A visual lamina in the medulla oblongata of the frog, Rana pipiens.

We have discovered a lamina of visually responsive units in the medulla oblongata of the frog. It spans the entire medial aspect of the rostrocaudal length of the medulla and extends dorsoventrally from the cell-dense dorsal zone into the cell-sparse ventral zone. Most visual units within this lamina have large receptive fields, with the majority extending bilaterally in the frontal visual field. Most of these neurons are binocular, have no apparent directional preference, respond equally well to stimuli of a variety of shapes and sizes, and exhibit strong habituation. More medial locations in the visual lamina represent ipsilateral visual space while more lateral locations within the lamina represent contralateral visual space. Many units in the caudal aspect of the visual lamina are bimodal, responding to both visual and somatosensory stimuli. HRP tracing reveals inputs to the lamina from many primary and secondary visual areas in the midbrain and diencephalon. There is no area-by-area segregation of the projections to the visual lamina. For example, most parts of the tectum project across the visual lamina. The only spatial order in the visual lamina is that at more medial sites there tends to be more input from contralateral tectum; and at more lateral sites there tends to be more input from ipsilateral tectum. There is bilateral input to the visual lamina from tectum, tegmentum, posterior nucleus of the thalamus, posterior tuberculum, and ventromedial thalamic nucleus. There is ipsilateral input to the visual lamina from torus semicircularis, pretectum, nucleus of Bellonci, and ventrolateral thalamic nucleus. There is contralateral input to the visual lamina from basal optic complex. Collectively, these results show the presence of visual influences in regions of the medulla that likely represent an important step in sensorimotor transformation.

Dramatic changes in gene expression in different forms of Crithidia fasciculata reveal potential mechanisms for insect-specific adhesion in kinetoplastid parasites.

Kinetoplastids are a group of parasites that includes several medically-important species. These human-infective species are transmitted by insect vectors in which the parasites undergo specific developmental transformations. For each species, this includes a stage in which parasites adhere to insect tissue via a hemidesmosome-like structure. Although this structure has been described morphologically, it has never been molecularly characterized. We are using Crithidia fasciculata, an insect parasite that produces large numbers of adherent parasites inside its mosquito host, as a model kinetoplastid to investigate both the mechanism of adherence and the signals required for differentiation to an adherent form. An advantage of C. fasciculata is that adherent parasites can be generated both in vitro, allowing a direct comparison to cultured swimming forms, as well as in vivo within the mosquito. Using RNAseq, we identify genes associated with adherence in C. fasciculata. As almost all of these genes have orthologs in other kinetoplastid species, our findings may reveal shared mechanisms of adherence, allowing investigation of a crucial step in parasite development and disease transmission. In addition, dual-RNAseq allowed us to explore the interaction between the parasites and the mosquito. Although the infection is well-tolerated, anti-microbial peptides and other components of the mosquito innate immune system are upregulated. Our findings indicate that C. fasciculata is a powerful model system for probing kinetoplastid-insect interactions.

Connections of Anterior Thalamic Visual Centers in the Leopard Frog, Rana pipiens.

The amphibian retina projects to two discrete regions of neuropil in the anterior thalamus: the neuropil of Bellonci and the corpus geniculatum. These retinorecipient areas are encompassed within a larger zone of surrounding neuropil we call the NCZ (for neuropil of Bellonci/corpus geniculatum zone). The NCZ is characterized electrophysiologically by a distinctive tonic oscillatory response to blue light; it appears to be a visual module involved in processing the stationary visual environment. Using horseradish peroxidase (HRP), we mapped the connections of the NCZ. Retrogradely labeled cell bodies are found in: (1) the contralateral anterior thalamus; (2) both retinas; and (3) the posterior medial dorsal thalamus (PMDT). Anterogradely labeled fibers are found in: (1) the contralateral anterior thalamus; (2) the ipsilateral PMDT; (3) the ipsilateral neuropil lateral to the posterior tuberculum in the ventrolateral posterior thalamus; and (4) the ipsilateral anterior medulla. There are no direct connections between the NCZ and the telencephalon, the tectum, or the suprachiasmatic nucleus. Applying HRP to the PMDT, we found that its inputs are limited to the contralateral and ipsilateral NCZ and the contralateral PMDT. Thus, PMDT appears to be a satellite of the NCZ. Blue light elicits tonic oscillatory electrical responses in the PMDT quite similar to the responses to blue light in the NCZ. We discuss how the leopard frog NCZ and the mammalian ventral lateral geniculate nucleus share anatomical and physiological properties.

Representation of the visual field in the anterior thalamus of the leopard frog, Rana pipiens.

We used physiological and anatomical methods to elucidate how the visual field is represented in the part of the dorsal anterior thalamus of the leopard frog that receives direct retinal projections. We recorded extracellularly while presenting visual stimuli, and characterized a physiologically defined region that encompasses the retinal projections as well as an extended zone beyond them. We probed the area systematically to determine if the zone is organized in a visuotopic map: we found that it is not. We found that units in this region respond only to stimuli in the contralateral half of the visual field, which is similar to what is seen in the dorsal lateral geniculate nucleus in mammals. When we backfilled retinal ganglion cells from application of HRP to the anterior thalamus, we found labeled cells only in those parts of the retina corresponding to the contralateral hemifield, confirming our physiological observations.

Light and shadow: visual recognition of the stationary environment by leopard frogs.

