Distributions of vesicular glutamate transporters 1 and 2 in the visual system of tree shrews (Tupaia belangeri).

Vesicular glutamate transporter (VGLUT) proteins regulate the storage and release of glutamate from synapses of excitatory neurons. Two isoforms, VGLUT1 and VGLUT2, are found in most glutamatergic projections across the mammalian visual system, and appear to differentially identify subsets of excitatory projections between visual structures. To expand current knowledge on the distribution of VGLUT isoforms in highly visual mammals, we examined the mRNA and protein expression patterns of VGLUT1 and VGLUT2 in the lateral geniculate nucleus (LGN), superior colliculus, pulvinar complex, and primary visual cortex (V1) in tree shrews (Tupaia belangeri), which are closely related to primates but classified as a separate order (Scandentia). We found that VGLUT1 was distributed in intrinsic and corticothalamic connections, whereas VGLUT2 was predominantly distributed in subcortical and thalamocortical connections. VGLUT1 and VGLUT2 were coexpressed in the LGN and in the pulvinar complex, as well as in restricted layers of V1, suggesting a greater heterogeneity in the range of efferent glutamatergic projections from these structures. These findings provide further evidence that VGLUT1 and VGLUT2 identify distinct populations of excitatory neurons in visual brain structures across mammals. Observed variations in individual projections may highlight the evolution of these connections through the mammalian lineage.

Selective photoreceptor damage in albino rats using continuous blue light. A protocol useful for retinal degeneration and transplantation research.

PURPOSE: To develop a retinal degeneration model with selective photoreceptor loss and RPE sparing, to be used as recipient for evaluating retinal transplants. METHODS: Albino rats were exposed to blue light, continuously, for 1-7 days (24-168 h) in a specially designed cage. Eyes were histologically analyzed at periods between 2 h and 8 months after the light exposure. Electroretinograms (ERGs) were recorded from some rats at 12-216 days after exposure. Using behavioral methods, visual thresholds of some rats were determined before exposure and re-measured between 18 and 52 days following exposure. RESULTS: Apoptotic nuclei appeared exclusively in the photoreceptor layer after 1-5 days exposure to blue light. Light microscopy revealed that 2-4 days of light exposure reduced the outer nuclear layer (normally eight to ten rows) to 1 row of cells in the central retina and to two to three rows in the periphery, both in the superior and the inferior retina. Average ERG a- and b-wave amplitudes of light-damaged rats were both reduced by about 98%. Visual performance in the behavioral test was substantially impaired. CONCLUSIONS: Continuous exposure of albino rats to moderate blue light for 2-5 days selectively eliminates most of the photoreceptors while leaving the RPE initially intact.

Psychophysical measurement of temporal modulation sensitivity in the tree shrew (Tupaia belangeri).

Temporal modulation sensitivity functions (MSFs) were measured behaviorally in three adult tree shrews (Tupaia belangeri). Shrews were trained to detect temporal sinusoidally-modulated full-field luminance variations in one of three stimuli, the two alternatives being static stimuli of equal size and time-averaged luminance (34 cd/m2). Modulation depth was varied trial-by-trial using a modified staircase technique under ambient illumination of 16 lux. Threshold modulation depths were determined for five temporal frequencies ranging from 3.7 to 47 Hz. Results revealed temporal MSFs that peaked at 15 Hz with a low-frequency roll-off and an extrapolated high-frequency cut-off beyond 50 Hz. These findings confirm the comparatively good temporal vision of Tupaia predicted by behavioral observations.

Noninvasive assessment of retinal function in rats using multifocal electroretinography.

