Faculty Collaboration Database

Circadian clockwork machinery in neural retina: evidence for the presence of functional clock components in photoreceptor-enriched chick retinal cell cultures. Mol Vis 2006 Mar 30;12:215-23

Date

04/11/2006

Pubmed ID

16604054

Scopus ID

2-s2.0-33645410050 (requires institutional sign-in at Scopus site)   37 Citations

Abstract

PURPOSE: Circadian clocks in retinas regulate a variety of biochemical and physiological processes. Retinal neurons, particularly photoreceptor cells, are thought to contain autonomous circadian clocks that control iodopsin expression, cFos expression, cAMP levels, and melatonin synthesis. Photoreceptor-enriched cell cultures prepared from chick embryo retina and entrained to a daily light-dark (LD) cycle exhibit circadian rhythms of cAMP levels and the activity of arylalkylamine N-acetyltransferase (AANAT), a key regulatory enzyme in melatonin synthesis. The present study was conducted to investigate the expression of circadian clockwork machinery comprised of clock genes; a clock-controlled gene, Aanat; and a clock output, melatonin, in the photoreceptor-enriched cultured retinal cells.

METHODS: Photoreceptor-enriched cell cultures were prepared from E6 neural retinas and incubated under 14 h:10 h light-dark cycle (LD) of illumination for 8 days and then transferred to constant (24 h/day) darkness (DD). Cells were collected every 4 h in LD and DD, and RNA was isolated. cDNA was prepared from each sample and transcripts of clock genes and Aanat were measured using real-time polymerase chain reaction (PCR). Melatonin release into the culture medium was assayed by HPLC with fluorescence detection at intervals of 3 h in LD and DD.

RESULTS: Cultured neural retina cells exposed to a light-dark cycle showed rhythmic expression of clock genes. Bmal1 and Npas2 (also known as Mop4) peaked late in the day in LD and in DD. Clock mRNA was high at night in LD, but arrhythmic in DD. Cry1 and Per2 transcripts increased rapidly in the early morning and were low at night. The rhythm of Per2 was reduced in amplitude in constant darkness (DD). Levels of Cry1 and Per2 transcripts were stimulated by light exposure at night. Melatonin release and Aanat mRNA were low during the day and high at night. Rhythmic expression of clock genes and Aanat was not observed in cultures not exposed to a LD cycle but treated otherwise identically to cultures described above.

CONCLUSIONS: Photoreceptor-enriched cell cultures derived from chick embryo neural retina contain a complete circadian clockwork system that is entrained by the light-dark cycle, and has a core timekeeping mechanism and circadian output in the form of melatonin synthesis.

Author List

Chaurasia SS, Pozdeyev N, Haque R, Visser A, Ivanova TN, Iuvone PM

Author

Shyam S. Chaurasia PhD Associate Professor in the Ophthalmology and Visual Sciences department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

ARNTL Transcription Factors
Animals
Arylalkylamine N-Acetyltransferase
Biological Clocks
Cells, Cultured
Chick Embryo
Circadian Rhythm
Cryptochromes
Eye Proteins
Flavoproteins
Gene Expression
Melatonin
Nerve Tissue Proteins
Photoperiod
Photoreceptor Cells, Vertebrate
Physical Conditioning, Animal
RNA, Messenger
Retina
Transcription Factors