Grant W. Balkema

Impaired visual thresholds in hypopigmented animals.

Ocular hypopigmentation is associated with neurological defects in structure and function. This paper investigates the absolute visual thresholds in dark-adapted hypopigmented animals compared to their normally pigmented controls. Here we asked (1) whether the threshold elevation found in hypopigmented animals is a general consequence of the reduction in melanin content; (2) if so, which melanin components in the eye are likely to influence visual thresholds; and (3) whether similar threshold defects can be detected in orders other than rodents. By single-unit recordings from the superior colliculus, we compared incremental thresholds of normal black mice of the C57BL/6J strain to hypopigmented mutants: beige (bg/bg), pale ear (ep/ep), and albino (c2J/c2J) mice, three mutants in which melanin pigment throughout the body is affected; and Steel (Sl/Sld) and dominant-spotting/W-mice (W/Wv), two mutants with normal pigmentation in the retinal pigment epithelium (RPE) but without any melanin in the choroid or the rest of the body. We found that all mutants had elevated thresholds that varied with the reduction in melanin. The albinos were 25 times less sensitive than black mice, pale ear mice 20 times, beige mice 11 times, and Steel and W-mice 5 times. The mean thresholds of dark-adapted black mice were 0.008 cd/m2. Recordings from rabbits showed a similar impairment of visual sensitivity; incremental thresholds were elevated 40 times in New Zealand-White albino rabbits (0.0008 cd/m2) compared to Dutch-Belted pigmented controls (0.00002 cd/m2).(ABSTRACT TRUNCATED AT 250 WORDS)

Diurnal variation in synaptic ribbon length and visual threshold

Previous work suggests that photoreceptor synaptic ribbon length and absolute dark-adapted threshold may vary during a 24-h diurnal cycle. To test this hypothesis, we examined the length of photoreceptor synaptic ribbons and the dark-adapted threshold in black (+/+) and albino (c2J/c2J) C57BL/6J mice at six times over a 24-h period. Testing began 2 h after light onset (ZT 2:00) and continued at successive 4-h intervals (12 h:12 h light:dark). We determined the length of the synaptic ribbons in frozen sections by labeling them with an antibody specific for synaptic ribbons. Synaptic ribbons vary in length at different points in the diurnal cycle in both types of mice, but the synaptic ribbons in black mice are longer than those in albino mice by an average of 0.33 microm. The synaptic ribbons of black mice also have a larger response to changes in the light cycle. Ribbon length in black mice ranges from 1.66 microm to 1.4 microm, whereas ribbon length in albino mice ranges from 1.32 microm to 1.25 microm. The shortest ribbons are evident 6 h after light onset in both types of mice, whereas the longest ribbons appear within 2 h after light onset. These changes in synaptic ribbon length support the idea that photoreceptor synaptic ribbons are dynamic structures whose length changes over a 24-h diurnal cycle. Examining black and albino mice with a water-maze behavioral assay showed that visual thresholds in black and albino mice vary over the 24-h diurnal cycle. The visual thresholds of albino mice are elevated compared with black mice at all times tested. This is consistent with previous findings of visual thresholds in hypopigmented mice. The lowest threshold (greatest sensitivity) is present 2 h after light onset (ZT 2:00) and corresponds to the time when synaptic ribbons are the longest. The highest threshold is observed 6 h after light onset, the time when synaptic ribbons are shortest. These results show that synaptic ribbon length and visual sensitivity vary together in relation to the time.

Biochemical Correlates of Epilepsy in the El Mouse: Analysis of Glial Fibrillary Acidic Protein and Gangliosides

