Mechanisms underlying synapse-specific clustering of GABAA receptors

Lead Research Organisation: School of Pharmacy
Department Name: Unlisted


To recognise things around us, process information and respond in a useful, safe and socially acceptable way, the brain performs extremely complex computations. Our brains contain millions of nerve cells (neurones) which process information and transfer it to other neurones via synapses. Since there are many types of neurones, there are many different types of synapse. Even subtle changes at one type of synapse can produce behavioural, or emotional changes and contribute to neurological or psychiatric disease. This project focusses on inhibitory synapses which reduce activity in other neurones, blocking their responses to other inputs. They select precisely which information is processed and control inappropriate perceptions, responses and behaviour patterns. Many drugs affect their function, eg. anaesthetics, sedatives and anti-anxiety drugs, while changes at some of these synapses caused by changing hormone levels contribute to premenstrual tension, increased epileptic seizure susceptibility at some times of the month and to ?postpartum blues?.

At each synapse, a minute, highly specialised region of the output fibre of one neurone comes very close to the surface of another making a functional connection. On each side of the synapse so formed, proteins cluster into highly specific, complex functional units. These synaptic proteins are highly specialised components. We know something of their structures, their interactions with each other as they control information transfer and that subtly different components are used by different types of synapse. What we do not yet understand is how each of them is selected and inserted at just the right place, or precisely how each combination of components leads to one set of distinctive functional properties. A first requirement for this level of precision is for two neurones, one on either side of the synapse, to recognise each other. Neurone A might receive inputs from twenty different types of neurones and might generate output onto twenty different types of other neurones. It must therefore construct its own half of each of these synapses with enormous precision using just the right components at each one. The first question to be answered is therefore - how does it recognise the neurone on the other side ? The sheer complexity has, until recently, precluded a deeper understanding. The tools needed to probe further are however, becoming available and with them, new insight into the mechanisms that underlie this precision. We will combine these tools in two parallel, novel and complementary experimental approaches to the problem.

Technical Summary

For two decades, neurophysiologists have documented selective expression of synaptic properties at different subclasses of central synapse. It is clear that postsynaptic neurones play a fundamental role in determining the properties of axon terminals innervating them and presynaptic terminals determine the receptors that cluster postsynaptically. This proposal asks how this is achieved.

Cleft-spanning proteins have recently become recognised as the elements mediating transynaptic recognition and with extensive alternative splicing, may provide the molecular diversity controlling functional diversity. Several proteins specific to glutamatergic, or GABAergic synapses have been identified and their importance in the development and stabilization of synapses demonstrated. However, few studies have attempted to go further to explore synapse specificity at subsets of excitatory or inhibitory synapses.

This project focusses on one key question; how is the specific clustering of only one subtype of GABA-A receptor at each class of GABAergic synapse achieved ? We propose two parallel approaches. First to explore the level of pre- and post-synaptic molecular complexity required to ensure selective clustering, by co-culturing neurones with non-neuronal cell lines expressing GABA-A receptors, neuroligin 2 and associated proteins. Immunofluorescence will indicate colocalisation of GABA-A receptors with synaptic proteins and dual whole cell recordings explore the functional integrity of synapses formed. Secondly, to identify receptor-associated proteins and splice variants that are selectively inserted at specific subsets of mature GABAergic synapses. Synaptosomes derived from GABAergic neurone subclasses will be purified, detergent-solubilised, affinity purified on a column decorated with GABA-A receptor extracellular domains and the proteins identified using Mass Spectroscopy.


