Michael S. Grace

Circadian rhythms of dopamine in mouse retina: The role of melatonin

Both dopamine and melatonin are important for the regulation of retinal rhythmicity, and substantial evidence suggests that these two substances are mutually inhibitory factors that act as chemical analogs of day and night. A circadian oscillator in the mammalian retina regulates melatonin synthesis. Here we show a circadian rhythm of retinal dopamine content in the mouse retina, and examine the role of melatonin in its control. Using high-performance liquid chromatography (HPLC), we measured levels of dopamine and its two major metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in retinas of C3H+/+ mice (which make melatonin) and C57BL/6J mice that are genetically incapable of melatonin synthesis. In a light/dark cycle both strains of mice exhibited daily rhythms of retinal dopamine, DOPAC, and HVA content. However, after 10 days in constant darkness (DD), a circadian rhythm in dopamine levels was present in C3H, but not in C57 mice. C57 mice given ten daily injections of melatonin in DD exhibited a robust circadian rhythm of retinal dopamine content whereas no such rhythm was present in saline-injected controls. Our results demonstrate that (1) a circadian clock generates rhythms of dopamine content in the C3H mouse retina, (2) mice lacking melatonin also lack circadian rhythms of dopamine content, and (3) dopamine rhythms can be generated in these mice by cyclic administration of exogenous melatonin. Our results also indicate that circadian rhythms of retinal dopamine depend upon the rhythmic presence of melatonin, but that cyclic light can drive dopamine rhythms in the absence of melatonin.

Infrared imaging in vipers: differential responses of crotaline and viperine snakes to paired thermal targets

Pit vipers use infrared-sensitive pit organs to accurately target homeothermic prey even in the absence of visual cues. It has been suggested that other vipers, including large ambush predators of the genus Bitis, also may use radiant infrared information for predatory targeting. We compared behavioral responses of pit vipers and snakes of the viperine genus Bitis to paired targets of different temperatures (i.e. cool and warm balloons), some scented with rodent odors. The rates of tongue flicking, head turning and approaches by pit vipers (Agkistrodon contortrix and Crotalus atrox) were significantly higher toward warm targets than toward cool ones. Moreover, they all were significantly higher in pit vipers than in the vipers Bitis arietans, Bitis gabonica, and Bitis nasicornis. Bitis sp. exhibited no significant differences in their behaviors toward warm versus cool targets. Pit vipers often struck at targets (always the warm target even when paired with a prey-scented cool target), but Bitis sp. never struck at either warm or cool targets. These results show that the behavioral correlates of infrared-based predatory and/or defensive targeting differ significantly between these two groups of viperid snakes, and suggest that the neural substrates of infrared imaging in crotaline snakes are either absent or functionally distinct in viperine snakes of the genus Bitis.

Expression of heat shock proteins 70 and 47 in tissues following short-pulse laser irradiation: assessment of thermal damage and healing.

In order to develop effective laser-based therapeutics, the extent of laser-induced damage must be quantified for given laser parameters. Therefore, we want to determine the spatiotemporal expression patterns of heat shock proteins, both to understand the roles of heat shock proteins in laser-induced tissue damage and repair and to develop heat shock proteins as tools to illustrate the extent of laser-induced damage and wound healing following irradiation. We exposed anesthetized mice to the focused beam of a short-pulse Nd:YAG laser (1064 nm; 200 ns pulsewidth) for 15s, while measuring temperature distribution in the skin using an infrared thermal camera. Following irradiation, we examined expression of HSP47 and HSP70 over time (0-24h) as indicators of the heat shock response and recovery from damage in the laser-irradiated region. Expression patterns of HSP70 and HSP47 as detected by immunohistochemistry and confocal microscopy delineate the extent of damage and the process of healing in tissue. Both HSP70 and HSP47 were expressed in dermis and epidermis following laser irradiation, and the spatial and temporal changes in HSP expression patterns define the laser-induced thermal damage zone and the process of healing in tissues. HSP70 may define biochemically the thermal damage zone in which cells are targeted for destruction, and HSP47 may illustrate the process of recovery from thermally induced damage. Studying the effects of different laser parameters on the expression of HSPs will allow development of effective laser therapies that provide accurate and precise tissue ablation and may promote rapid wound healing following laser-based surgery.

The role of thermal contrast in infrared-based defensive targeting by the copperhead, Agkistrodon contortrix

The facial pit organs of the copperhead are the end organs of a complex infrared-imaging system that allows accurate and precise strikes on potential prey. Anecdotal and recent experimental observations show that pit vipers can use their infrared-imaging systems to discriminate between cool and warm features in the environment. We tested the hypothesis that the infrared-imaging system is a thermal contrast detector and determined whether behavioural responses of copperheads differ with contrast type. We found that blindfolded copperheads responded behaviourally towards both warm objects moving against cool backgrounds and cool objects moving against warm backgrounds. Responses towards the former were more robust than those towards the latter. Furthermore, oscillating warm targets generated rhythmic following behaviour in-phase with target motion, while oscillating cool targets generated rhythmic behavioural movement in antiphase to target motion. The results show that the infrared-imaging system of pit vipers operates on the basis of thermal contrast, but the differences in behavioural responses with respect to contrast type indicate that pit vipers preferentially target the warm aspect of thermal differentials. Our results also demonstrate that the infrared-imaging system allows defensive targeting of potential endothermic predators.

