Biomedical subjects

M E Geusz

Publications and source records attributed to M E Geusz.

13 recordsLinked to original sources

PubMed2003

Luciferase expression controlled by a viral gene promoter in a mammalian circadian pacemaker.

Retinal light exposure induces several immediate-early genes in the hypothalamic suprachiasmatic nucleus (SCN), which contains the major circadian pacemaker of mammals. Clock-controlled and light-induced genes expressed in the SCN such as c- and contain upstream regulatory elements similar to those of the major immediate-early gene (IE-1) of the human cytomegalovirus. IE-1 expression is critical for viral reactivation from latency and increases in response to agents acting through depolarization or the cAMP response element. To test whether IE-1 could be under circadian control, bioluminescence was imaged in individual SCN cells of brain slice cultures from transgenic mice containing the IE-1 enhancer/promoter upstream from the firefly luciferase gene. A small percentage of the cells in neonatal and adult cultures displayed circadian transgene expression, particularly ones near the dorsomedial edge of the SCN. Single-cell bioluminescence imaging revealed that the circadian pacemaker can regulate exogenous viral genes and could play a role in viral diseases.

Animals↗

PubMed2001

Long-term recordings of networks of immortalized GnRH neurons reveal episodic patterns of electrical activity.

The CNS controls reproduction through pulsatile secretion of gonadotropin-releasing hormone (GnRH). Episodic increases in the firing rate of unidentified hypothalamic neurons have been associated with downstream markers of GnRH secretion. Whether this episodic electrical activity is intrinsic to GnRH neurons, intrinsic to other "pulse generator" neurons that drive GnRH neurons, or a combination of these is unknown. To determine if GnRH neurons display episodic firing patterns in isolation from other cell types, immortalized GnRH neurons (GT1-7 cells) were cultured on multiple microelectrode arrays. Long-term, multi-site recordings of GT1-7 cells revealed repeated episodes of increased firing rate with an interval of 24.8 +/- 1.3 (SE) min that were completely eliminated by tetrodotoxin, a sodium channel blocker. This pattern was comprised of active units that fired independently as well as coincidentally, suggesting the overall pattern of electrical activity in GT1-7 cells emerges as a network property. The A-type potassium-channel antagonist 4-aminopyridine (1 mM) increased both firing rate and GnRH secretion, demonstrating the presence of A-type currents in these cells and supporting the hypothesis that electrical activity is associated with GnRH release. Physiologically relevant episodic firing patterns are thus an intrinsic property of immortalized GnRH neurons and appear to be associated with secretion. The finding that overall activity is derived from the sum of multiple independent active units within a network may have important implications for the genesis of the GnRH secretory pattern that is delivered to the target organ. Specifically, these data suggest not every GnRH neuron participates in each secretory pulse and provide a possible mechanism for the variations in GnRH-pulse amplitude observed in vivo.

4-Aminopyridine↗

PubMed1997

Long-term monitoring of circadian rhythms in c-fos gene expression from suprachiasmatic nucleus cultures.

BACKGROUND: The AP-1 family of transcription factors has been implicated in the control of the expression of many genes in response to environmental signals. Previous studies have provided temporal profiles for c-fos expression by taking measurements from many animals at several points in time, but these studies provide limited information about dynamic changes in expression. Here, we have devised a method of continuously measuring c-fos expression. RESULTS: A transgenic mouse line expressing the human c-fos promoter linked to the firefly luciferase reporter gene (fos/luc) was generated to continuously monitor c-fos gene expression. A second transgenic mouse line expressing luciferase under the control of the cytomegalovirus promoter (CMV/luc) served as a control. Luminescence originating from identifiable brain regions was imaged from fos/luc brain slice cultures. Expression of the fos/luc transgene accurately reflected transcriptional responses of the endogenous c-fos gene. Dynamic changes in fos/luc expression in suprachiasmatic nuclei (SCN) explant cultures were monitored continuously, and luminescence showed almost 24 hour rhythms lasting up to five circadian cycles. In contrast, bioluminescence monitored from CMV/luc SCN explant cultures was not rhythmic. CONCLUSION: The fos/luc transgenic mouse will be useful for long-term, non-invasive monitoring of c-fos transcriptional responses to the changing cellular environment. Circadian rhythms in c-fos expression can be monitored non-invasively in real time from the SCN, clearly demonstrating that c-fos transcription is regulated by the circadian clock.

Animals↗

PubMed1997

Circadian rhythms in mouse suprachiasmatic nucleus explants on multimicroelectrode plates.

The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus functions as a circadian pacemaker. This study used multimicroelectrode plates to measure extracellular action potential activity simultaneously from multiple sites within the cultured mouse SCN. Neurons within the isolated mouse SCN expressed a circadian rhythm in spontaneous firing rate for weeks in culture.

Action Potentials↗

PubMed1997

Opsin-like immunoreactivity in the circadian pacemaker neurons and photoreceptors of the eye of the opisthobranch mollusc Bulla gouldiana.

