts of electrical and chemical information. We have developed both experimental and theoretical models of neuronal function, and are applying them to address a number of different problems.

Synaptic Transmission at a Single Terminal

In the past year we have investigated modulation of synaptic strength at a single synaptic contact point between two neurons (a synaptic terminal). This study was performed in collaboration with Professor Grantyn, and employed an exciting new technique for stimulating a single visualised synaptic terminal, and simultaneously recording the response in the postsynaptic neuron. This approach provides unprecedented insights into the function of small central synapses, and permits correlations to be made between a synaptic terminal's structure and function. We studied paired pulse depression (PPD), an important form of short-lived synaptic modulation. We discovered that PPD had two distinct components: PPD(fast) and PPD(slow). The properties of these two components were unexpectedly complex. In particular, PPD(slow) showed no dependence on extracellular Ca²⁺ concentration, and probably reflects a release-independent inhibition of exocytosis. We concluded that PPD is produced by at least two release-independent presynaptic mechanisms and one release-dependent postsynaptic mechanism.

Pharmacological Management of an Inherited Neurological Disease

The glycine pathway of the brainstem and spinal cord is another important research theme in the Brain Modelling lab. Glycine mediates reflex inhibition of motoneurons (neurons that control muscle contraction). Mutations that damage the glycine receptor on motoneurons produce a hereditary disease named hyperekplexia (startle disease). The symptoms are similar to a mild case of strychnine poisoning. These include an exaggerated startle response characterized by muscle rigidity, and in some cases convulsions. Symptoms often peak early in life (stiff baby syndrome), but persist into adulthood.

At present, the only treatment for hyperekplexia is to administer benzodiazepines. Unfortunately, these drugs act via an indirect mechanism, and have unpleasant side-effects including drowsiness and depression. What is needed is a drug that acts directly on the mutant glycine receptors to enhance their activity. In the past year we tested many different drugs for their ability to enhance the response of these receptors to applied glycine. These drugs included experimental benzodiazepines, compounds related to atropine, barbiturates and anesthetics. Compounds known to modulate the closely-related 5HT receptor were also tested. Unfortunately, none of them appears promising in the treatment of hyperekplexia, however our study provided a useful guide post for what kind of compounds to test in a future round of drug trials.

Cerebral Cortex Laboratory

Dr John Bekkers

Research in the Cerebral Cortex Laboratory (formerly Synaptic Biophysics Group) aims to answer basic questions about the mechanisms of synaptic transmission and integration in the mammalian central nervous system. Our main experimental approach is to make electrical recordings from single, identified neurons in tissue obtained from rodents. We study neurons in dissociated cell cultures, in brain slices, and in vivo in intact brains. By studying the brain at all these different levels of reductionism, we hope to contribute to an understanding of how the healthy brain carries out its computations, and what goes wrong in disease states like epilepsy.

During 2002 we have worked on four main projects.

1. Synaptic transmission and integration in the piriform cortex. Previous work in the lab has focused on synaptic integration in large Layer 5 pyramidal neurons in the sensorimotor cortex. We have recently started a new area of research that extends this approach to the piriform (primary olfactory) cortex. The piriform cortex is a primitive cortical region with a relatively simple anatomy, making it highly attractive for neurophysiological study. We plan to characterise the electrical properties of the different types of neurons in slices of piriform cortex, initially focusing on the pyramidal cells and a subtype of parvalbumin-positive interneuron. We shall also examine synaptic connectivity and plasticity, as well as calcium dynamics in the dendrites using a two-photon microscope located in the Division. So far we have done a basic characterization of the firing properties of different classes of neurons, and have made synaptic recordings from connected neuronal pairs. This project is the subject of a successful NHMRC project grant application.

