Cyclin D1 induction preceding neuronal death via the excitotoxic NMDA pathway involves selective stimulation of extrasynaptic NMDA receptors and JNK pathway.

The N-methyl-D-aspartate subtype of ionotropic glutamate receptors (NMDARs) signals both prosurvival and death-inducing (excitotoxic) neuronal responses via synaptically (synaptic NMDAR) and extrasynaptically (extrasynaptic NMDAR) located receptor pools, respectively. Both receptor pools share similar, though not identical, postreceptor signaling molecules. The activation of the extrasynaptic NMDAR pathway is predominant. Therefore, in order to inhibit the extrasynaptic death pathway while sparing synaptic responses, it is critical to identify selective postreceptor effectors of extrasynaptic NMDARs. The present study addressed these issues by using primary cultures of rat hippocampal neurons and a pharmacological protocol of selective NMDAR stimulation for Western blot and immunocytochemistry analyses. We found that the activation of extrasynaptic NMDARs, either alone or together with synaptic NMDARs, triggers cyclin-D1-associated re-entry into the cell cycle, which does not proceed beyond the S-phase. This aberrant cell cycle re-entry is particularly associated with neuronal death triggered specifically via extrasynaptic NMDAR-induced c-Jun N-terminal protein kinase (JNK). In addition, NMDA-elicited neuronal death was significantly inhibited by pharmacological blockade of JNK-mediated cyclin D1 expression or by silencing cyclin D1 RNA. Taken together, these data suggest a causal relationship between cyclin D1 induction and extrasynaptic NMDAR-triggered neuronal death along the excitotoxic NMDA pathway. Therefore, cyclin D1 may be a putative target for the development of neuroprotective strategies sparing physiological synaptic NMDAR signaling.

Polyphenols as potential inhibitors of amyloid aggregation and toxicity: possible significance to Alzheimer's disease.

Beta-amyloid (Abeta)likely plays a pivotal role in the etiology of Alzheimer's disease (AD). Consequently, Abeta associated pathways are targets for the development of possible effective AD therapies. This review first updates strategies aimed at the inhibition of Abeta formation and then discusses the role of food-derived polyphenols as putative anti-amyloid drugs.

Developmental profile of neuregulin receptor ErbB4 in postnatal rat cerebral cortex and hippocampus.

We investigated the cellular and subcellular distributions of neuregulin tyrosine kinase receptor ErbB4 in the postnatal rat frontal cortex and hippocampus by light-, confocal- and electron-microscopic immunocytochemistry. At birth, ErbB4-immunoreactivity (ErbB4-IR) was prominent in the apical cytoplasm and dendrites of cortical plate neurons and hippocampal pyramidal cells. Throughout postnatal development and in adulthood, ErbB4-IR in both regions remained confined to the somatodendritic compartment of neurons, which increased in number to reach the adult pattern by the end of the first postnatal month (P30). At all ages examined, double-labeling experiments revealed that ErbB4-IR always co-localized with the neuronal marker neuronal nuclei (NeuN) and never with glial markers Nestin or glial fibrillary acidic protein (GFAP). Immunoperoxidase labeling at the ultrastructural level confirmed the exclusive localization of ErbB4-IR in somatodendrites, and notably in dendritic spines. Immunogold labeling showed preponderant ErbB4-IR in the cytoplasm, where it was associated with microtubules. Furthermore, ErbB4-IR was abundant in the nucleus of adult cortical and hippocampal neurons, suggesting a role for ErbB4 nuclear signaling in the brain beyond embryonic development. Taken together, these results show that ErbB4 is expressed by neuronal somatodendrites in cerebral cortex and hippocampus from birth to adulthood, and support a role for neuregulins in dendritic growth and plasticity.

Applications and current challenges of proteomic approaches, focusing on two-dimensional electrophoresis.

Since the formulation of the concept of "proteomics" in 1995, a plethora of proteomic technologies have been developed in order to study proteomes of tissues, cells and organelles. The powerful new technologies enabled by proteomic approaches have lead to the application of these methods to an exponentially increasing variety of biological questions for highly complex protein mixtures. Continuous technical optimization allows for an ever-increasing sensitivity of proteomic techniques. In this review, a brief overview of currently available proteomic techniques and their applications is given, followed by a more detailed description of advantages and technical challenges of two-dimensional electrophoresis (2-DE). Some solutions to circumvent currently encountered technical difficulties for 2-DE analyses are proposed.

