Exercise-induced Nogo-A influences rodent motor learning in a time-dependent manner.

The adult, mature central nervous system (CNS) has limited plasticity. Physical exercising can counteract this limitation by inducing plasticity and fostering processes such as learning, memory consolidation and formation. Little is known about the molecular factors that govern these mechanisms, and how they are connected with exercise. In this study, we used immunohistochemical and behavioral analyses to investigate how running wheel exercise affects expression of the neuronal plasticity-inhibiting protein Nogo-A in the rat cortex, and how it influences motor learning in vivo. Following one week of exercise, rats exhibited a decrease in Nogo-A levels, selectively in motor cortex layer 2/3, but not in layer 5. Nogo-A protein levels returned to baseline after two weeks of running wheel exercise. In a skilled motor task (forelimb-reaching), administration of Nogo-A function-blocking antibodies over the course of the first training week led to improved motor learning. By contrast, Nogo-A antibody application over two weeks of training resulted in impaired learning. Our findings imply a bimodal, time-dependent function of Nogo-A in exercise-induced neuronal plasticity: While an activity-induced suppression of the plasticity-inhibiting protein Nogo-A appears initially beneficial for enhanced motor learning, presumably by allowing greater plasticity in establishing novel synaptic connections, this process is not sustained throughout continued exercise. Instead, upregulation of Nogo-A over the course of the second week of running wheel exercise in rats implies that Nogo-A is required for consolidation of acquired motor skills during the delayed memory consolidation process, possibly by inhibiting ongoing neuronal morphological reorganization to stabilize established synaptic pathways. Our findings suggest that Nogo-A downregulation allows leaning to occur, i.e. opens a 'learning window', while its later upregulation stabilizes the learnt engrams. These findings underline the importance of appropriately timing of application of Nogo-A antibodies in future clinical trials that aim to foster memory performance while avoiding adverse effects.

How to time the time - A preface to the special issue Circadian Rhythms in the Brain.

In this Preface to the Journal of Neurochemistry special issue "Circadian Rhythms in the Brain", we summarize recent insights into connections between circadian rhythms and societal concerns related to aging and food intake, with consequences for healthy or aberrant metabolic homeostasis. The articles in this special issue were written by leading authors who presented their research at the 2019 Congress of the European Biological Rhythm Society, and are thus reflective of a broad variety of state-of-the-art research on all levels of chronobiology, from circadian rhythm generators in various tissues (including astrocytes) and the molecular mechanisms they base on, such as GABAergic regulation or ubiquitination, to the systems and behavioral level effects of chrono-nutrition and aging. Cover Image for this issue: https://doi.org/10.1111/jnc.15058.

Melatonin modulates daytime-dependent synaptic plasticity and learning efficiency.

Mechanisms of hippocampus-related memory formation are time-of-day-dependent. While the circadian system and clock genes are related to timing of hippocampal mnemonic processes (acquisition, consolidation, and retrieval of long-term memory [LTM]) and long-term potentiation (LTP), little is known about temporal gating mechanisms. Here, the role of the neurohormone melatonin as a circadian time cue for hippocampal signaling and memory formation was investigated in C3H/He wildtype (WT) and melatonin receptor-knockout ( MT 1 / 2 - / - ) mice. Immunohistochemical and immunoblot analyses revealed the presence of melatonin receptors on mouse hippocampal neurons. Temporal patterns of time-of-day-dependent clock gene protein levels were profoundly altered in MT 1 / 2 - / - mice compared to WT animals. On the behavioral level, WT mice displayed better spatial learning efficiency during daytime as compared to nighttime. In contrast, high error scores were observed in MT 1 / 2 - / - mice during both, daytime and nighttime acquisition. Day-night difference in LTP, as observed in WT mice, was absent in MT 1 / 2 - / - mice and in WT animals, in which the sympathetic innervation of the pineal gland was surgically removed to erase rhythmic melatonin synthesis. In addition, treatment of melatonin-deficient C57BL/6 mice with melatonin at nighttime significantly improved their working memory performance at daytime. These results illustrate that melatonin shapes time-of-day-dependent learning efficiency in parallel to consolidating expression patterns of clock genes in the mouse hippocampus. Our data suggest that melatonin imprints a time cue on mouse hippocampal signaling and gene expression to foster better learning during daytime.

