Biomedical subjects

M R Ralph

Publications and source records attributed to M R Ralph.

At least 37 records · Page 2Linked to original sources

PubMed1995

Growth hormone-releasing hormone mediates feeding-specific feedback to the suprachiasmatic circadian clock.

Growth hormone-releasing hormone (GHRH) is known to stimulate food intake in a circadian phase-dependent manner in rats. The suprachiasmatic nucleus (SCN) is an important site of action for this effect. In light of the central role played by the SCN in the control of circadian rhythms, together with the phase-dependent nature of GHRH-induced feeding, we sought to determine the possible involvement of SCN GHRH activity in the regulation of circadian rhythmicity. Two studies were conducted using hamsters as subjects. Study one replicated the daytime feeding-stimulatory effects of GHRH in hamsters, thereby validating its appetitive effects in this species. Study two showed that, in free-running hamsters, intra-SCN microinjections of GHRH produced phase advances when injected during the subjective day while having little effect during the subjective night. The GHRH phase-response curve was found to resemble that observed for nonphotic influences on the clock. It is suggested that GHRH input to the SCN is a neural representation of a nonphotic influence (perhaps feeding specific) on the clock.

Animals↗

PubMed1995

Circadian modulation in the rat acoustic startle circuit.

The acoustic startle reflex (ASR) in rats exhibits robust circadian modulation, with ASR amplitudes greater during subjective night. To identify the location of this modulation, startle reactions were evoked either acoustically or electrically via electrodes implanted in the primary ASR circuit. Startle amplitudes were compared at different times in the circadian cycle. In constant environmental conditions, startle amplitudes were greater in subjective night for acoustically evoked and for electrically evoked reactions from the ventral lateral lemniscus and medial longitudinal fasciculus. The results show that at least 1 site of circadian modulation must occur at some point in the circuit after the last brainstem synapse in the caudal pontine reticular formation, at the level of spinal interneurons or motoneurons or at the neuromuscular junction.

Animals↗

PubMed1995

Circadian locomotor rhythms in aged hamsters following suprachiasmatic transplant.

Circadian activity rhythms that have been eliminated by lesions of the suprachiasmatic nucleus (SCN) can be restored by fetal SCN grafts. Partial lesions of the host allow simultaneous expression of both donor and host rhythms. Because partial SCN ablation produces characteristic changes in activity rhythms that are similar to those that occur with age, including shortened period, reduced amplitude, and fragmentation, we investigated the extent to which fetal SCN grafts may be expressed by an animal whose activity rhythm exhibits these age-dependent changes. The results indicate that expression of a transplanted clock is possible in an unlesioned aged host. Grafts of fetal SCN into young hosts and cortical tissue grafts into intact aged hosts have no effect. In those aged animals that received SCN grafts, three patterns of expression emerged in the subsequent locomotor activity record: complete dominance of locomotor rhythmicity by the donor; relative coordination between donor and host rhythms; and spontaneous switching between host and donor phenotypes. The results suggest that the expression of rhythmicity by the grafted SCN may depend on the relative amplitude or strength of signals produced by the host and donor SCN.

Aging↗

PubMed1994

KN-62, an inhibitor of Ca2+/calmodulin kinase II, attenuates circadian responses to light.

Expression of immediate early genes and phosphorylation of the transcription factor CREB are induced in the suprachiasmatic nucleus after light pulses that cause phase shifts of circadian rhythms. To test for a direct role of this signalling pathway in mediating circadian responses to light in hamsters, we used KN-62 to inhibit the activity of CaM kinase II (known to phosphorylate CREB) prior to giving light pulses at times that would normally induce phase shifts. Central administration of KN-62 significantly inhibited phase delays and advances induced by bright pulses of light. The data support a model for photic responses of the circadian clock in the SCN that includes the phosphorylation of CREB by activation of CaM kinase II.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1994

Inhibition of GABA transaminase enhances light-induced circadian phase delays but not advances.