Cellular distribution of insulin-like growth factor-II/mannose-6-phosphate receptor in normal human brain and its alteration in Alzheimer's disease pathology.

The insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor is a multifunctional membrane glycoprotein, which binds different classes of ligands including IGF-II and M6P-bearing lysosomal enzymes. Besides participating in the process of endocytosis this receptor functions in the trafficking of lysosomal enzymes from the trans-Glogi network (TGN) or the cell surface to lysosomes. In Alzheimer's disease (AD) brain, marked overexpression of certain lysosomal enzymes in vulnerable neuronal populations and their association to beta-amyloid (Abeta) containing neuritic plaques has been correlated to altered metabolic functions. In the present study, we measured the levels of IGF-II/M6P receptor and characterized its distribution profile in selected regions of AD and age-matched normal postmortem brains. Western blot analysis revealed no significant alteration in the levels of IGF-II/M6P receptor either in the hippocampus, frontal cortex or cerebellum between AD and age-matched control brains. However, a significant gene dose effect of apolipoprotein E (APOE) epsilon4 allele on IGF-II/M6P receptor levels was evident in the hippocampus of the AD brain. At the cellular level, immunoreactive IGF-II/M6P receptors were localized in the neurons of the frontal cortex, hippocampus and cerebellum of control brains. In AD brains, the labeling of the neurons was less intense in the frontal cortex and hippocampus than in the age-matched control brains. Additionally, IGF-II/M6P receptor immunoreactivity was observed in association with a subpopulation of Abeta-containing neuritic plaques as well as tau-positive neurofibrillary tangles both in the frontal cortex and the hippocampus. Reactive glial cells localized adjacent to the plaques also occasionally exhibited IGF-II/M6P receptor immunoreactivity. These results, when analyzed in context of the established role of the IGF-II/M6P receptor in the regulation of the intracellular trafficking of lysosomal enzymes, suggest that alterations in IGF-II/M6P receptor levels/distribution are possibly associated with altered functioning of the lysosomal enzymes and/or loss of neurons observed in AD brains, especially in patients carrying APOE epsilon4 alleles.

Beta-amyloid peptides as direct cholinergic neuromodulators: a missing link?

Beta-Amyloid peptide (Abeta) is found in diffuse and focal deposits throughout the brain from Alzheimer's disease (AD) patients. Another feature of AD is the widespread degeneration and dysfunction of the basal-forebrain cholinergic system. Until now, it has been unclear how these features of AD might be related. Recent reports, however, suggest that Abeta can potently inhibit various cholinergic neurotransmitter functions independently of apparent neurotoxicity. This capacity of Abeta might contribute to the vulnerability of selected cholinergic neuronal populations in AD. Moreover, the high potency (picomolar to nanomolar concentrations) of these effects and the secretion of Abeta by brain cells indicate that Abeta-induced cholinergic hypoactivity might have physiological in addition to pathological significance.

Rediscovering an old friend, IGF-I: potential use in the treatment of neurodegenerative diseases.

Insulin-like growth factor-I (IGF-I) is a pleiotropic protein that acts on many tissues and organs. As it is one of the major trophic factors in the circulation, its actions in peripheral tissues are well established. It has been used for the treatment of several diseases, including growth deficiency, osteoporosis, catabolic disorders and diabetes. Recent evidence supports the significance of IGF-I in the maintenance of the integrity and homeostasis of the nervous system. The widespread distribution of its receptor allows IGF-I to affect the survival of numerous populations of neurones and glial cells in both the CNS and the PNS. Most recently, a clinical trial has revealed the beneficial effects of IGF-I in amyotrophic lateral-sclerosis (ALS), a degenerative disease of the motoneurones. We review briefly here experimental and clinical information that suggests the potential usefulness of IGF-I in the treatment of certain neurodegenerative diseases, including ALS, Alzheimer's disease, various neuropathies and brain trauma. The rather unique propensity of IGF-I to act on a variety of neuronal cells might provide a general means of reducing or slowing down neuronal losses that occur following various brain insults.

Neurotensin receptor levels as a function of brain aging and cognitive performance in the Morris water maze task in the rat.