PURPOSE: To assess the applicability of multifocal electroretinograms (mfERGs) for evaluation of function in this small-eyed animal with a rod-dominant retina that is often used to model retinal diseases. METHODS: Noninvasive monocular mfERGs were recorded in anesthetized albino (Sprague-Dawley) and pigmented (Long Evans) rats. Achromatic stimuli subtending a 49 degrees by 53 degrees field consisted of 61 hexagons that were generated and presented (at varying rates and luminances) using a Visual Evoked Response Imaging System (VERIS; EDI, San Mateo, CA). The VERIS also was used to calculate individual responses and for analysis. RESULTS: mfERGs were recorded from pigmented and albino rats by slowing the rate of stimulus presentation to allow for the slow recovery time of the rod system. In each rat strain, responses varied systematically with changes in stimulus parameters. Peak response amplitude increased as the rate of stimulation was slowed and as stimulus luminance was increased. Response latency decreased as stimulus intensity was increased. The local nature of the response was assessed by several independent measures. CONCLUSIONS: The present work demonstrated the feasibility and limitations of using mfERG to assess topographical changes in the rat retina. It showed that despite the problems of the unavoidable self-adapting nature of the stimulus, the small eye of the animal, and the high potential for light scatter within the retina, multifocal responses with a good signal-to-noise ratio can be obtained from the rat.

Preliminary report: indications of improved visual function after retinal sheet transplantation in retinitis pigmentosa patients.

PURPOSE: To report indications of new visual function after retinal transplantation in two blind patients with retinitis pigmentosa. METHODS: Intact sheets of fetal retina (15 and 17 weeks gestational age) were transplanted subretinally (between the neurosensory retina and the retinal pigment epithelium) near the fovea in the left eye of a 23-year-old white man (Patient A) and in the left eye of a 72-year-old white woman (Patient B), both with autosomal-recessive retinitis pigmentosa. RESULTS: Postoperatively, at 6 and 5 months, respectively, both patients reported new visual sensation in the visual field corresponding to the transplant. In both patients, the visual sensation continued to be present after transplantation, at 12 and 8 months, respectively. In Patient A, a transient multifocal electroretinography (mfERG) response was observed in the transplant area 4 months postoperatively but was not detectable in Patient A at 6.0 and 9.5 months post-retinal transplantation. In Patient B, no positive mfERG responses were seen up to 5 months postoperatively. No rejection (presenting as cystoid macular edema, macular pucker, and extensive intraretinal edema with disrupted retinal pigment epithelium) to the transplanted tissue was seen up to 13 months in Patient A and 9 months in Patient B by fluorescein angiography. CONCLUSION: Transplantation of intact sheets of fetal human retina in two patients with retinitis pigmentosa was not associated with evidence of transplant rejection. Subjective improvement and an indication of objective improvement 4 months postoperatively were seen in Patient A, and subjective improvement only was seen in Patient B.

Localization of nitric oxide synthase in the tree shrew retina.

Nitric oxide (NO) is a novel neuronal messenger that likely influences retinal function by activating retinal guanylyl cyclase to increase levels of cGMP. In the present study, the localization of neuronal nitric oxide synthase (nNOS, Type I NOS) in the cone-dominant tree shrew retina was studied using NADPH-d histochemistry and nNOS immunocytochemistry. Both NADPH-d and nNOS-immunoreactivity (IR) labeled the inner segments of rods and the myoids of a regular subpopulation of cones, with their corresponding nuclei outlined. The labeled cone myoids were co-localized with a marker for short-wave-sensitive (SWS) cones (S-antigen) and also displayed the regular triangular packing and density (7%) characteristic of SWS cones in tree shrew and other mammalian retinas. These measures confirmed the identity of the labeled cones as SWS cones. Photoreceptor ellipsoids of all cones were strongly labeled by NADPH-d reactivity, but lacked nNOS-IR. Another novel finding in tree shrew retina was that both NADPH-d and nNOS-IR labeled Muller cells, which have not been labeled by nNOS-IR in other mammalian retinas. Consistent with findings in rod-dominant retinas, two types of amacrine cells at the vitreal edge of the inner nuclear layer and a subpopulation of displaced amacrine cells at the scleral edge of the ganglion cell layer were labeled by both NADPH-d and nNOS-IR. Processes of these labeled cells were seen to extend into the inner plexiform layer, where dense punctate label was seen, especially in the central sublamina. These results show that localization of NOS in the cone-dominant tree shrew retina shares some common properties with rod-dominant mammalian retinas, but also shows some species-specific characteristics. The new finding of nNOS localization in tree shrew SWS cones and rods, but not in other cones, raises interesting questions about the roles of NO in the earliest level of visual processing.

