Glial and light-dependent glutamate metabolism in the suprachiasmatic nuclei.

The suprachiasmatic nuclei, the main circadian clock in mammals, are entrained by light through glutamate released from retinal cells. Astrocytes are key players in glutamate metabolism but their role in the entrainment process is unknown. We studied the time dependence of glutamate uptake and glutamine synthetase (GS) activity finding diurnal oscillations in glutamate uptake (high levels during the light phase) and daily and circadian fluctuations in GS activity (higher during the light phase and the subjective day). These results show that glutamate-related astroglial processes exhibit diurnal and circadian variations, which could affect photic entrainment of the circadian system.

Disease correction by combined neonatal intracranial AAV and systemic lentiviral gene therapy in Sanfilippo Syndrome type B mice.

Mucopolysaccharidosis type IIIB (MPS IIIB) or Sanfilippo Syndrome type B is a lysosomal storage disease resulting from the deficiency of N-acetyl glucosaminidase (NAGLU) activity. We previously showed that intracranial adeno-associated virus (AAV)-based gene therapy results in partial improvements of several aspects of the disease. In an attempt to further correct the disease, MPS IIIB mice were treated at 2-4 days of age with intracranial AAV2/5-NAGLU (IC-AAV), intravenous lentiviral-NAGLU (IV-LENTI) or the combination of both (BOTH). The BOTH group had the most complete biochemical and histological improvements of any treatment group. Compared with untreated MPS IIIB animals, all treatments resulted in significant improvements in motor function (rotarod) and hearing (auditory-evoked brainstem response). In addition, each treatment group had a significantly increased median life span compared with the untreated group (322 days). The combination arm had the greatest increase (612 days), followed by IC-AAV (463 days) and IV-LENTI (358 days). Finally, the BOTH group had nearly normal circadian rhythm measures with improvement in time to activity onset. In summary, targeting both the systemic and central nervous system disease of MPS IIIB early in life appears to be the most efficacious approach for this inherited metabolic disorder.

Learning chronobiology by improving Wikipedia.

Although chronobiology is of growing interest to scientists, physicians, and the general public, access to recent discoveries and historical perspectives is limited. Wikipedia is an online, user-written encyclopedia that could enhance public access to current understanding in chronobiology. However, Wikipedia is lacking important information and is not universally trusted. Here, 46 students in a university course edited Wikipedia to enhance public access to important discoveries in chronobiology. Students worked for an average of 9 h each to evaluate the primary literature and available Wikipedia information, nominated sites for editing, and, after voting, edited the 15 Wikipedia pages they determined to be highest priorities. This assignment (http://www.nslc.wustl.edu/courses/Bio4030/wikipedia_project.html) was easy to implement, required relatively short time commitments from the professor and students, and had measurable impacts on Wikipedia and the students. Students created 3 new Wikipedia sites, edited 12 additional sites, and cited 347 peer-reviewed articles. The targeted sites all became top hits in online search engines. Because their writing was and will be read by a worldwide audience, students found the experience rewarding. Students reported significantly increased comfort with reading, critiquing, and summarizing primary literature and benefited from seeing their work edited by other scientists and editors of Wikipedia. We conclude that, in a short project, students can assist in making chronobiology widely accessible and learn from the editorial process.

Circadian rhythms in firing rate of individual suprachiasmatic nucleus neurons from adult and middle-aged mice.

The suprachiasmatic nucleus contains a biological clock that drives circadian rhythms in vivo and in vitro. It has been suggested that the suprachiasmatic nucleus is a primary target of the aging process, because age-related changes in behavioral rhythms are mirrored in alterations in circadian pacemaker function. Using long-term, single-cell recording, we assessed the effect of age on firing-rate patterns of individual suprachiasmatic nucleus neurons of young adult (2-4 months) and middle-aged (9-11 months) C3H mice. Individual suprachiasmatic nucleus neurons from adult mice maintained in culture for at least one week exhibited robust circadian rhythms in spontaneous activity that were similar in the free-running period (23.7+/-0.3 h mean+/-S.E.M.) to recordings from neurons dispersed from neonatal tissue, and showed evidence of entrainment to prior light cycles by exhibiting peak activity, in vitro, approximately 4.0+/-0.3 h (mean+/-S.E.M.) after the time of expected light onset. Aging led to a decreased amplitude of impulse activity in dispersed suprachiasmatic nucleus neurons and increased variability in the circadian waveform. From these results we suggest that age-related deterioration in circadian clock function occurs at the level of individual cells, which may account for some of the age-related deficits observed in the expression of behavioral rhythmicity.

