Effects of altered Clock gene expression on the pacemaker properties of SCN2.2 cells and oscillatory properties of NIH/3T3 cells.

While peripheral tissues and serum-shocked fibroblasts express rhythmic oscillations in clock gene expression, only the suprachiasmatic nucleus (SCN) is capable of endogenous, self-sustained rhythmicity and of functioning as a pacemaker by imposing rhythmic properties upon other cells. To differentially examine the molecular elements necessary for the distinctive rhythm-generating and pacemaking properties of the SCN, the effects of antisense inhibition of Clock expression on the rhythms in 2-deoxyglucose uptake and Per gene expression were compared in immortalized SCN cells and a fibroblast cell line. Similar to changes in molecular and physiological rhythmicity observed in the SCN of Clock mutant mice, the rhythmic pattern of Per2 expression was disrupted and the period of metabolic rhythmicity was increased in SCN2.2 cells subjected to antisense inhibition of Clock. NIH/3T3 fibroblasts cocultured with antisense-treated SCN2.2 cells showed metabolic rhythms with comparable increases in period and decreases in rhythm amplitude. Per2 expression in these cocultured fibroblasts exhibited a similar reduction in peak levels, but was marked by non-24 h or irregular peak-to-peak intervals. In serum-shocked NIH/3T3 fibroblasts, oscillations in Per2, Bmal1, and Cry1 expression persisted with some change in rhythm amplitude during antisense inhibition of CLOCK, demonstrating that feedback interactions between Clock and other core components of the clock mechanism may be regulated differently in SCN2.2 cells and fibroblasts. The present results suggest that CLOCK is differentially involved in the generation of endogenous molecular and metabolic rhythmicity within SCN2.2 cells and in the regulation of their specific outputs that control rhythmic processes in NIH/3T3 cells.

Developmental alcohol and circadian clock function.

Studies in rats found that alcohol exposure during the early postnatal period, particularly during the brain-growth-spurt period, can result in cell loss in various brain regions and persistent behavioral impairments. Some investigators have speculated that the body's internal clock, which is located in the suprachiasmatic nuclei (SCN) in the brain, may also be affected by developmental alcohol exposure. For example, alcohol-induced damage to the SCN cells and their function could result in disturbances of the circadian timekeeping function, and these disturbances might contribute to the behavioral impairments and affective disorders observed in people prenatally exposed to alcohol. Preliminary findings of studies conducted in rats suggest that developmental alcohol exposure may indeed interfere with circadian clock function as evidenced by a shortened circadian sleep-wake cycle and changes in the release of certain brain chemicals (i.e., neuropeptides) by SCN cells.

Oscillating on borrowed time: diffusible signals from immortalized suprachiasmatic nucleus cells regulate circadian rhythmicity in cultured fibroblasts.

The capacity to generate circadian rhythms endogenously and to confer this rhythmicity to other cells was compared in immortalized cells derived from the suprachiasmatic nucleus (SCN) and a fibroblast line to differentiate SCN pacemaker properties from the oscillatory behavior of non-clock tissues. Only SCN2.2 cells were capable of endogenously generating circadian rhythms in 2-deoxyglucose uptake and Per gene expression. Similar to SCN function in vivo, SCN2.2 cells imposed rhythms of metabolic activity and Per gene expression on cocultured NIH/3T3 fibroblasts via a diffusible signal. The conferred rhythms in NIH/3T3 cells were phase delayed by 4-12 hr relative to SCN2.2 circadian patterns, thus resembling the phase relationship between SCN and peripheral tissue rhythms in vivo. Sustained metabolic rhythmicity in NIH/3T3 cells was dependent on continued exposure to SCN2.2-specific outputs. In response to a serum shock the NIH/3T3 fibroblasts exhibited recurrent oscillations in clock gene expression, but not in metabolic activity. These molecular rhythms in serum-shocked fibroblasts cycled in a phase relationship similar to that observed in the SCN in vivo; peak Per1 and Per2 mRNA expression preceded the rhythmic maxima in Cry1 and Cry2 mRNA levels by 4 hr. Despite these clock gene oscillations the serum-shocked NIH/3T3 cells failed to drive circadian rhythms of Per1 and Per2 expression in cocultures of untreated fibroblasts, suggesting that expression and circadian regulation of the Per and Cry genes are not sufficient to confer pacemaker function. Therefore, SCN-specific outputs are necessary to drive circadian rhythms of metabolic activity, and these output signals are not a direct product of clock gene oscillations.

Overlapping and divergent actions of estrogen and the neurotrophins on cell fate and p53-dependent signal transduction in conditionally immortalized cerebral cortical neuroblasts.