2. Mechanisms of neurotransmitter release at hippocampal synapses in cell culture. Neurotransmitter release is known to involve an array of specialised presynaptic proteins, but the exact function of each these proteins remains unclear. We are addressing this issue by using molecular techniques to substitute a non-native form of one of these proteins, Syntaxin, at hippocampal synapses in culture. We will then use electrophysiological methods to test our hypotheses about the effect of this substitution on synaptic transmission. This project has been delayed by the move of a collaborator, Dr Catherine Morgans, from JCSMR to the Oregon Health Sciences University, Portland, OR, USA. However, we have successfully transfected our cultures and are now preparing to do patch clamp experiments. In a separate, but related, project we are studying the turnover of presynaptic vesicles using a novel electrophysiological assay. This technique involves loading the vesicles of single, isolated glutamatergic neurons with GABA by stimulating them in the presence of external GABA. By measuring the subsequent release of GABA onto subsynaptic GABA-A receptors, it is possible to measure the kinetics of cycling of presynaptic vesicles using GABA as a quasi-physiological tracer. This year we have continued these experiments, characterising further the kinetics of vesicle turnover.

3. The effect of the dendritic tree on the firing of cerebellar Purkinje cells. This project was the subject of a two and a half month sabbatical this year by John Bekkers in the laboratory of Professor Michael Häusser, University College London. We established a new 'dendrotomy' technique for occluding or removing the Purkinje cell dendritic tree while recording from the soma. We found striking changes in the firing properties of the neurons following removal of the electrical 'load' of the dendrites, and are currently doing further analysis and modelling. The project was funded by a travel grant to JB from the Wellcome Trust.

4. Neurophysiology of a GalR1 knockout mouse with an epileptic phenotype. Galanin is a neuropeptide that is widespread in the central nervous system, but its exact function in the brain remains unclear. Drs Arie Jacoby and Tiina Iismaa at the Garvan Institute, Sydney, recently generated a knockout mouse that lacks the Galanin-1 receptor, and made the surprising discovery that these animals are spontaneously epileptic. In a collaboration with Jacoby and Iismaa, we have this year begun neurophysiological studies with the aim of identifying the mechanisms of this epilepsy. So far we have focused on pyramidal neurons in the ventral hippocampus, which contains high levels of GalR1 in wildtype animals. By comparing synaptic and firing properties of these neurons in wildtype and mutant mice, we have identified an alteration in the firing behaviour of neurons in the mutants. We are also collaborating with Dr Craig McColl, a neurologist and PhD student with Professor Stephen Redman, JCSMR, to make EEG recordings from the spontaneously fitting mice. We hope that this will allow us to identify the seizure focus, which we can then target with patch electrodes.

Developmental Neurobiology Laboratory

Professor Ian Hendry

Nerve cells are dependent on information they receive from the postsynaptic cell in order to survive and mature. One of the major problems for the survival of nerve cells is their extreme length, sometimes over a meter from the cell body along the axon to the terminal. Survival of neurons during development of the nervous system is dependent upon trophic factors signalling from the nerve terminals to the cell body. These factors act on receptors at the nerve terminal and are then faced with the problem of transducing their signal along the axon to the cell body. Understanding the processes that control the flow of information from the nerve terminal to the cell body is essential if we are to understand neuronal development and apply this understanding to enhance nerve regeneration. The results of our studies will suggest alternate ways to enhance neuronal regeneration by perturbing the second messenger cascades promoting axonal transport.

The main players in this signaling process are a family of neurotrophic factors that include nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and neurotrophin 4 (NT-4). These factors promote the survival of sympathetic and sensory neuronal populations by binding to receptors present both in the cell body and on the nerve terminal. There are two classes of receptors, one is a low affinity neurotrophin receptor named p75^NTR, which binds all the neurotrophins. The other class consists of the high affinity tyrosine kinase receptors: TrkA that binds NGF, TrkB that binds NT-4 and BDNF and TrkC that bind NT-3. At the nerve terminal the neurotrophins bind to their receptors, and the neurotrophin-receptor complex is then internalised into a vesicle and transported to the cell body. While vesicular retrograde transport of neurotrophins in vivo is well established, relatively little is know about the mechanisms that underlie vesicle endocytosis and formation prior to transport from the varicosities. We have demonstrated that in vivo not all retrograde transport-vesicles are alike, nor are they all formed using the same mechanisms. As characterised by density, at least two populations of vesicles are present in the synaptic terminal and retrogradely transported along the axon; these are neurotrophin-containing vesicles and synaptic-derived vesicles. Neurotrophin-containing vesicles labelled with iodinated NGF, NT-3 or NT-4 had similar densities of about 1.05 g/ml. Iodinated anti-DBH was used to identify the membrane of synaptic-derived vesicles which had a density of about 1.16 g/ml, corresponding closely to previously published data for synaptic-derived vesicles densities. Using the anterior eye chamber as an elegant model of nerve terminal functionality we were able to measure the effects of pharmacological agents in vivo. The GTPase dynamin is a key element in synaptic vesicle recycling and is responsible for vesicle endocytosis after neurotransmitter release. Dynamin was axonally transported in both a retrograde and anterograde direction and the endocytosis of synaptic vesicles was shown to be dynamin-dependent. In contrast to studies using PC-12 cells in vitro, endocytosis of NGF was found to be dynamin-independent. Although different vesicle populations may arise via different endocytic mechanisms, we found that inhibitors of PI3 kinase and of actin function blocked the transport of both anti-DBH and NGF demonstrating the essential role of these molecules in the successful retrograde transport of either type of vesicle. Lastly, we found an intriguing result with the use of okadaic acid, which resulted in a significant decrease in the retrograde axonal transport anti-DBH and an increase in NGF transport. This suggests that vesicle translocation-mechanisms may be present which can discriminate between vesicle types.

