THE ROLE OF GLUTAMATE IN AXONAL PHYSIOLOGY
Year of Dissertation:
The information within the spinal cord and peripheral nervous system propagates along the nerves in the form of Compound Action Potentials (CAP). Although CAPs were always considered to be steady signals, with constant amplitude and velocity as determined by the conductive properties of the nerves our data show that exogenous glutamate increased the CAP. This increase in CAP was blocked after the addition of the general glutamate receptor antagonist kynurenic acid, the specific glutamate receptor antagonist MK801 and CNQX and prevented when these experiments were performed in a calcium-free medium. The goal of this thesis was to examine the changes in axonal physiology in response to electrical stimulation and to pharmacological manipulation. We found that high frequency stimulation, or addition of exogenous glutamate (100 µM) increases the amplitude of compound action potentials (CAPs) in sciatic nerve preparations. These results were further extended and supported by immunohistochemical experiments showing that axolemma contains glutamate receptors (NMDA, AMPA/kainate and mGluR2), the excitatory amino acid transporter responsible for glutamate uptake (Excitatory Amino Acid Transporter-EAAT), and voltage-gated sodium and calcium channels. Thus, the axolemma of peripheral nerves expresses several proteins important for neuronal communication and modulation of the membrane excitability. Apparently, these proteins embedded into the axonal membrane, can under the influence of electrical stimulation or exogenous glutamate change membrane permeability and ionic conductance leading to an increase in the amplitude of the compound action potentials observed in our experiments. Our results demonstrate of existence of axonal plasticity expressed as a change in the amplitude of the action potential following periods of changed activity accompanied by release of neurotransmitters. Therefore we suggest a mechanism of the process whereby electrical stimulation leads to increased axonal activity and subsequent release of glutamate that through activation of the glutamate receptors results in changes in the amplitude of CAPs. We term this phenomenon as axonal plasticity, which would represent one of the forms of neuronal plasticity. Neuronal plasticity is defined as a treatment-induced change in the neuronal response in spite of unchanged strength of the test stimulation. This observation was long described as a property of central synapses and thought to be the basis of learning (Malenka, 1994). Axonal plasticity , would constitute exclusive property of the axon and could contribute together with synaptic plasticity to modification of the efficiency of neuronal connections. This type of plasticity would be fundamentally different from the synaptic plasticity expressed in CNS in the form of Long-Term Potentiation-LTP (Bliss and Collingridge, 1993), Long-Term Depression-LTD (Dudek et al; 1992), and Spike Timing Dependent Plasticity (STDP) (Markram et al., 1997) which has been intensively investigated for last several decades. We assume that high frequency electrical stimulation induces the release of glutamate from stimulated axons. Subsequent increase in the extracellular glutamate concentration would be responsible for observed increase in CAP. Increase in the amplitude of CAP may be a result of: An increase in the number of activated axons (recruitment), 2) and/or increase in the amplitudes of individual potentials generated by single axons. The mechanisms responsible for each of these changes are very different. In the case of recruitment one can suggest paracrine action of glutamate which released from group of axons would enhance the CAP of their neighbors. The increase in the action potential generated by individual axon could be due to a change in the threshold of this individual axon. Our novel data together with published results clearly indicate that in spite of prevailing notion about "all-or-nothing" property of the action potential, axons and action potentials are capable of conveying the information in an analog manner (Clark and Hausser 2006). Presented results convincingly demonstrate that the amplitude of subsequently generated action potentials can change in a way correlated with the frequency of stimulation, or pharmacological treatment. In both cases the change occurred gradually with each evoked action potential slightly larger than its predecessor. This indicates that the effect was building step by step as the intraaxonal mechanisms have been recruited to contribute to the final effect. We have also observed reduced latency and increased area of CAP after glutamate application. The most obvious explanation for both phenomena would be a recruitment of additional, fast conducting axons which would shorten the latency and increase the area of CAP. Simultaneously this would increase the duration of the entire CAP, as slower conducting axons which contributed to CAP before the treatment would be activated as well.
The Role of Striatal Neuropeptides on Glutamate and Methamphetamine-Induced Neurotoxicity in the Murine Brain
ABSTRACT THE ROLE OF STRIATAL NEUROPEPTIDES ON GLUTAMATE AND METHAMPHETAMINE-INDUCED NEUROTOXICITY IN THE MURINE BRAIN by Lauriaselle Afanador Adviser: Dr. Jesus A. Angulo
Year of Dissertation:
The rising worldwide epidemic in addiction to methamphetamine (METH) and the well-documented neurological detriments it causes emphasizes the importance of elucidating the mechanisms by which METH causes widespread and prolonged damage. Also, METH's pathophysiology resembles a number of neurodegenerative diseases. Therefore a better understanding of the mechanisms involved would provide more effective therapeutic targets for the treatment of these neurological disorders.
METH toxicity is a complex interplay of various factors however a number of necessary components have been identified such as dopamine overflow (DA), glutamate signaling, and oxidative stress. Although METH-induced DA overflow is the initiating event, it is not the direct cause of damage. Oxidative stress is thought to be the mediator of METH toxicity and nitric oxide (NO) as a contributor.
We have found that substance P (SP) exacerbates METH-induced NO. Inhibition of SP signaling mitigated NO synthesis and conferred protection. Considering the role SP is playing in METH toxicity we wanted to investigate the role that other striatal neuropeptides play in these events, notably the inhibitory peptides neuropeptide Y (NPY) and somatostatin (SST).
We hypothesized that SP is augmenting NMDA signaling and thus magnifying NO production. Whereas NPY and SST would serve as a counteracting force thus dampening oxidative stress and conferring protection. Overall, our data demonstrated that SP does augment NMDA signaling as inhibition of the neurokinin-1 receptor (NK-1R) decreased NMDA-induced striatal cell loss. We found that SP was potentiating NMDA-induced NO production. Although the predominant source of NO was the inducible form of nitric oxide synthase (NOS).
In support of our hypothesis, NPY and SST proved to attenuate NO. Also, they were protective from METH-induced cell death although SST failed to protect DA terminals. However, an agonist for the NPY-Y2 receptor was successful in maintaining DA terminal viability. Of interest is that neither NPY nor SST modulated NMDA-induced NO or cell loss suggesting that their protective mechanism does not include modulation glutamate signaling within the striatum.