The mammalian circadian clock shop.

In mammals, a master circadian pacemaker driving daily rhythms in behavior and physiology resides in the suprachiasmatic nucleus (SCN). The SCN contains multiple circadian oscillators that synchronize to environmental cycles and to each other in vivo. Rhythm production, an intracellular event, depends on more than eight identified genes. The period of the rhythms within the SCN also depends upon intercellular communication. Many other tissues also retain the ability to generate near 24 -h periodicities although their place in the organization of circadian timing is still unclear. This paper focuses on the tissue-, cellular- and molecular-level events that generate and entrain circadian rhythms in behavior in mammals and emphasizes the apparent differences between the SCN and peripheral oscillators.

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.

Lithium lengthens the circadian period of individual suprachiasmatic nucleus neurons.

Lithium treatment lengthens the period of circadian rhythms in most organisms. In the present study, we tested whether lithium acts directly on the mammalian suprachiasmatic nucleus (SCN) to lengthen rhythms of individual neurons. Lithium increased the circadian period of firing rate rhythms of cultured SCN neurons in a concentration-dependent manner. Lithium had no effect on the amplitude of these rhythms, but did affect the period of some cells more than others. The results indicate that lithium acts directly on the SCN to lengthen the free-running period of individual neurons.

The role of Clock in the developmental expression of neuropeptides in the suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the dominant circadian pacemaker in mammals. To understand better the ontogeny of mouse SCN and the role of the pacemaker in peptide expression, the authors examined the distribution of cells that were immunoreactive for vasopressin (AVP) or vasoactive intestinal polypeptide (VIP) in wild type and Clock mutant mice at two developmental stages. Clock homozygous mice failed to show the dramatic increase in the number of VIP-immunoreactive (VIP-ir) neurons from postnatal day 6 (P6) to P30 that was found in the SCN of wild type mice. The number of AVP-ir neurons was relatively constant in the postnatal SCN but was significantly reduced in Clock/Clock mice. The effects of the Clock mutation varied with position in the SCN for both peptides. Densitometry of immunolabeled brains indicated that the Clock mutation reduced AVP expression specifically in the SCN and not in other brain areas. The SCN did not significantly change shape or size with age or Clock genotype. Taken together, these results indicate that the neonatal mouse SCN has its full complement of cells, some of which are not yet mature in their neuropeptide content. Furthermore, the observation that the Clock mutation appears to act on a subset of AVP and VIP cells suggests heterogeneity within these cell classes in the SCN.

Keeping an eye on retinal clocks.

Circadian pacemakers that drive rhythmicity in retinal function are found in both invertebrates and vertebrates. They have been localized to photoreceptors in molluscs, amphibians, and mammals. Like other circadian pacemakers, they entrain to light, oscillate based on a negative feedback between transcription and translation of clock genes, and control a variety of physiological and behavioral rhythms that often includes rhythmic melatonin production. As a highly organized and accessible tissue, the retina is particularly well suited for the study of the input-output pathways and the mechanism for rhythm generation. Impressive advances can now be expected as researchers apply new molecular techniques toward looking into the eye's clock.

Clock controls circadian period in isolated suprachiasmatic nucleus neurons.

The suprachiasmatic nucleus (SCN) is the master circadian pacemaker in mammals, and one molecular regulator of circadian rhythms is the Clock gene. Here we studied the discharge patterns of SCN neurons isolated from Clock mutant mice. Long-term, multielectrode recordings showed that heterozygous Clock mutant neurons have lengthened periods and that homozygous Clock neurons are arrhythmic, paralleling the effects on locomotor activity in the animal. In addition, cells in dispersals expressed a wider range of periods and phase relationships than cells in explants. These results suggest that the Clock gene is required for circadian rhythmicity in individual SCN cells and that a mechanism within the SCN synchronizes neurons and restricts the range of expressed circadian periods.

Retinal anatomy of Chorocaris chacei, a deep-sea hydrothermal vent shrimp from the Mid-Atlantic Ridge.