The present study evaluated whether neurotensin (NT) binding sites were altered in the aged rat brain and if these alterations were related to the cognitive status of the animal. Aged (24-25 months old) Long-Evans rats were behaviorally screened using the Morris water maze task and were classified as either aged, cognitively impaired (AI) or cognitively unimpaired (AU) based on their relative performances in the task compared to young control (Y) animals. Decreases in specific [125I]NT binding were observed in the hippocampal formation, namely the dentate gyrus (DG), as well as in the septum and hypothalamus. Both aged groups also showed significant reductions in specific [125I]NT binding levels compared to the Y animals in the hippocampal CA3 sub-field, with the AI animals exhibiting the lowest levels. In the Substantia Nigra Zona Compacta (SNc) and the ventral tegmental area (VTA), specific [125I]NT binding was decreased as a function of age while binding in the paraventricular nucleus of the hypothalamus (PVNh) was decreased as a function of age and cognitive status. These alterations in the level of specific [125I] NT binding in the aged animals suggest decreases in NT receptor signaling as a function of age and potential involvement of NT-ergic systems in the etiology of age-related cognitive deficits.

Nerve growth factor rapidly induces prolonged acetylcholine release from cultured basal forebrain neurons: differentiation between neuromodulatory and neurotrophic influences.

Long-term exposure to nerve growth factor (NGF) is well established to have neurotrophic effects on basal forebrain cholinergic neurons, but its potential actions as a fast-acting neuromodulator are not as well understood. We report that NGF (0.1-100 ng/ml) rapidly (<60 min) and robustly enhanced constitutive acetylcholine (ACh) release (148-384% of control) from basal forebrain cultures without immediate persistent increases in choline acetyltransferase activity. More ACh was released in response to NGF when exposure was coupled with a higher depolarization level, suggesting activity dependence. In a long-term potentiation-like manner, brief NGF exposure (10 ng/ml; 60 min) induced robust and prolonged increases in ACh release, a capacity that was shared with the other neurotrophins. K252a (10-100 nm), BAPTA-AM (25 microm), and Cd(2+) (200 microm) prevented NGF enhancement of ACh release, suggesting the involvement of TrkA receptors, Ca(2+), and voltage-gated Ca(2+) channels, respectively. Forskolin (10 microm), a cAMP generator, enhanced constitutive ACh release but did not interact synergistically with NGF. Tetrodotoxin (1 microm) and cycloheximide (2 microm) did not prevent NGF-induced ACh release, indicative of action at the level of the cholinergic nerve terminal and that new protein synthesis is not required for this neurotransmitter-like effect, respectively. In contrast, after a 24 hr NGF treatment, distinct protein synthesis-dependent and independent effects on choline acetyltransferase activity and ACh release were observed. These results indicate that neuromodulator/neurotransmitter-like (protein synthesis-independent) and neurotrophic (translation-dependent) actions likely make distinct contributions to the enhancement of cholinergic activity by NGF.

The molecular pharmacology of CGRP and related peptide receptor subtypes.

Calcitonin gene-related peptides (alpha and beta isoforms), better known as CGRPalpha and CGRPbeta, were isolated twenty years ago. In fact, these were the first peptides to be characterized using a molecular cloning strategy, which is not the traditional approach of biochemical extraction and purification. Paradoxically, progress in the characterization of CGRP receptor subtypes has been extremely slow as a result of difficulties in their cloning and the lack of selective receptor subtype agonists and antagonists. However, exciting progress has been made overthe pasttwo years and is briefly reviewed here.

DNA microarrays in neuropsychopharmacology.

Recent advances in experimental genomics, coupled with the wealth of sequence information available for a variety of organisms, have the potential to transform the way pharmacological research is performed. At present, high-density DNA microarrays allow researchers to quickly and accurately quantify gene-expression changes in a massively parallel manner. Although now well established in other biomedical fields, such as cancer and genetics research, DNA microarrays have only recently begun to make significant inroads into pharmacology. To date, the major focus in this field has been on the general application of DNA microarrays to toxicology and drug discovery and design. This review summarizes the major microarray findings of relevance to neuropsychopharmacology, as a prelude to the design and analysis of future basic and clinical microarray experiments. The ability of DNA microarrays to monitor gene expression simultaneously in a large-scale format is helping to usher in a post-genomic age, where simple constructs about the role of nature versus nurture are being replaced by a functional understanding of gene expression in living organisms.

Chemokines and chemokine receptors in the CNS: a possible role in neuroinflammation and patterning.