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Description GABAaRs can initiate and support synapse formation and maturation in the absence of postsynaptic neuronal adhesion molecules
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
Title 17 mutated GABA(A) receptor subunits 
Description Mutated GABA(A) receptor subunits (alpha 1, beta 3, gamma 2S) containing an added myc tag, a histidine (6x) tag and/or a thrombin clevage site in the extracellular domain have been made. When co-expressed, many of these mutant receptor subunits can form pentomers, but do not transfer well to the cell surface, indicating that the sites used for the insertions or mutations are novel sites important for plasma membrane insertion. Entry in 2014 [additional research undertaken using alternative funding] - Given the inability of these mutated receptors to insert into the plasma membrane, this part of the project - to produce near pure pentomeric GABAaR extracellular domains that could be used as "blocking reagents", or to pull down synaptic cleft proteins - failed. The related finding of the study, that the mutated site is important for normal receptor trafficking was pursued and it is expected that the results of this additional project will be published next year. A different approach has been used to determine whether the presence of an excess of GABAa receptors in the extracellular space can influence the formation of synapses, by interfering with an interaction between the membrane-bound receptors and a presynaptic binding partner. Single subunit extracellular domains have been generated and used as 'blocking reagents' and to pull down potential synaptic cleft binding partners. Results so far demonstrate that the presence of these extracellular domains does indeed influence synapse formation. Studies of potential binding partners continue. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact Results of co-culture experiments demonstrate that the GABAa receptors themselves play a pivotal role in the recognition of a postsynaptic target by a presynaptic nerve terminal (and vice versa) and the formation of functional synapses between them. Without the appropriate combination of alpha, beta and gamma subunits in the pentomeric postsynaptic GABAa receptors, functional synapses either did not form at all, or far fewer formed. Moreover, the addition of appropriate GABAaR extracellular domains to the medium bathing the developing cultures, strongly influenced the formation of synapses. It is hoped that these data will be published next year. 
Title Cell line co-cultured with striatal neurones 
Description Stably transfected HEK 294 cell line expressing alpha 1, beta 2, gamma2. Transient transfection of this cell line with neuroligin 2. The development of neuronal/HEK co-cultures was the first important step in analyzing the signals that mediate cell-cell recognition in the formation and maturation of inhibitory synapses. Striatal neurones are now successfully cultured with HEK 394 cells that stably express pentameric GABA(A) receptors containing the alpha1 subunit (plus beta and gamma). Stable connections are formed that exhibit electrophysiological and structural properties similar to those of synapses. Comparisons of these connections with and without the addition of neuroligin 2 are underway. Entry made in 2014 - these cultures have since been used to test which GABAa receptor subunits support functional synapse formation. The gamma 2 subunit has been found to be essential, without this subunit, synapses do not form. The specific beta and alpha subinits included in the pentomeric receptors expressed by the HEK 294 cells also influenced the formation of synapses. 
Type Of Material Cell line 
Year Produced 2009 
Provided To Others? Yes  
Impact It is too early to judge the impact since the results from this work have only recently been presented at symposia. The data presented were, however, extremely well received and triggered many questions. Entry made in 2014 - the findings from these studies continue to excite interest. The first major study was published in 2013 (Fuchs et al, 2013) and more are in preparation. 
Title Developments since year 4 - Mechanisms underlying synapse-specific clustering of GABAA receptors 
Description New Reagents We have produced two new stable lines of HEK293 cells expressing alpha2/beta2 and alpha2/beta3 subunits, which were used in our most recent study in which we have directly compared the efficacy of different GABAA receptor subtypes in promoting synapse formation. These experiments have revealed clear differences between different subtypes of GABAA receptors, with alpha1/beta2/gamma2 combination being the most efficient and alpha2/beta 3/gamma2 combination being the least efficient in synapse formation. These experiments have also revealed that the presence of gamma2 subunit is a prerequisite for synapse formation with any of alpha/beta subunit combinations. The most recent experiments have also indicated that the extracellular domains of alpha1, beta 2 and gamma2 subunits play a direct role(s) in synapse formation because this process is significantly inhibited in the presence of exogenous purified extracellular domains of these subunits applied individually in co-culture experiments. Together, the data suggests that different subtypes of GABAA receptors promote synapse formation with different efficacy but within each subtype all incorporated subunits contribute to this process to some extent and may therefore play very specific roles. This is a manuscript in preparation. Fuchs et al : GABAA receptors, in addition to fulfilling an essential functional role in synaptic transmission at GABAergic synapses, also participate in the assembly of these synapses by promoting the adhesion of inhibitory axons and their functional maturation. Brown et al : In this manuscript we have described a novel in vitro co-culture model system, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. The protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs were first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts. 