In collaboration with the Gene Targeting Group, we have made mice deficient in the heterotrimeric GTP-binding protein, Ga_z. The group has been studying the phenotype of these animals in order to discover the role of this protein in neuronal signalling mechanisms. Morphine induced hypothermia was significantly attenuated in the Ga_z knockout mouse, and there were strain and species differences in the mechanism of morphine hypothermia. The development of morphine tolerance on the hotplate test demonstrated the existence of a relationship between the quantity of Ga_z present, and the amount of tolerance that the mutant mouse subsequently develops, thus showing a clear gene dosage effect. We hypothesized that Ga_z may be coupled to the opioid receptor and since morphine lethality is mediated by the opioid receptor we tested whether morphine lethality is altered in tolerant Ga_z deficient mice. The results showed a clear gene dose dependent rightward shift in morphine lethality as a result of the loss of Ga_z.

The liver plays an important role in morphine metabolism and Ga_z has been reported to be expressed in the liver. Therefore, we examined whether pharmacokinetic differences could account for the development of greater morphine tolerance in the Ga_z knockout mouse. Serum levels of morphine, as well as the major morphine metabolites, M3G and Nor-M3G were measured in tolerant wildtype and Ga_z deficient mice. Despite the Ga_z knockout animals showing greater tolerance, there was no significant difference in the levels of morphine or its metabolites in the serum. These results suggest a biochemical mechanism is likely to underlie the greater development of morphine tolerance in the Ga_z knockout mice.

The morphine analgesic tolerance that develops was shown to be non-associative and not due to behavioural learning differences. When physical dependence on morphine was examined in mutant mice, a gene dose dependent reduction in naloxone precipitated jumping was observed without significant differences in the other withdrawal signs. This suggests a dissociation of not only between morphine tolerance and physical dependence in the Ga_z knockout mouse, but also between the different symptoms of physical dependence.

The absence of Ga_z results in a complex alteration of morphine stimulated locomotor responses, suggesting the possible alteration in function of more than one receptor in this intricate behavioural response. The locomotor responses of the Ga_z knockout mouse to amphetamine show an increase in locomotor activity. Dopamine D2-like receptors in the brain couple to G_z, and a subtle impairment of function of one of the dopamine D2-like receptors may potentially explain the altered locomotor response profiles of the Ga_z knockout mouse to psychostimulants and morphine as described above.

Learning and Emotions Laboratory

Dr Pankaj Sah

The amygdala is a part of the limbic system that is involved in assigning emotional significance to cognitive events. In particular, it is involved in the processing of fear-producing stimuli. One simple form of learning the amygdala is involved in is fear conditioning.   Dr John Power

Fear conditioning is the process during which a normally innocuous stimulus such as a flashing light becomes associated with a fear producing stimulus (like an electric shock) so that the innocuous stimulus itself subsequently produces a fear response. It represents a form of learning and involves the storage of "emotional" memories. Fear conditioning has been shown to occur in every species that has been examined from flies to humans and its expression shows a remarkably conserved pattern of symptoms which include increases in heart rate and blood pressure, reduction in salivation and freezing of ongoing movement. Associated with the autonomic symptoms of the fear response there are, in humans, cognitive effects such as feelings of dread and despair. Disorders of the storage or expression of fear responses are thought to underlie such mental disorders as panic attacks, anxiety and post traumatic stress disorder.