The CNS neurotransmitter GABA is distributed extensively throughout the suprachiasmatic nucleus, the site of circadian pacemaker cells in mammals. Pharmacological agents that act at GABAA receptors alter specific circadian responses to light and may induce phase shifts of circadian rhythms. In the present study, the role of endogenously released GABA in rhythm regulation was investigated using vigabatrin (gamma-vinyl GABA), an agent that has been shown to increase chronically or acutely the CNS levels of this neurotransmitter by inhibiting GABA transaminase. In Experiment 1, hamsters in constant darkness (DD) received a saline or a vigabatrin injection 1 hr before a 15-min, 700-lux light pulse. Vigabatrin increased photic phase delays but did not affect advances. In Experiment 2, vigabatrin delivered chronically via osmotic minipump treatment did not affect locomotor activity period in DD. However, after 14 days of infusion, photic phase delays (but not advances) were greatly increased in the vigabatrin group. In Experiment 3, in constant light (LL), chronic vigabatrin-treated animals showed an increased period that returned to pretreatment values after the 14-day drug infusion. The results are consistent with the phase-dependent effects of other agents that alter GABA neurotransmission. The data support the general hypothesis that GABA modulates the circadian responses to light in a phase-dependent manner, and may participate in entrainment to light-dark cycles by influencing the relative responsiveness to light in the early and late subjective night.

4-Aminobutyrate Transaminase↗

PubMed1993

The role of extracellular calcium in generating and in phase-shifting the Bulla ocular circadian rhythm.

Since extracellular calcium is known to be involved in the entrainment of the circadian pacemaker in the retina of Bulla gouldiana, we have assessed the requirement for extracellular calcium in the generation of the circadian rhythm. To enable us to assay the state of the pacemaker during low-calcium treatment, which often obscures rhythmicity, long-duration pulses of low-calcium artificial seawater (no added calcium, 10 mM EGTA, calculated calcium concentration = 4.5 x 10(-10) M) were applied, and the phase of the subsequent rhythm was measured. Pulse treatments started at zeitgeber time (ZT) 6, and durations ranged from 4 to 72 hr. Although no phase shifts followed pulses ending before the next projected dawn (ZT 24), phase delays of up to 4 hr followed pulses ending after projected dawn, and delays of up to 8 hr followed pulses spanning two dawns. Some activity records exhibited unequivocal circadian rhythmicity during the long low-calcium treatments, with phases and periods similar to untreated control eye records; this finding suggests that the phase delays observed following long low-calcium pulses are attributable to the pulsatile nature of the treatment. These data suggest that extracellular calcium is not an essential requirement for the pacemaker in generating the circadian rhythm.

Action Potentials↗

PubMed1993

Light-induced phase shifts and Fos expression in the hamster circadian system: the effects of anesthetics.

In the present study, we examined the effect of administration of anesthetics on light-induced phase shifts of the circadian system. This information is of critical importance, because many studies of light input to the mammalian suprachiasmatic nucleus (SCN) have been performed on anesthetized animals. We found that light-induced phase shifts were blocked by all drugs used at anesthetic doses. We then determined the effect of two of these agents on light induction of Fos-like immunoreactivity in the SCN. We found that the administration of sodium pentobarbital prevented light induction of Fos expression in the SCN, whereas the administration of urethane did not. These results raise cautions about the use of anesthetized animals to answer questions about the photic regulation of neuronal activity in the SCN.

Animals↗

PubMed1993

Culture and transplantation of the mammalian circadian pacemaker.

In transplantation studies using the tau mutation in the golden hamster, it has been demonstrated that suprachiasmatic nucleus (SCN) pacemaker cells and mechanisms of communication with the host brain are retained even after tissue dissociation and maintenance for many weeks in primary cell culture. Brain grafts of cultured SCN cells are capable of restoring overt rhythms of locomotor activity, and preliminary studies where cells from two tau genotypes are combined in a single graft demonstrate that pacemaker cells may communicate with each other to produce coherent rhythms with intermediate periods. The opportunity is presented, therefore, to study pacemaker-pacemaker communication in circadian chimeras produced by SCN transplantation. Immunocytochemical analysis of graft-host interactions requires the positive identification of host versus donor cells. Although grafted blocks of tissue are easily recognized during immunocytochemical analysis, implants of dissociated and cultured cells may be more diffusely located and are not as readily identified. Unless distinct strain- or species-specific markers are available, it is difficult to identify connections that may carry timing information to the host organism. We have taken an anatomical approach that utilizes cell-labeling techniques for hamster tissue along with foreign protein expression in transgenic mice to identify patterns of communication among graft and host cells, focusing specifically on SCN-SCN communication. The data indicate the usefulness of these transgenes as markers in transplantation studies where communication between graft and host is addressed.