Exploration of deep-sea hydrothermal vents over the past quarter century has revealed that they support unique and diverse biota. Despite the harsh nature of the environment, vents along the Mid-Atlantic Ridge are dominated by large masses of highly motile Bresiliid shrimp. Until 1989, when it was discovered that the vent shrimp Rimicaris exoculata possesses a hypertrophied dorsal eye, many believed that animals populating hydrothermal vents were blind. Chorocaris chacei (originally designated Rimicaris chacei) is a Bresiliid shrimp found at hydrothermal vent fields along the Mid-Atlantic Ridge. Like R. exoculata, C. chacei has a hypertrophied retina that appears to be specialized to detect the very small amount of light emitted from the orifices of black smoker hydrothermal vent chimneys. C. chacei lacks the sophisticated compound eyes common to other decapod crustaceans. Instead, it has a smooth cornea, with no dioptric apparatus, apposed by a tightly packed, massive array of photosensitive membrane. Photoreceptors in the C. chacei retina are segmented into a hypertrophied region that contains the photosensitive membrane and an atrophied cell body that is roughly ten times smaller in volume than the photosensitive segment. The microvillar photosensitive membrane is consistent in structure and ultrastructure with the rhabdoms of decapod and other invertebrate retinas. However, the volume density of photosensitive membrane (> or =60%) exceeds that typically observed in invertebrate retinas. The reflecting pigment cells commonly found in decapod retinas are represented in the form of a matrix of white diffusing cells that exhibit Tyndall scattering and form an axial sheath around the photoreceptors. All photoreceptor screening pigment granules and screening pigment cells are restricted to the region below the photoreceptor nuclei and are thereby removed from the path of incident light. No ultrastructural evidence of rhythmic cycling of photosensitive membrane was observed. The morphological adaptations observed in the C. chacei retina suggest that it is a high-sensitivity photodetector that is of functional significance to the animal.

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.

Retinal anatomy of a new species of bresiliid shrimp from a hydrothermal vent field on the Mid-Atlantic Ridge.

A new species of shrimp (Rimicaris sp.) was recently collected from the Snake Pit hydrothermal vent field on the Mid-Atlantic Ridge. Until the discovery in 1989 that the deep-sea, hydrothermal vent species, Rimicaris exoculata, possessed a hypertrophied dorsal eye, everyone believed that animals recovered from vent environments were blind. Like R. exoculata, Rimicaris sp., a small orange bresiliid shrimp, has an enlarged dorsal eye specialized for detecting light in a very dim environment instead of the expected compound eye. The individual lenses characteristic of a compound eye adapted for imaging have been replaced in Rimicaris sp. by a smooth cornea underlain by a massive array of photosensitive membrane. The number of ommatidia in this species is about the same as in shrimp species that live at the surface; however, the photoreceptors are larger in the deep-sea species and the shape of the photoreceptors is markedly different. The light-sensitive region of the photoreceptor is much larger than those of other shrimp and the rest of the receptor is much smaller than normal. All screening pigment has moved out of the path of incident light to a position below the retina, and the reflecting pigment cells have adapted to form a bright white diffusing screen between and behind the photoreceptors. The ultrastructure of the microvillar array comprising the rhabdom is typical for decapod crustaceans; however, there is a much greater volume density of rhabdom (80% to 85%) than normal. There is no ultrastructural evidence for cyclic rhabdom shedding or renewal. Rimicaris sp. has apparently adapted its visual system to detect the very dim light emitted from the throats of the black smoker chimneys around which it lives.

Limulus vision in the ocean day and night: effects of image size and contrast.

Male horseshoe crabs, Limulus polyphemus, use their eyes to locate mates day and night. We investigated their ability to detect targets of different size and contrast in a mating area of Buzzards Bay, Cape Cod, MA. We found that males can see large, high-contrast targets better than small, low-contrast ones. For targets of the same size, animals must be about 0.1 m closer to a low-contrast target to see it as well as a high-contrast one. For targets of the same contrast, animals must be approximately 0.2 m closer to a small target to see it as well as one twice as large. A decrease of 0.05 steradians in the size of the retinal image of a target can be compensated by a four-fold increase in contrast. About 60% of the animals detect black targets subtending 0.110 steradians (equivalent to an adult female viewed from about 0.56 m), while only 20% detect targets subtending 0.039 steradians. This study shows that horseshoe crabs maintain about constant contrast sensitivity under diurnal changes in light intensity in their natural environment. As a consequence of circadian and adaptive mechanisms in the retina, male horseshoe crabs can detect female-size objects about equally well day and night.