Endogenous modulation of human visual cortex activity improves perception at twilight.

Perception, particularly in the visual domain, is drastically influenced by rhythmic changes in ambient lighting conditions. Anticipation of daylight changes by the circadian system is critical for survival. However, the neural bases of time-of-day-dependent modulation in human perception are not yet understood. We used fMRI to study brain dynamics during resting-state and close-to-threshold visual perception repeatedly at six times of the day. Here we report that resting-state signal variance drops endogenously at times coinciding with dawn and dusk, notably in sensory cortices only. In parallel, perception-related signal variance in visual cortices decreases and correlates negatively with detection performance, identifying an anticipatory mechanism that compensates for the deteriorated visual signal quality at dawn and dusk. Generally, our findings imply that decreases in spontaneous neural activity improve close-to-threshold perception.

Period1 gates the circadian modulation of memory-relevant signaling in mouse hippocampus by regulating the nuclear shuttling of the CREB kinase pP90RSK.

Memory performance varies over a 24-h day/night cycle. While the detailed underlying mechanisms are yet unknown, recent evidence suggests that in the mouse hippocampus, rhythmic phosphorylation of mitogen-activated protein kinase (MAPK) and cyclic adenosine monophosphate response element-binding protein (CREB) are central to the circadian (~ 24 h) regulation of learning and memory. We recently identified the clock protein PERIOD1 (PER1) as a vehicle that translates information encoding time of day to hippocampal plasticity. We here elaborate how PER1 may gate the sensitivity of memory-relevant hippocampal signaling pathways. We found that in wild-type mice (WT), spatial learning triggers CREB phosphorylation only during the daytime, and that this effect depends on the presence of PER1. The time-of-day-dependent induction of CREB phosphorylation can be reproduced pharmacologically in acute hippocampal slices prepared from WT mice, but is absent in preparations made from Per1-knockout (Per1(-/-) ) mice. We showed that the PER1-dependent CREB phosphorylation is regulated downstream of MAPK. Stimulation of WT hippocampal neurons triggered the co-translocation of PER1 and the CREB kinase pP90RSK (pMAPK-activated ribosomal S6 kinase) into the nucleus. In hippocampal neurons from Per1(-/-) mice, however, pP90RSK remained perinuclear. A co-immunoprecipitation assay confirmed a high-affinity interaction between PER1 and pP90RSK. Knocking down endogenous PER1 in hippocampal cells inhibited adenylyl cyclase-dependent CREB activation. Taken together, the PER1-dependent modulation of cytoplasmic-to-nuclear signaling in the murine hippocampus provides a molecular explanation for how the circadian system potentially shapes a temporal framework for daytime-dependent memory performance, and adds a novel facet to the versatility of the clock gene protein PER1. We provide evidence that the circadian clock gene Period1 (Per1) regulates CREB phosphorylation in the mouse hippocampus, sculpturing time-of-day-dependent memory formation. This molecular mechanism constitutes the functional link between circadian rhythms and learning efficiency. In hippocampal neurons of wild-type mice, pP90RSK translocates into the nucleus upon stimulation with forskolin (left), whereas in Period1-knockout (Per1(-/-) ) mice (right) the kinase is trapped at the nuclear periphery, unable to efficiently phosphorylate nuclear CREB. Consequently, the presence of PER1 in hippocampal neurons is a prerequisite for the time-of-day-dependent phosphorylation of CREB, as it regulates the shuttling of pP90RSK into the nucleus. Representative immunofluorescence images show a temporal difference in phosphorylated cAMP response element-binding protein (pCREB; green color) levels in all regions of the dorsal hippocampus between a wild-type C3H mouse (WT; left) and a Period1-knockout (Per1(-/-) ; right) mouse. Images were taken 2 h after lights on, thus, when fluctuating levels of pCREB peak in WT mouse hippocampus. Insets show a representative hippocampal neuron, in response to activating cAMP signaling, stained for the neuronal marker NeuN (red), the nuclear marker DAPI (blue) and the activated CREB kinase pP90RSK (green). The image was taken 2 h after light onset (at the peak of the endogenous CREB phosphorylation that fluctuates with time of day). Magnification: 100X, inset 400X. Read the Editorial Highlight for this article on page 650. Cover image for this issue: doi: 10.1111/jnc.13332.