The developing cerebral cortex undergoes overlapping periods of neurogenesis, suicide, and differentiation to generate the mature cortical plate. The following experiments examined the role of the gonadal hormone estrogen in comparison to the neurotrophins, in the regulation of p53-dependent cortical cell fate. To synchronize choices between neurogenesis, apoptosis, and neural differentiation, embryonic rat cerebral cortical neuroblasts were conditionally immortalized with the SV40 large T antigen containing the tsA58/U19 temperature-sensitive mutations. At the nonpermissive temperature, cessation of large T antigen expression was accompanied by induction of p53, as well as the p53-dependent proteins, wild-type p53-activated fragment-1/Cdk (cyclin-dependent kinase)-interacting protein-1 (p21/Waf1), Bcl (B-cell lymphoma)-associated protein (Bax), and murine double minute 2 (MDM2), that lead to cell cycle-arrest, suicide, and p53 inhibition, respectively. Simultaneously, neuroblasts exit cell cycle and die apoptotically or differentiate primarily into astrocytes and immature postmitotic neuroblasts. At the nonpermissive temperature, estrogen specifically induced an antagonist-independent increase in phosphorylated p53 expression, while increasing p21/Waf1 and decreasing Bax. Coincidentally, estrogen rapidly increased and then decreased MDM2 relative to controls, suggesting temporal modulation of p53 function. Both estrogen and neurotrophins prevented DNA fragmentation, a marker for apoptosis. However, estrogen also induced a transient increase in released lactate dehydrogenase, suggesting that estrogen simultaneously induced rapid cell death in a subpopulation of cells. In contrast to the neurotrophins, estrogen also increased cell proliferation. Both estrogen and the neurotrophins supported neuronal differentiation. However, in contrast to the neurotrophins, estrogen only supported the expression of a subset of oligodendrocytic markers. These results suggest that estrogen and the neurotrophins support overlapping and distinct aspects of differentiation in the developing cerebral cortex.

Establishment and characterization of adenoviral E1A immortalized cell lines derived from the rat suprachiasmatic nucleus.

Primary cultured cells from the presumptive anlage of the rat suprachiasmatic nucleus (SCN) were immortalized by infection with a retroviral vector encoding the adenovirus 12S E1A gene. After drug selection, the resulting neural cell lines (SCN1.4 and SCN2.2) displayed (a) extended growth potential without evidence of transformed or tumorigenic properties, (b) expression of E1A protein within all cell nuclei, and (c) heterogeneous cell types in various stages of differentiation. A large proportion of the SCN1.4 and SCN2.2 cells were characterized by gliallike morphologies, but showed limited expression of corresponding cell type-specific antigens. In addition, both lines exhibited a stable population of cells with neuronlike characteristics. When treated so as to enhance differentiation, these cells were often distinguished by fine, long processes and immunocytochemical expression of neuronal markers and peptides found within SCN neurons in situ. Observations on SCN neuropeptide immunostaining, content, release, and mRNA expression followed a concordant pattern in which somatostatin and vasopressin cells were the most and least common peptidergic phenotypes in both lines, respectively. Since these results indicate that constituents of E1A-immortalized lines derived from the primordial SCN can differentiate into cells with phenotypes resembling parental peptidergic neurons, it will be critical to explore next whether these lines also retain the distinctive function of the SCN to generate circadian rhythms. Cloning of immortalized cell types could subsequently yield useful tools for studying the development of SCN glial and peptidergic cell types and delineating their distinct roles in mammalian circadian time-keeping.

Immortal time: circadian clock properties of rat suprachiasmatic cell lines.

Cell lines derived from the rat suprachiasmatic nucleus (SCN) were screened for circadian clock properties distinctive of the SCN in situ. Immortalized SCN cells generated robust rhythms in uptake of the metabolic marker 2-deoxyglucose and in their content of neurotrophins. The phase relationship between these rhythms in vitro was identical to that exhibited by the SCN in vivo. Transplantation of SCN cell lines, but not mesencephalic or fibroblast lines, restored the circadian activity rhythm in arrhythmic, SCN-lesioned rats. Thus, distinctive oscillator, pacemaker, and clock properties of the SCN are not only retained but also maintained in an appropriate circadian phase relationship by immortalized SCN progenitors.

Circadian rhythm of brain-derived neurotrophic factor in the rat suprachiasmatic nucleus.

