Single channel recording from small mammalian presynaptic nerve terminals.
Lead Research Organisation:
University College London
Department Name: Neuroscience Physiology and Pharmacology
Abstract
The human brain is composed of nerve cells that communicate with each other by sending and receiving chemical signals, called neurotransmitters. This form of communication is tightly controlled by a set of proteins called ion channels. The activity of these ion channel proteins determines when chemical signals are sent. Thus, if we are to understand chemical signalling in the human brain we must understand how ion channels control that signalling. Although there are technologies that allow us to measure the activity of ion channel proteins, in most cases they cannot be applied directly at the most important area for chemical signalling: the synapse. The synapse is a tiny area (about one millionth of a metre across) which has the special function of sending and receiving chemical signals between nerve cells. The reason we cannot measure ion channel activity at most synapses is simply because they are too small. However, we have made a major breakthrough in this area by applying methods used in nanotechnology (the science concerned with things that are about one thousand millionth of a metre in size). By applying methods adopted from nanotechnology we have been able to show that for the first time it is possible to record the activity of ion channel proteins at synapses and thus to radically improve our understanding of chemical signalling in the brain. This technology will eventually have extensive implications for researchers studying the brain in both health and disease. This proposal thus seeks funding to refine our methods for general use and to begin the enormous task of measuring the array of ion channel activity that controls chemical signalling in the brain.
Technical Summary
The ion channels that control neurotransmitter release at presynaptic terminals play a vital role in human and animal physiology, in both health and disease. Unfortunately, technical limitations associated with the small size of most CNS synapses prevent the most informative approach towards characterizing these channels; direct single channel recording. As a consequence of this, presynaptic channels at small terminals are largely uncharacterised at the single channel level. We are proposing the use and further development of an approach that will radically improve our understanding by allowing us to make single channel recordings from small, intact presynaptic boutons. Our method is based on a combination of patch clamp recording, high resolution topographical imaging (using scanning ion conductance microscopy; SICM), and confocal microscopy. By combining these techniques we will be able to locate and patch clamp small presynaptic terminals. We thus plan to characterise both voltage-gated and ligand-gated ion channels in synaptic terminals from two key brain regions; the cerebellum and the hippocampus. We have provided proof-of-principle for our approach in cultured neurons but we also plan to extend this technology so that it can also be used in brain slice preparations.
Publications
Caldwell M
(2012)
Method for estimating the tip geometry of scanning ion conductance microscope pipets.
in Analytical chemistry
Del Linz S
(2014)
Contact-free scanning and imaging with the scanning ion conductance microscope.
in Analytical chemistry
Duguid IC
(2007)
Somatodendritic release of glutamate regulates synaptic inhibition in cerebellar Purkinje cells via autocrine mGluR1 activation.
in The Journal of neuroscience : the official journal of the Society for Neuroscience
Lalo U
(2018)
Role for Astroglia-Derived BDNF and MSK1 in Homeostatic Synaptic Plasticity
in Neuroglia
Lalo U
(2020)
Astroglia-Derived BDNF and MSK-1 Mediate Experience- and Diet-Dependent Synaptic Plasticity.
in Brain sciences
Novak P
(2009)
Nanoscale live-cell imaging using hopping probe ion conductance microscopy.
in Nature methods
Novak P
(2013)
Nanoscale-targeted patch-clamp recordings of functional presynaptic ion channels.
in Neuron
Description | The human brain is composed of nerve cells that communicate with each other by sending and receiving chemical signals, called neurotransmitters. This form of communication is tightly controlled by a set of proteins called ion channels. The activity of these ion channel proteins determines when chemical signals are sent. Thus, if we are to understand chemical signalling in the human brain we must understand how ion channels control that signalling. The most important area for chemical signalling is the synapse. The synapse is a tiny area (about one millionth of a metre across) which has the special function of sending and receiving chemical signals between nerve cells. We made a major breakthrough in this area by applying methods used in nanotechnology (the science concerned with things that are about one thousand millionth of a metre in size). By applying methods adopted from nanotechnology we have been able to show that for the first time it is possible to record the activity of ion channel proteins at synapses and thus to radically improve our understanding of chemical signalling in the brain. This technology will eventually have extensive implications for researchers studying the brain in both health and disease. |
Exploitation Route | By charactereising synapses in disease states and by looking at a wider range of synapses. |
Sectors | Pharmaceuticals and Medical Biotechnology |
Description | We began a collaboration with Ionscope Ltd, the UK-based internationally leading manufacturer and supplier of SICM systems. Ionscope is an SME. We started work on a calibration standard for SICM and were awarded a grant application for a PhD studentship to continue. We have developed PDMS casting skills during the project and have trained Ionscope staff in these methods. |
Sector | Manufacturing, including Industrial Biotechology |
Impact Types | Economic |
Description | BBSRC Case Award |
Amount | £100,173 (GBP) |
Funding ID | BB/J01253X/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2012 |
End | 09/2016 |
Title | A method for estimating the geometry of SICM probes |
Description | This tool relates to scanning ion conductance microscopy (SICM), a type of scanning probe, 3D imaging technique. The resolution of this technique and the ability to scan without contacting the cell are determined by the geometry of the probe. However, determining this geometry has traditionally been done by electron microscopy, which is difficult and time-consuming. We found a simple way to determine probe geometry without the use of an electron microscope. |
Type Of Material | Technology assay or reagent |
Year Produced | 2012 |
Provided To Others? | Yes |
Impact | This technology is being introduced into the Ionscope range of scanning ion conductance microscopes, which will make this UK-based SEM more competitive in the global market. |
Description | CASE studentship partnership with Ionscope Ltd |
Organisation | Ionscope Ltd |
Country | United Kingdom |
Sector | Private |
PI Contribution | The calibration technique and other developments we made during the grant led to further collaboration with Ionscope Ltd, manufacturers of scanning ion conductance microscopes. We now have a BBSRC CASE award supporting this work. |
Start Year | 2012 |
Description | Schools talk (based at UCL) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Schools |
Results and Impact | As above - extensive discussions afterwards. Another school has since contacted me about involvment in the Gifted and Talented Programme. |
Year(s) Of Engagement Activity | 2010 |