Ras Activity Oscillates in the Mouse Suprachiasmatic Nucleus and Modulates Circadian Clock Dynamics.

Circadian rhythms, generated in the mouse suprachiasmatic nucleus (SCN), are synchronized to the environmental day-night changes by photic input. The activation of the extracellular signal-regulated kinases 1 and 2 (ERK1,2) and cAMP response element-binding protein (CREB)-mediated transcription play a critical role in this photoentrainment. The small GTPase Ras is one of the major upstream regulators of the ERK1,2/CREB pathway. In contrast to the well-described role of Ras in structural and functional synaptic plasticity in the adult mouse brain, the physiological regulation of Ras by photic sensory input is yet unknown. Here, we describe for the first time a circadian rhythm of Ras activity in the mouse SCN. Using synRas transgenic mice, expressing constitutively activated V12-Ha-Ras selectively in neurons, we demonstrate that enhanced Ras activation causes shortening of the circadian period length. We found upregulated expression and decreased inhibitory phosphorylation of the circadian period length modulator, glycogen synthase kinase-3 beta (GSK3ß), in the SCN of synRas mice. Conversely, downregulation of Ras activity by blocking its function with an antibody in oscillating cell cultures reduced protein levels and increased phosphorylation of GSK3ß and lengthened the period of BMAL1 promoter-driven luciferase activity. Furthermore, enhanced Ras activity in synRas mice resulted in a potentiation of light-induced phase delays at early subjective night, and increased photic induction of pERK1,2/pCREB and c-Fos. In contrast, at late subjective night, photic activation of Ras/ERK1,2/CREB in synRas mice was not sufficient to stimulate c-Fos protein expression and phase advance the clock. Taken together, our results demonstrate that Ras activity fine tunes the period length and modulates photoentrainment of the circadian clock.

Ribosomal RNA - a tail wagging the dog?

It is an intriguing hypothesis that the complex organization of neuronal dynamics important for a memory engram is largely underpinned by the regulation of nucleolar functioning. This Editorial highlights a study by Capitano and coworkers in this issue of the Journal of Neurochemistry, in which the authors tackle this hypothesis with a behavioral approach. The study investigates the role of axo-dendritic mRNAs within learning-induced plasticity and in vivo modulation of rRNA transcription in response to spatial learning. The authors confirm with their in vivo approach what is known from many earlier in vitro experiments: efficient learning and memory requires a proper homeostasis of hippocampal neurons in general, which, however, depends crucially on proper integrity of the nucleolus. Read the highlighted article 'RNA polymerase I transcription is modulated by spatial learning in different brain regions' on page 706.

PERIOD1 coordinates hippocampal rhythms and memory processing with daytime.

In species ranging from flies to mammals, parameters of memory processing, like acquisition, consolidation, and retrieval are clearly molded by time of day. However, mechanisms that regulate and adapt these temporal differences are elusive, with an involvement of clock genes and their protein products suggestive. Therefore, we analyzed initially in mouse hippocampus the daytime-dependent dynamics of parameters, known to be important for proper memory formation, like phosphorylation of the "memory molecule" cyclic adenosine monophosphate (cAMP) responsive element binding protein (CREB) and chromatin remodeling. Next, in an effort to characterize the mechanistic role of clock genes within hippocampal molecular dynamics, we compared the results obtained from wildtype (WT) -mice and mice deficient for the archetypical clock gene Period1 (Per1(-/-) -mice). We detected that the circadian rhythm of CREB phosphorylation in the hippocampus of WT mice disappeared completely in mice lacking Per1. Furthermore, we found that the here for the first time described profound endogenous day/night rhythms in histone modifications in the hippocampus of WT-mice are markedly perturbed in Per1(-/-) -mice. Concomitantly, both, in vivo recorded LTP, a cellular correlate for long-term memory, and hippocampal gene expression were significantly altered in the absence of Per1. Notably, these molecular perturbations in Per1(-/-) -mice were accompanied by the loss of daytime-dependent differences in spatial working memory performance. Our data provide a molecular blueprint for a novel role of PER1 in temporally shaping the daytime-dependency of memory performance, likely, by gating CREB signaling, and by coupling to downstream chromatin remodeling.