Differentiation of short-wavelength-sensitive cones by NADPH diaphorase histochemistry.

NADPH diaphorase (NADPH dehydrogenase; EC 1.6.99.1) histochemistry labels neurons that synthesize the neurotransmitter nitric oxide (NO). In retina, it has been demonstrated that NO can affect the metabolism of cGMP in rod photoreceptors. To investigate potential involvement of NO in cone photoreceptor activity, we utilized NADPH diaphorase histochemistry to study the cone-dominated retina of the tree shrew (Tupaia belangeri). Unexpectedly, our results revealed different NADPH diaphorase activity in the cellular subcompartments of the spectral classes of cone photoreceptors. Although all cones showed intense labeling of inner segment ellipsoids, the short-wavelength-sensitive (SWS or "blue-sensitive") cones and the rods displayed intense staining of the myoid inner segment subcompartment as well. Furthermore, only SWS cones and rods displayed surface labeling of their nuclei. These findings indicate a manner in which SWS cones differ biochemically from other cone types and in which they are more similar to rods. Such differences may underlie some of the unusual functional properties of the SWS cone system, which have been attributed to postreceptoral processes.

Immunocytochemical identification of photoreceptor populations in the tree shrew retina.

The presence and retinal distributions of short-wavelength-sensitive cones, long-wavelength-sensitive cones, and rods were assessed in the retina of the tree shrew Tupaia belangeri using visual pigment antibodies OS-2, COS-1 and anti-rhodopsin, respectively. Results demonstrated a clear immunocytochemical differentiation of the three photoreceptor types with each showing regional variation in density across the retina.

Immunohistochemical organization of the ventral lateral geniculate nucleus in the ground squirrel.

The ventral lateral geniculate nucleus (vLGN) of the thirteen-lined ground squirrel (Citellus tridecemlineatus) is a highly differentiated nucleus that is divisible into five major subdivisions on the basis of retinal projections and cytoarchitecture. To pursue the likelihood that these subdivisions (the dorsal cap, intergeniculate leaflet, external magnocellular lamina, internal magnocellular lamina, and parvicellular segment) correlate with the functional diversity of this complex, the present study examined the neurochemical composition of the vLGN with regard to substances that have previously proved useful in distinguishing functionally distinct subregions within nuclei (i.e., neuropeptide Y (NPY), substance P (SP), leucine and methionine enkephalins, gamma-aminobutyric acid (GABA), cytochrome oxidase (CO), acetylcholinesterase (AChE), and NADPH-diaphorase). The results showed a clear differential neurochemical distribution within the nucleus. Neuropeptide Y immunoreactive perikarya were found predominantly in the intergeniculate leaflet and external magnocellular lamina, with only a few present in the internal magnocellular lamina and dorsal cap, and none observed in the parvicellular segment. NPY+ fibers, however, were present in all divisions except the parvicellular segment. The highest concentration of SP immunoreactive cells was observed in the internal magnocellular lamina, and substantial numbers also were scattered in the external magnocellular lamina and parvicellular segment. SP+ fibers were seen predominantly in the intergeniculate leaflet and the magnocellular laminae. The heaviest concentration of enkephalinergic fibers occurred in the internal magnocellular lamina and dorsal cap, but fibers were also observed in the external magnocellular lamina and intergeniculate leaflet. GABA reactivity was widespread throughout the vLGN, with the dorsal cap and external magnocellular lamina most heavily labeled, followed by the intergeniculate leaflet and the internal magnocellular lamina. Cytochrome oxidase, AChE, and NADPH-diaphorase histochemistry revealed rich reactivity within the dorsal cap, and external and internal magnocellular laminae and paler reactivity in the intergeniculate leaflet and parvicellular segment. The external magnocellular lamina was more reactive for CO and NADPH-diaphorase than AChE, while the internal magnocellular lamina showed the opposite pattern of reactivity. In addition, NADPH-diaphorase reactive cells were present in caudal intergeniculate leaflet and lateral external magnocellular lamina. These local differences in the neurochemical character of the vLGN support its parcellation into multiple subdivisions. Taken in conjunction with the differences in cytoarchitecture and retinal projections, these results suggest substantial functional diversity within the ventral lateral geniculate complex.

