Treatment of age-related visual impairment with a peptide acting on mitochondria.

Age-related visual decline and disease due to neural dysfunction are major sources of disability that have resisted effective treatment. In light of evidence that visual impairment and mitochondrial dysfunction advance with age, we characterized age-related decline of spatial visual function in mice and investigated whether treatment of aged mice with the mitochondrion-penetrating peptide elamipretide that has been reported to improve mitochondrial function, would improve it. Impaired photopic acuity measured by using a virtual optokinetic system emerged near 18 months and declined to ∼40% below normal by 34 months. Daily application of the synthetic peptide elamipretide, which has high selectivity for mitochondrial membranes that contain cardiolipin and promotes efficient electron transfer, was able to mitigate visual decline from 18 months onwards. Daily application from 24 months onwards, i.e. when acuity had reduced by ∼16%, reversed visual decline and normalized function within 2 months. Recovered function persisted for at least 3 months after treatment was withdrawn and a single treatment at 24 months delayed subsequent visual decline. Elamipretide applied daily from 32 months onwards took longer to take effect, but substantial improvement was found within 2 months. The effects of age and elamipretide treatment on contrast sensitivity were similar to those on acuity, systemic and eye drop applications of elamipretide had comparable effects, scotopic spatial visual function was largely unaffected by age or treatment, and altered function was independent of variation in optical clarity. These data indicate that elamipretide treatment adaptively alters the aging visual system. They also provide a rationale to investigate whether mitochondrial dysfunction is a treatable pathophysiology of human visual aging and age-related visual disease.

Perception of visual motion coherence by rats and mice.

The coherence thresholds to discriminate the direction of motion in random-dot kinematograms were measured in rats and mice. Performance was best in the rats when dot displacement from frame-to-frame was about 2 degrees, and frame duration was less than 100 ms. Mice had coherence thresholds similar to those of rats when tested at the same step size and frame duration. Although the lowest thresholds in the rats and mice occasionally reached human levels, average rodent values ( approximately 25%) were 2-3 times higher than those of humans. These data indicate that the rodent and primate visual systems are similar in that both have local motion detectors and a system for extracting global motion from a noisy signal.

Independent visual threshold measurements in the two eyes of freely moving rats and mice using a virtual-reality optokinetic system.

Slow horizontal head and body rotation occurs in mice and rats when the visual field is rotated around them, and these optomotor movements can be produced reliably in a virtual-reality system. If one eye is closed, only motion in the temporal-to-nasal direction for the contralateral eye evokes the tracking response. When the maximal spatial frequency capable of driving the response ("acuity") was measured under monocular and binocular viewing conditions, the monocular acuity was identical to the binocular acuity measured with the same rotation direction. Thus, the visual capabilities of each eye can be measured under binocular conditions simply by changing the direction of rotation. Lesions of the visual cortex had no effect on the acuities measured with the virtual optokinetic system, whereas perceptual thresholds obtained previously with the Visual Water Task are. The optokinetic acuities were also consistently lower than acuity estimates from the Visual Water Task, but contrast sensitivities were the same or better. These data show that head-tracking in a virtual optokinetic drum is driven by subcortical, lower frequency, and contralateral pathways.

Characterization of mouse cortical spatial vision.

Little is known about the spatial vision of mice or of the role the visual cortex plays in mouse visual perception. In order to provide baseline information upon which to evaluate the spatial vision of experimentally and genetically altered mice, we used the visual water task to assess the contrast sensitivity and grating acuity of normal C57BL/6 mice. We then ablated striate cortex (V1) bilaterally and re-measured the same visual functions. Intact mice displayed an inverse "U"-shaped contrast sensitivity curve with a maximum sensitivity near 0.2 cycles/degree (c/d). Grating acuity, measured either by discriminating a sine-wave grating from an equiluminant gray, or vertical from horizontal sine wave gratings, was near 0.55 c/d. Grating acuity and contrast sensitivity were reduced significantly following aspiration of V1. The mouse visual system exhibits fundamental mammalian characteristics, including the feature that striate cortex is involved in processing visual information with the highest sensitivity and spatial frequency.

Preservation of vision following cell-based therapies in a model of retinal degenerative disease.

This study examines whether subretinal transplantation of a human retinal pigment epithelial cell line (ARPE19) or human Schwann cells early in the course of degeneration can limit the loss of visual acuity that normally occurs in dystrophic Royal College of Surgeons (RCS) rats as they age. The acuity of both transplanted groups was significantly better than controls at all ages tested, though the rescue profile of each cell type was unique. These data indicate that the transplantation of cells with very different phenotypes can be used to limit the deterioration of spatial vision in an animal model of retinal degenerative disease.