Animals↗

PubMed1992

Phase response curve to anisomycin in tau mutant hamsters.

Administration of the protein synthesis inhibitor, anisomycin, to wild type hamsters produces phase shifts in their circadian rhythms that have similarities to shifts produced by non-photic behavioral stimulation. A mutation that shortens the period of rhythms in hamsters results in altered responsiveness to non-photic input. However, responses of the mutants to anisomycin are unaffected: their phase response curve (PRC) for anisomycin is similar to that of wild types. This suggests that 1) anisomycin is not acting on mechanisms specifically involved in non-photic behavioral phase shifting, and 2) the mutation affects the non-photic input pathway or the pacemaker itself at a point that is upstream from anisomycin's site of action.

Animals↗

PubMed1992

The Drosophila per gene homologs are expressed in mammalian suprachiasmatic nucleus and heart as well as in molluscan eyes.

This study presents evidence for the conservation of Drosophila per gene homologs in mammalian DNA and for their expression in a number of tissues which are involved in various aspects of circadian timekeeping. Distinct 5 kb sequences, which hybridized to a non repetitive fragment of the Drosophila per gene under stringent conditions, were detected by Southern blotting. Sequences homologous to per gene of Drosophila were also amplified from rat and mouse brain cDNA libraries and from a mouse anterior hypothalamus and human hypothalamus libraries. Degenerate PCR primer design was based on conserved segments of the per protein. The per homologs were shown directly (by RT-PCR) to be expressed in hamster and mouse SCN, in hamster heart and in Aplysia and Bulla eyes.

Animals↗

PubMed1992

Protein differences in tau mutant hamsters: candidate clock proteins.

In the tau mutant hamster, the period of the circadian rhythm is shortened from about 24 h to about 22 h in heterozygotes and to about 20 h in homozygotes. Understanding the biochemical basis of the period changes in the tau mutant may elucidate the regulation of the vertebrate pacemaker. Using two-dimensional gel electrophoresis, we have found two sets of proteins that differ between the different genotypes. P33tau (about 33 kDa; pI 6.5) was found in all gels from wild type and heterozygous animals, but was absent in gels from all except one of the homozygous mutant animals. P32tau (about 32 kDa; pI 4.8) was a chain of spots, which showed a striking difference in pattern between gels from wild type animals and from mutant animals. P33tau was greatly enriched in soluble cellular fractions, whereas P32tau was found only in insoluble fractions. These differences between P33tau and P32tau were apparent in gels from both SCN and cortical tissue, suggesting that both proteins are distributed throughout the brain. These proteins should be useful as new tools to explore the biochemistry of circadian pacemakers.

Animals↗

PubMed1992

Nonphotic phase shifting in hamster clock mutants.

Golden hamsters with the tau mutation were kept in the dark and induced to become active through confinement to a novel running wheel for 3 hr. The response of the mutants to this nonphotic phase-shifting stimulus differed from that of wild-type hamsters. The mutants showed larger phase shifts, and their phase response curves differed in shape, with an advance portion at about circadian time 24, a phase at which wild types show delays. The results establish that the tau mutation, in addition to its already known effects, alters the response of the circadian system to nonphotic events.

Animals↗

PubMed1992

Behavioral inhibition of circadian responses to light.

Circadian locomotor rhythms in rodents may be synchronized by either photic or nonphotic events that produce phase shifts of the rhythm. Little is known, however, about how these two types of stimuli interact to produce entrainment. The well-characterized circadian photic response of the golden hamster was examined in situations where a short light pulse and locomotor activity, a nonphotic event, occurred simultaneously. Light-induced phase advances were attenuated when animals were active during light exposure. The results show that circadian responses to light depend upon the environmental situation in which the light is given, and call into question the implicit assumption in circadian rhythm research that phase shifting and entrainment to light-dark cycles depend simply on photic activation of well-known retinofugal pathways. Moreover, since light therapy is becoming an important component in the treatment of circadian-based disorders in humans, the results emphasize the need for evaluation of the behavioral aspects of light therapy protocols.

