Abstract
Throughout life the brain is faced with the challenge of maintaining its stability, while also being sufficiently flexible to respond to environmental changes and to make modifications required for storage of memories. The process of maintaining brain functions near some set point in the face of these challenges is called homeostasis. Homeostasis regulates the electrical activity of neurons and is likely to be exceptionally important for life-long health and for healthy aging. It is required to maintain a neuron's activity within an optimal range. If neurons have too much or too little activity, neurons will be damaged or information will be lost. Deficits in homeostasis are believed to play critical roles in a spectrum of brain disorders that are targets for pharmaceutical and biotechnology industries. Yet, we know very little about the basic cellular or molecular mechanisms that stabilize neural activity in the adult brain. We propose a new approach to establish fundamental cellular and molecular mechanisms that mediate homeostasis in the adult brain. Our approach uses molecular tools that we have recently developed to specifically manipulate activity of identified populations of neurons in the brains of adult mice. With these tools we can either increase or reduce neuronal activity and then directly measure homeostatic responses that return key neuronal functions to previous set points. We will focus on a brain area called the dentate gyrus (DG), which is important for spatial memory and is implicated in age-related memory loss. This is a good model as it has well defined anatomical and physiological properties. Our preliminary data demonstrate that neurons in the dentate gyrus homeostatically adapt to manipulations that cause their activity to be increased or reduced. We now propose to use this new approach to to identify molecules that are important for homeostasis in the adult brain and to understand the underlying mechanisms. We will use our new molecular tools to induce homeostatic responses in neurons in the DG of adult mice. We will then use electrophysiological recordings to measure the functional changes that have occurred to return neural activity to its previous set point. These experiments will determine if neurons compensate homeostatically for changes in their activity levels by altering their communication with each other or by changing the way they process incoming information. We will develop computational models to reconcile data from different experiments and to make testable predictions for further experiments. Using gene expression profiling technology we will identify which genes have become more or less active during the homeostatic response. We will then examine how these genes contribute to the cellular changes that are associated with homeostasis. By linking gene expression, cellular changes and computational models of neuronal activity, we aim to predict the impact of homeostasis on the function of circuits in the brain and ultimately on cognitive processes and behaviour. The models and experimental results generated by this study will be of benefit and application in several areas. 1) By establishing basic links between genes, communication between neurons and neural homeostasis, the study will provide important insight into how neurons function in the healthy brain. It will form a basis for further investigations of how specific genes influence brain function. 2) The results of the study will give a foundation for investigation of the roles of homeostasis during aging and in disease. Identification of cellular changes underpinning homeostasis will provide potential targets for drug discovery and our approach to altering excitability in adult neurons will provide a useful model for drug testing and validation. 3) The computational models that we build will enable dry lab testing of potential therapeutic strategies in development by pharmaceutical or biotechnology companies.
Technical Summary
This study proposes to address the mechanisms for in vivo homeostatic adaptation in adult neurons. We have developed an experimental system to suppress or enhance excitability of granule cells in the adult dentate gyrus. These neurons are a good model because they are easily accessible for experimental manipulation and have relatively well defined roles in learning and memory. To perturb activation of these neurons we stereotaxically inject lentivirus expressing either a wild-type potassium channel, to make neurons less excitable, or a dominant negative potassium channel, to make neurons more excitable. We have found that these manipulations induce opposing forms of homeostatic adaptation. This approach is the first to permit investigation of homeostasis following manipulation of identified adult neurons in intact functioning circuits. The proposed work will use electrophysiological, computational and molecular methods to address the following objectives. First, we will establish the contribution of changes in synaptic transmission and synaptic integration to homeostasis in the dentate gyrus. These experiments will use electrophysiological recordings and computational models. Second, to examine the molecular pathways responsible for adaptation we will isolate mRNA from transduced granule cells following fluorescence activated cell sorting and then profile gene expression changes using microarray profiling and quantitative PCR. Targets will be further validated by in situ hybridization. Third, to determine the roles of identified candidate genes we will knock down their expression and examine the consequences for homeostatic adaptation. By identifying candidate genes, developing new computational tools and beginning to establish cellular mechanisms, the proposed project will direct new understanding of the cellular and molecular basis for homeostatic maintenance of neural function in the adult brain.
Planned Impact
Users and beneficiaries of the proposed work are in several areas of importance to long-term UK economic growth, health and well being. 1) The proposed project will contribute to UK capacity building in life long health and well being, and in systems biology. These areas have been identified by the BBSRC as strategic priorities of long-term benefit to the UK. The proposed work will provide training for the postdoctoral research associates employed to work on the project and for PhD, Masters and undergraduate students who will have the opportunity to contribute to work that develops from the project. The University of Edinburgh is particularly well placed for the project to contribute to postgraduate training, with several successful PhD and Masters programmes, both within the host School (Biomedical Sciences) and within the School of Informatics. 2) A major beneficiary outside the academic community will be the commercial private sector, in particular pharmaceutical and biotechnology companies. The molecular pathways identified by the study and the experimental approaches used to investigate homeostatic mechanisms in adult neurons will be of use for drug discovery and drug safety evaluation. 3) The proposed project will also benefit the wider general public, both through the products of commercial ventures discussed above, and by increased understanding and awareness of mechanisms of brain function. The proposed project includes plans for communication and engagement that take advantage of the strong infrastructure in place at the University of Edinburgh. Engagement in capacity building will primarily be through the framework of the University of Edinburgh undergraduate and postgraduate training programs. Communication and engagement with pharmaceutical and bioscience industries will be both through conventional channels, such as publication and conferences, and by taking advantage of the University of Edinburgh commercial liaison unit, Edinburgh Research and Innovation (ERI). The ERI publicity team will also provide resources for communication with the media and general public. The principal applicant's website will carry details of the project in a form accessible to a wide audience. The proposed work will generate experimental data, experimental tools and models that will be suitable for further exploitation and application. Data and tools will be deposited in publicly accessible repositories (see Data Sharing statement). Opportunities for further exploitation come from the potential to apply the data, models and expertise to areas of commercial interest, such as drug discovery. These will be evaluated by the Principal Applicant during the project and at the end of the grant life-cycle. When significant opportunities arise they will be exploited using resources provided by ERI, which has extensive experience of working with external organisations to develop commercial opportunities arising from new and innovative developments in academic research within the University of Edinburgh. ERI has established a co-ordinated strategy to protect intellectual property and determine the most appropriate commercialisation route for discoveries on all projects.
