The role of the GluN2A NMDAR subunit in determining synaptic function and plasticity

The human brain contains upwards of 100 billion neurons (nerve cells) which form a network that throughout life acts as our own personal 'information superhighway' that continuously sends and receives signals, processing these to control every aspect of our behaviour - from fundamental, tasks such as breathing and walking to higher functions such as perceiving emotion, interpreting our senses and storing and recalling memories. Neurons 'communicate' with each other at specialized sites known as synapses - a site where one neuron releases a chemical (known as a neurotransmitter) that binds to specific proteins (receptors) on the second neuron. There are many different neurotransmitters in the brain but the most common one that causes excitation is glutamate. Glutamate binds to several different types of receptors but one in particular has been the focus for much research over the past 20 years and is known as the NMDA receptor (NMDARs). This receptor plays a very important role in brain function throughout life. For example, in early life this receptor helps to ensure that the correct wiring pattern is laid down in the developing forebrain, without it we wouldn't be able to learn how to see or walk normally and cognition would be severely impaired. If it is over-activated it causes epilepsy while following a stroke the glutamate released from dying cells acts on NMDARs to cause more neurons to die and its altered function may play a role in diseases such as Alzheimer's, Parkinson's and schizophrenia. The NMDAR protein falls into a class of neurotransmitter receptors which contain within their structure a pore which opens when the neurotransmitter binds to it. This pore allows ions to flow into the neuron generating an electrical signal which we can measure and which is essential for the process of synaptic transmission. To further complicate matters there are four different NMDAR subtypes which all are subtlety different in their function and their properties. In particular, different NMDAR subtypes respond for different durations of time when glutamate binds to trigger their opening. Of particular focus for this proposal is the role and function of NMDARs of the GluN2A subtype, a major subtype in the forebrain. GluN2A NMDARs give rise to responses which are the shortest in duration of all NMDARs - we want to know in the developing brain what processes require these short-duration responses and importantly what differences in brain function are observed in the absence of GluN2A NMDAR subunits. It might be thought that an easy way to assess the role played by a particular NMDAR subtype would be to use a drug to selectively block its effects - unfortunately this is not possible as either they do not show sufficient discrimination for GluN2A NMDARs and other subtypes or they are difficult to use because they are not sufficiently soluble and hence effective concentrations cannot be used in slice preparations or in vivo. We have taken an alternative approach and created a genetically engineered rat that lacks the GluN2 NMDA subunit - this is not a lethal mutation and such animals live and breed with no overt signs of the consequences of the loss of this protein. This makes this model ideal to study the role played by the GluN2A NMDAR subunit in processes such as synaptic transmission, learning and memory, sensory manipulation and behavioural tasks which are dependent on brain regions where this receptor is normally expressed abundantly from the third postnatal week. Our proposal uses a variety of cutting-edge techniques that allow us to record and visualize the activity of either individual or groups of neurons as they function both in preparations of brain slices and in behaving animals. Our work will inform how during ageing this particular NMDAR subtype sculpts and refines brain function.

We have recently created a transgenic rat using CRISPR/Cas9 technology which lacks the GluN2A NMDAR subunit. Together with GluN2B (and the obligatory GluN1 subunit) GluN2A is expressed at high levels in the adult forebrain. Unlike GluN1 or GluN2B, complete knock-out of GluN2A protein is not lethal and as such our transgenic rat model is well suited to assess the role played by this subunit in the maturation of neuronal function. The approaches we will adopt in our programme of work, all of which exist in the investigators' laboratories, are summarized below:

1. Whole-cell patch-clamp recording from identified pyramidal cells and interneurons in hippocampal and visual cortical slices will assess the impact of GluN2A loss on the biophysical and pharmacological properties of EPSCs while multielectrode array recording will assess synaptic network activity. To assess GluN2A-dependency on structural organisation of function we will use laser photo-uncaging and 2P imaging to conduct a morphological and functional analysis of synaptic spines.

2. The vulnerability of GluN2A-null neurons to excitotoxic insults be assessed in vitro. Excitotoxic events will be monitored by imaging and linked to the degree of NMDAR-dependent Ca2+ influx.

3. Deficits in synaptic plasticity in GluN2A-null neurons will be assessed using spike-timing dependent plasticity (STDP) paradigms that in WT animals induce (i) LTP (post- preceded by presynaptic stimulation) and (ii) LTD (pre- preceded by postsynaptic stimulation). Such protocols are ideally suited to assess the impact of GluN2A-null altered NMDAR kinetic behaviour on the temporal dependencies of STDP paradigms.

4. Visual cortical imaging, hippocampal CA1 place field electrophysiology and spontaneous exploration hippocampal-dependent tasks in GluN2A-null rats will assess the extent to which neuronal function is compromised at key developmental time-points in the maturation of CNS network function and age-acquired behaviours.

The work outlined in our proposal is basic science that addresses a fundamental question in the area of glutamatergic synaptic physiology and function - namely what are the consequences for neuronal and network function and for synaptic plasticity and behaviour when the central nervous system does not express one of the two major GluN2 subunits present in NMDARs found in the mammalian forebrain? Elucidating the roles played by specific proteins and understanding why their expression patterns are temporally and spatially regulated are key if we are to understanding how biological systems work. For neurotransmitters there are many examples of receptor subunits that share common properties but each of which have their own distinct set of pharmacological and biophysical properties. Our particular focus on NMDAR function is mirrored in the work of others where, inter alia, their focus could be on another family of neurotransmitter receptor subtypes or a key regulatory isoform of an enzyme in a signalling pathway. For example nicotinic and GABAA receptor subunits are examples where unique receptor-subtype specific properties of the members of these families have been exploited to assess their therapeutic potential.

The immediate beneficiaries of our research will be the community of neurophysiolgists engaged in related research whether this be focussed on nervous system development, glutamatergic synaptic function and plasticity, neuronal network activity or the study of behaviour. Thus the initial impact of our work will be within this very large and broad stakeholder community. While a metrics-based approach to define the impact of research endeavours has its flaws it can be noted that analysis of the combined publication record of the Principal Investigators named in this proposal shows that their work has been cited >11,000 times and they have a joint H-index of >60. Included in this combined output are a subset of approximately 90 papers in the topic area of NMDARs which have been cited >6,500 times.

We anticipate that our work will have impact for those involved in drug discovery and the identification of potential therapeutic targets for drug action. NMDAR hypofunction is considered to be a contributing factor to the aetiology of some psychiatric disorders, notably schizophrenia. In addition is it recognized that both inherited and de novo mutations in NMDARs are likely to underlie the biological bases of some forms of epilepsy, aphasia and intellectual disability. Understanding the roles played by NMDARs in the maturation of synaptic and network function and behaviour provides insight into their dysfunction as occurs in disease. Nevertheless it is likely that such impact will take a considerable time since the drug discovery process and successful drug to market timescales are measured in many years, if not decades.

More immediately our work can have impact in broadening the knowledge base and understanding of patients suffering from diseases affecting brain function, their carers and the wider public. As highlighted in our "Pathways to Impact" statement, the Principal Investigators engage frequently with such groups while exhibitions held at science festivals and awareness events allow the impact of basic research to be highlighted.

The impact of our research is disseminated to those we specifically teach and train: under-graduate and post-graduate students and post-doctoral researchers and fellows. These individuals benefit from the environment we foster within our laboratories where they can experience cutting-edge research, see its output and recognize its potential for providing insight to understanding fundamental biological processes underlying behaviour.