Circadian pacemaker cells in the eyes of the opisthobranch mollusc Bulla gouldiana generate a near 24-h rhythm in the frequency of optic nerve impulses. Previous electrophysiological studies suggest that these basal retinal neurons are intrinsically photosensitive and transduce light signals that shift the phase of their pacemaker mechanism. To test whether the pacemaker neurons contain opsin-like proteins, several polyclonal antibodies that recognize opsins of vertebrate photoreceptors have been tested on histological sections of the eye and on the neurons in primary cell culture. The antibodies label both the pacemaker cells and the large distal photoreceptors that surround the lens. Immunoblot analyses of the proteins of the eye have identified a single band at 62+/-4 kDa. These opsin antibodies may label the photopigment used in the entrainment of the circadian pacemaker.

Animals↗

PubMed1994

Calcium imaging in organotypic cultures of the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) of the hypothalamus contains a circadian pacemaker responsible for several circadian rhythms. Retinal cell projections to the SCN carry light information that phase shifts the pacemaker through the release of excitatory amino acids. To study this pathway, the Ca(2+)-sensitive dyes Fluo-3 and Fura-2 were used in organotypic slice cultures of rat SCN to visualize changes in intracellular Ca2+ of individual cells. After at least two weeks of culture, Ca2+ responses were measured in response to agonists of glutamate receptors in the presence of tetrodotoxin (TTX). Cells that showed a Ca2+ increase in response to N-methyl-D-aspartate (NMDA) and non-NMDA agonists also showed immunoreactivity towards vasoactive intestinal peptide (VIP), providing further evidence that VIP-containing neurons receive direct retinal input. The cells differed in their responses to the NMDA and non-NMDA agonists, suggesting that the cells contain differing densities of glutamate receptor subtypes.

Action Potentials↗

PubMed1994

Intracellular calcium responses of circadian pacemaker neurons measured with fura-2.

The circadian pacemaker in the eye of the mollusk Bulla gouldiana is located within basal retinal neurons (BRNs) that express a circadian rhythm in cell culture. Light and other depolarizing stimuli shift the phase of the pacemaker in the eye through a process that requires extracellular calcium and is blocked by Ni2+. To test directly if an influx of Ca2+ is present throughout depolarizing treatments that produce phase shifts, dissociated BRNs in cell culture were loaded with a membrane-permeable form of the calcium-sensitive dye fura-2, and then depolarized with elevated levels of extracellular K+. Calcium levels in the BRNs remained elevated during treatments with 50 mM K+ lasting 1 h, a sufficient duration to phase shift the circadian pacemaker. Lowering extracellular free Ca2+ (approx. 1.7 x 10(-7) M) during depolarization blocked the rise in intracellular Ca2+, verifying that a Ca2+ influx is required. The sustained Ca2+ elevation during depolarization was also prevented with 50 mM Ni2+, which blocks phase shifts of the rhythm to depolarization, but not with 5 mM Ni2+, which does not block phase shifts. The initial rise in [Ca2+]i in response to 50 mM K+ was largest on average during the subjective night. The results show that a critical portion of the entrainment pathway persists in pacemaker neurons during cell culture, and that the phase-shifting stimulus may depend on a prolonged Ca2+ signal.

Animals↗

PubMed1994

Intracellular calcium in the entrainment pathway of molluscan circadian pacemakers.

Circadian clock systems contain three components--an entrainment pathway, a pacemaker mechanism, and an output or expressed rhythm. The entrainment pathway for light stimuli can be studied by separating steps involved in light transduction and subsequent events acting on the pacemaker mechanism from the steps critical for continued motion of the pacemaker. Studies indicate that calcium entry across the plasma membrane is a required step in the light entrainment pathway of the ocular circadian pacemaker of the marine snail Bulla gouldiana. A calcium influx due to phase-shifting stimuli has recently been measured using the calcium-sensitive dye Fura-2 in dissociated pacemaker neurons from Bulla. Studies preceding these calcium imaging experiments are presented together with a simple model of the role of Ca2+ influx in entrainment and a discussion of problems in demonstrating that calcium influx alone is a sufficient step in the entrainment pathway.

Animals↗

PubMed1993

Circadian rhythm in membrane conductance expressed in isolated neurons.

Although isolated neurons can generate rhythmic activity, they have not yet been shown to generate rhythms with a period in the circadian range (near 24 hours). The eye of the mollusk Bulla gouldiana expresses a circadian rhythm in optic nerve impulses that is generated by electrically coupled cells known as basal retinal neurons (BRNs). Daily fluctuations in the membrane potential of the BRNs appear to be driven by a rhythm in membrane conductance. Isolated BRNs exhibited spontaneous conductance changes similar to those observed in the intact retina. Membrane conductance was high in the late subjective night and decreased approximately twofold near projected dawn during at least two circadian cycles in culture. The persistence of daily conductance changes in isolated BRNs indicates that individual neurons can function as circadian pacemakers.

Action Potentials↗

PubMed1992

The retinal cells generating the circadian small spikes in the Bulla optic nerve.