Animals↗

PubMed1991

Transplantation: a new tool in the analysis of the mammalian hypothalamic circadian pacemaker.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of pacemaker cells that generate circadian rhythmicity in mammals. Transplantation of the nucleus into animals whose own nucleus has been ablated results in the restoration of overt rhythmicity to the arrhythmic host. By using donors and hosts with genetically different circadian characteristics, the unambiguous recognition of the donor rhythm expressed in a transplant recipient is possible. The reappearance of a rhythm indicates that not only has the grafted tissue survived the transplantation procedure, but that pacemaker cells that generate circadian rhythms were included in the graft; this is essential in interpreting results of such transplantation experiments. The restoration of circadian function by neural transplantation has become an important tool for studying the generation and expression of biological rhythms in mammals, and is being used in the investigation of basic questions in this field.

Animals↗

PubMed1991

Does low intracellular pH stop the motion of the Bulla circadian pacemaker?

The eye of the mollusk Bulla has proven itself useful as an in vitro neural circadian pacemaker. Here, we report that treatments applied to lower intracellular pH may stop the motion of this circadian pacemaker in a phase-dependent manner. Lowering the extracellular pH of the artificial seawater bath to 6.9, or application of the stilbene derivatives 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) or 4,4-di-isothiocyanostilbene-2,2'-disulfonic acid (DIDS), abolishes the circadian rhythm in optic nerve compound action-potential frequency. Because these treatments are known to lower intracellular pH, these data suggest that the pacemaker may be inhibited by low intracellular pH. In order to assess the state of the pacemaker during low extracellular pH treatment, pulses of seawater at pH 6.8 were applied, and the phase of the rhythm subsequent to the pulse was observed. All pulses started 1 hr after subjective dusk [circadian time (CT) 13] and were applied to eyes in constant darkness; pulse lengths varied from 4 to 47 hr for different preparations. The phases of the eye rhythms following pulses that ended before subjective dawn (about CT 24) were not different from untreated preparations. However, for pulses longer than 11 hr and therefore ending after subjective dawn, the subsequent phase of the rhythm was a function of the ending time of the pulse. These data suggest that the pacemaker's motion was stopped at dawn during the low-pH treatment and resumed following restoration of normal pH. To distinguish between phase and duration dependence of this effect in the above experiment, phase shifts were obtained to 14-hr pulses of pH 6.8 seawater applied at three different phases.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1990

Do NMDA receptors mediate the effects of light on circadian behavior?

We report here the results of experiments designed to evaluate whether a specific NMDA receptor antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine maleate (MK-801), blocks the phase shifting effects of light on the circadian rhythm of wheel-running activity in golden hamsters. Intraperitoneal administration of (+)-MK-801 produced a dose-dependent blockade of both light-induced phase advances and delays. The effect was stereoselective and treatment with related compounds, phenylcyclidine and ketamine, also blocked light-induced phase shifts. MK-801, by itself, did not cause any consistent effect on the phase of the rhythm. These data, coupled with previous findings, indicate that excitatory amino acid receptors play an important role in the transmission of light information from the retina to the circadian system.

Animals↗

PubMed1990

Chloride conductance contributes to period determination of a neuronal circadian pacemaker.

The isolated eye of Bulla gouldiana, a marine mollusc, is a circadian pacemaker. Previous studies have shown that membrane potential changes of neurons at the base of the Bulla retina play a critical role in the expression of the circadian rhythm and that the free-running period can be modified by chronic alteration of the resting membrane potential. We now report that treatments which inhibit CI- conductance shorten the free-running period. Substitution of CI- with the anions SO4(2-), isethionate and glutamate significantly shorten the period of the ocular rhythm in vitro. Furthermore, addition of the CI- channel blocker 9-anthracene-carboxylic acid (9-AC) is also effective at shortening the period of the circadian rhythm. These data suggest that a CI- conductance participates in determining the free-running period of the circadian pacemaker cells. This is the first report of CI- conductance involvement in a circadian system and the effect is remarkable in that few treatments are known which reliably shorten the period of circadian clocks.

Animals↗

PubMed1990

Transplanted suprachiasmatic nucleus determines circadian period.

The pacemaker role of the suprachiasmatic nucleus in a mammalian circadian system was tested by neural transplantation by using a mutant strain of hamster that shows a short circadian period. Small neural grafts from the suprachiasmatic region restored circadian rhythms to arrhythmic animals whose own nucleus had been ablated. The restored rhythms always exhibited the period of the donor genotype regardless of the direction of the transplant or genotype of the host. The basic period of the overt circadian rhythm therefore is determined by cells of the suprachiasmatic region.

Animals↗