Sulfide as a Chemical Stimulus for Deep-Sea Hydrothermal Vent Shrimp.

Organisms dependent on deep-sea hydrothermal vents for their existence face extinction when their vents expire, unless they can establish populations on neighboring vents or on new vent sites. Propagules, including larvae and motile adults, are readily dispersed broadly by seafloor currents, but how they recognize active hydrothermal sites is problematical. Compelling evidence that vent organisms can find and colonize hydrothermal sites has been provided by a series of observations on the East Pacific Rise (1). New hydrothermal vents created there following a volcanic eruption on the seafloor in March 1991 were colonized by sessile invertebrates in less than one year. On the Mid-Atlantic Ridge, shrimp that normally cluster on sulfide surfaces have been observed to swim directly back to the surfaces when displaced from them. How do vent animals locate new or existing vents? Passive transport by currents (2) or active swimming without guidance by some physical cue is not likely to result in success (3). Chemicals present in hydrothermal fluids have been proposed as attractants. We provide the first evidence of a chemosensory response in a vent invertebrate to sulfides, which are prevalent in vent fluids and provide the energy,for chemosynthetic primary production at vents.

The morphology of the dorsal eye of the hydrothermal vent shrimp, Rimicaris exoculata.

The bresiliid shrimp, Rimicaris exoculata, lives in large masses on the sides of hydrothermal vent chimneys at two sites on the Mid-Atlantic Ridge. Although essentially no daylight penetrates to depths of 3500 m, very dim light is emitted from the hydrothermal vents themselves. To exploit this light, R. exoculata has evolved a modified compound eye on its dorsal surface that occupies about 0.5% of the animal's body volume. The eye's morphology suggests that it is extremely sensitive to light. The cornea of the dorsal eye is smooth with no dioptric apparatus. The retina consists of two wing-shaped lobes that are fused across the midline anteriorly. The rhabdomeral segments of the 7000 ommatidia form a compact layer of photosensitive membrane with an entrance aperture of more than 26 mm2. Within this layer, the volume density of rhabdom is more than 70%. Below the rhabdomeral segments, a thick layer of white diffusing cells scatters light upward into the photoreceptors. The arhabdomeral segments of the five to seven photoreceptors of each ommatidium are mere strands of cytoplasm that expand to accommodate the photoreceptor nuclei. The rhabdom is comprised of well-organized arrays of microvilli, each with a cytoskeletal core. The rhabdomeral segment cytoplasm contains mitochondria, but little else. The perikaryon contains a band of mitochondria, but has only small amounts of endoplasmic reticulum. There is no ultrastructural indication of photosensitive membrane cycling in these photoreceptors. Vestigial screening pigment cells and screening pigment granules within the photoreceptors are both restricted to the inner surface of the layer of the white diffusing cells. Below the retina, photoreceptor axons converge in a fanshaped array to enter the dorsal surface of the brain. The eye's size and structure are consistent with a role for vision in shrimp living at abyssal hydrothermal vents.

The Limulus-eye view of the world.

The compound lateral eye of the adult horseshoe crab, Limulus polyphemus, views the world with approximately 1000 ommatidia. Their optical properties and orientation determine the eye's resolution, field of view, and light collecting ability. Optic axes of adjacent ommatidia diverge from 1-15 deg with an average value of 5.5 deg yielding an average resolution of 0.1 cycles/deg. Resolution is not uniform across the eye: along horizontal planes, it is maximal in the anterior region of the eye (0.22 cycle/deg) and minimal in the posterior region (0.07 cycle/deg); along vertical planes, it is maximal near or just below the horizon (0.23 cycle/deg) and minimal above the horizon (0.04 cycle/deg). Together the ommatidia of one eye view approximately 60% of the hemispheric world on one side of the body. There is little binocular overlap (< 1% of total field). Ommatidial facets of up to 320 microns in diameter (among the largest known in the animal kingdom) make the eye a superb light collector. Limulus are known to use vision to find mates both day and night. Apparently, the optics of the lateral eye sample a large enough part of the world with sufficient resolution and light-collecting ability for the animal to succeed at this essential task.