Abstract: The El (epileptic) mouse is considered a model for complex partial seizures in humans. Seizures in El mice begin around 7–8 weeks of age and persist throughout life. To determine if astrocytic gliosis was present in adult seizing El mice, the distribution of glial fibrillary acidic protein (GFAP) was studied in the hippocampus using an antibody to GFAP. The mean number of GFAP‐positive cells per square millimeter of hippocampus was approximately 15‐ to 40‐fold higher in adult El mice than in nonseizing control C57BL/ 6J (B6) mice or in young nonseizing El mice. Relative GFAP concentration (expressed per milligram of total tissue protein) in hippocampus and cerebellum was estimated by densitometric scanning of peroxidase‐stained western blots. GFAP concentration was 2.7‐fold greater in hippocampus of adult seizing El mice than in the control B6 mice. No differences in GFAP content were detected between the strains in the cerebellum. Because gangliosides can serve as cell surface markers for changes in neuronal cytoarchitecture, they were analyzed to determine if the gliotic response in El mice was associated with changes in neural composition. Although the total ganglioside concentration of hippocampus, cerebral cortex, and cerebellum was similar in adult El and control B6 mice, a synaptic membrane enriched ganglioside, GD1a, was elevated in the adult El cerebral cortex and hippocampus. The findings indicate that El mice express a type of gliosis that is not accompanied by obvious neuronal loss.

Elevated dark-adapted thresholds in hypopigmented mice measured with a water maze screening apparatus

In previous electrophysiological experiments from hypopigmented animals (mice, rats, rabbits), single-unit recordings from both retinal ganglion axons and cells in the superior colliculus have demonstrated an increase in threshold in the dark-adapted state which is roughly proportional to the ocular melanin concentration. In the present study we compared an albino mouse strain which is relatively resistant to light damage and the beige mouse mutant to their wild-type controls in a situation that involved unanesthetized, unrestrained mice as a control to the electrophysiological single unit experiments. We used a six-chambered water maze. Animals were trained to swim to an illuminated ramp until their performances leveled off (about 10 days). The animals were then dark-adapted for 24 h and tested after reducing the luminance level of the water maze. We found that the albino mice failed to find the ramp when the luminance fell to 1.58×10−3 cd/m2 (p≤.0001), the beige mice failed at 2.00×10−4 cd/m2 (p≤.0001), and the normally pigmented controls performed to 5.00×10−5 cd/m2 (p≤.0001). These results support our previous findings that the sensitivity defect in hypopigmented animals is proportional to the degree of ocular hypopigmentation.

Visual thresholds in mice: Comparison of retinal light damage and hypopigmentation

In previous electrophysiological experiments from hypopigmented animals (mice, rats, rabbits), single-unit recordings from both retinal ganglion axons and cells in the superior colliculus have demonstrated an increase in threshold in the dark-adapted state which is roughly proportional to the animals ocular melanin concentration. We have examined the thresholds in hypopigmented mice by using a behavioral water maze screening test and found similar threshold elevations to the electrophysiology. In the present study, we investigated the contribution of retinal light damage to the threshold elevation in an albino mouse strain which is relatively resistant to light damage (C57BL/6J c2J/c2J) and mice with profound retinal degeneration (C57BL/6J rd/rd). Black or albino littermates (C57BL/6J +/c2J or c2J/c2J) were placed in either constant light (350 cd/m2) or dim cycling light (0.001 cd/m2) for 21 days before testing. The normally pigmented animals had thresholds of 1.00 x 10(-5) cd/m2 regardless of their light history. The albino mice (c2J/c2J) maintained in constant light had a slight 0.30 log unit elevation compared to their controls that were maintained in dim cycling light 6.3 x 10(-4) cd/m2 (similar to previously published reports). We examined the retinal morphology of representative animals in semi-thin plastic sections. We could not detect any light damage (overall morphology or cell counts in the outer-nuclear layer) in either the normally pigmented animals or the albino mice (c2J/c2J) maintained in dim cycling light. We found extensive light damage in the albino mice (c2J/c2J) maintained in constant light (virtual absence of photoreceptor outersegments) that corresponded to the slight elevation in threshold.(ABSTRACT TRUNCATED AT 250 WORDS)

