Electrophysiological and Biochemical Studies on the Effects of RNA and Protein Synthesis Inhibitors on the Circadian Rhythm of the Isolated Aplysia Eye

Author: Rothman, Barry Samuel

Year: 1976

Degree: Dissertation (Ph.D.)

Advisor: Strumwasser, Felix

Committee Members: Strumwasser, Felix; Benzer, Seymour; Davidson, Eric H.; Mitchell, Herschel K.; Wiersma, Cornelis A. G.

Option: Biochemistry; Neurobiology

DOI: 10.7907/ay8x-wp67

Abstract

The isolated eye of Aplysia californica produces a circadian rhythm of optic nerve activity. In filtered sea water, at 15°C, in constant darkness, the free-running period of the circadian rhythm is 23.4 hrs. Optic nerve activity is recorded by means of suction electrode, and is in the form of spontaneous compound action potentials (CAPs) which vary in frequency from 0 to 200 per hr.

Experiments were designed to test the necessity of macromolecular synthesis for the production of the circadian rhythm. Eyes were given a pulse of an inhibitor of RNA or protein synthesis and the effects on the circadian rhythm, biochemistry or electrophysiology assayed.

When eyes were given a 3 hr pulse of actinomycin D (AMD)(4 ug/ml the circadian rhythm was inhibited without blocking spontaneous activity altogether. Eyes receiving a 3 hr pulse of aflatoxin B1(AFTX) (16 ug/ml) revealed similar effects in half the cases studied, while in the other half a reduced amplitude phase delayed circadian rhythm was found. Eyes given a 12 hr pulse of puromycin (PURO) (20-134 ug/ml) or cycloheximide (CHX) (500-2000 ug/ml) beginning in mid-subjective night had their circadian rhythms phase delayed by 12-16 hrs and 6-12 hrs, respectively, after the drug pulse was washed out. A phase-response curve determined for the effects of a 6 hr PURO (125 ug/ml) pulse showed that maximum phase delays were caused by pulses given in late subjective night, and maximum phase advances caused by pulses given in early subjective day.

In biochemical studies, incorporation of 3H-uridine and 14C-leucine were measured 1-9, 9-17, 49-57 and 73-81 hrs after the removal of a 3 hr pulse of AFTX (16 ug/ml) or AMD (4 ug/ml). Uridine incorporation was inhibited by 50-75% from 1 to 17 hrs after an AFTX or AMD pulse, while leucine incorporation was inhibited by 40-70% from 1 to 81 hrs after an AFTX pulse, and by about 20% from 49 to 57 hrs after an AMD pulse. At all other times measured, uridine and leucine incorporation were not significantly different from controls. In other biochemical studies the· effects of ?URO and CHX on leucine incorporation were tested by means of a double-label 303-polyacrylamide gel system. When eyes were labeled during the last 5 hrs of a 12 hr pulse of PUR.O (20 ug/ml) or CHX (500 ug/ml), incorporation was inhibited by about 50%. The distribution of label in the gels of PURO-treated eyes showed increasing inhibition of incorporation with increasing molecular weight above 75,000 daltons, while in the gels of CHX-treated eyes, incorporation was almost equally inhibited at all molecular weights. A 12 hr PURO (125 ug/ml) inhibited leucine incorporation by about 85%, while the distribution of label in the gels showed increasing inhibition of incorporation with increasing molecular weight above 12,000 daltons. At 12-20 hrs and 20-28 hrs after the end of the PURO pulse, incorporation was normal except for a small peak at 20,000 daltons.

The electrophysiological properties of eyes were tested by recording spontaneous CAP activity and responses to light pulses at various times before, during and after the administration of a drug pulse. Eight electrophysiological parameters were measured and compared quantitatively between experimental and control eyes. They were the latency, amplitude and frequency of both the phasic and tonic light responses; and the amplitude and frequency of spontaneous CAP activity. AFTX (3 hrs, 16 ug/ml) induced multiphasic tonic light responses during the drug pulse; and when applied during the peak of an activity cycle, increased the frequency of spontaneous CAP activity by 35% for the remainder of the cycle. AMD (3 hrs, 4 ug/ml) caused a 13% increase in spontaneous CAP amplitude and a 10% decrease in tonic light response latency subsequent to its removal. PURO (12 hrs, 20 ug/ml) increased the amplitude of the tonic light response by 23% when measured more than. 7 hrs after the end of the pulse. CHX (12 hrs, 500 ug/ml) caused a 32% increase in the tonic light response frequency measured 0-7 hrs after the end of the pulse, and a 33% decrease in the duration of spontaneous CAP bursts during the pulse.

The results of these experiments indicate that doses of four inhibitors of macromolecular synthesis capable of modifying the circadian rhythm of the eye reduce the incorporation of uridine and/or leucine and cause only small changes in the electrophysiology of the eye. These data suggest that the production of the circadian rhythm of the Aplysia eye is dependent on macromolecular synthesis.

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