A circadian rhythm in the frequency of compound action potentials (CAPs) in the optic nerve of the mollusc Bulla gouldiana is believed to be generated by the basal retinal neurons (BRNs) of the eye. Along with the CAPs, which are about 100 microV in amplitude, there are 10- to 40-microV impulses from an undetermined cell type in records from the optic nerve. These impulses, called "small spikes," are generated spontaneously in darkness and show a circadian rhythm in frequency that is about 12 hr out of phase with the CAP rhythm. To enable us to determine the origin of the small spikes, intracellular recordings were made from retinal cells while optic nerve activity was monitored. The cells were identified by their light responses and then injected with the fluorescent dye Lucifer Yellow CH or the tracer biocytin. It was found that the large photoreceptors of the distal retina generated graded depolarizations in response to light, and had axons in the optic nerve, but did not show impulses at the level of the photoreceptor layer. By contrast, the spiking retinal cells of the photoreceptor layer generated depolarizations and impulses in response to light. In addition, the spiking cells were found to be dye-coupled to a series of retinal cells approximately 7 microns in diameter, connected to a single axon in the optic nerve. Impulses from the spiking cells occurred spontaneously and correspond with the small spikes in the optic nerve. The BRNs appear to inhibit the retinal cells that generate the small spikes. Hyperpolarization of the BRNs, through constant-current injection, increased the number of small spikes in the optic nerve. Release from hyperpolarization led to a decrease in small spikes. This could explain how circadian changes in BRN membrane potential might modulate spontaneous firing of the spiking cells, resulting in the circadian rhythm in small-spike frequency.

Animals↗

PubMed1991

An opsin-based photopigment mediates phase shifts of the Bulla circadian pacemaker.

1. The spectral response of the circadian pacemaker of the eye of the mollusk Bulla gouldiana was examined in two ways: by using the latency of the first light-evoked compound action potential (CAP) as an acute photoresponse of the putative pacemaker cells of the eye, the basal retinal neurons (BRNs), and by measuring the effectiveness of monochromatic light pulses at resetting the pacemaker. 2. Through measurements of the spectral sensitivity of the acute response of the BRNs, a photopigment absorbing maximally near 490 nm (lambda max) was described. Action spectra of the acute response following isolation of the BRNs, by surgical removal of the distal photoreceptor layer or the use of low Ca2+ media to block chemical synapses on the BRNs, further suggested that a 490 nm lambda max photopigment is used in generating the acute light response. The spectral sensitivity of eyes adapted to a dim background illumination also agreed with the expected absorption of a 490 lambda max rhodopsin. 3. The effectiveness of monochromatic light pulses at shifting the phase of the circadian rhythm in CAP frequency suggested that the photopigment used in the entrainment of the pacemaker is the opsin based molecule identified through acute response measurements.

Animals↗

PubMed1990

The circadian rhythm and photosensitivity of small impulses of the Bulla eye.

The eye of the mollusk Bulla gouldiana contains a pacemaker that generates a circadian rhythm in compound action potentials (CAPs) in the optic nerve. In this paper, we present evidence of a second circadian rhythm in the optic nerve of the eye maintained in darkness at 15 degrees C. This is a rhythm in the frequency of small (10-40 microV) neural impulses that occurs about 12 h out-of-phase with the rhythm in CAPs. Typically, the small-spike frequency is at a minimum within an hour of the peak in CAP frequency and is maximal during the subjective night. Like the CAP rhythm, the phase of the small-spike rhythm is determined by the prior light/dark cycle. A rebound in small-spike activity following the end of a light pulse and the presence of photoinhibited impulses in surgically reduced eyes suggests that the cells that generate the small-spikes may be photoreceptors that are inhibited by light. In addition, by using isolated nervous system preparations, we have found that small-spikes occur in the two optic nerves in a one-for-one relationship immediately following a light-to-dark transition. This inter-eye communication may be involved in the coupling of the ocular pacemakers.

Action Potentials↗

PubMedDate not supplied

Circadian rhythms.

Circadian rhythms are a ubiquitous adaptation of eukaryotic organisms to the most reliable and predictable of environmental changes, the daily cycles of light and temperature. Prominent daily rhythms in behavior, physiology, hormone levels and biochemistry (including gene expression) are not merely responses to these environmental cycles, however, but embody the organism's ability to keep and tell time. At the core of circadian systems is a mysterious mechanism, located in the brain (actually the suprachiasmatic nucleus of the hypothalamus) of mammals, but present even in unicellular organisms, that functions as a clock. This clock drives circadian rhythms. It is independent of, but remains responsive to, environmental cycles (especially light). The interest in temporal regulation--its organization, mechanism and consequences--unites investigators in diverse disciplines studying otherwise disparate systems. This diversity is reflected in the brief reviews that summarize the presentations at a meeting on circadian rhythms held in New York City on October 31, 1992. The meeting was sponsored by the Fondation pour l'Etude du Système Nerveux (FESN) and followed a larger meeting held 18 months earlier in Geneva, whose proceedings have been published (M. Zatz (Ed.), Report of the Ninth FESN Study Group on 'Circadian Rhythms', Discussions in Neuroscience, Vol. VIII, Nos. 2 + 3, Elsevier, Amsterdam, 1992). Some speakers described progress made in the interim, while others addressed aspects of the field not previously covered.

Aging↗