ULTRASTRUCTURAL LOCALIZATION OF A SYNAPTIC RIBBON PROTEIN RECOGNIZED BY ANTIBODY B16

SummaryThis study examines the immunolabelled structures in the mouse retina following incubation with a monoclonal antibody (B16) that recognizes a highly conserved antigen found in retinas from lizards, frogs, fish, birds, mice, rats, rabbits, cats, and monkeys. This paper focuses on observations in the murine retina. The B16 labelling pattern in the retina was compared with that of two synaptic vesicle antigens: SV2 and anti-synaptophysin in the outer plexiform layer were more diffuse and apparently filled the entire presynaptic terminal whereas B16 labelling was more restricted and labelled a discrete structure resembling a semi-ellipse or an arc with the ends pointing to the inner nuclear layer and the middle curve facing the outer nuclear layer (1–2 μm long by <0.05 μm in width). The structure labelled in the inner plexiform layer resembles a short bar (0.8 μm long by <0.05 μm in width) that is confined to the inner half the inner plexiform layer. Cryo-ultra microtomy was used to examine the ultrastructural distribution of the labelling, because the B16 epitope is sensitive to fixation and plastic embedding. The tissue was incubated with the B16 antibody and visualized with goat-anti-mouse 10 nm gold particles. In all cases label was found to be confined to the ribbon structure in the photoreceptor terminal. This is consistent with previous evidence linking B16 to an epitope associated with the synaptic ribbon. The labelling is confined to the ribbon structure and does not appear to be associated with synaptic vesicles.

Antigenic epitopes of the photoreceptor synaptic ribbon

The purpose of this study was twofold: 1) to purify and identify a protein containing an epitope recognized by an anti‐synaptic ribbon antibody B16 and 2) to identify and sequence the epitope. B16 recognizes several unrelated proteins in retina immunoblots. Purification and microsequencing of the strongest band (88 kDa) demonstrate 94% identity to aconitase over 111 amino acids. Polyclonal antibodies against aconitase recognize aconitase on Western blots, but not synaptic ribbons in sections. We conclude that although aconitase contains the epitope, aconitase is not the synaptic ribbon protein. The B16 epitope was identified to be 542DTYQHPPKDS551. A synthetic peptide to this sequence absorbs B16 activity in both Western blots and immunohistochemistry studies, whereas partial peptides fail to absorb activity. Additional antibodies against this peptide label synaptic ribbons. When mouse retina were double labeled with B16 and anti‐α‐actinin, B16 was found to label synaptic ribbons in the outer plexiform layer that partially enclosed the α‐actinin label. We have determined the amino acid sequence of the B16 epitope and found that the B16 labeling colocalizes with α‐actinin at the photoreceptor synapse. J Comp Neurol 413:209–218, 1999.

Increased absolute light sensitivity in Himalayan mice with cold-induced ocular pigmentation.

Controversy over the relationship between ocular pigmentation and absolute dark-adapted light sensitivity has persisted for over two decades. Previous electrophysiological experiments in hypopigmented mammals (mice, rats, rabbits) show increased thresholds in the dark-adapted state proportional to the deficit in ocular melanin. Animals with the least amount of ocular melanin have the most elevated thresholds. Dark-adapted thresholds in hypopigmented mice show similar threshold elevations in behavioral tests. The present study extends these findings to show that a specific increase in ocular pigmentation results in the converse effect, lowered absolute dark-adapted thresholds. The increase in ocular melanin was accomplished by keeping Himalayan mice in the cold (4 degrees C) for 6 weeks. Himalayan mice (C57BL/6J cH/cH) were compared to black mice (C57BL/6J (+/+)) and albino mice (C57BL/6J c2J/c2J) after 6 weeks at either 4 degrees C or 20 degrees C in 12-h cycling light (<1 cd/m2). The Himalayan mice that were kept in the cold exhibited a 44% increase in ocular melanin compared to Himalayan mice kept at room temperature. Cold rearing did not effect ocular melanin or visual thresholds in control animals (black mice = 10(-5.9) cd/m2 and albino mice = 10(-4.4) cd/m2). In contrast, the Himalayan mice maintained at 4 degrees C had thresholds of 10(-5.7) cd/m2 compared to 10(-5.1) cd/m2 for Himalayan mice kept at 20 degrees C. This represents compelling evidence of a direct relationship between ocular melanin concentration and absolute dark-adapted light sensitivity.