This study was conducted to determine whether the rat suprachiasmatic nucleus (SCN) is characterized by circadian expression of brain-derived neurotrophic factor (BDNF). In constant darkness, SCN content of both BDNF mRNA and protein oscillated in a circadian fashion. BDNF mRNA and protein levels in the SCN reached peak values during the early subjective day and during the subjective night, respectively. In contrast, the hippocampus showed no sign of circadian rhythmicity in its expression of BDNF mRNA and protein. Since BDNF enhances synaptic transmission in other brain regions, the coincidence between peak expression of BDNF protein in the SCN and the known interval of circadian pacemaker sensitivity to the phase-shifting effects of light may have some implications for the role of BDNF in the circadian regulation of the SCN pacemaker by photic signals from the retinohypothalamic tract.

Light induces expression of fos-related proteins within gastrin-releasing peptide neurons in the rat suprachiasmatic nucleus.

The present study was conducted to determine whether the photic induction of c-fos expression in the rat suprachiasmatic nucleus (SCN) occurs within neurons containing gastrin-releasing peptide (GRP) and/or vasoactive intestinal polypeptide (VIP) because these peptidergic cells are closely associated with retinal projections to the ventrolateral subfield. Using dual immunostaining and thin sectioning techniques, the ventrolateral SCN of light-exposed rats was examined for evidence of individual neurons coexpressing nuclear immunostaining for c-fos proteins (Fos) with cytoplasmic immunoreactivity for GRP or VIP. In all animals, the photic induction of Fos expression in the SCN was mainly confined to cells segregated within the ventrolateral subfield and was evident in approximately 40% of the SCN neurons with cytoplasmic immunoreactivity for GRP. However, neurons coexpressing Fos and GRP comprised only a small fraction of the total number of cells within the ventrolateral SCN exhibiting light-induced Fos immunoreactivity. No sign of Fos expression was detected within VIP-immunoreactive perikarya in the ventrolateral SCN of light-treated rats. These results demonstrate that light induces Fos expression in a number of GRP-containing neurons within the SCN, suggesting that these peptidergic cells may process photic information mediating circadian entrainment.

Circadian regulation of c-fos expression in the suprachiasmatic pacemaker by light.

The primary objective of this research was to examine expression of the immediate-early gene c-fos within the suprachiasmatic nucleus (SCN) for evidence of circadian regulation by photic stimuli. In vivo and in vitro analyses demonstrate that photic signals have an inductive effect on c-fos expression in the SCN, but only at critical times when light is capable of phase-shifting circadian rhythms. This evidence for correlative relations between the effects of light signals in inducing c-fos gene expression in the SCN and modulating the circadian period of the SCN pacemaker suggests that immediate-early genes may be components of the signal transduction cascade by which light entrains circadian rhythms. In addition, dual-immunostaining methods were utilized to examine neurochemical identity of SCN cells that exhibit this circadian induction of c-fos expression in response to light. Within the ventrolateral SCN, the photic induction of Fos expression occurred in neurons expressing gastrin-releasing peptide (GRP), but not in those containing vasoactive intestinal polypeptide (VIP). This finding suggests that SCN neurons containing GRP may be involved in the transduction of photic signals mediating circadian entrainment.

Rhythmic expression of Fos-related proteins within the rat suprachiasmatic nucleus during constant retinal illumination.

Within the retinorecipient or ventrolateral subfield of suprachiasmatic nuclei (SCN) in rodents, expression of the protein product of the c-fos proto-oncogene, Fos, is regulated by light. In the present study, the expression of Fos and Fos-related proteins within the SCN was examined immunocytochemically for evidence of rhythmic variation in rats sacrificed at different circadian times during exposure to constant retinal illumination (LL). In all animals, nuclear Fos immunoreactivity was mainly confined to an area of the SCN that was coextensive with neuropeptide Y-immunopositive fibers distinguishing the ventrolateral subfield of the nucleus. Moreover, Fos-immunostaining within the ventrolateral SCN of rats exposed to LL fluctuated over the course of the circadian cycle, such that the density of immunopositive cells within this subfield was 2 times greater during the subjective night than during the subjective day. Since Fos expression within the SCN oscillates in the absence of photoperiodic time cues and since the peak of this oscillation coincides with the circadian times when light modulates the periodicity of the SCN pacemaker, these data provide further evidence that expression of the c-fos gene may be a molecular signal in the circadian timekeeping mechanism in the SCN and its regulation by photic stimuli.

Effects of tetrodotoxin on the circadian pacemaker mechanism in suprachiasmatic explants in vitro.