Immunohistochemical organization of the ventral lateral geniculate nucleus in the tree shrew.

The ventral lateral geniculate nucleus (vLGN) of the tree shrew (Tupaia belangeri) was differentiated into multiple subdivisions (dorsal cap, intergeniculate leaflet, parvicellular segment, and internal and external magnocellular laminae, the latter being further divisible into a lateral and medial division) on the basis of retinal projections, immunochemistry, and histochemistry. Retinal projections traced with intravitreal injections of wheat germ agglutinin conjugated horseradish peroxidase revealed direct bilateral input to all subregions of the vLGN, except for the internal magnocellular lamina (which received only contralateral input) and the parvicellular segment (which was not retinorecipient). Furthermore, retinal inputs clearly distinguished the relatively heavily retinorecipient intergeniculate leaflet from the less prominently labeled dorsal cap. Immunohistochemical localization of Neuropeptide Y (NPY) perikarya revealed their prominence in the intergeniculate leaflet and the external magnocellular laminae with a concentration along the optic tract. NPY immunoreactive fibers were seen in all but the parvicellular subregion. Gamma amino butyric acid immunoreactivity was seen throughout the vLGN, but was most concentrated in the dorsal cap and the magnocellular laminae, followed by the intergeniculate leaflet. Histochemical studies of cytochrome oxidase and nicotinamide adenosine dinucleotide phosphate (NADPH)-diaphorase localization revealed similar patterns of dense reactivity within the external magnocellular lamina, intergeniculate leaflet and dorsal cap, and somewhat less dense, but substantial reactivity in the internal magnocellular lamina. Within the external magnocellular lamina, cells reactive for cytochrome oxidase were noted in the lateral portion bordering the optic tract, whereas those specific for NADPH-diaphorase were dispersed throughout the lamina. Poor reactivity for both histochemical markers was evident in the parvicellular segment. Overall, the markedly different patterns of retinal input and neurochemical organization between the subdivisions of the tree shrew vLGN suggest their involvement in diverse functions. Furthermore, the basic similarity of the organization of the tree shrew vLGN to that of the taxonomically unrelated ground squirrel may indicate a common mammalian scheme.

Green-sensitive cone photoreceptors are selectively labeled by Procion yellow dye in goldfish retina.

Selective labeling of intravitreal Procion yellow dye by presumed blue-sensitive cone photoreceptors has been demonstrated in primate retina. To determine whether Procion yellow is selective for this cone type in an unrelated vertebrate species, labeling by this dye was studied in goldfish retina, where cone pigment type can be directly inferred from photoreceptor morphology. At low vitreal concentrations of the dye (less than 0.4%), only cone outer segments were labeled. At vitreal concentrations of 0.4-0.5%, the inner segments of short-double cones and a subset of long single cones (presumed green-sensitive cones) were selectively stained. At still higher vitreal concentrations (0.6-0.7%), the inner segments of short-single cones and miniature short-single cones (presumed blue-sensitive cones) showed evidence of Procion label, but were not as heavily labeled. The inner segments of long-double cones and a subset of long-single cones (presumed red-sensitive cones) did not label at any of these concentrations. These results show that Procion yellow is not a selective marker for blue-sensitive cones in the goldfish retina. In addition, stained rod and cone nuclei were observed at each dye concentration, including those concentrations at which no inner segments were labeled.