We determined how leopard frogs respond to non-moving aspects of the environment. We have discovered that these frogs are attracted to dark, stationary, opaque objects. This attraction depends on the relative reflectance of the object, i.e., the darker the block, the more attractive it is, and the attraction is found under both bright and dim ambient light levels. Larger blocks are more attractive than smaller blocks, but frogs are still attracted to blocks much smaller than themselves. Previous studies have shown that frogs are also attracted to sources of light. Using a choice experiment, we show that the probability a frog will choose a dark object versus a light source depends on the intensity of the light source relative to the intensity of the ambient light. The frog only moves toward a light source when it is at least 20 times brighter than the brightest object in the environment. These findings help to clarify the frog's "phototactic" nature.

Leopard frog priorities in choosing between prey at different locations.

Frogs are able to respond to a prey stimulus throughout their 360° ground-level visual field as well as in the superior visual field. We compared the likelihood of frogs choosing between a more nasally located, ground-level prey versus a more temporally located ground-level prey, when the prey at the nasal location is further away from the frog. Two crickets were presented simultaneously at 9 pairs of angles that included both crickets in the binocular visual field, both crickets in the monocular visual field, or one cricket in the binocular field and one in the monocular field. Frogs chose the more nasally located prey at least 71% of the time when the more temporal prey was in the monocular field; and 64% of the time when both prey were in the binocular field. Frogs tended to choose the more nasally located prey, even though it takes the frog longer to reach the prey. In addition, when given a choice between a prey located at ground level versus a prey located in the superior field, frogs tend to choose the prey at ground-level. These results suggest that there is a neural mechanism that biases frogs' responses to prey stimuli.

Conformational change induced by ATP binding correlates with enhanced biological function of Arabidopsis cryptochrome.

Cryptochromes are widely distributed blue light photoreceptors involved in numerous signaling functions in plants and animals. Both plant and animal-type cryptochromes are found to bind ATP and display intrinsic autokinase activity; however the functional significance of this activity remains a matter of speculation. Here we show in purified preparations of Arabidopsis cry1 that ATP binding induces conformational change independently of light and increases the amount and stability of light-induced flavin radical formation. Nucleotide binding may thereby provide a mechanism whereby light responsivity in organisms can be regulated through modulation of cryptochrome photoreceptor conformation.

Combining visual information from the two eyes: the relationship between isthmotectal cells that project to ipsilateral and to contralateral optic tectum using fluorescent retrograde labels in the frog, Rana pipiens.

The frog nucleus isthmi (homolog of the mammalian parabigeminal nucleus) is a visually responsive tegmental structure that is reciprocally connected with the ipsilateral optic tectum; cells in nucleus isthmi also project to the contralateral optic tectum. We investigated the location of the isthmotectal cells that project ipsilaterally and contralaterally using three retrograde fluorescent label solutions: Alexa Fluor 488 10,000 mw dextran conjugate; Rhodamine B isothiocyanate; and Nuclear Yellow. Dye solutions were pressure-injected into separate sites in the superficial optic tectum. Following a 6-day survival, brains were fixed, sectioned, and then photographed. Injection of the different labels at separate, discrete locations in the optic tectum result in retrograde filling of singly labeled clusters of cells in both the ipsilateral and contralateral nucleus isthmi. Generally, ipsilaterally projecting cells are dorsal to the contralaterally projecting cells, but there is a slight overlap between the two sets of cells. Nonetheless, when different retrograde labels are injected into opposite tecta, there is no indication that individual cells project to both tecta. The set of cells that project to the ipsilateral tectum and the set of cells that project to the contralateral tectum form a visuotopic map in a roughly vertical, transverse slab. Our results suggest that nucleus isthmi can be separated into two regions with cells in the dorsolateral portion projecting primarily to the ipsilateral optic tectum and cells in the ventrolateral nucleus isthmi projecting primarily to the contralateral optic tectum.

Novel ATP-binding and autophosphorylation activity associated with Arabidopsis and human cryptochrome-1.

Cryptochromes are blue-light photoreceptors sharing sequence similarity to photolyases, a class of flavoenzymes catalyzing repair of UV-damaged DNA via electron transfer mechanisms. Despite significant amino acid sequence similarity in both catalytic and cofactor-binding domains, cryptochromes lack DNA repair functions associated with photolyases, and the molecular mechanism involved in cryptochrome signaling remains obscure. Here, we report a novel ATP binding and autophosphorylation activity associated with Arabidopsis cry1 protein purified from a baculovirus expression system. Autophosphorylation occurs on serine residue(s) and is absent in preparations of cryptochrome depleted in flavin and/or misfolded. Autophosphorylation is stimulated by light in vitro and oxidizing agents that act as flavin antagonists prevent this stimulation. Human cry1 expressed in baculovirus likewise shows ATP binding and autophosphorylation activity, suggesting this novel enzymatic activity may be important to the mechanism of action of both plant and animal cryptochromes.

Nucleus isthmi enhances calcium influx into optic nerve fiber terminals in Rana pipiens.

We examined the role of nucleus isthmi in enhancing intracellular calcium concentrations in retinotectal fibers in the frog optic tectum in vitro. The intracellular calcium levels were measured using the fluorescent calcium-sensitive dye, Calcium Green-1 3000 mw dextran conjugate (CG-1), which was injected into one optic nerve. Electrical stimulation of the labeled optic nerve alone increased tectal CG-1 fluorescence whereas electrical stimulation of nucleus isthmi alone had no effect on CG-1 fluorescence. Electrical stimulation of the nucleus isthmi ipsilateral to the labeled tectum, followed by electrical stimulation to the optic nerve can enhance calcium uptake more than a double pulse stimulation of the optic nerve alone. Maximum enhancement of the calcium signal by nucleus isthmi occurs when optic nerve stimulation follows the ipsilateral nucleus isthmi stimulation by 10 ms. These results suggest that nucleus isthmi input can facilitate retinotectal neurotransmission, and the mechanism could be used to allow the frog to attend to a single prey stimulus in an environment of several prey stimuli.