PSYCH C113 - Midterm1

PSYCH C113 - Midterm1 - P g 93-100 The Mammalian Ci rcadian...

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Pg 93-100 The Mammalian Circadian Clock in the Suprachiasmatic Nuclei Is Reset in vitro by cAMP (Prosser and Gillette 1989) Oscillatory pattern of electrical activity of SCN neurons in vivo can be seen in vitro for up to 60 hours after the brain removal. 1 hour pulses of cAMP induced rhythm advances for two cycles; the rhythm was unaffected when using non-cyclic AMP analog. This proves 1) the SCN contains a self sustaining pacemaker that survives in vitro. 2) this pacemaker is rapidly reset by daytime increases in cAMP. Furthermore, this shows that the cAMP pathways have access to entraining mechanisms. SCN isolated from CNS afferents from the environment. Brain slice shows neuron activity and glucose utilization, peak activity, are high in the subjective day and low at subjective night. Time of peak was altered during subjective night, when the animal is sensitive to the resetting effects of light. GOAL OF STUDY: 1. determine whether circadian rhythm in SCN neuronal firing rate was maintained in vitro. (Prove it is endogenous). 2. Test the ability of specific biochemical agents to induce phase shifts. 3. Are the phase shifts stable? MATERIALS AND METHODS: Brain slices of LD 12:12 reared rats. 500 μ m coronal slices of hypothalamus containing paired SCN, optic chiasm, third ventricle, and surronding tissue were perfused at 35 ml/hr with a salt solution, supplemented with 24.6 mM glucose and 26.2 mM sodium bicarbonate. pH adjusted to 7.2 and 37*C, saturated with 95% O_2 and 5%CO_2. Medium is sterilized by filtration. Extracellular recording by microelectrodes filled with 5M NaCl. Electrical signals achieving or exceeding twice the level of background noise were isolated, observed, and recorded. Slices were allowed to equilibrate for one hour in recording chamber. 8- benzylamino-cyclic adenosine monophosphate (BA-cAMP) and 8-bromocyclic adenosine monophosphate (Br-cAMP) were both used and are both membrane permeable cAMP analogs that are potent stimulators of cAMP-dependant protein kinases. To receive treatment, test substance was dissolved into the normal perfusion medium, pH adjusted to 7.2, 95%0_2 and 5%CO_2. After one hour, perfusion of test substance is stopped and normal medium perfusion is resumed. Electrical activity monitored again. Analysis of time of peak determined by visual inspection for the symmetrically highest point, using one
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hour lags. Second analysis similarly conducted with 15 min lags. The reliability of this method was reinforced do to and average difference between the two analysises was less than 15 minutes per experiment for 80 experiments. RESULTS: After monitoring in vitro brain slices during the subjective day for 3 circadian cycles, the time-of-peak was determined at CT (circadian time) 7.1. (CT 0 corresponds to lights-on in donor’s LD cycle and continues for 24 hours before starting over) After 60 hours, they continued same patterns exhibited by day 1 in vitro. Daily period observed in the slices appear slightly less than 24 hours. SCN not effected by cAMP pulse during early subj day, but was
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