Psychophysical measurement of spectral sensitivity and color vision in red-light-reared tree shrews (Tupaia belangeri).

The role of the spectral lighting environment on the post-natal development of spectral sensitivity and color vision was studied in tree shrews (Tupaia belangeri) that were born and reared to adulthood in cyclic red light. Normal tree shrews are dichromats, possessing short-wavelength-sensitive (SWS) and long-wavelength-sensitive (LWS) cone receptors and a small population of rods. Red-light-rearing (RLR) produced differential stimulation of the cone types by effectively eliminating photic stimulation of the SWS cones, without depriving the LWS cones. Spectral sensitivity and color vision were measured behaviorally for RLR shrews and normal shrews under different ambient light levels. Spectral sensitivity functions were deutan-like, exhibiting maxima at ca 450 and 550 nm and a minimum at 510 nm. No significant differences in spectral sensitivity were observed between RLR and control animals. Furthermore, all animals demonstrated deutan-type dichromatic color vision evidenced by their ability to discriminate monochromatic lights from equally-bright achromatic lights except for a "neutral point" near 505 nm. These results demonstrate that a population of functional SWS cones survived the lack of post-natal photic stimulation. However, RLR shrews differed from controls in that they were poorer at making chromatic/achromatic discriminations. While no severe disorganization of color vision was evident, the poorer discrimination displayed by the RLR animals is likely the result of changes in post-receptoral visual mechanisms.

Visual pigments of the tree shrew (Tupaia belangeri) and greater galago (Galago crassicaudatus): a microspectrophotometric investigation.

Optical density, linear dichroism and bleaching difference spectra were measured in photoreceptors from the cone-dominated retina of the tree shrew (Tupaia belangeri) and from the rod-dominated retina of the greater galago (Galago crassicaudatus) using a single-beam, wavelength-scanning, dichroic microspectrophotometer. In Tupaia, we obtained spectral records from 272 cone receptors (from 10 eyes), of which 264 were long-wave sensitive (lambda max = 555 +/- 6 nm) and 8 were short-wave sensitive (lambda max = 428 +/- 15 nm). Also, one anatomically-recognizable rod receptor was encountered and showed a peak absorption at approx. 496 nm. No mid-wave sensitive cone pigment was found, as would be expected in deutan-type dichromats like the tree shrew. Pre-retinal absorption by the cornea and lens was maximal at 370 nm and negligible beyond 430 nm. In Galago, all outer segments measured were rod-like in appearance (lambda max near 501 nm). Measurements of pre-retinal absorption yielded a single-peaked function with a maximum at 363 nm.

Retinal projections in the ground squirrel (Citellus tridecemlineatus).

The retinal projections of the thirteen-lined ground squirrel were determined by tracing anterograde transport of intravitreally injected horseradish peroxidase (HRP) or wheat-germ conjugated horseradish peroxidase (WGA-HRP). Label was seen in the suprachiasmatic nucleus and adjacent anterior hypothalamic area, the accessory optic system (the medial, dorsal, and lateral terminal nuclei), the dorsal and ventral lateral geniculate nuclei, the intergeniculate leaflet, the pretectal nuclei (the anterior, posterior, and olivary pretectal nuclei and the nucleus of optic tract), and the superior colliculus. Most of these structures were labeled bilaterally, with dense contralateral label and sparse ipsilateral label, a pattern typical for animals with laterally placed eyes. However, the suprachiasmatic nucleus and the nucleus of the optic tract received input only from the contralateral eye. In contrast to previous degeneration studies, the sensitive HRP tracers (in conjunction with cytochrome-oxidase reactivity) revealed an elaborate organization within the lateral geniculate nucleus (dorsal LGN, ventral LGN, and intergeniculate leaflet) that is consistent with existing organizational schemes for other mammalian species.

Striped pattern of labeling in ground squirrel superior colliculus following intraocular HRP injections.