Developmental shifts in functional morphology of the retina in Atlantic tarpon, Megalops atlanticus (Elopomorpha: Teleostei) between four ecologically distinct life-history stages

The Atlantic tarpon, Megalops atlanticus, is a large piscivorous fish that supports economically important recreational fisheries in the Gulf of Mexico, Caribbean, and Florida Atlantic coast. Megalops atlanticus undergoes ontogenetic shifts in morphology, hatching in the open ocean as larvae (less than 1 cm in length), moving into hypoxic turbid mangrove marshes as juveniles (around 10 cm in length), and then moving into coastal oceanic waters as adults (over 100 cm in length). In this study, photoreceptor distributions, opsin distributions, and photoreceptor absorbance characteristics were studied with light microscopy, transmission electron microscopy, antiopsin immunofluorescence, and microspectrophotometry, respectively, at four ecologically distinct life-history stages--premetamorphic larva, settlement stage, juvenile, and adult. The purposes of this study were 1) to determine the extent to which the retina of M. atlanticus changes over the course of development and 2) to relate these retinal changes with ecological shifts between developmental stages. The new data presented here indicate that the M. atlanticus retina changes substantially in rod and cone distributions and absorbance characteristics over the course of development and that these changes correlate closely with those in habitat and behavior. We show that M. atlanticus has a rod-dominated retina at the larval stage (which is unusual for teleost larvae) and that the scotopic visual system becomes far better developed with maturity, adding a substantial tapetum and high densities of small, bundled, and stacked rod cells. We also show that there are shifts in cone and rod spectral sensitivities and an increase in the diversity of spectrally distinct cone classes, including the addition of ultraviolet cones as fish mature into adults.

Evolutionary loss of cone photoreception in balaenid whales reveals circuit stability in the mammalian retina

The classical understanding of mammalian vision is that it occurs through “duplex” retinae containing both rod and cone photoreceptors, the signals from which are processed through rod‐ and/or cone‐specific signaling pathways. The recent discovery of rod monochromacy in some cetacean lineages provides a novel opportunity to investigate the effects of an evolutionary loss of cone photoreception on retinal organization. Sequence analysis of right whale (Eubalaena glacialis; family Balaenidae) cDNA derived from long‐wavelength sensitive (LWS) cone opsin mRNA identified several mutations in the opsin coding sequence, suggesting the loss of cone cell function, but maintenance of non‐photosensitive, cone opsin mRNA‐expressing cells in the retina. Subsequently, we investigated the retina of the closely related bowhead whale (Balaena mysticetus; family Balaenidae) to determine how the loss of cone‐mediated photoreception affects light signaling pathways in the retina. Anti‐opsin immunofluorescence demonstrated the total loss of cone opsin expression in B. mysticetus, whereas light microscopy, transmission electron microscopy, and bipolar cell (protein kinase C‐α [PKC‐α] and recoverin) immunofluorescence revealed the maintenance of cone soma, putative cone pedicles, and both rod and cone bipolar cell types. These findings represent the first immunological and anatomical evidence of a naturally occurring rod‐monochromatic mammalian retina, and suggest that despite the loss of cone‐mediated photoreception, the associated cone signaling structures (i.e., cone synapses and cone bipolar cells) may be maintained for multichannel rod‐based signaling in balaenid whales. J. Comp. Neurol. 524:2873–2885, 2016.

A rod-dominated visual system in leptocephalus larvae of elopomorph fishes (Elopomorpha: Teleostei)

The nature and distributions of photoreceptor cell types were investigated in the retinas of 12 species (5 families) of elopomorph anguilliform leptocephalus larvae. Anti-opsin immunofluorescence, light microscopy and transmission electron microscopy (TEM) were used to assess opsin distribution across the retinas and to associate photoreceptor morphology and opsin content. Retinas of all species were immunoreactive with anti-rhodopsin throughout, while anti-cone opsin immunoreactivity was restricted only to the ventral region of the retina in all specimens. Rod and cone photoreceptors were morphologically indistinguishable at low magnifications; TEM revealed that nearly all photoreceptors had rod-like ultrastructure, with only rare examples of cone-like cells identified in the ventral retina. These results indicate a rhodopsin/rod-dominated retina in leptocephalus larvae of anguilliform eels in the teleost subdivision Elopomorpha, contrasting with the cone-dominated retinas of nearly all other species of teleost larvae. This distinctive developmental pattern shared among elopomorph larvae has important evolutionary and ecological implications, indicating a shared ancestor and/or ecological characteristics that are very different from most other teleost larvae.

