Intrinsic plasticity of neuronal excitability in the auditory brainstem and neocortex: nitrergic signalling to voltage-gated potassium channels

Lead Research Organisation: University of Leicester
Department Name: Cell Physiology and Pharmacology

Abstract

Many people suffer from brain diseases caused by over-excitability (e.g. tinnitus, epilepsy or attention deficit hyperactivity disorder) exemplifying a fundamental problem: How does the brain keep a balance between too much and too little activity (e.g. epilepsy vs coma)? Maintaining this balance or equilibrium is known as "Homeostasis" (like keeping the correct body temperature: too hot or too cold is bad for you). In the brain too an imbalance of activity is associated with disease and dementia.

Brain cells (neurons) receive information from their neighbours via chemical messengers (neurotransmitters) released at specialised contacts called synapses. We know a lot about how synapses work: excitatory synapses release a messenger called glutamate and this excites the target neuron to trigger an electrical pulse (action potential, AP). This AP then propagates along the neuronal process (axon) to the next set of synapses on other neurons. Thus networks of interconnected neurons pass information to each other; neurons use electrical pulses for internal information transmission and chemical messengers at synapses to communicate with each other.

If a neuron receives lots of synapses then it should fire many APs, and scientists have shown that changing the strength of the synapses (giving more or less excitation) underlies learning and memory - this is often called synaptic plasticity. But how does a neuron know when to fire an AP? The action potential is generated by a class of proteins known as Voltage-Gated Ion Channels: sodium channels start the AP and potassium channels terminate it. Potassium channels are crucial regulators of neuronal excitability; they determine when it fires APs, how many it fires and how long they last. There are around 40 genes specifying different potasium channels, so they are difficult to study in 'real' neurons. The summed activity of all ion channels in a neuron determines its "intrinsic excitability" and changes in this are called "intrinsic plasticity" (in analogy with synaptic plasticity). So the brain can modify synaptic strength by a process of synaptic plasticity and adjust its ability to fire APs by changing intrinsic plasticity.

Although scientists know a lot about synaptic plasticity, intrinsic plasticity has only recently been recognised as playing a significant role.

We have shown that one messenger for this intrinsic plasticity is the chamical nitric oxide (NO). NO has many physiological roles in the immune, cardiovascular, reproductive and alimentary systems. NO acts on its receptor, guanylyl cyclase to generate cGMP and activates protein kinase G (PKG). These and other kinases change protein structure, activity or trafficking by adding phosphate groups.

My laboratory has studies two channel families called Kv2 and Kv3 (KvX - stands for voltage-gated potassium channel family 2 or 3, respectively). These channels 'pull' the voltage back down to the resting voltage (around -70mV) after an AP so as to prepare the neuron for the next AP. We have discovered that an excitatory synaptic messenger (glutamate) causes some neurons to make NO and this signals to surrounding neurons to change from using Kv3 to Kv2 channels; i.e. the synapse has triggered intrinsic plasticity. We have spent 6 years tacking down how this is achieved at one type of synapse in the auditory pathway. Recently we showed that the same process is occurring in the hippocampus (which is the old part of the neocortex). We have developed many molecular tools and are now ready to determine the broader significance of this phenomenon for cortical function (using that part concerned with hearing - the auditory cortex) and how it contributes to disease and injury processes such as deafness (in the auditory brainstem) and tinnitus or epilepsy in the higher brain areas.

Our work is an example of how fundamental research is necessary to understand mechanisms of disease.