Type Of Material Cell line 
Provided To Others? No  
Impact There has been considerable interest in the methods used and in the results obtained, when presented at conferences. 
Title HEK 294 cells stably transfected with mouse alpha 1 (or alpha 2) plus beta 2, and gamma 2. 
Description To allow mouse neurones (from trangenic GFP-expressing mice) to be co-cultured with mouse GABA(A) receptor-expressing HEK cells, two new cell lines stably expressing alpha1 (or alpha 2), beta 2 and gamma 2L have been developed. New cell lines with improved stability have been developed since. 
Type Of Material Cell line 
Provided To Others? No  
Impact Ability to test and compare the importance of several GABA(A) subunits in the formation of functional synaptic contacts, in co-culture systems. 
Title Installation of FACS Machine 
Description Installation of the FACS Aria purchased with this grant. Currently methods for sorting neurones or synaptosomes labelled with fluorescent markers are being developed. 
Type Of Material Improvements to research infrastructure 
Year Produced 2009 
Provided To Others? Yes  
Impact Other scientists in the School of Pharmacy can have access to this machine as part of the purchase price was donated by the School's SRIF fund. 
Title New materials developed in yrs 3 and 4 
Description 1. 4 new bacmid DNA constructs expressing the extracellular domains of alpha1, alpha2, beta2 and gamma2 subunits of GABAA receptors 2. Setting up the first baculovirus expression system and insect cell cultures at the School of Pharmacy 3. Setting up a new protocol for purification of extracellular domains of alpha1, alpha2, beta2 and gamma2 subunits from insect cells 4. Setting up and running the proteomics and mass spec analysis of proteins specifically binding to the extracellular domains of alpha1, alpha2, beta2 and gamma2 subunits 5. Setting up a new protocol for isolation of alpha1, alpha2, beta2 and gamma2 extracellular domains and their use as blocking reagents in neuronal cell cultures and neuron-HEK293 co-cultures 6. Three new HEK293 cell lines expressing mouse alpha1/beta3, alpha1/beta3/gamma2 and alpha1/beta2 subunits 7. Setting up a new protocol for isolation of GFP-positive precursors of parvalbumin interneurons from embryonic brains of G42 transgenic mice line. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact Preliminary data suggest that GABAaRs may bind to subunit-specific synaptic cleft proteins. Entry made in 2014 - extracellular domain constructs have been used to determine whether their presence in the extracellular fluid can affect interactions (and synapse formation) between striatal cells and HEK 395 cells expressing GABAa receptors. Evidence so far suggests that the presence of these domains can significantly influence these interactions. 
Description Contribution to EC funded Flagship Programme Application 
Organisation Swiss Federal Institute of Technology in Lausanne (EPFL)
Country Switzerland 
Sector Public 
PI Contribution This project - the Human Brain Project - has passed the first two rounds of selection and the final application has been submitted. Funding of successful programmes is expected to be in the order of 100EUM for each of 10 years. We contributed to planning and preparation of the application.
Collaborator Contribution EPFL and multiple additional research centres in Europe and around the world. Major facilities (several new) for brain research will become available to members of this consortium, as well as funding for appropriate projects if the programme is funded.
Impact No output as yet , apart from the development of the consortium and the application - results of the funding competition due in early 2013. Multidisciplinary - neurophysiology, neuroanatomy, neurochemistry, psychology, neuronal and circuit modelling, neuromorphic modelling, novel chip design, large scale clinical and scientific data-basing and data-mining, super-computing, robotics, assessment of societal impact etc...
Start Year 2010
Description Modelling of mutated receptors and proteomics 
Organisation University College London
Department School of Pharmacy
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration in the generation of the models required to develop this project.
Collaborator Contribution 3D structural models that predict the structure of the mutated receptor subunits and pentomeric receptors that have been generated for this project.
Impact Molecular biology/genetics/computational modelling Outcome so far - further insight into the mechanisms involved in assembly and trafficking of receptors.
Start Year 2009
Description Lay abstracts are on the School's website 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Available to prospective undergraduate and postgraduate students, as well as the general public.

None tangible
Year(s) Of Engagement Activity 2009,2010,2011,2012