Selective targeting of synapses to specific dendritic locations and their modulation by voltage-gated channels

Lead Research Organisation: School of Pharmacy
Department Name: Pharmacology

Abstract

The neocortex is the part of the brain that has expanded most dramatically in size and complexity in parallel with the development of human intellectual abilities. Here, countless thousands of pieces of information about our own bodies and the world around us are integrated and processed to generate perceptions and memories and to initiate appropriate responses. These pieces of information arrive from all over the nervous system as electrical signals. These electrical signals are transferred from one nerve cell, or neurone, to another via tiny chemical signals that in turn generate electrical and chemical events in each follower neurone. The receiving neurones are often very complex, in their shapes and in their electrical and chemical properties. Each one is made up of many different compartments into which information can be channelled. The properties of these different compartments, the way that events in them interact with events in other compartments and how they work together to transform the many different pieces of information that constantly bombard each neurone, into the output of that cell, is the subject of this project. We focus particularly on properties that are affected in neurological diseases such as the several different types of epilepsy, in some cases as the possible cause, in others as the brain?s response to another change or insult.

Technical Summary

Evidence for specificity in the properties of synaptic connections is growing, though few studies have examined this issue directly in the smaller dendritic branches of pyramidal cells. Fewer still have studied how the properties of postsynaptic dendritic compartments shape individual synaptic inputs and their summation with events from neighbouring synapses. Many different mechanisms contribute to dendritic properties; mechanisms that may be differentially expressed in different pyramidal cells, different dendrites and dendritic compartments.



We will test the hypothesis that each class of synaptic input to a given class of postsynaptic cell is distributed over a highly specific region of dendritic space that is homogeneous in its active and passive properties, involves only certain types of dendrites and is at a given electrotonic distance from the soma.



This project will compare the properties of two families of neocortical pyramidal cell dendrites, the apical oblique and the basal dendrites of layer 3 pyramidal cells and how their properties shape the synaptic inputs that impinge upon them. Inputs from layer 4 spiny cells selectively innervate the basal dendrites of layer 3 pyramids, while layer 3 inputs also innervate apical obliques. Calcium transients in the dendrite and voltage responses at the soma produced by the release of caged-glutamate onto different dendritic compartments will be recorded. These will be compared with responses to the glutamate released by the synaptic boutons of a single identified presynaptic neurone in paired recording experiments; the postsynaptic dendritic compartment(s) involved being identified by the close apposition of fluorescently labelled axon and dendrite and co-localised calcium signals. How voltage-gated K+ and cation-selective (HCN) ion channels modify glutamatergic inputs to different dendritic compartments will be explored with ion channel blockers and by comparison of wild type mice with mice lacking HCN or Kv4 channels. These data and 3D reconstructions of the layer 3 neurones will tune a detailed compartmental layer 3 pyramidal cell model and predictions eg. about summation tested experimentally.



How both pre- and post-synaptic GABAergic inhibition in these same dendritic compartments interacts with excitatory inputs will also be directly assessed. In one block of paired recording experiments, the effect of uncaging GABA at different positions relative to a synapse(s) from a single presynaptic excitatory cell will be studied. In the other, responses to glutamate uncaged at different positions along a dendrite will be challenged with the input from a single inhibitory interneurone innervating the same dendritic branch.

Publications

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Title Action potential 
Description As part of an exhibition AR has created a short film illustrating the basic functioning of synapses and the brain. 
Type Of Art Film/Video/Animation 
Year Produced 2016 
Impact Workshops for primary and secondary schools, printed information booklet with games and illustrations about the brain and how it works. 
 
Title Multi-photon set up - first 18 months of project. 
Description Setting up and development of a multi-photon microscope in parallel with electrophysiological recording. Tuning of this sophisticated and versatile system. A lot of work was involved - both building and tuning the system and writing algorithms for data-collection and analysis The first component of the project is near completion and will be presented at SfN in 2013. Entry in 2014 - the system continues to be tuned and refined to allow additional protocols to be implemented. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This machine enables the combination of state of the art imaging in parallel with state of the art electrophysiological recordings. 
 