Clock gene expression in the human pituitary gland.

Pituitary function relies on strictly timed, yet plastic mechanisms, particularly with respect to the daytime-dependent coordination of hormone synthesis and release. In other systems, clock genes and their protein products are well-described candidates to anticipate the daily demands in neuroendocrine coupling and to manage cellular adaptation on changing internal or external circumstances. To elucidate possible mechanisms of time management, a total of 52 human autoptic pituitary glands were allocated to the 4 time-of-day groups, night, dawn, day, and dusk, according to reported time of death. The observed daytime-dependent dynamics in ACTH content supports a postmortem conservation of the premortem condition, and thus, principally validates the investigation of autoptic pituitary glands. Pituitary extracts were investigated for expression of clock genes Per1, Cry1, Clock, and Bmal1 and corresponding protein products. Only the clock gene Per1 showed daytime-dependent differences in quantitative real-time PCR analyses, with decreased levels observed during dusk. Although the overall amount in clock gene protein products PER1, CRY1, and CLOCK did not fluctuate with time of day in human pituitary, an indication for a temporally parallel intracellular translocation of PER1 and CRY1 was detected by immunofluorescence. Presented data suggest that the observed clock gene expression in human pituitary cells does not provide evidence for a functional intrinsic clockwork. It is suggested that clock genes and their protein products may be directly involved in the daytime-dependent regulation and adaptation of hormone synthesis and release and within homeostatic adaptive plasticity.

A survey of molecular details in the human pineal gland in the light of phylogeny, structure, function and chronobiological diseases.

The human pineal gland is a neuroendocrine transducer that forms an integral part of the brain. Through the nocturnally elevated synthesis and release of the neurohormone melatonin, the pineal gland encodes and disseminates information on circadian time, thus coupling the outside world to the biochemical and physiological internal demands of the body. Approaches to better understand molecular details behind the rhythmic signalling in the human pineal gland are limited but implicitly warranted, as human chronobiological dysfunctions are often associated with alterations in melatonin synthesis. Current knowledge on melatonin synthesis in the human pineal gland is based on minimally invasive analyses, and by the comparison of signalling events between different vertebrate species, with emphasis put on data acquired in sheep and other primates. Together with investigations using autoptic pineal tissue, a remnant silhouette of premortem dynamics within the hormone's biosynthesis pathway can be constructed. The detected biochemical scenario behind the generation of dynamics in melatonin synthesis positions the human pineal gland surprisingly isolated. In this neuroendocrine brain structure, protein-protein interactions and nucleo-cytoplasmic protein shuttling indicate furthermore a novel twist in the molecular dynamics in the cells of this neuroendocrine brain structure. These findings have to be seen in the light that an impaired melatonin synthesis is observed in elderly and/or demented patients, in individuals affected by Alzheimer's disease, Smith-Magenis syndrome, autism spectrum disorder and sleep phase disorders. Already, recent advances in understanding signalling dynamics in the human pineal gland have significantly helped to counteract chronobiological dysfunctions through a proper restoration of the nocturnal melatonin surge.

Dynamics in enzymatic protein complexes offer a novel principle for the regulation of melatonin synthesis in the human pineal gland.