Chemokines constitute a growing family of structurally and functionally related small (8-10 kDa) proteins associated with inflammatory-cell recruitment in host defence. In addition to their well-established role in the immune system, recent data suggest their involvement in the maintenance of CNS homeostasis, in neuronal patterning during ontogeny and as potential mediators of neuroinflammation, playing an essential role in leukocyte infiltration into the brain. Chemokines and their G protein-coupled receptors are constitutively expressed at low-to-negligible levels in various cell types in the brain. Their expression is rapidly induced by various neuroinflammatory stimuli, implicating them in various neurological disorders such as trauma, stroke and Alzheimer's disease, in tumour induction and in neuroimmune diseases such as multiple sclerosis or acquired immunodeficiency syndrome (AIDS). Here, F. Mennicken, R. Maki, E. B. De Souza and R. Quirion briefly summarize recent exciting findings in the field.

Muscarinic receptor subtypes in human neurodegenerative disorders: focus on Alzheimer's disease.

Much evidence has clearly revealed the existence of marked cholinergic deficits in cortical and hippocampal areas in Alzheimer's disease. Although not necessarily of etiological origin, these deficits have been associated with learning and memory disabilities observed in this neurogenerative disorder. We report here that in addition to deficits in choline acetyltransferase (ChAT) activity, the maximal densities of high affinity [3H]acetylcholine and [3H]AF-DX 116 (possibly M2), but not M1 muscarinic receptor binding sites are decreased in cortex and hippocampus in Alzheimer's disease. Similar findings are also observed in Parkinson's disease with Alzheimer's type dementia. Additionally, animal studies suggest that a population of M2 receptors is presynaptically located on cholinergic nerve terminals where they can act as negative autoreceptors to decrease acetylcholine release. Interestingly, blockade of these sites facilitates acetylcholine release and learning in rats. This may be relevant for the design of more appropriate therapeutic approaches toward the treatment of certain symptoms of Alzheimer's disease.

Acetylcholine release is elicited in the visual cortex, but not in the prefrontal cortex, by patterned visual stimulation: a dual in vivo microdialysis study with functional correlates in the rat brain.

By its projections to the primary visual and the prefrontal cortices, the basal forebrain cholinergic system is involved in cognitive processing of sensory stimuli. It has been suggested that visual stimulus-induced cholinergic activation of the visual cortex may exert a permissive role on thalamocortical inputs. However, it is not known if visual stimulation elicits cholinergic activation of high-order brain areas in the absence of attentional need. In the present study, we measured the effects of patterned visual stimulation (horizontal grating) on the release of acetylcholine with dual-probe in vivo microdialysis in the visual and the prefrontal cortices of anesthetized rats. We also used retrograde tracing to determine the anatomical relationships of cholinergic neurons with neurons of the visual system and the prefrontal cortex. Finally, we evaluated a functional correlate of this stimulation, namely c-fos immunolabeling. Patterned visual stimulation elicited significant increases in acetylcholine release in the visual cortex, accompanied by an increased number of c-fos immunoreactive neurons in this brain area. In contrast, in the prefrontal cortex, neither the level of acetylcholine release nor the number of c-fos immunoreactive neurons was significantly changed because of the stimulation. Cholinergic basal forebrain neurons projecting to the visual or the prefrontal cortices were both localized within the horizontal limb of the diagonal band of Broca but were not immunoreactive for c-fos during visual stimulation. No parts of the visual system were found to directly project to these basal forebrain neurons. These results suggest the differential involvement of cholinergic projections in the integration of sensory stimuli, depending on the level of activity of the targeted cortical area.

Alterations in dendritic morphology of prefrontal cortical and nucleus accumbens neurons in post-pubertal rats after neonatal excitotoxic lesions of the ventral hippocampus.

Neonatal ventral hippocampal (nVH) lesions in rats result in adult onset of a number of behavioral and cognitive abnormalities analogous to those seen in schizophrenia, including hyperresponsiveness to stress and psychostimulants and deficits in working memory, sensorimotor gating and social interaction. Molecular and neurochemical alterations in the prefrontal cortex (PFC) and nucleus accumbens (NAcc) of nVH-lesioned animals suggest developmental reorganization of these structures following neonatal lesions. To determine whether nVH lesions lead to neuronal morphological changes, we investigated the effect of nVH lesion on dendritic structure and spine density of pyramidal neurons of the PFC and medium spiny neurons of the NAcc. Bilateral ibotenic acid-induced lesion of the VH was made in Sprague-Dawley pups at postnatal day 7 (P7); and at P70, neuronal morphology was quantified by modified Golgi-Cox staining. The results show that length of basilar dendrites and branching and the density of dendritic spines on layer 3 pyramidal neurons were significantly decreased in rats with nVH lesions. Medium spiny neurons from the NAcc showed a decrease in the density of dendritic spines without significant changes in dendritic length or arborization. The data, comparable to those observed in the PFC of schizophrenic patients, suggest that developmental loss of excitatory projections from the VH may lead to altered neuronal plasticity in the PFC and the NAcc that may contribute to the behavioral changes in these animals.