Title Selective targeting of synapses to specific dendritic locations and their modulation by voltage-gated channels - progress in last 24 months 
Description The technical approach required for this project pushes current technology almost to the limit. We have worked closely with the engineers from the companies who supplied the equipment (multi-photon microscope, lasers, electrophysiological equipment) to optimise performance and significant progress has been made. Improving stability and reproducibility has required extensive testing and modification of the system, both of hardware and software. Novel protocols for diagnosis of the sources of image- and uncaging-drift have been developed and the equipment and software modified significantly to minimise drift arising from several different components. We are awaiting the most recent technical improvement, once Prairie has tested it 'in house'. The project has required development of a range of protocol components including: - Single, dual and paired whole-cell recordings - Cell-loading with fluorescent dyes and calcium indicators of different colours e.g. Alexa594 (10-100 uM), Alexa 488 (100 uM), OGB-1 (100 uM) and biocytin (0.4%) - Two-photon imaging of postsynaptic dendritic spines - Post-hoc recovery of biocytin-filled neurones and their 3D reconstruction in Neurolucida - followed by... - Statistical comparison of morphological parameters (Matlab) across reconstructed cells - Uncaging of caged compounds with sub-micron accuracy, and with stability over multiple trials. - Testing different caged compounds. - Electrophysiological characterization of recorded cells (for data correlation and future modelling) and .... - Development of semi-automated analysis procedures (IgorPro and Neuromatic) and ... - Statistical comparison of 51 electrophysiological parameters in anatomically reconstructed cells (Matlab) Ongoing model development : - Importation of detailed 3D reconstructed cells into simulation software - NEURON - Exploration of steady-state and PSP-like signal attenuation across the dendritic tree in a passive model (NEURON) - Adjustment of modelled conductance kinetics and densities in a multi-compartmental model to match experimental data In the last 24 months, the contributions made by different K+ channels to the strength and shape of synaptic inputs onto single, or multiple spine synaptic sites have been studied, with a focus on IA. Modelling studies are exploring the relative contributions of the nature of the conductances and the changes to passive properties of the neurones, when channel blockers are applied. 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2012 
Provided To Others? No  
Impact As highlighted in the previous submission, the PI fell ill and was no longer able to cope with the demands for research. Hence the co-PI (AR) took leadership of the project to take it to completion. Notably, we have a manuscript under revision that has received extremely positive comments and for which we are performing additional experiments. An editorial decision is expected to come by the end of April 2018. 
 
Title Selective targeting of synapses to specific dendritic locations and their modulation by voltage-gated channels - progress in parallel projects 
Description Mercer, Eastlake et al : Two types of PV-immunopositive basket cells were identified in this study. A minority resembled those in CA1, with fast spiking behaviour, vertically oriented dendrites and axons confined to SP of the region of origin. In contrast, the majority of PV-immunopositive CA2 basket cells had long, horizontally oriented, sparsely spiny dendrites extending into all CA subfields in stratum oriens, adapting firing patterns and a pronounced "sag" in voltage responses to hyperpolarising current injection. Broad CA2 basket cells innervated all three CA-subfields and could thus provide CA1 and CA2 with feed-forward and CA3 with feed-back inhibition. Mercer, Botcher et al : This study revealed a subclass of dendrite-preferring interneurones in CA2 that had not been previously described in any CA regions, SP-SR interneurones. The dendritic trees of these cells had a similar pattern to those of CA1 and CA2 bistratified cells and Ivy cells in CA1. SP-SR interneurones, however, had a more complex dendritic tree. These cells also had horizontal dendrites that extended into the CA1 and CA3 regions resembling those of CA2 broad basket and bistratified cells. The major difference between these interneurones and previously described classes, was the region-specificity of their axonal arbours and their unique electrophysiological features. Botcher et al : This study describes the distributions of interneurones expressing widely used markers in the CA2 region and compares these distributions with those in CA1 and CA3. The CA2 region contains interneurones that express, from the most abundant to the least abundant, Reelin, NPY, SOM, CR, CB, CCK, PV and VIP and their distribution in CA2 is layer-specific. Most distributions were similar to those in CA1 and CA3. However, GAD-positive cells were most likely to be PV-, reelin-, SOM-, CB- and VIP-immunopositive interneurones in Stratum Pyramidale in CA2 than in other areas. Stratum Radiatum of CA2 had a higher density of reelin- and NPY-positive cells than SR in CA1 and/or CA3, but fewer NPY- and CR-immunopositive cells were found in Stratum Lacunosum Moleculare in CA2 than in CA1. Interestingly, analysis revealed that the two classes of interneurones that are most likely to be involved in neurological diseases, PV- and Reelin- immunopositive cells, are more abundant in this region than in CA1 and CA3. Mercer (review) : An overview of the known properties of electrical synapses between pyramidal cells was given, focusing on a study in the CA1 region of the hippocampus. 
Type Of Material Physiological assessment or outcome measure 
Provided To Others? No  
Impact Recognition of the distinctive properties of the CA2 region of the hippocampus and of the neuronal classes it contains has been slow to emerge. Indeed, only a few years ago, there was uncertainty and much debate, as to whether the rodent hippocampus had a CA2 region. Interest in this region has been growing in recent years with evidence that it has distinct profiles of change in a range of diseases and that it receives inputs that other CA regions do not receive. These studies have added novel findings and added significantly to this growth of interest in this important region. 
 