A converging body of literature over the last forty years indicates that the amygdala is critically involved in assigning emotional significance or value to events through associative learning. Stimulation of the amygdala can elicit the same constellation of symptoms as fear, and lesions of the amygdala reduce the acquisition and expression of fear. An understanding of the function of this structure is thus essential in the development of rational therapies for a range of related anxiety disorders. The anatomical organisation of the amygdala is now fairly well understood. However, its physiology is just beginning to be elucidated. The main focus of the cellular neurophysiology group is to elucidate the basic electrophysiological properties of cells within the amygdala and study its synaptic connectivity.

It is known that the major inputs to the amygdala use glutamate as the principal transmitter. Glutamatergic synapses can undergo a type of plasticity which has been implicated in the storage of memories. In the amygdala, these synapses are likely to be involved in the acquisition of fear conditioning. The amygdala is broadly divided into three main subnuclei: the lateral, basal and central. Sensory and cortical inputs enter the amygdala at the level of the lateral and basal nuclei. The different subnuclei are extensively interconnected and finally project to the central nucleus. Cells within the central nucleus project to brainstem and hypothalamic nuclei responsible for evoking the physiological responses associated with fear.

One project in our group is involved with examining the properties of cells in the input side of the amygdala. We have shown that cells within the lateral and basal nuclei can be divided into two broad categories: pyramidal cells and interneurones. Pyramidal cells form the major type of cell (93%) and are similar to excitatory cells found throughout the cortex. The remaining cells (7%) are interneurons which are inhibitory and form extensive connections with the excitatory cells in the amygdala. We have found that the pyramidal neurons fall into a large continuum of cells with distinct firing properties. Surprisingly we found that the properties of synaptic inputs onto interneurons were quite different from those onto pyramidal cells. These findings indicate that the modulation of inhibitory pathways may be an important control mechanism within the amygdala.

Another project is studying the output side of the amygdala - the central nucleus. This structure is divided into two main parts the medial and lateral. It has recently been shown that cells in the lateral division are inhibitory and make local circuits which cells in the medial division project out of the amygdala. We have been examining the effects of a class of drugs called benzodiazepines (eg diazepam or valium). These drugs are widely used as anxiolytics and their role in the amygdala is of great interest. These drugs are thought to work by potentiating the actions of the major inhibitory transmitter in the brain gamma amino butyric acid (GABA). We have found that the central nucleus also contains a second type of GABA receptor which is inhibited by benzodiazepines. This finding may have potential therapeutic implications as it might be a potential target for new classes of drugs. We are currently looking for the molecular identity of this new type of GABA receptor using molecular techniques.

A third project is studying the properties of calcium dynamics in neurons in the basolateral amygdala. Calcium ions are involved in a range of physiological processes from controlling cells firing properties to gene transcription. Using a new multiphoton imaging system that was acquired by the Division of Neuroscience through a major equipment grant from the Wellcome Trust, we are looking at calcium signals that are associated with action potentials and synaptic stimulation in these neurons.

Dr Louise Faber

Movement and Memory Laboratory

Professor Steve Redman

The Role of Different Calcium Sources in the induction of Long-Term Potentiation at CAI Hippocampus Synapses

Dr Clarke Raymond & Professor Stephen Redman

Long-term potentiation (LTP) is the maintained enhancement of synaptic strength following activation of the synapses combined with depolarization of the post-synaptic neurone. The essential trigger for this mechanism is an elevation of post-synaptic calcium. This calcium can be derived from a number of sources. These are voltage dependent calcium channels (VDCCs), ryanodine sensitive stores, inositol triphosphate (IP₃) activated calcium release and NMDA receptors. With the exception of the pivotal role for calcium via NMDA receptors, the relative importance of calcium derived from each of these stores during LTP induction is unknown. We have shown that LTP induced by weak conditioning stimulation is sensitive to the release of calcium from ryanodine sensitive stores, but insensitive to supply from VDCCs and IP₃ sensitive stores. Stronger conditioning results in more pronounced and persistent LTP that is insensitive to calcium derived from ryanodine sensitive stores and VDCCs, but is sensitive to calcium release from IP₃ sensitive stores. Maximal LTP, induced by strong conditioning stimuli, requires calcium entry via VDCCs, but does not require calcium to be released from either store. These results were obtained initially from field recordings, and are currently being investigated with whole cell recordings of CA1 pyramidal neurones.

