AMPA- and GABA(A) receptor signalling and corticospinal motor neuron excitability in mouse models of ALS

The proper function of our bodies relies on the chemical communication that takes place among nerve cells in the brain and between nerve cells and peripheral tissues. Nerve cells that convey information to muscles are known as motor neurons. The progressive and selective death of these neurons is a defining feature of the debilitating, and ultimately fatal, neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS). Unable to function without correct input, the muscles weaken and waste, leaving the patient incapable of voluntary movement. Among the mechanisms thought contribute to this motor neuron loss, 'excitotoxic damage' triggered by elevation of intracellular calcium ions is held to be especially important. Like other nerve cells, motor neurons receive excitatory signals mediated by the neurotransmitter glutamate, which acts by binding to receptor proteins at points of contact between the cells. A combination of poor calcium buffering capacity and the expression of one subclass of AMPA-type glutamate receptors that allow the entry of calcium ions, is thought to contribute to the vulnerability of motor neurons in ALS. However, details of calcium-permeable AMPA receptor function and regulation in motor neurons are poorly understood. Transgenic rodents provide valuable models of the disease, but the study of spinal motor neurons using patch-clamp recording (a technique allowing high-resolution examination of a cell's electrical activity) is possible only in immature, asymptomatic, animals. Unlike motor neurons in the spinal cord, 'upper' motor neurons in the cortex, which also undergo degeneration in ALS, are amenable to patch-clamp recording in slices from mature animals. We plan to take advantage of this, and selectively label these corticospinal motor neurons (CSMNs), allowing us to record from identified motor neurons in acute brain slices from 'ALS' mice at different stages of disease progression. We wish to examine the prevalence and regulation of calcium-permeable AMPA receptors in CSMNs and determine how changes in inhibitory input mediated by a different neurotransmitter receptor, the GABA(A) receptor, contribute to motor neuron excitability, and thus excitotoxic damage, in ALS. By performing electrophysiological studies of motor neurons in both control and symptomatic animals, this project will shed light on normal brain function and provide answers to outstanding questions regarding the mechanisms underlying vulnerability of motor neurons in ALS.

We will determine functional characteristics of excitatory and inhibitory influences on identified CSMNs in brain slices prepared from SOD1(G93A) 'ALS' mice at different stages of disease progression. Cells will be retrogradely labelled with FluoroGold or latex beads applied to the cervical spinal cord. We will determine the active and passive electrical properties of the neurons and establish any general alterations in their properties at different stages of the disease. We will then examine the contribution of calcium-permeable AMPARs and their auxiliary proteins (TARPs and CNIHs) to synaptic and extrasynaptic signalling. We will record pharmacologically isolated AMPAR-mediated macroscopic currents and synaptic currents. The contribution of specific proteins will be determined by comparing the kinetics, voltage-dependence, calcium-permeability, conductance and pharmacology of synaptic and extrasynaptic receptors with those of recombinant receptors expressed with specific auxiliary subunits in tsA201 cells. To determine how GABAergic signalling is altered during disease progression, we will quantify the relative contribution to charge transfer of phasic and tonic GABA(A)R activation. We will distinguish between pre- and postsynaptic changes by examining pharmacologically isolated mIPSCs (amplitude and kinetics, and the conductance of the underlying channels). To determine the origin of changes in tonic GABA(A)R-mediated currents will examine the properties of native receptors in outside-out somatic patches and compare these with those of recombinant GABA(A)Rs expressed in tsA201 cells. We will assess the impact of GABA(A)R activation on cell excitability and potential changes in chloride homeostasis in recordings using the gramicidin perforated-patch technique to preserve intracellular chloride. Our research will contribute to the understanding of normal brain function and highlight potential strategies for limiting excitotoxic damage of motor neurons in ALS.

Academic impact: this work has the potential to enhance the knowledge economy by providing new knowledge and scientific advancement. The principal beneficiaries of this project will be neuroscience researchers. By providing a clearer understanding of the properties of calcium-permeable AMPARs in cortical motor neurons and the impact of GABA(A)R-mediated inhibition in regulating cortical motor neuron excitability during disease progression in ALS mice, our work may held identify research avenues that will ultimately lead to strategies to limit motor neuron damage. In addition, academic benefit may come from our development of any new methodologies and techniques to achieve our goals. In the broader sense, our work will also be of benefit to neuroscience researchers wishing to understand normal brain function. Immediate benefits (through exchange of scientific knowledge) are likely to accrue in the short to medium term, and certainly within the lifetime of the project. The research will also have capacity-building impact through the training of highly skilled researchers, who will acquire specific competencies and broader transferable skills.

Economic and societal impact: ALS and related neurodegenerative conditions cause an important economic and social burden. In the long term, by informing drug development or otherwise helping to facilitate rational approaches to therapy, our studies may ultimately lead to enhanced health and well-being.