Technical Summary

This project explores a new role for nitric oxide (NO) in brain signalling: as the mediator which adapts the intrinsic excitability of target neurons to their excitatory synaptic input. This is distinct from a role in synaptic plasticity but complimentary to it. Postsynaptic calcium influx through NMDAR or AMPAR activates neuronal nitric oxide synthase; NO activates the guanylyl cyclase receptor, generating cGMP and downstream signalling to potassium channels. We postulate that NO signalling increases Kv2 (and decrease Kv3) potassium currents through phosphorylation, so effecting trafficking to, or activity of channels in the plasma membrane. Increased current speeds repolarization giving short action potentials (AP) and enhanced high frequency firing. This homeostatic function maintains transmission during extreme signalling, which would otherwise result in AP failure. We will first explore the role of this conductance in protecting the cochlea from noise trauma by studying the neurones that give rise to the medial olivocochlear efferent projection (which express high levels of Kv2.2 and are located adjacent to an NO source in the superor olivary complex). Second we will explore how nitrergic signalling regulates Kv2.1 and Kv2.2 channels in the primary auditory cortex. Several reports have noted how glutamatergic synaptic inputs increase potassium currents in cortical neurons. We have evidence linking this to nitrergic signalling. We postulate that NO-mediated modulation of Kv2 is a general mechanism by which neuronal excitability is tuned to incoming synaptic activity. This research links the wealth of molecular evidence for NO involvement in neurological disease and neurodegeneration to the dynamic control of neuronal excitability; where NO is a trans-neuronal messenger by which synaptic input tunes the target to maintain effective physiological network function, and where aberrant cortical excitability underlies disease.

Planned Impact

The short-term beneficiaries of this research will be other academics.

The first part of the project will demonstrate how Kv2.2 contributes to protection of the ear during loud sounds. This will be of broad relevance to understanding mechanisms of auditory damage and give new insights into potential ways to protect hearing in the young (the iPod generation) and will have clinical implications. We meet with members of the Institute for Hearing Research (IHR, Nottingham) and discuss collaborations and highlight clinical implications of our research. Recent examples are discussions with clinicians from Sheffield about nitric oxide signalling in the risks of deafness in neonatal jaundice following our recent evidence for NO-induced degeneration of the auditory brainstem (Haustein et al., 2010).

The work will have impact on understanding cognitive function and for academics studying voltage-gated potassium channels, nitrergic signalling, auditory processing, the neocortex and intrinsic plasticity. The demonstration of nitric oxide-mediated intrinsic plasticity is a crucial finding of very broad implications for our understanding of brain function. It strongly suggests that volume transmission cannot be ignored in modelling of cortical function.

In the medium term, computational neuroscientists will benefit from new data on activity-dependent modulation of voltage-gated conductances and in demonstrating volume transmission mechanisms, which will influence modelling of neuronal networks.

In the long term this work will impact on design of neural prosthetics in terms of cochlear nucleus implants and influence development of brain-machine interfaces. Medical implications will be in terms of insights into mechanisms of injury in the brain induced by damaging levels of sound and for possible pharmacological agents which will mitigate hyperexcitability in diseases such as epilepsy and tinnitus. So these studies will be conducted on the auditory cortex, but the inplications of the results will be much broader across all areas of the cortex and hence to dementia and therapeutic mechanisms of maintaining cognitive function by enhancing excitability.

The postdoc and technician will benefit from advanced training neuroscience techniques and recombinant virus production, transgenic animals and molecular biology; insight into how to design experiments and in writing and presenting work for publication. The Technician will directly benefit from learning the recombinant viral technologies through our collaborative links and will become expert at single cell PCR through close collaboration with the electrophysiology postdoc.

A further important benefit will be as advisors and collaborators in the Company Autifony (a spin-out from GSK and led by Charles Large), in which we are involved in describing mechanisms by which lead compounds act on potassium channels, with particular relevance to auditory diseases such as tinnitus and in controlling hyper-excitable conditions.

Milestones:
1. Year 1. Role of Kv2.2 and nitrergic signalling in protection from auditory over-exposure, this will be completed in about 12 months and a publication submitted. Generation of a conditional Kv2.1KO mouse.

2. Year 2. The auditory cortex slice has been set up and the basic electrophysiology and molecular tools are already validated, so this work will start toward the end of year 1 and take about 24 months. The first paper will concern NO-induced plasticity in layer 5 neurons. The second paper, toward the end of year 2 will define the different roles of Kv2.1 and Kv2.2 in the Pyramidal neuron. Generation of a conditional Kv2.2KO mouse.

3. Year 3. The third paper will concern the differences between intrinsic plasticity in layer 2 versus layer 5 cells and will be prepared for publication from the middle of year 3. We will aim for a Neuron impact level Journal.

We expect to publish 3 or 4 research papers based on this project.

