Michael H. Hastings

mCRY1 and mCRY2 Are Essential Components of the Negative Limb of the Circadian Clock Feedback Loop

We determined that two mouse cryptochrome genes, mCry1 and mCry2, act in the negative limb of the clock feedback loop. In cell lines, mPER proteins (alone or in combination) have modest effects on their cellular location and ability to inhibit CLOCK:BMAL1 -mediated transcription. This suggested cryptochrome involvement in the negative limb of the feedback loop. Indeed, mCry1 and mCry2 RNA levels are reduced in the central and peripheral clocks of Clock/Clock mutant mice. mCRY1 and mCRY2 are nuclear proteins that interact with each of the mPER proteins, translocate each mPER protein from cytoplasm to nucleus, and are rhythmically expressed in the suprachiasmatic circadian clock. Luciferase reporter gene assays show that mCRY1 or mCRY2 alone abrogates CLOCK:BMAL1-E box-mediated transcription. The mPER and mCRY proteins appear to inhibit the transcriptional complex differentially.

Differential Functions of mPer1, mPer2, and mPer3 in the SCN Circadian Clock

The role of mPer1 and mPer2 in regulating circadian rhythms was assessed by disrupting these genes. Mice homozygous for the targeted allele of either mPer1 or mPer2 had severely disrupted locomotor activity rhythms during extended exposure to constant darkness. Clock gene RNA rhythms were blunted in the suprachiasmatic nucleus of mPer2 mutant mice, but not of mPER1-deficient mice. Peak mPER and mCRY1 protein levels were reduced in both lines. Behavioral rhythms of mPer1/mPer3 and mPer2/mPer3 double-mutant mice resembled rhythms of mice with disruption of mPer1 or mPer2 alone, respectively, confirming the placement of mPer3 outside the core circadian clockwork. In contrast, mPer1/mPer2 double-mutant mice were immediately arrhythmic. Thus, mPER1 influences rhythmicity primarily through interaction with other clock proteins, while mPER2 positively regulates rhythmic gene expression, and there is partial compensation between products of these two genes.

The timed infusion paradigm for melatonin delivery: what has it taught us about the melatonin signal, its reception, and the photoperiodic control of seasonal responses?

Abstract: This review summarizes the evidence showing that the duration of the nocturnal secretory profile of pineal melatonin (MEL) is critical for eliciting seasonally appropriate reproductive physiological and behavioral responses in mammals. We review experiments using the timed infusion paradigm (TIP) to deliver MEL either systemically or centrally to pinealectomized hamsters and sheep. In this paradigm, MEL is infused, usually once daily, for a specific number of hours and at a predetermined time of day. This experimental strategy tests most directly those features of the MEL signal that are necessary to trigger photoperiodic responses. The data suggest that the duration of the MEL stimulation is the critical feature of the MEL signal for both inhibitory and stimulatory effects of the hormone on the photoperiodic control of reproductive development in juvenile Siberian hamsters, and for the photoperiodic control of reproductive and metabolic responses in adult Siberian and Syrian hamsters and sheep. The use of the TIP reveals the importance of the frequency of the signal presentation of MEL and suggests the importance of a period of low‐to‐absent circulating concentrations of the hormone. The TIP also reveals that the characteristics of the MEL signal that regulate male sexual behavior are similar to those that are critical for reproductive and metabolic responses in Syrian hamsters. We summarize the locations of possible functional MEL target sites identified by combining the TIP with traditional brain lesion techniques. Evidence from such studies suggests that the integrity of the suprachiasmatic nucleus (SCN) region in Siberian hamsters and the anterior hypothalamus in Syrian hamsters is necessary for the response to short‐day MEL signals. The TIP has been used to deliver MEL to putative target sites for the hormone in the brain of juvenile and adult Siberian hamsters. The results of these preliminary experiments suggest that the regions of specific MEL binding in this species, especially the SCN, are effective sites where MEL may stimulate short‐day‐type responses. In contrast, results from intracranial application of MEL in sheep suggest the medial basal hypothalamus as a critical site of action. Finally, we also discuss potential applications of the TIP for identification of brain MEL target sites, understanding of other photoperiodic phenomena and responses, and resolution of the cellular/molecular basis underlying the reception and interpretation of MEL signals. It is our collective view that the TIP has played, and will continue to play, a pivotal role in elucidation of the function of MEL in the photoperiodic control of seasonal mammalian responses and that the duration of the MEL signal is the critical parameter of the nocturnal secretion profile of the hormone for the photoperiodic control of several seasonally adaptive responses in mammalian species as diverse as hamsters and sheep.