Thermal Ablation of Mouse Skin Tissue Using Ultra-Short Pulse 1552 nm Laser

Analysis of biological tissue ablation by an ultra-short pulse laser and the corresponding mathematical modeling of ablation are of fundamental importance to the understanding of laser-tissue interaction for advancing surgical application of lasers. The objective of this paper is to analyze the thermal ablated damage zones during irradiation of freshly excised mouse skin tissue samples by a novel approach of using a focused laser beam from an ultra-short pulse laser source. Experiments are performed using Raydiance Desktop Laser having a wavelength of 1552 nm and a pulse width of 1.3 ps. Mouse tissue samples are translated in a direction perpendicular to the laser beam using three-axis automated motion-controlled stages. Scanning of the tissue sample ensures a fresh region of the tissue is irradiated each time. The surface temperature distribution is measured using a thermal imaging camera. It is observed that use of focused beam results in minimal radial heat spread to the surrounding tissue regions. The ablation phenomenon is analytically modeled by the use of two-phase transient heat conduction model. After completion of tissue irradiation experiments, histological studies are performed using frozen sectioning technique to observe morphological changes in tissue samples in response to laser irradiation. The ablation depth measurements obtained using histological studies are compared with the modeling results. A parametric study of various laser parameters such as time-average power, pulse repetition rate, and pulse energy, and as well as irradiation time and scanning velocity is performed to determine the necessary ablation threshold. Analytical modeling results are in very good agreement with experimentally measured ablation depth. The goal of this research is to develop a tool for selection of appropriate laser parameters for precise clean tissue ablation.Copyright

Ontogenic retinal changes in three ecologically distinct elopomorph fishes (Elopomorpha:Teleostei) correlate with light environment and behavior.

Unlike the mammalian retina, the teleost fish retina undergoes persistent neurogenesis from intrinsic stem cells. In marine teleosts, most cone photoreceptor genesis occurs early in the embryonic and larval stages, and rods are added primarily during and after metamorphosis. This study demonstrates a developmental paradigm in elopomorph fishes in which retinas are rod-dominated in larvae, but undergo periods of later cone genesis. Retinal characteristics were compared at different developmental stages among three ecologically distinct elopomorph fishes-ladyfish (Elops saurus), bonefish (Albula vulpes), and speckled worm eel (Myrophis punctatus). The objectives were to improve our understanding of (1) the developmental strategy in the elopomorph retina, (2) the functional architecture of the retina as it relates to ecology, and (3) how the light environment influences photoreceptor genesis. Photoreceptor morphologies, distributions, and spectral absorption were studied at larval, juvenile, and adult stages. Premetamorphic retinas in all three species are rod-dominated, but the retinas of these species undergo dramatic change over the course of development, resulting in juvenile and adult retinal characteristics that correlate closely with ecology. Adult E. saurus has high rod densities, grouped photoreceptors, a reflective tapetum, and longer-wavelength photopigments, supporting vision in turbid, low-light conditions. Adult A. vulpes has high cone densities, low rod densities, and shorter-wavelength photopigments, supporting diurnal vision in shallow, clear water. M. punctatus loses cones during metamorphosis, develops new cones after settlement, and maintains high rod but low cone densities, supporting primarily nocturnal vision. M. punctatus secondary cone genesis occurs rapidly throughout the retina, suggesting a novel mechanism of vertebrate photoreceptor genesis. Finally, in postsettlement M. punctatus, the continuous presence or absence of visible light modulates rod distribution but does not affect secondary cone genesis, suggesting some degree of developmental plasticity influenced by the light environment.

Spectral Sensitivity Change May Precede Habitat Shift in the Developing Retina of the Atlantic Tarpon (Megalops atlanticus)

Fish that undergo ontogenetic migrations between habitats often encounter new light environments that require changes in the spectral sensitivity of the retina. For many fish, sensitivity of the retina changes to match the environmental spectrum, but the timing of retinal change relative to habitat shift remains unknown. Does retinal change in fish precede habitat shift, or is it a response to encountered changes in environmental light? Spectral sensitivity changes were examined over the development of the Atlantic tarpon (Megalops atlanticus) retina relative to ontogenetic shifts in habitat light. Opsin gene isoform expression and inferred chromophore use of visual pigments were examined over the course of M. atlanticus development. Spectral sensitivity of the retina was then determined by electroretinography and compared to the spectroradiometric measurements of habitat light encountered by M. atlanticus from juveniles to adults. These data, along with previously known microspectrophotometric measurements of sensitivity in M. atlanticus, indicate retinal spectral sensitivity that matches the dominant wavelengths of environmental light for juvenile and adult fish. For the intervening subadult stage, however, spectral sensitivity does not match the dominant wavelength of light it occupies but better matches the dominant wavelengths of light in the habitat of its forthcoming migration. These results first indicate that the relationship between environmental light spectrum and spectral sensitivity of the retina changes during M. atlanticus development and then suggest that such changes may be programmed to support visual anticipation of new photic environments.