Time of day is communicated to the body through rhythmic cues, including pineal gland melatonin synthesis, which is restricted to nighttime. Whereas in most rodents transcriptional regulation of the arylalkylamine N-acetyltransferase (Aanat) gene is essential for rhythmic melatonin synthesis, investigations into nonrodent mammalian species have shown post-transcriptional regulation to be of central importance, with molecular mechanisms still elusive. Therefore, human pineal tissues, taken from routine autopsies were allocated to four time-of-death groups (night/dawn/day/dusk) and analyzed for daytime-dependent changes in phosphorylated AANAT (p31T-AANAT) and in acetyl-serotonin-methyltransferase (ASMT) expression and activity. Protein content, intracellular localization, and colocalization of p31T-AANAT and ASMT were assessed, using immunoblotting, immunofluorescence, and immunoprecipitation techniques. Fresh sheep pineal gland preparations were used for comparative purposes. The amount of p31T-AANAT and ASMT proteins as well as their intracellular localization showed no diurnal variation in autoptic human and fresh sheep pineal glands. Moreover, in human and sheep pineal extracts, AANAT could not be dephosphorylated, which was at variance to data derived from rat pineal extracts. P31T-AANAT and ASMT were often found to colocalize in cellular rod-like structures that were also partly immunoreactive for the pinealocyte process-specific marker S-antigen (arrestin) in both, human and sheep pinealocytes. Protein-protein interaction studies with p31T-AANAT, ASMT, and S-antigen demonstrated a direct association and formation of robust complexes, involving also 14-3-3. This work provides evidence for a regulation principle for AANAT activity in the human pineal gland, which may not be based on a p31T-AANAT phosphorylation/dephosphorylation switch, as described for other mammalian species.

Temporal dynamics of mouse hippocampal clock gene expression support memory processing.

Hippocampal plasticity and mnemonic processing exhibit a striking time-of-day dependence and likely implicate a temporally structured replay of memory traces. Molecular mechanisms fulfilling the requirements of sensing time and capturing time-related information are coded in dynamics of so-called clock genes and their protein products, first discovered and described in the hypothalamic suprachiasmatic nucleus. Using real-time PCR and immunohistochemical analyses, we show that in wildtype mice core clock components (mPer1/PER1, mPer2/PER2, mCry1/CRY1, mCry2/CRY2, mClock/CLOCK, mBmal1/BMAL1) are expressed in neurons of all subregions of the hippocampus in a time-locked fashion over a 24-h (diurnal) day/night cycle. Temporal profiling of these transcriptional regulators reveals distinct and parallel peaks, at times when memory traces are usually formed and/or consolidated. The coordinated rhythmic expression of hippocampal clock gene expression is greatly disordered in mice deficient for the clock gene mPer1, a key player implicated in both, maintenance and adaptative plasticity of circadian clocks. Moreover, Per1-knockout animals are severely handicapped in a hippocampus-dependent long-term spatial learning paradigm. We propose that the dynamics of hippocampal clock gene expression imprint a temporal structure on memory processing and shape at the same time the efficacy of behavioral learning.

Exploiting endogenous anti-apoptotic proteins for novel therapeutic strategies in cerebral ischemia.

The acute neuronal degeneration in the ischemic core upon stroke is followed by a second wave of cell demise in the ischemic penumbra and neuroanatomically connected sites. This temporally delayed deleterious event of programmed cell death ('secondary degeneration') often exceeds the initial damage of stroke and, thus, contributes pivotally to significant losses in neurological functions. In fact, it is the injured neurons in these regions around the ischemic core zone that neuropharmacological prevention is targeting to preserve. Clinical and pre-clinical studies have focussed on neuroprotective interventions with caspase inhibitors, but it remains ambiguous whether diminishing or even silencing these aspartate-specific cysteine proteases are in sum beneficial for the clinical outcome. It is often ignored that caspase inhibitors are able to antagonize calpain and cathepsins, thereby protecting the cytoskeleton from damage. Moreover, there is a point of no return, beyond which interfering with caspases cannot rescue the cell, but spoil the obligate and necessary suicide program such that the cellular environment suffers from by-products of necrosis and secondary inflammation. Here we discuss novel alternative strategies to abrogate the death cascade at the level of the genomic response (transcription factors, NF-kappaB, CREB, ICER, HIF), of mitochondrial effectors (cytochrome c, Bcl-2, Smac/DIABLO, HtrA2), and of inhibitor of apoptosis proteins (IAPs). IAPs are the only known endogenous proteins that inhibit specifically and with high affinity the activity of both initiator and effector caspases. Based on compelling biochemical evidence, we argue that patronizing the neuronal endogenous anti-apoptotic machinery could be superior to the pharmacological inhibition of caspases at various levels, with regard to specificity, side effects, and the 'therapeutic window of opportunity'.