Analysis of Clock Proteins in Mouse SCN Demonstrates Phylogenetic Divergence of the Circadian Clockwork and Resetting Mechanisms

The circadian clock in the suprachiasmatic nuclei (SCN) is comprised of a cell-autonomous, autoregulatory transcriptional/translational feedback loop. Its molecular components include three period and two cryptochrome genes. We describe circadian patterns of expression of mPER2 and mPER3 in the mouse SCN that are synchronous to those for mPER1, mCRY1, and mCRY2. Coimmunoprecipitation experiments demonstrate in vivo associations of the SCN mPER proteins with each other and with the mCRY proteins, and of mCRY proteins with mTIM, but no mPER/mTIM interactions. Examination of the effects of weak and strong resetting light pulses on SCN clock proteins highlights a central role for mPER1 in photic entrainment, with no acute light effects on either the mCRY or mTIM proteins. These clock protein interactions and photic responses in mice are divergent from those described in Drosophila.

Do Corticosteroids Damage the Brain

Corticosteroids are an essential component of the bodys homeostatic system. In common with other such systems, this implies that corticosteroid levels in blood and, more importantly, in the tissues remain within an optimal range. It also implies that this range may vary according to circumstance. Lack of corticosteroids, such as untreated Addisons disease, can be fatal in humans. In this review, we are principally concerned with excess or disturbed patterns of circulating corticosteroids in the longer or shorter term, and the effects they have on the brain.

Non-photic phase shifting of the circadian activity rhythm of Syrian hamsters: the relative potency of arousal and melatonin.

This study investigated the relative potency of melatonin and arousal as Zeitgebers in the non-photic phase shifting of circadian rhythmicity in the adult Syrian hamster. Animals held under dim red light (DD) exhibited robust free-running rhythms of wheel-running activity. Melatonin (1 mg/kg) or ethanolic saline vehicle, delivered manually by subcutaneous injection after removing the animal from its cage, resulted in phase advances of the activity rhythm. This effect was phase dependent, injections at CT 8 and 10 being effective (CT 12 = anticipated activity onset), whereas injection at CT 2, 6, 14 and 20 did not cause a shift. There was no significant difference between the magnitude or timing of phase shifts in response to injections of saline or melatonin. To determine whether the observed shifts were related to arousal of the animals induced by handling, a second group held under DD were fitted with chronic s.c. cannulae so that melatonin solution or vehicle could be delivered remotely at projected CT 10. Neither solution had any effect upon the free-running rhythm. However, when these animals received manual s.c. injection of saline or melatonin solution, they exhibited phase advances similar to those observed in Expt. 1. These results fail to support the hypothesis that melatonin can exert a chemically specific, acute phase-shifting action in the adult Syrian hamster. They do, however, demonstrate the potent effect of arousing stimuli upon the circadian clock in this species.

The circadian cycle of mPER clock gene products in the suprachiasmatic nucleus of the Siberian hamster encodes both daily and seasonal time

The circadian clock in the hypothalamic suprachiasmatic nuclei (SCN) regulates the pattern of melatonin secretion from the pineal gland such that the duration of release reflects the length of the night. This seasonally specific endocrine cue mediates annual timing in photoperiodic mammals. The aim of this study was to investigate how changes in photoperiod influence the cyclic expression of recently identified clock gene products (mPER and mTIM) in the SCN of a highly seasonal mammal, the Siberian hamster (Phodopus sungorus). Immunocytochemical studies indicate that the abundance of both mPER1 and mPER2 (but not mTIM) in the SCN exhibits very pronounced, synchronous daily cycles, peaking approximately 12 h after lights‐on. These rhythms are circadian in nature as they continue approximately under free‐running conditions. Their circadian waveform is modulated by photoperiod such that the phase of peak mPER expression is prolonged under long photoperiods. mPER1 protein is also expressed in the pars tuberalis of Siberian hamsters. In hamsters adapted to long days, the expression of mPER1 is elevated at the start of the light phase. In contrast, there is no clear elevation in mPER1 levels in the pars tuberalis of hamsters held on short photoperiods. These results indicate that core elements of the circadian clockwork are sensitive to seasonal time, and that encoding and decoding of seasonal information may be mediated by the actions of these transcriptional modulators.