Publications

10 25 50
 
Description Deafness Research UK PhD Studentship to study Jaundice
Amount £60,000 (GBP)
Organisation Action on Hearing Loss 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2006 
End 10/2010
 
Description Deafness and central auditory injury by sound exposure 
Organisation University of Leicester
Department Department of Cell Physiology and Pharmacology
Country United Kingdom 
Sector Academic/University 
PI Contribution We have used techniques such as auditory brainstem response measurements and collaborated in immunohistochemistry as well as Journal Clubs.
Collaborator Contribution Expertise and manpower
Impact Papers are in preparation one is submitted
Start Year 2007
 
Description Megakaryocyte potassium channels 
Organisation University of Leicester
Department Department of Cell Physiology and Pharmacology
Country United Kingdom 
Sector Academic/University 
PI Contribution We collaborated on voltage-clamp and transgenic animals to study Kv1.3 potassium channels
Collaborator Contribution manpower and expertise
Impact Paper. Showed the importance of this channel in platelet activation. Multidisciplinary: electrophysiology; cardiovascular system.
Start Year 2009
 
Description Modelling of neuronal function and dysfunction 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution We conducted experiments on AMPA glutamate receptors in the auditory brainstem. Our collaborators built and refined a kinetic model to explain the temperature dependence observed in our experiments.
Collaborator Contribution It has allowed us to develop increased collaboration between experimentalists and engineers.
Impact A paper Multidisciplinary, computational neurosciecne and experimental neuroscience Modelling of nitric oxide volume transmission. Published in Neuron.
Start Year 2006
 
Description Modelling of neuronal function and dysfunction 
Organisation University of Stirling
Country United Kingdom 
Sector Academic/University 
PI Contribution We conducted experiments on AMPA glutamate receptors in the auditory brainstem. Our collaborators built and refined a kinetic model to explain the temperature dependence observed in our experiments.
Collaborator Contribution It has allowed us to develop increased collaboration between experimentalists and engineers.
Impact A paper Multidisciplinary, computational neurosciecne and experimental neuroscience Modelling of nitric oxide volume transmission. Published in Neuron.
Start Year 2006
 
Description Presynaptic Calcium Channels in Migraine 
Organisation University of Buenos Aires
Country Argentina 
Sector Academic/University 
PI Contribution We make presynaptic recordings from the calyx of held synaptic terminal in wt and KO mice lacking the P-type calcium channel
Collaborator Contribution Good training opportunity for staff
Impact Inchauspe CG, Forsythe ID, Uchitel OD (2007) Changes in synaptic transmission properties due to the expression of N-type calcium channels at the calyx of Held synapse of mice lacking P/Q-type calcium channels. J Physiol. 584: 835-51. Inchauspe CG, Pilati N, Di Guilmi MN, Urbano FJ, Ferrari MD, van den Maagdenberg AM, Forsythe ID, Uchitel OD. Familial hemiplegic migraine type-1 mutated cav2.1 calcium channels alter inhibitory and excitatory synaptic transmission in the lateral superior olive of mice. Hear Res. 2015 Jan;319:56-68. doi: 10.1016/j.heares.2014.11.006. PubMed PMID: 25481823. Di Guilmi MN, Wang T, Inchauspe CG, Forsythe ID, Ferrari MD, van den Maagdenberg AM, Borst JG, Uchitel OD. Synaptic gain-of-function effects of mutant Cav2.1 channels in a mouse model of familial hemiplegic migraine are due to increased basal [Ca2+]i. J Neurosci. 2014 May 21;34(21):7047-58. doi: 10.1523/JNEUROSCI.2526-13.2014. PubMed PMID: 24849341; PubMed Central PMCID: PMC4028489.
Start Year 2008
 
Description Brain Awareness Week 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact We run whole day outreach talking about the brain and its relevance to society using short lectures, demonstrations and posters. In the afternoon it is aimed at A-level students and in the evening it is aimed at the general public.
Year(s) Of Engagement Activity 2013,2014,2015,2016,2017,2018
 
Description School Visit to Animal Research Facility (CRF, Central Research Facility) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Around 70 school children visited and had tours of the CRF to see animal care in situ. Part of our transparency and institutional policy of openness in animal research. I gave a short talk about my research and shy we used animals.

They were amazed at the facilities and interested to ask questions and talk about the value of research.
Year(s) Of Engagement Activity 2014