Using perifused explants of the rat suprachiasmatic nucleus (SCN), the effects of tetrodotoxin (TTX) on vasopressin (VP) release and its circadian profile were studied at various times throughout the circadian cycle. VP release from SCN explants was consistently attenuated during TTX treatment, with the amplitude of this effect depending on the time of administration. In addition, the effect of TTX on the circadian pattern of VP release was also time-dependent, such that treatment during the late subjective day was followed by a disruption of circadian rhythmicity in which peptide output remained at basal levels without notable variation whereas treatment at all other times caused no measurable perturbation in the circadian VP rhythm in succeeding cycles. In SCN explants experiencing this TTX-induced arrest of circadian VP output, subsequent exposure to KCl induced acute increases in VP release, suggesting that VP neurons retain the capacity to actively release peptide in spite of this effect of TTX. These results indicate that the interruption of electrical impulses at a critical phase may compromise the circadian function or output of the pacemaker in the SCN. In addition, the present observations provide further evidence that the overt expression of circadian rhythmicity is dependent on sodium-generated action potentials and that disruption of these electrical signals does not alter the precision of the SCN pacemaker, at least in instances where the phase of the VP rhythm can be determined after treatment.

Photic regulation of c-fos expression in neural components governing the entrainment of circadian rhythms.

The rapid and transient induction of the proto-oncogene c-fos in mature neurons within the brain occurs in response to a variety of extracellular stimuli. To determine whether lighting conditions influence c-fos gene expression in the primary neural structures mediating the photoentrainment and generation of mammalian circadian rhythms, the expression of the c-fos protein (Fos) and related proteins in the retina and suprachiasmatic nuclei (SCN) of the anterior hypothalamus was examined immunohistochemically in rats exposed to a light-dark cycle of 12 h of light and 12 h of darkness (LD 12:12), constant light (LL), or constant dark (DD). The retina exhibited clear light-dark differences in the expression of Fos protein(s), such that immunopositive nuclei were readily evident during exposure to light (i.e., during the day of diurnal lighting or in LL), but were absent during exposure to darkness. In the SCN, the distribution of Fos immunoreactivity within specific subfields was differentially affected by photic conditions. Following exposure to light, a dense population of Fos-immunopositive cells was found in close association with the immunohistochemically distinct cell and fiber populations distinguishing the ventrolateral subfield of the SCN. In dark-exposed animals, Fos-immuno-reactive profiles were distributed throughout the SCN in areas coextensive with the immunohistochemical localization of peptidergic neural elements in both the ventrolateral and dorsomedial subfields. As a consequence of this light-dark difference in the distribution of Fos immunoreactivity, the density of labeled cells was increased within the ventrolateral SCN, but was decreased within the dorsomedial subfield, as a result of exposure to light versus darkness.(ABSTRACT TRUNCATED AT 250 WORDS)

Specificity of circadian function in transplants of the fetal suprachiasmatic nucleus.

Fetal tissues obtained from specific regions of the developing hypothalamus were transplanted to determine whether the precursor neurons of the suprachiasmatic nucleus (SCN) can be distinguished from those of the presumptive paraventricular nucleus (PVN) on the basis of the functional capacity to generate circadian rhythms. The presumptive SCN, the PVN, and a portion of the neocortical primordium were dissected from the developing forebrains of normal Long-Evans fetuses, separated, and selectively transplanted into the periventricular-third ventricle region of adult, vasopressin (VP)-deficient Brattleboro rats. In host animals that received grafts containing the precursor population of SCN neurons, the temporal profile of VP levels in the cerebrospinal fluid (CSF) oscillated with a circadian periodicity in a manner similar to that observed in normal Long-Evans rats. CSF collected serially from animals with grafts of the presumptive PVN also contained VP, but no circadian variation was manifested in peptide levels. VP was undetectable in CSF samples obtained from Brattleboro rats with cortical grafts. In association with their circadian functional capacity, grafts of the SCN primordium were characterized by clusters of parvicellular neurons immunopositive for VP or vasoactive intestinal polypeptide (VIP) that resembled the cell groups of the in situ SCN. In contrast, transplants of the presumptive PVN did not contain neurons immunoreactive for VIP, and the VP neurons in these grafts resembled the neurosecretory cells of the PVN.(ABSTRACT TRUNCATED AT 250 WORDS)

Circadian vasopressin release from perifused rat suprachiasmatic explants in vitro: effects of acute stimulation.

Using a perifusion culture technique, the pattern of vasopressin release from the suprachiasmatic nucleus was studied in vitro under basal and stimulated conditions. Individual rat suprachiasmatic explants released vasopressin in a rhythmic fashion and these rhythms persisted with a circadian period for up to 4 cycles. One-hour exposures to KCl on days 2 and 4 in culture induced acute increases in vasopressin output and altered the circadian pattern of release from perifused suprachiasmatic explants. These results provide evidence that the release of vasopressin in an acute as well as a circadian fashion from isolated suprachiasmatic neurons is indicative of active release processes. Furthermore, it is anticipated that this method of studying circadian rhythms generated by suprachiasmatic explants may provide a basis for elucidating the neural organization of mammalian circadian pacemakers.