Visual memory task for rats reveals an essential role for hippocampus and perirhinal cortex.

Visual recognition memory is subserved by a distributed set of neural circuits, which include structures of the temporal lobe. Conflicting experimental results regarding the role of the hippocampus in nonspatial forms of such memories have been attributed to species, task, and lesion discrepancies. We have overcome obstacles that have prevented a direct evaluation of the role of the hippocampus in this type of memory by developing for rats a nonspatial, picture-based, trial-unique, delayed matching-to-sample task that is a procedural analogue of standard visual recognition memory tasks used in primates. With this task, we demonstrate that rats have a visual memory profile, which is analogous to that in primates and depends on the function of perirhinal cortex. We also find that selective lesions of hippocampus impair delay-dependent visual memory with a profile different from that produced by damage to the perirhinal cortex. These data demonstrate that rats have a visual recognition memory system fundamentally similar to primates that depends on the function of the hippocampus.

Reduced visual acuity impairs place but not cued learning in the Morris water task.

The Morris water task is a standard method for testing spatial learning in rodents. In a place version of the task, animals utilize multiple visual cues to learn the location of a hidden platform. The ability of animals to locate a cued platform is often used to qualitatively test for possible non-cognitive contributions to deficient place learning, including reduced visual function. We investigated the role of visual acuity in water maze performance quantitatively by depriving rats of pattern vision during a critical period for visual plasticity, which reduced their acuity by approximately 27% and then tested them in typical place and cued platform configurations of the Morris water task. Animals with reduced visual acuity had a significant deficit in place learning, but eventually reached the same escape latency as non-deprived animals. Deprived and non-deprived animals, however, did not differ in their ability to locate a cued platform following place learning. These data indicate that reduced visual acuity in rats can influence measurement of their place learning and that a typical cued platform version of the task cannot detect a modest, but significant, visual deficit.

Experience-dependent plasticity of visual acuity in rats.

Rats have become a popular model for investigating the mechanisms underlying ocular dominance plasticity; however, no quantitative assessment of the effects of visual deprivation on behavioural acuity has been reported in this species. We measured the spatial acuity of monocularly and binocularly deprived rats with a visual discrimination task. The average spatial acuity of normal rats and rats deprived of vision after postnatal day 40 was approximately 1 cycle/degree. Monocular deprivation up to postnatal day 40 resulted in a 30% decrease in acuity and there was no recovery after 8 months. Identical binocular deprivation produced a comparable but significantly smaller reduction in acuity. The deleterious effects of monocular and binocular deprivation on visual acuity indicate that the development of cortical receptive field properties related to spatial tuning are affected by both monocular and binocular deprivation. The similarities in the effects of visual deprivation on visual acuity between rats and other mammals confirm that rats are a good model system for studying the cellular and molecular mechanisms underlying experience-dependent visual plasticity.

Environmental enrichment from birth enhances visual acuity but not place learning in mice.

The effect of richness of the environment on behavioral function was investigated in C57B6 mice. Animals were raised in either enriched (group-housed in large clear plexiglas cages with stimulating objects) or restricted (group housed in opaque white plastic cages with no stimulating objects) environmental conditions and their spatial learning and visual acuity were measured as adults. The performance of enriched and restricted groups were indistinguishable in place and cued versions of the Morris water task; however, the visual acuity of the enriched group, measured in a grating versus gray version of the visual water task, was 18% higher than the restricted group. These data demonstrate that the function of the mouse visual system can be significantly influenced by the nature of early visual input. They also indicate that the effects of environmental enrichment are manifested differently in behavioral measures of spatial learning and visual acuity.

Behavioral assessment of visual acuity in mice and rats.

We have developed a simple computer-based discrimination task that enables the quick determination of visual acuities in rodents. A grating is displayed randomly on one of two monitors at the wide end of a trapezoidal-shaped tank containing shallow water. Animals are trained to swim toward the screens, and at a fixed distance, choose the screen displaying the grating and escape to a submerged platform hidden below it. Both mice and rats learn the task quickly. Performance falls below 70% when the spatial frequency is increased beyond 0.5 cycles in most C57BU6 mice, and around 1.0 cycles per degree (cpd) in Long-Evans rats.

Cervical motoneuron topography reflects the proximodistal organization of muscles and movements of the rat forelimb: a retrograde carbocyanine dye analysis.