Transcription factor dynamics in pineal gland and liver of the Syrian hamster (Mesocricetus auratus) adapts to prevailing photoperiod.

The anticipation of day length and duration of darkness is necessary and advantageous for animals to survive and requires a photoperiodic memory. In the Syrian hamster this adaptation to photoperiod is mirrored by seasonal changes in the animal's reproductive state and its liver metabolism. Both events are linked to season-dependent alterations of the nocturnally elevated synthesis of the pineal hormone melatonin. To decipher molecules that are involved in this temporal gating, hamsters were exposed to long photoperiod (16 hr light:8 hr darkness; LP), or short photoperiod (8 hr light:16 hr darkness; SP). Dynamics in gene expression was investigated in the pineal gland [inducible cAMP early repressor (ICER)], and in the liver (ICER; C/EBPdelta; clock genes) using immunochemistry and reverse transcriptase PCR. While in the pineal, ICER rhythms tightly follow the prior duration of light and dark with decreasing levels at the beginning of the dark period in both LP and SP, ICER is not rhythmic in liver. In the liver, clock genes and their protein products reflect differences in photoperiodic history, with enhanced rhythm amplitudes of PER, CRY, CLOCK, and BMAL1 under SP conditions. Thus, in the Syrian hamster transcription factor expression patterns lock onto the prevailing photoperiod in two peripheral oscillators, the pineal gland and the liver, to function as mediators of suprachiasmatic nucleus-derived information on environmental light and dark. This tissue-specific gating in gene transcription represents a strategy to ameliorate consequences of altering environmental lighting conditions on endocrine and metabolic parameters that endow a strong circadian bias.

Day-night expression patterns of clock genes in the human pineal gland.

Rhythm generation within the mammalian circadian system is achieved by clock genes and their protein products. As an integral part of this system, the pineal gland serves the need to tune the body to the temporal environment by the rhythmic synthesis and release of melatonin. A number of human disorders and syndromes are associated with alterations in circadian rhythms of clock genes and their protein products and/or a dysfunction in melatonin synthesis. In the human, little is known about the molecular signature of time management. Pineal tissue from regular autopsies was allocated to asserted time-of-death groups (dawn, day, dusk, night), and analyzed by RT-PCR, immunoblotting, immunohistochemistry, and confocal laser scanning microscopy for expression of clock genes. Despite the observed diurnal rhythms in activity of the arylalkylamine N-acetyltransferase and in melatonin content, mRNA levels for the clock genes Period1, Cryptochrome1, Clock, and Bmal1, and also amounts of corresponding clock gene proteins showed no differences between time- of-death groups. In contrast, a time-of-day-dependent nucleocytoplasmic shuttling of clock gene proteins was detected. These data confirm the minor importance of a transcriptional regulation for dynamics in the human pineal gland, and offer a novel twist in the molecular competence of clock gene proteins.

The mammalian pineal gland: known facts, unknown facets.

In the mammalian pineal gland, information on environmental lighting conditions that is neuronally encoded by the retina is converted into nocturnally elevated synthesis of the hormone melatonin. Evolutionary pressure has changed the morphology of vertebrate pinealocytes, eliminating direct photoreception and the endogenous clock function. Despite these changes, nocturnally elevated melatonin synthesis has remained a reliable indicator of time throughout evolution. In the photo-insensitive mammalian pineal gland this message of darkness depends on the master circadian pacemaker in the hypothalamic suprachiasmatic nuclei. The dramatic change in vertebrate pinealocytes has received little attention; here, we therefore link the known evolutionary morphodynamics and well-investigated biochemical details responsible for rhythmic synthesis of melatonin with recently characterized patterns of gene expression in the pineal gland. We also address the enigmatic function of clockwork molecules in mammalian pinealocytes.

Melatonin synthesis in the human pineal gland: advantages, implications, and difficulties.