A cholinergic antagonist, mecamylamine, blocks the phase-shifting effects of light on the circadian rhythm of locomotor activity in the golden hamster.

Despite the well known role of the light-dark cycle in the entrainment of circadian rhythms, very little is known about the neurochemical events that mediate the effects of light on the mammalian circadian clock. Recent anatomical and pharmacological data support the hypothesis that acetylcholine may be involved in relaying light-dark information from the retina to, or within, the circadian clock of rodents. If acetylcholine is required for this response, it should be possible to block the phase-shifting effects of a light pulse by blocking cholinergic neurotransmission. To test this possibility, hamsters free-running in constant darkness received an intraventricular injection of the anticholinergic drug, mecamylamine (450 micrograms), 10 min before being exposed to a 5-min pulse of light known to induce sub-maximal phase shifts in the circadian rhythm of wheel-running behavior. Compared to vehicle-injected control animals, mecamylamine treatment blocked or reduced both the phase-advancing and phase-delaying effects of light. These results support the hypothesis that acetylcholine is involved in mediating the phase-shifting effects of light on the mammalian circadian clock.

Circadian rhythms of vasopressin release from individual rat suprachiasmatic explants in vitro.

A culture system was developed to study intrinsic oscillations within rat suprachiasmatic nuclei for long periods of time. After isolation in static organ cultures, individual rat suprachiasmatic explants released vasopressin in a circadian fashion for at least 4 cycles. This finding suggests that the suprachiasmatic nucleus contains one or more self-sustained circadian oscillators, and that this method of measuring circadian rhythms from individual suprachiasmatic organ cultures may prove to be a valuable model for elucidating the physiology and biochemistry of mammalian circadian pacemakers.

Neurochemical basis for the photic control of circadian rhythms and seasonal reproductive cycles: role for acetylcholine.

A pharmacological approach was used to examine the role of acetylcholine in the photic control of circadian rhythms and seasonal reproductive cycles. The experimental protocol was designed to determine whether the administration of carbachol, a cholinergic agonist, could mimic the effects of brief light pulses on gonadal function and/or the circadian rhythm of wheel-running activity in golden hamsters. Intraventricular injections of carbachol, administered singularly at discrete phase points throughout the circadian cycle, induced phase-dependent shifts in the free-running rhythm of activity similar to those caused by a brief light exposure. Injections of carbachol once every 23.33 hr for 9 weeks entrained the activity rhythm and stimulated the neuroendocrine-gonadal axis in a manner similar to that observed after the presentation of 1-hr light pulses at this frequency. In contrast, the administration of carbachol once every 24 hr did not consistently provide an entraining signal for the activity rhythm and did not stimulate reproductive function. Importantly, the effects of carbachol on the seasonal reproductive response were correlated with the timing of the injections relative to the activity rhythm. These findings suggest that acetylcholine may play an important role in the mechanism by which light regulates circadian rhythms and seasonal reproductive cycles.

Periodic exposure to a brief light signal stimulates neuroendocrine-gonadal activity in golden hamsters.

The photoperiodic effects of a periodic light pulse on neuroendocrine-gonadal activity in the male golden hamster was examined using a night interruption paradigm. Adult male hamsters that had been housed 3 to 5 per cage on LD 14:10 were placed either in individual cages, each equipped with a running wheel, or maintained in communal housing conditions, without access to a running wheel. Animals were then transferred to either LD 6:18 or to a LD 6:18 light cycle with a periodic 10-second night interruption (8 hours after lights off) occurring once every two, four, or seven days. As expected, exposure to LD 6:18 for 11 weeks induced complete regression of the testes and seminal vesicles, accompanied by low serum levels of LH and FSH, in both individually and communally-housed animals. However, in individually-housed animals receiving a 10-second night interruption every other day on LD 6:18, paired testis and seminal vesicle weights, as well as serum gonadotropin levels, were maintained at values comparable to those normally observed in hamsters exposed to photostimulatory long days. Furthermore, the presentation of a periodic 10-second night interruption once every four or seven days to individually-housed animals with access to a running wheel was sufficient to partially or totally block the inhibitory effects of short days on neuroendocrine-gonadal activity. Communally-housed hamsters receiving a 10-second light pulse once every two, four, or seven days also exhibited paired testis and seminal vesicle weights, as well as serum gonadotropin levels, that were consistently higher than the values obtained for animals exposed only to LD 6:18.(ABSTRACT TRUNCATED AT 250 WORDS)