Lydia Engel

Daily oscillation of gene expression in the retina is phase-advanced with respect to the pineal gland.

The photoreceptive retina and the non-photoreceptive pineal gland are components of the circadian and the melatonin forming system in mammals. To contribute to our understanding of the functional integrity of the circadian system and the melatonin forming system we have compared the daily oscillation of the two tissues under various seasonal lighting conditions. For this purpose, the 24-h profiles of the expression of the genes coding for arylalkylamine N-acetyltransferase (AA-NAT), nerve growth factor inducible gene-A (NGFI-A), nerve growth factor inducible gene-B (NGFI-B), retinoic acid related orphan receptor beta (RORbeta), dopamine D4 receptor, and period2 (Per2) have been simultaneously recorded in the retina and the pineal gland of rats under short day (light/dark 8:16) and long day (light/dark 16:8) conditions. We have found that the cyclical patterns of all genes are phase-advanced in the retina, often with a lengthened temporal interval under short day conditions. In both tissues, the AA-NAT gene expression represents an indication of the output of the relevant pacemakers. The temporal phasing in the AA-NAT transcript amount between the retina and the pineal gland is retained under constant darkness suggesting that the intrinsic self-cycling clock of the retina oscillates in a phase-advanced manner with respect to the self-cycling clock in the suprachiasmatic nucleus, which controls the pineal gland. We therefore conclude that daily rhythms in gene expression in the retina are phase-advanced with respect to the pineal gland, and that the same temporal relationship appears to be valid for the self-cycling clocks influencing the tissues.

Daily Profile in Melanopsin Transcripts Depends on Seasonal Lighting Conditions in the Rat Retina

The retinal photopigment melanopsin (Opn4) mediates photoentrainment of the circadian system. In the present study, seasonal regulation of the melanopsin gene was investigated in comparison with the arylalkylamine N‐acetyltransferase (AA‐NAT) gene as an indicator of retinal pacemaker output. For this purpose, the daily profiles in the amount of melanopsin mRNA and AA‐NAT mRNA were monitored under 8 : 16 h light/dark, 12 : 12 h light/dark and 16 : 8 h light/dark photoperiods using real‐time polymerase chain reaction analysis. We found that, under all of the lighting regimes, melanopsin and AA‐NAT expression oscillated with a peak around dark onset and the middle of the dark phase, respectively. The lighting regime influenced both genes, but in an opposing manner. Under long photoperiods, the duration of peak expression was prolonged for melanopsin, whereas it was shortened for AA‐NAT. Under constant darkness, the rhythm of mRNA was abolished for melanopsin, but persisted for AA‐NAT whereas, under constant light, the rhythm of mRNA was abolished for both genes. Our findings suggest that, in contrast to the AA‐NAT gene, the daily and photoperiod‐dependent regulation of the melanopsin gene does not rely on a circadian oscillator but is directly illumination‐dependent.

Unique clockwork in photoreceptor of rat.

J. Neurochem. (2010) 115, 585–594.

The photoperiod entrains the molecular clock of the rat pineal.

The suprachiasmatic nucleus‐pineal system acts as a neuroendocrine transducer of seasonal changes in the photoperiod by regulating melatonin formation. In the present study, we have investigated the extent to which the photoperiod entrains the nonself‐cycling oscillator in the Sprague–Dawley rat pineal. For this purpose, the 24‐h expression of nine clock genes (bmal1, clock, per1, per2, per3, cry1, cry2, dec1 and dec2) and the aa‐nat gene was monitored under light‐dark 8 : 16 and light‐dark 16 : 8 in the rat pineal by using real‐time RT‐PCR. The 24‐h pattern of the expression of only per1, dec2 and aa‐nat genes was affected by photoperiod. In comparison with the short photoperiod, the duration of elevated expression under the long photoperiod was elongated for per1 and shortened for dec2 and aa‐nat. For each of the genes, photoperiod‐dependent variations partly persisted under constant darkness. Therefore, the pineal clockwork appears to memorize the photoperiod of prior entrained cycles. The findings of the present study indicate that the nonself‐cycling oscillator of the rat pineal is entrained by photoperiodic information and therefore that it participates in seasonal timekeeping.