Strategies in neuropeptide receptor binding research.

Strategies and general approaches used in neuropeptide receptor binding assays are described. Special attention is given to the nature of the ligand, its physical and chemical stability and the demonstration of an appropriate ligand selectivity pattern. Examples are given to illustrate critical aspects of neuropeptide receptor binding assays. Strong correlation between binding and bioassay data is also stressed.

Neuroanatomical localization, pharmacological characterization and functions of CGRP, related peptides and their receptors.

Calcitonin generelated peptide (CGRP) is a neuropeptide discovered by a molecular approach over 10 years ago. More recently, islet amyloid polypeptide or amylin, and adrenomedullin were isolated from human insulinoma and pheochromocytoma respectively, and revealed between 25 and 50% sequence homology with CGRP. This review discusses findings on the anatomical distributions of CGRP mRNA, CGRP-like immunoreactivity and receptors in the central nervous system, as well as the potential physiological roles for CGRP. The anatomical distribution and biological activities of amylin and adrenomedullin are also presented. Based upon the differential biological activity of various CGRP analogs, the CGRP receptors have been classified in two major classes, namely the CGRP1 and CGRP2 subtypes. A third subtype has also been proposed (e.g. in the nucleus accumbens) as it does not share the pharmacological properties of the other two classes. The anatomical distribution and the pharmacological characteristics of amylin binding sites in the rat brain are different from those reported for CGRP but share several similarities with the salmon calcitonin receptors. The receptors identified thus far for CGRP and related peptides belong to the G protein-coupled receptor superfamily. Indeed, modulation of adenylate cyclase activity following receptor activation has been reported for CGRP, amylin and adrenomedullin. Furthermore, the binding affinity of CGRP and related peptides is modulated by nucleotides such as GTP. The cloning of various calcitonin and most recently of CGRP1 and adrenomedullin receptors was reported and revealed structural similarities but also significant differences to other members of the G protein-coupled receptors. They may thus form a new subfamily. The cloning of the amylin receptor(s) as well as of the other putative CGRP receptor subtype(s) are still awaited. Finally, a broad variety of biological activities has been described for CGRP-like peptides. These include vasodilation, nociception, glucose uptake and the stimulation of glycolysis in skeletal muscles. These effects may thus suggest their potential role and therapeutic applications in migraine, subarachnoid haemorrhage, diabetes and pain-related mechanisms, among other disorders.

Hypothalamic-pituitary-adrenal activity during chronic central administration of interleukin-2.

The cytokine interleukin-2 (IL-2) exerts numerous effects within the immune as well as the central nervous system and is thought to serve as a humoral signal in their communication. Brain-derived or blood-borne IL-2 may also control the activity of the hypothalamic-pituitary-adrenal (HPA) axis at various levels of regulation. In this study we investigated whether persistently elevated levels of central IL-2, which are associated with several diseases or induced during immunotherapeutic use of this cytokine, could induce long term activation of the HPA axis. Adult male Sprague-Dawley rats received an intracerebroventricular infusion of the recombinant cytokine at a rate of 5 U/h (equivalent to 2.5 ng/h or 162 fmol/h) by means of osmotic minipumps. Control animals received heat-inactivated IL-2. After 7 days of continuous infusion, blood samples were taken at intervals of 4 h over a period of 24 h, and plasma levels of ACTH and corticosterone (CORT) were determined. IL-2 caused a significant increase in ACTH levels during the later portion of the dark phase of the cycle. Plasma CORT concentrations were significantly elevated over almost the whole diurnal cycle. Measurements of CORT-binding globulin concentrations revealed IL-2-induced decreases during the dark phase, resulting in a marked increase in free CORT. Additionally, after 11 days of chronic infusion, both groups of animals underwent a 20-min restraint stress. IL-2-treated animals showed stress-induced increases in plasma ACTH and CORT that were not significantly different from those of animals treated with heat-inactivated IL-2. Along with the alteration of HPA activity seen in the IL-2-treated animals, chronic delivery of the cytokine caused periventricular tissue damage and gliosis. Taken together, the data reflect the capacity of IL-2 to modulate neuroendocrine activity over an extended period of treatment. Moreover, the IL-2-induced effects on HPA activity seen here may help to explain some of the endocrine disturbances seen in patients undergoing IL-2 immunotherapy.