In the 13-lined ground squirrel (Citellus tridecemlineatus), intravitreal injections of horseradish peroxidase (HRP) or wheat germ-agglutinated HRP (WGA-HRP) tracers produced a striped pattern of labeling in the contralateral superficial gray (SGS) of the superior colliculus in most, but not all, cases. The stripes extended dorsoventrally throughout the SGS in a rostral-medial to caudal-lateral orientation. In general, the striped labeling was more distinct and was observed more frequently with the HRP, rather than the WGA-HRP, tracer.

Contrast-sensitivity functions of W-, X-, and Y-like relay cells in the lateral geniculate nucleus of bush baby, Galago crassicaudatus.

1. This paper represents a continuation of our effort to examine the relationship between the physiology of distinct classes of primate lateral geniculate nucleus (LGN) cells and spatial vision. The present study focuses on modeling the contrast-sensitivity functions (CSFs) of separate LGN cell classes, examining differences in the CSFs of different classes of LGN cells and comparing the results with behaviorally defined CSFs. 2. CSFs to drifting sinusoidal gratings were obtained from single LGN relay cells in the nocturnal primate, Galago crassicaudatus. The CSFs of 14 X-like, 27 Y-like, and 6 W-like cells with standard center-surround organization were well fit by a difference of Gaussians (DOG) model with small residual errors (mean error per data point +/- SEM = 0.008 +/- 0.002). The larger residual errors shown by a few of the Y-like cells were not due to nonlinearity of spatial summation. 3. The CSFs of eight cells that appeared to have nonstandard center-surround organization (primarily, a silent, suppressive surround) were also well fit by the DOG model. 4. The DOG curves that best fitted the data differed considerably between the three groups. As a group, X-like cells had a small center mechanism (Rc = 0.19 degrees) with high sensitivity (Kc = 76.53) and a small, sensitive surround (Rs = 0.71 degrees; Ks = 5.50). These parameters produced CSFs with high cutoff frequencies (Vcutoff = 2.5 c/deg) and low peak sensitivities (CSpk = 6.1) that occurred at 0.8 c/deg. 5. Y-like cells had a large center mechanism (Rc = 0.46 degrees) with low sensitivity (Kc = 21.16) and a large, insensitive surround (Rs = 2.38 degrees; Ks = 0.81). These parameters produced CSFs with lower cutoff frequencies (Vcutoff = 1.2 c/deg) and higher peak sensitivities (CSpk = 12.5) that occurred at 0.2 c/deg. 6. The few W-like cells that responded to gratings well enough to determine a CSF were quite variable. As a group they had a large center mechanism (Rc = 0.38 degrees) with intermediate sensitivity (Kc = 34.55) and a surround with intermediate size and sensitivity (Rs = 1.59 degrees; Ks = 1.59). These produced CSFs with intermediate cutoffs (Vcutoff = 1.6 c/deg) and low peak sensitivities (CSpk = 5.0) occurring at 0.4 c/deg.(ABSTRACT TRUNCATED AT 400 WORDS)

A retinal smear technique for varying grating contrast.

We present here a technique for modulating contrast in gratings seen in Maxwellian-view or on a projection screen. The method can be used with achromatic or chromatic grating stimuli. It is based on a high frequency oscillatory displacement of the grating image, which in turn produces a smearing of the image on the observer's retina. The waveform of the displacement and its amplitude determine the appearance and contrast of the image produced on the retina.

A method for determining threshold from single-unit neural activity.

A computationally straightforward method is described for determining the latency, duration and magnitude of stimulus-evoked single-unit neural activity. A unique feature of the method is its ability to define the neural response without reference to stimulus parameters. First, the temporal component of the spike train that represents the response is located and then that component is analyzed to determine the magnitude of the response. Intensity-response functions can then be constructed, using the number of extra spikes above baseline activity as a measure of response magnitude. Threshold can be defined as any point on the intensity-response function.