Title Modelling dendritic integration in layer 3 neurons 
Description The model is a simulation of dendritic integration in a sample layer 3 cortical neuron reconstructed in 3D. Using a novel computational approach we have examined the how changes in A-type K(+) channel distribution along fine basal and oblique dendrites affected the integrative properties of these neurons. The model is currently under consideration for publication as part of a revised manuscript. 
Type Of Material Computer model/algorithm 
Year Produced 2015 
Provided To Others? Yes  
Impact Latest impact relates to the submission of code of the model to the ModelDB platform (modeldb.yale.edu), a freely accessible database of published computational neuroscience models that seeks to promote model discoverability and attributed reuse. This is ongoing work and URL will appear later Notable impact was made when presenting data from the model at national and international conferences, including the meeting of the Society for Neuroscience in 2015, the UCL Neuroscience Symposium in 2016, and invited seminars - e.g. University of San Antonio, Texas, in 2017. 
 
Description King's College 
Organisation King's College London
Department King's College London, Graduate School
Country United Kingdom 
Sector Academic/University 
PI Contribution I have pioneered an assay and obtained proof-of-principle data suggesting that the membrane-bound zinc transporter ZnT8 is electrogenic, i.e. generates a membrane current. This protein is involved in the packaging of zinc in insulin-containing granules released by beta pancreatic cells. The research underlying this collaboration has potential important ramifications in the field of diabetes research. We found that loading zinc into cells expressing ZnT8 generated a transporter current that lasted ~100 seconds. Untransfected cells exhibited a much smaller fraction of this current. These results demonstrate that ZnT8 is expressed at the plasma membrane enabling direct electrical measurements. Now we want to 1) characterise the biophysical properties of human ZnT8 and 2) analyse zinc transporter currents in cells expressing human ZnT8 variants. These experiments shed-light on previously unknown biophysical parameters of the ZnT8 protein including its sensitivity to zinc, the charge transfer and kinetics of transport.
Collaborator Contribution The collaborative team at King's College provides the human clones and cell reagents necessary for transfection of cultured cells. Recordings are performed in my laboratory with assistance of a postdoctoral researcher from team based at King's College.
Impact This collaboration consolidated into a Seed Award application to the Wellcome Trust. Preliminary data were obtained using the imaging system sponsored by the present award. The grant had multiple layers of collaborative approaches including electrophysiology, 2-photon microscopy, biochemistry and structural biology, as well as chemistry. Unfortunately, the outcome of the grant was negative although judged with merit. A BBSRC application in responsive mode has been submitted February 2019. This was unsuccessful An MRC project grant application will be submitted during the call opening 12 March 2020
Start Year 2016
 
Description Saudi Arabia 
Organisation King Saud University
Country Saudi Arabia 
Sector Academic/University 
PI Contribution Training in electrophysiology and imaging. Providing laboratory space and equipment, computer software. Training and help with analysis of research data. Thesis guidance and corrections
Collaborator Contribution Access to laboratory equipment. Generation of new human cDNA receptor clones. Electrophysiology, immuno and imaging experiments
Impact Phd thesis
Start Year 2017
 
Description Neuroscience exhibition and workshops, Swiss Cottage art gallery, London 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Action Potential - Exploring the Neuron ( 2016, January 21th - March 8th) profiled work done in our laboratories at the UCL School of Pharmacy. This exhibition allowed the public to join me Dr Arnaud Ruiz (AR) as I explained how neurones work and what they do. Utilising film, hologram and other displays to showcase neurones within a context of current scientific understanding, the exhibition let the viewer explore the cells that allow us to think, see and feel. While learning about neurones, the public could appreciate the different media types and visuals showcased to create a learning experience accessible by people of all ages. This was enhanced by the location of the gallery within the space allocate to the Swiss Cottage library in the borough of Camden. More than 15 primary schools from North London attended a series of workshops with educational games, craft activities, and live experiments with the UCL scientists and artists behind the show. Additional funding by the National Lottery and Camden Council was obtained to set up the exhibition and activities. A substantial impact on the interest for our exhibition and for Neuroscience in general was achieved as evidenced in the following links:
Year(s) Of Engagement Activity 2016
URL https://akx95.wordpress.com/2016/02/13/action-potential/