There are two possible interpretations of these results. The first is that each level of LTP is achieved by a different signalling pathway, and that the critical calcium source for each level of LTP is specifically coupled to that pathway. The alternative explanation is that each level of LTP requires a different calcium concentration to be achieved.

Direct imaging of calcium concentration changes in spines and dendrites during the conditioning stimulation is being used to gain further insight into these phenomena.

Calcium Buffering in terminals of mossy fibre axons in the hilus

Professors Meyer Jackson and Stephen Redman

Calcium entry into the nerve terminals of mossy fibre axons in the hilus was investigated by whole cell recording from dentate granule cells, with the electrode containing the required concentration of calcium indicator. This indicator diffused along the axon and reached equilibrium concentrations over the first few hundred microns of the axon. The fluorescence changes following a single impulse and trains of impulses of sufficient frequency and duration to saturate the indicator were measured and converted to calcium concentration changes. A single impulse results in an increase in free calcium of approximately 1mM in the terminal, with about 20-30 times that amount of calcium entering the terminal but being buffered by endogenous buffers. The endogenous buffers begin to saturate after a few nerve impulses. The endogenous buffer has a K_d of 450-850mM, and a concentration of 75-160mM. This K_d is similar to that of calbindin -28D, which is known to be plentiful in dentate granule cells. Saturation of the endogenous buffer following a few impulses has implications for facilitation of transmitter release, and for calcium dependent plasticity following repetitive stimulation.

Calcium currents in motoneurones

Dr Olfat Zeid and Professor Stephen Redman

Motoneurones have long dendrites, and voltage clamp recordings at the soma are unable to provide an accurate account of the types of calcium current present in motoneurone membrane, and where they are located. Calcium imaging, using line scans across dendrites at various distances from the soma, in response to back-propagating impulses initiated at the soma, and somatic voltage clamp, are being used to identify the type and location of calcium channels. This work will lead on to an investigation of endogenous calcium buffering in motoneurones of different types.

Schizophrenia in a culture dish

Dr Zan-Min Song

Schizophrenia is a severe and disabling brain disease that afflicts approximately 1% of the population throughout the world.

In the last 20 years, mounting evidence suggests that schizophrenia is a neurodevelopmental disorder related to various conditions during early pregnancy. This notion is substantiated by evidence documenting significant structural abnormalities in the brains of schizophrenia patients. The structural changes generated during early development may lie dormant until puberty when the first episode occurs. In this context, we investigated a2 adrenoceptors that are both involved in neurodevelopment and implicated in clinical aspects of schizophrenia.

a2 adrenoceptors are associated with various aspects of schizophrenia, such as cognitive impairment, pharmacological treatment and genetics. Application of a2 adrenoceptor agonists or a2A selective agonists significantly increases neurite lengths in primary neuronal cultures. Our data are consistent with other reports showing that a2 adrenoceptors play a neurotrophic and neuroprotective role in the central nervous system. We further tested the hypothesis that a2A adrenoceptors act through intracellular pathways that lead to alterations in the phosphorylation state of a cytoskeletal protein, microtubule-associated protein 2 (MAP2). MAP2 phosphorylation controls its ability to maintain dendritic structure. It turns out that MAP2 phosphorylation is significantly reduced shortly after application of a2A agonists and maintained at low level for several days in culture. Our findings that a2 adrenoceptors regulate neurodevelopment provide a further link to the understanding of the etiolology of schizophrenia.