The Role of N‐Methyl‐D‐Aspartate‐Type Glutamatergic Neurotransmission in the Photic Induction of Immediate‐Early Gene Expression in the Suprachiasmatic Nuclei of the Syrian Hamster

This study investigated the role of N‐methyl‐D‐aspartate (NMDA)‐type glutamatergic neurotransmission in mediating the photic induction of immediate‐early gene expression in the Suprachiasmatic nucleus (SCN) of the Syrian hamster. Activation of c‐fos, c‐jun and egr‐1 was assessed by immunocytochemical detection of their protein products. To characterize the circadian basis to the inductive effects of light, hamsters were allowed to free‐run in constant dim red light and received a 1 h light pulse at different circadian phases relative to activity onset (defined as CT 12). In control animals which did not receive light pulses, c‐fos and egr‐1 expression was absent or restricted to a small area of the dorsolateral region of the SCN, and expression of c‐jun could not be detected in the SCN. In hamsters killed after presentation of a light pulse at either CT 14 or CT 20, there was a marked increase in c‐fos and egr‐1 immunoreactivities throughout the ventrolateral division of the SCN. In contrast, light pulses given at CT4 or CT 8 failed to activate immediate‐early gene expression. Light pulses did not induce c‐jun immunoreactivity at any circadian phase tested. Staining for c‐fos was maximal 1 h after the start of the light pulse and had started to decline by 2 h. At this later time, c‐jun expression was still undetectable. To compare the distribution of retinal afferents with that of c‐fos induction, hamsters held on a light schedule of 16 h light: 8 h dark received an intraocular injection of cholera toxin‐horseradish peroxidase conjugate 3 days before exposure to a 1 h light pulse given 2 h after lights off. Comparison of adjacent sections processed for c‐fos immunoreactivity or for cholera toxin‐horseradish peroxidase revealed that light‐induced c‐fos expression was precisely restricted to retinal terminal fields in the SCN. Light pulses also induced c‐fos expression in the retinoreceptive ventral lateral geniculate nucleus and intergeniculate leaflet but not in the retinal fields of the dorsal lateral geniculate nucleus, indicating that the expression of cfos in response to light is spatially specific.

Entrainment of the circadian system of mammals by nonphotic cues

Although light is the principal zeitgeber to the mammalian circadian system, other cues can be shown to have a potent resetting effect on the clock of both adult and perinatal mammals. Nonphotic entrainment may have both biological and therapeutic significance. This review focuses on the effect of behavioral arousal as a nonphotic cue and the neurochemical circuitry that mediates arousal-induced entrainment in the adult rodent. In addition, it considers the role of nonphotic entrainment of the developing circadian system in perinatal life prior to the establishment of retinal input to the clock.

Melatonin Receptor Sites in the Syrian Hamster Brain and Pituitary. Localization and Characterization Using [125|]lodomelatonin*

A high‐affinity, discretely localized melatonin receptor has been characterized and mapped within the brain and pituitary of the Syrian hamster using the high specific activity ligand [125|]iodomelatonin and a combination of in vitro autoradiography and membrane homogenate receptor assays. Specific binding of radioligand was found in regions of the epithalamus and hypothalamus in the brain and the pars tuberalis of the pituitary. Excitatory amino‐acid lesions destroyed [125|]iodomelatonin binding within the brain, demonstrating that binding sites are located on neurons. Analysis of [125|]iodomelatonin binding to membrane homogenates of the pars tuberalis revealed a linear relationship between specific ligand binding and the amount of tissue. The time‐course of specific binding at 37°C reached equilibrium after 30 min and remained stable thereafter. The addition of increasing concentrations of [125|]iodomelatonin alone and in the presence of 1 μM melatonin showed that specific binding reached equilibrium at 80 to 100 pM. Analysis of the saturation isotherm using a one‐site binding model was consistent with a single receptor site with a Kd of 29.3 (±5.9 SEM) pM and Bmax of 2.54 (±0.19 SEM) fmol/mg protein.