Masters Theses

Date of Award

5-2007

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Rebecca A. Prosser

Committee Members

Jimmy C. Hall, Jae H. Park

Abstract

Light is the primary entraining signal for the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Light entering the eye leads to release of glutamate directly onto SCN neurons where it binds to N-methyl D-aspartate (NMDA) receptors initiating a cascade of cellular processes that ultimately modulates clock phase. SCN neurons show a 24-hour rhythm in neuronal activity that peaks in the middle of the day when isolated in a brain slice preparation. Treatments that phase-shift the SCN clock in vivo have been shown similarly to shift this rhythm of neuronal activity in vitro. Here, I have investigated the effects of ethanol on circadian rhythms in SCN brain slices.

My experiments have found that acute application of glutamate [1mM] in the early night to mouse SCN brain slices causes a mean phase-delay of 2.7 hours. Co-application of ethanol blocks this phase-delay in a dose dependent manner, with a maximum effect at 20mM. Ethanol could affect the glutamate-initiated pathway through one or more mechanisms including the prevention of glutamate and/or its co-agonists from binding to the NMDA receptor. Experiments, however, show that high levels of glutamate are not able to overcome ethanol’s blocking effects. Similarly, excess concentrations of glycine and D-serine, the two potential co-agonist candidates, do not prevent ethanol’s block.

Additional experiments showed that high concentrations of glycine applied in the early subjective night caused a phase-advance of 3.4 hours, indicating the presence of glycine receptors in the SCN that are capable of modulating clock phase. Experiments involving strychnine, a glycine receptor antagonist, revealed that when applied alone in the early subjective night resulted in a phase delay of 2 hours. The mechanism behind strychnine’s phase shifting abilities is unknown.

This study has shown that ethanol at a physiologically relevant level has the ability to block glutamate induced phase delays. Though the specific mechanism through which it acts has not been identified, this study suggests ethanol does not interfere with binding of glutamate, nor its co-agonist, to the NMDA receptor. In addition to the ethanol work, my study has shown the phase-shifting effects of glycine and strychnine, two previously unknown phenomena.

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