Nicotinic Modulation of Cortical Excitability

Dr Craig McColl, Professor Stephen Redman, Professor Malcolm Horne, and Dr John Drago

The physiological function of nicotinic receptors in the brain remains uncertain but they are known to play an important role in a number of medical conditions including cigarette addiction and dementia. Nicotinic receptors are also mutated in a rare form of inherited epilepsy, Autosomal Dominant Nocturnal Frontal Lobe Epilepsy, and previous work has shown that mice lacking the alpha-4 subunit of the neuronal nicotinic receptor have a greatly enhanced seizure response to proconvulsants. In an attempt to understand the role of nicotinic receptors in the modulation of cortical excitability, we are studying the effects of nicotine in an in vitro thalamocortical slice preparation. Results to date indicate that, in normal mice, nicotine facilitates thalamocortical synapses, increasing the amplitude of excitatory post-synaptic potentials in cortical pyramidal cells. It also has a profound effect on thalamocortical field potentials, increasing the activity of populations of cortical neurons in response to stimulation of the thalamus. These results will be compared to those obtained in mutant mice lacking the alpha-4 subunit of the nicotinic receptor. The same preparation will be used to study the effect of nicotine on inhibitory cortical interneurons and on epileptiform field potentials produced by stimulating the thalamus in the presence of proconvulsants.

Neuronal Signalling Laboratory

Dr Greg Stuart

As a first step to understanding how the brain works the focus of my group is to understand how individual neurons process information. To do this we make electrical recordings from single neurons in slices of living brain tissue from rats. As nerve cells receive most of their synaptic input onto their "dendrites", much of our research is focused on understanding how dendrites influence the way single neurons process information.

In the past year S Williams and G Stuart have investigated how effective inhibition placed at different locations is in regulating neuronal output. These experiments showed that the local amplitude of inhibitory postsynaptic potentials (IPSPs) in cortical pyramidal neurons increases exponentially with distance from the soma, while the somatic amplitude of these IPSPs showed pronounced dendro-somatic attenuation. The somatic half-width of IPSPs was independent of the site of IPSP generation due to the presence of the hyperpolarisation-activated conductance Ih. The attenuation of IPSPs was also voltage-dependent, decreasing when IPSPs were generated from potentials depolarized to IPSP reversal potential; an affect that increased in a site-dependent manner and was sensitive to block of both Ih and voltage-activated Na channels. These date indicate that the somatic impact of dendritic IPSPs is constrained in a voltage-dependent manner by both Ih and voltage-dependent sodium channels.

In a related project M Häusser & G Stuart have investigated how the output signal of neurons - the action potential - interacts with IPSPs. This study showed that action potentials in cortical pyramidal neurons shunt IPSPs in a distance-dependent manner, with somatic IPSPs shunted much more than dendritic IPSPs. Dendritic IPSPs could actually be boosted due to a significant increase in IPSP driving force generated by backpropagating dendritic action potentials. These findings indicate that shunting of inhibition by action potentials is less pronounced than for excitation, suggesting that the balance between excitation and inhibition may be influenced by firing rate. This has relevance to epilepsy, where an imbalance between excitation and inhibition is thought to lead to epileptic seizures.

A Gulledge & G Stuart have also been investigating the strength and regulation of dendritic excitability in pyramidal neurons from the prefrontal cortex, which receives a dense dopaminergic projection for the ventral tegmental area. Action potentials were found to backpropagate in an active manner, showing modest attenuation with distance. Strong dendritic current injection or distal synaptic stimulation initiated dendritic spikes that preceded somatic action potentials. Bath-applied dopamine depolarised the soma and dendrites but did not modify action potential backpropagation or dendritic spike initiation. Imaging experiments using calcium-sensitive dyes revealed that backpropagating action potentials produced substantial dendritic calcium signals, which were not sensitive to dopamine. These data demonstrate active, dopamine-independent propagation and initiation of action potential in the dendrites of prefrontal pyramidal neurons.

Finally, B Kampa and G Stuart have investigated the cellular mechanism underlying long-term changes in synaptic strength during pairing of pre- and postsynaptic activity. We have found direct evidence that repetitive activation of action potentials in cortical pyramidal neurons can enhance the activation of synaptic NMDA receptors - a finding that presumably explains why pairing of presynaptic activity with postsynaptic action potentials can lead to the induction of synaptic plasticity. Furthermore, our results suggest that dendritic electrogenesis associated with bursts of somatic cation potentials can significantly increase NMDA receptor channel activation. This finding may underlie the importance of burst firing for induction of synaptic plasticity in the cortex. This research increases our understanding of how our brains make memories, and should in the long run help in the development of therapies to treat conditions associated with memory loss (eg Alzheimer's disease).