Rhythms in the mammalian pineal organ depend on afferent information that is derived from the endogenous clock residing in the hypothalamic suprachiasmatic nucleus (SCN). The best characterized function of the pineal gland is the nocturnally elevated synthesis of the hormone melatonin, which provides the body with the signal of the duration of the night period. The rate-limiting enzyme for melatonin synthesis is arylalkylamine N-acetyltransferase (AANAT). In contrast to the transcriptional regulation of the Aanat gene in rodents, a post-translational shaping of the melatonin pattern is indicated in the human pineal gland. Despite the fact that melatonin levels can be determined easily in various body fluids, the molecular elements involved in shaping the rhythmic hormone synthesis cannot be analyzed experimentally in the living organism. However, the use of post-mortem pineal material seems to constitute a valid approach to decipher the regulation of human melatonin synthesis.

Characterization of human melatonin synthesis using autoptic pineal tissue.

The mammalian pineal gland synthesizes rhythmically the hormone melatonin, which provides the body with a signal coding the duration of the night period. The ultimate enzymatic step in melatonin synthesis is achieved by the hydroxyindole O-methyltransferase (HIOMT); the rate-limiting enzyme is, however, the arylalkylamine N-acetyltransferase (AA-NAT). In contrast to the central importance of a transcriptional regulation of the Aa-nat gene for rodent melatonin synthesis, mechanisms in the human pineal gland are elusive. Therefore, pineal tissue, taken from regular autopsies (n = 69; postmortem intervals ranging from 9 to 147 h) was analyzed simultaneously for Aa-nat and Hiomt mRNA levels by PCR, AA-NAT activity using (14)C-acetyl-coenzyme A, HIOMT activity using S-adenosyl-l-[(14)C]-methionine, and melatonin content using an ELISA. Results were allocated to asserted time-of-death groups (day, 1,000 to 1,630 h; dusk, 1,630 to 2,200 h; night, 2,200 to 0730 h; dawn, 0730 to 1,000 h). RNA degradation rates of genes of interest ran in parallel, and, therefore, data normalization could be established, regardless of postmortem delay in tissue sampling. Aa-nat and Hiomt mRNA and HIOMT activity showed no diurnal rhythm. In contrast, a significant rhythm was found for the correlation between time of death and both AA-NAT activity and melatonin content, with elevated values during dusk and night. Presented data demonstrate that postmortem brain tissue can be used to detect the remnant of premortem adaptive changes in neuronal activity. In particular, our results give strong experimental support for the idea that transcriptional mechanisms are not dominant for the generation of rhythmic melatonin synthesis in the human pineal gland.

Rhythms in clock proteins in the mouse pars tuberalis depend on MT1 melatonin receptor signalling.

Melatonin provides a rhythmic neuroendocrine output, driven by a central circadian clock that encodes information about phase and length of the night. In the hypophyseal pars tuberalis (PT), melatonin is crucial for rhythmic expression of the clock genes mPer1 and mCry1, and melatonin acting in the PT influences prolactin secretion from the pars distalis. To examine further the possibility of a circadian clockwork functioning in the PT, and the impact of melatonin on this tissue, we assessed circadian clock proteins by immunohistochemistry and compared the diurnal expression in the PT of wild type (WT), and MT1 melatonin receptor-deficient (MT1-/-) mice. While in the PT of WT mice mPER1, mPER2, and mCRY1 showed a pronounced rhythm, mCRY2, CLOCK, and BMAL1 were constitutively present. Despite reported differences in maximal levels and timing of mCry1, mPer1, and mPer2 RNAs, the corresponding protein levels peaked simultaneously during late day, suggesting a codependency for their stabilization and/or nuclear entry. MT1-/- mice had reduced levels of mPER1, mCRY1, CLOCK and BMAL1, consistent with the earlier reported reduction in mRNA expression of these clock genes. Surprisingly, mPER2-immunoreaction was constitutively low, although mPer2 was rhythmically expressed in the PT of MT1-/- mice. This suggests that mPER2 is degraded due to the reduced levels of its stabilizing interaction partners mPER1 and mCRY1. The results show that melatonin, acting through the MT1, determines availability of the circadian proteins mPER1, mPER2 and mCRY1 and thus plays a crucial role in regulating rhythmicity in PT cells.