Active zone proteins are dynamically associated with synaptic ribbons in rat pinealocytes.

Synaptic ribbons (SRs) are prominent organelles that are abundant in the ribbon synapses of sensory neurons where they represent a specialization of the cytomatrix at the active zone (CAZ). SRs occur not only in neurons, but also in neuroendocrine pinealocytes where their function is still obscure. In this study, we report that pinealocyte SRs are associated with CAZ proteins such as Bassoon, Piccolo, CtBP1, Munc13–1, and the motorprotein KIF3A and, therefore, consist of a protein complex that resembles the ribbon complex of retinal and other sensory ribbon synapses. The pinealocyte ribbon complex is biochemically dynamic. Its protein composition changes in favor of Bassoon, Piccolo, and Munc13–1 at night and in favor of KIF3A during the day, whereas CtBP1 is equally present during the night and day. The diurnal dynamics of the ribbon complex persist under constant darkness and decrease after stimulus deprivation of the pineal gland by constant light. Our findings indicate that neuroendocrine pinealocytes possess a protein complex that resembles the CAZ of ribbon synapses in sensory organs and whose dynamics are under circadian regulation.

Influence of photoperiodic history on clock genes and the circadian pacemaker in the rat retina.

The influence of seasonal lighting conditions on expression of clock genes and the circadian pacemaker was investigated in the rat retina. For this purpose, the 24‐h profiles of nine clock genes (bmal1, clock, per1, per2, per3, dec1, dec2, cry1 and cry 2) and the arylalkylamine N‐acetyltransferase gene as an indicator of the circadian pacemaker output were compared between light–dark periods of 8 : 16 and 16 : 8 h. The photoperiod influenced the daily patterns of the amount of transcript for per1, per3, dec2 and arylalkylamine N‐acetyltransferase. This indicates that photoperiodic information modulates clock gene expression in addition to the circadian pacemaker of the retina. Under constant darkness, photoperiod‐dependent changes in the daily profile of the level of transcript persisted for the arylalkylamine N‐acetyltransferase gene but not for any of the clock genes. Hence, quantitative expression of each clock gene is influenced by the photoperiod only under the acute light–dark cycle, whereas the pacemaker is capable of storing photoperiodic information from past cycles.

Cyclic AMP-inducible genes respond uniformly to seasonal lighting conditions in the rat pineal gland.

The encoding of photoperiodic information ensues in terms of the daily profile in the expression of cyclic AMP (cAMP)-inducible genes such as the arylalkylamine N-acetyltransferase (AA-NAT) gene that encodes the rate-limiting enzyme in melatonin formation. In the present study, we compared the influence of the photoperiodic history on the cAMP-inducible genes AA-NAT, inducible cyclic AMP early repressor (ICER), fos-related antigen-2 (FRA-2), mitogen-activated protein kinase phosphatase-1 (MKP-1), nerve growth factor inducible gene-A (NGFI-A) and nerve growth factor inducible gene-B (NGFI-B) in the pineal gland of rats. For this purpose, we monitored the daily profiles of each gene in the same pineal gland under a long (light/dark 16:8) and a short (light/dark 8:16) photoperiod by measuring the respective mRNA amounts by real-time polymerase chain reaction analysis. We found that, for all genes under investigation, the duration of increased nocturnal expression is lengthened and, in relation to light onset, the nocturnal rise is earlier under the long photoperiod (light/dark 16:8). Furthermore, with the exception of ICER, all other cAMP-inducible genes tend to display higher maximum expression under light/dark 8:16 than under light/dark 16:8. Photoperiod-dependent changes persist for all of the cAMP-inducible genes when the rats are kept for two cycles under constant darkness. Therefore, all cAMP-inducible genes are also influenced by the photoperiod of prior entrained cycles. Our study indicates that, despite differences regarding the expressional control and the temporal phasing of the daily profile, cAMP-inducible genes are uniformly influenced by photoperiodic history in the rat pineal gland.