Synapse and Hearing Laboratory

Dr Bruce Walmsley

Hearing is one of our most important and highly developed senses. The brain is required to process auditory information at an astonishing rate. In order to achieve this, the synaptic connections between nerve cells in those parts of the brain which process auditory signals contain some of the fastest and most powerful synaptic connections in the brain. In the Synapse and Hearing Laboratory, we study the fundamental mechanisms underlying the generation of synaptic signals and the processing of these signals in neurons of the auditory brainstem. Our experiments involve studies of the structure of synapses using electron-microscopy and fluorescent labelling of receptors in the target cell, and functional studies using electrical recording from individual neurons in living slices of the auditory brainstem. In addition, due to their large size, we are able to visualize individual synaptic terminals and obtain direct recordings from them. This allows us to perform molecular studies of the fundamental (quantal) nature of neurotransmitter release from these terminals.

Our immediate goal is to understand the role of presynaptic nerve activity and development in the regulation of synaptic strength. The auditory system provides an excellent model system in which to study these synaptic mechanisms.   Dr Sharon Oleskevich

Our investigations have shown that the synapses between auditory nerve fibers and neurons in the cochlear nucleus are stronger in deaf mice than in normal hearing mice. A similar finding has been reported for neurons grown in tissue culture, where connections are strengthened if neuronal activity is abolished. Over the past year we have extended our studies to investigate the effect of altered neuronal activity in deaf mice. One of these studies has examined inhibitory synaptic connections in the auditory brainstem. In conjunction with these physiological experiments, we have initiated a collaborative study with Professor REW Fyffe at Wright State University in Ohio, USA. Professor Fyffe is using immunohistochemistry to selectively label receptors, ion channels and other neuronal proteins to examine the effects on the expression of these molecules of a lack of auditory nerve activity in deaf mice. The level of expression of glycine receptors and glycine receptor clustering proteins is being compared with functional measures of inhibitory glycinergic transmission using electrophysiological recordings of synaptic currents.

The communication between central neurons depends not only on synaptic properties, but also on the response properties of the target neurons, which are determined by ion channels in the target cell membrane. We are studying the membrane properties of neurons in deaf and normal mice, since previous studies have suggested that activity during development may determine not only the properties of synapses, but also the properties of the target cells. In collaboration with Professor Fyffe, we are also examining the expression of voltage-activated channels, leak channels and other proteins involved in neuronal signalling in normal and congenitally deaf mice. Our preliminary results show that there are striking differences in the membrane properties of deaf versus normal mice.

A study was completed during the past year which investigated the sources of variability in the amplitude of spontaneous synaptic currents in auditory neurons. Nerve terminals spontaneously release individual quanta of neurotransmitter, which can be measured in the form of spontaneous miniature synaptic currents. Until recently it was thought that these spontaneously occurring events represented the spontaneous release of the neurotransmitter contents of individual synaptic vesicles from the presynaptic terminal. Several recent studies have shown that, at some synapses, there may be spontaneous release of multiple vesicles, leading to large postsynaptic currents. This synchronous release is triggered by spontaneously occurring calcium sparks released from intraterminal calcium stores. We have investigated this possibility at glycinergic synapses in auditory neurons, and find no involvement of terminal calcium stores. Instead, our results support the proposal that the large amount of variability in the amplitude of spontaneous glycinergic synaptic currents is due to site to site variability in the number of available glycine receptors at different synapses on the same postsynaptic neuron.

During the past year we have also initiated collaborative studies with Professor T Takahashi at Tokyo University to examine the effects of cochlear ablation on central synaptic transmission, and with Professor R Shepherd and Dr A Paolini at the University of Melbourne, to investigate the effects of congenital deafness on spontaneous activity in brainstem auditory neurons. It is hoped that these experiments will provide a better understanding of the central consequences of congenital deafness, and a wider understanding of the role of neuronal activity in the formation and regulation of synaptic connections in the brain.