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

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.

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.

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.