Hopper, Amy J.
(2023)
Axon diameter determines the mouse optic nerve compound action potential profile in response to varying stimulus frequency.
MRes thesis, University of Nottingham.
Abstract
The mouse optic nerve (MON) is a central white matter tract, whose axons range in diameter from 0.2 to 2.7 µm. In the adult animal all the axons are myelinated, conferring increased conduction velocity. Increased conduction velocity can also be achieved by increasing axon diameter. However, current thinking considers that although large axons conduct quickly, this is unlikely to be the primary function of large axons in the MON. The surface area to volume considerations, and the consistent mitochondrial density in the axoplasm, result in larger axons containing an increased number of mitochondria, capable of efficiently dealing with energy perturbations and maintaining ion equilibration.
The MON is an ideal model to investigate the effect of high frequency firing on the ability of axons to sustain firing. There is a clear differentiation between the three peaks of the compound action potential (CAP), which is predicted to relate to large, intermediate, and small axon, based on the premise that axon diameter dictates conduction velocity. The basal firing rate of the MON is 5 Hz. Larger axons can propagate action potentials at much higher frequencies with no loss of fidelity, whereas smaller axons cannot. This distinction between axons in the same tract is an intriguing observation worthy of investigation. The separation of the CAP profile into distinct peaks allows us to measure how the peaks respond to the same stimulus. Indeed, under metabolic challenge achieved via high frequency firing, we measured a differential fall in the third peak (small axons), compared to the second (intermediate), and then the first (large). Following 5 minutes of 100 Hz stimulus, peak 1 fell to 70%, peak 2 to 40%, and peak 3 to 15%, of the baseline peak amplitude.
Based on the classic electrophysiology experiments which modelled the depolarising effects of elevated potassium on membrane potential, it is reasonable to assume K+ efflux from the axons underlies the fall in the CAP. To investigate this, we used a two-pronged approach. First, we used a direct method to measure the extracellular potassium ([K+]o) with ion sensitive microelectrodes, supporting previous data showing that [K+]o does not significantly increase in response to high frequency firing. Second, we used an indirect method, increasing the artificial cerebrospinal fluid [K+] to determine the concentration at which the CAP peak amplitude started to reduce. We show that the CAP peaks fell at [K+] higher than those achieved in response to 5 minutes stimulus of 100 Hz stimulus: 4.5 mM. This suggests an alternate cause of CAP failure.
High intracellular sodium ([Na+]i) is a likely candidate, however technical considerations limit the ability to measure this directly in individual axons. Therefore, we used an indirect method by decreasing the extracellular [Na+], which caused the CAP to fall in amplitude. We quantified this failure using the Nernst equation, which subsequently allowed us to estimate the elevations in [Na+]i that would occur as a result of high frequency firing, when extracellular [Na+] remains constant. Following 5 minutes of 100 Hz, we predicted that [Na+]i rises to 23 mM for peak 1, 42 mM for peak 2, and 64 mM for peak 3.
We propose that the differential effect of frequency firing on large axons (compared to small axons) is due to the surface area to volume ratio providing an increased mitochondrial count, conferring an increased capacity to support firing under a metabolic challenge.
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