Evidence that the inhibition of luteinizing hormone secretion exerted by central administration of neuropeptide Y (NPY) in the rat is predominantly mediated by the NPY-Y5 receptor subtype.

A number of studies have indicated that neuropeptide Y (NPY) is a central regulator of the gonadotropic axis, and the Y1 receptor was initially suggested to be implicated. As at least five different NPY receptor subtypes have now been characterized, the aim of the present study was to reinvestigate the pharmacological profile of the receptor(s) mediating the inhibitory action of NPY on LH secretion by using a panel of NPY analogs with different selectivity toward the five NPY receptor subtypes. When given intracerebroventricularly (icv) to castrated rats, a bolus injection of native NPY (0.7-2.3 nmol) dose-dependently decreased plasma LH. Peptide YY (PYY; 2.3 nmol) was as potent as NPY, suggesting that the Y3 receptor is not implicated. Confirming previous data, the mixed Y1, Y4, and Y5 agonist [Leu31,Pro34]NPY (0.7-2.3 nmol) inhibited LH release with potency and efficacy equal to those of NPY. Neither the selective Y2 agonist C2-NPY (2.3 nmol) nor the selective Y4 agonist rat pancreatic polypeptide affected plasma LH, excluding Y2 and Y4 subtypes for the action of NPY on LH secretion. The mixed Y4-Y5 agonist human pancreatic polypeptide (0.7-7 nmol) as well as the mixed Y2-Y5 agonist PYY3-36 (0.7-7 nmol) that displayed very low affinity for the Y1 receptor, thus practically representing selective Y5 agonists in this system, decreased plasma LH with potency and efficacy similar to those of NPY, indicating that the Y5 receptor is mainly involved in this inhibitory action of NPY on LH secretion. [D-Trp32]NPY, a selective, but weak, Y5 agonist, also inhibited plasma LH at a dose of 7 nmol. Furthermore, the inhibitory action of NPY (0.7 nmol) on LH secretion could be fully prevented, in a dose-dependent manner (6-100 microg, icv), by a nonpeptidic Y5 receptor antagonist. This antagonist (60 microg, icv) also inhibited the stimulatory action of NPY (0.7 nmol) on food intake. The selectivity of PYY3-36, human PP, [D-Trp32]NPY, and the Y5 antagonist for the Y5 receptor subtype was further confirmed by their ability to inhibit the specific [125I][Leu31,Pro34]PYY binding to rat brain membrane homogenates in the presence of the Y1 receptor antagonist BIBP3226, a binding assay system that was described as being highly specific for Y5-like receptors. With the exception of [D-Trp32]NPY, all analogs able to inhibit LH secretion were also able to stimulate food intake. Taken together, these results indicate that the Y5 receptor is involved in the negative control by NPY of the gonadotropic axis.

Circadian rhythms of melatonin and cortisol in aging.

The relationship of age to the circadian rhythms of melatonin and cortisol was investigated in 44 men and 27 women (age range 19-89 years). Subjects were physically and psychiatrically normal. Four hourly serial blood samples were drawn from 8:00 AM until 8:00 AM the next day, with additional samples at 10:00 PM and 2:00 AM. The indoor illumination was restricted to 300 lux during day and 50 lux during the night. Plasma melatonin and cortisol were estimated by radioimmunoassay. Results show that the means of melatonin and cortisol values decreased significantly with age when the subjects were divided into three age groups, i.e., 19-25 years, 42-65 years, and 66-89 years. They also showed a significant negative correlation with age. The acrophases of the two hormonal rhythms, however, showed different relationships to age. The acrophase of melatonin rhythm showed a positive correlation with age (r = 0.38, p less than 0.001), and cortisol showed a negative correlation with age (r = -0.56, p greater than 0.001). It is suggested that this may indicate a weakened responsiveness of the circadian system in the elderly to the day-night cycle and an altered relationship between the pacemakers driving melatonin and cortisol circadian rhythms. This may thus represent a biomarker for the intrinsic process of the aging of the brain.