Behavioral evidence reveals that the laboratory rat and other rodent species display skilled paw and digit use in handling food during eating and skilled limb use in reaching for food in formal laboratory skilled reaching tests that is comparable to that described in carnivores and primates. Because less is known about the central control of skilled movements in rodents than in carnivores or primates, the purpose of the current study was to examine the relation between the rat's spinal motoneurons and the individual forelimb muscles that they innervate. In two experiments, 14 forelimb muscles (in the shoulder and the upper and lower arm segments) were injected with carbocyanine dye tracers. The topography of spinal motoneurons was reconstructed by using fluorescence microscopy. Motor neurons were found to be organized in columns throughout the length of the cervical and upper thoracic area, with 1) extensor motoneurons located more laterally than flexor motoneurons, 2) rostral motoneurons innervating more proximal muscles than caudal motoneurons, and 3) more dorsally located motoneurons innervating more distal muscles. These results reveal that the topography of rodent cervical spinal cord motoneurons is very similar to that of carnivores and of primates, which also are characterized by well-developed, skilled movements. In addition, the proximal-distal organization of motoneuron columns parallels the proximal-to-distal pattern of forelimb movement used by the rat when reaching. The data from this study enable the development of predictions about the specific movements that would be compromised by experimental transections or other injuries at different levels of the spinal cord in rat models of spinal injury.

The role of spontaneous retinal activity before eye opening in the maturation of form and function in the retinogeniculate pathway of the ferret.

During early mammalian development, inputs from the two retinas intermix within the lateral geniculate nucleus (LGN), then segregate during the first postnatal week into layers that receive input from a single retina. Functionally, the LGN also changes markedly during the first postnatal month; early geniculate responses to retinal input are mainly excitatory, then inhibitory circuits mature within the LGN. These remarkable changes in form and function of the retinogeniculate pathway occur at a time when patterned visual activity is not present, but retinal ganglion cells already manifest spontaneous action potential activity. To examine the role of early retinal activity in these critical developmental processes, we placed the slow release polymer Elvax embedded with tetrodotoxin (TTX) into the vitreous chamber of one or both eyes of neonatal ferrets. Animals receiving monocular injection of TTX had the other eye treated with Elvax containing control citrate buffer. Intraocular injection of horseradish peroxidase was made at the end of the period of TTX treatment to reveal the retinal terminals in the LGN. Chronic monocular or binocular blockade of retinal activity during the first postnatal week did not prevent eye-specific segregation, although it made the boundaries between layers less distinct. Retinal terminals ended preferentially in the appropriate layer, but a large number of terminals were also present in the inappropriate layer. Further segregation was achieved during the second postnatal week of activity blockade, when most retinal terminals ended preferentially in the appropriate geniculate layer and sharper layer boundaries were present. However, a small but significant number of terminals still extended into the inappropriate layer. Together, these findings indicate that monocular as well as binocular blockade of retinal activity resulted in some anomalous retinogeniculate projections and delayed eye-specific patterning, but segregation was largely intact at the end of the second postnatal week. We also report here that intraocular tetrodotoxin had a marked effect on the maturation of intrinsic geniculate circuits prior to eye opening. Whole-cell patch-clamp recordings in the LGN slice preparation revealed that activity blockade prevented the maturation of the slow, but not the fast, hyperpolarizing potential of LGN neurons during the first postnatal month and up to P38, the oldest age studied. In conclusion, these results indicate that spontaneous retinal activity modulates the time course of binocular segregation but does not alone account for the segregation of retinogeniculate terminals. However, early retinal activity plays an important role in developing the intrinsic circuitry of the LGN.

Novel method of chronically blocking retinal activity.

The ethylene-vinyl acetate copolymer Elvax has been used as a vehicle to deliver bioactive substances to discrete areas of the nervous system. Here we report a novel use of Elvax to chronically block retinal activity. Small pieces of Elvax containing the sodium channel blocker tetrodotoxin (TTX) were surgically implanted into the vitreous humor of ferret eyes. Observations of the light-induced pupillary reflex combined with electrophysiological assays of vitreous humor confirmed that these implants completely blocked retinal activity for up to 25 days without apparent retinal damage. The advantages of this procedure over previous methods requiring multiple daily injections of TTX, and alternative experimental applications are discussed.

Serial sectioning of thick tissue with a novel vibrating blade microtome.

Vibrating blade microtomes are used extensively in biological research to section non-frozen tissue. There are a wide variety of commercial instruments available for this purpose, however, they are designed to cut thin sections primarily from a tissue block less than one centimeter in height. Herein is described a simple modification of a microscope frame that creates a vibrating blade microtome capable of producing a sequential series of sections through three centimeters of tissue. We illustrate the use of this device to identify and reconstruct a column of rat spinal motor neurons retrogradely labeled from a peripheral muscle.

Intracellular filling and reconstruction of identified neurons in fixed rat brain slices.

A simple technique for the morphological characterization of pre-labelled neurons in fixed brain slices is described. Neurons are retrogradely-labeled with a carbocyanine dye and the tissue is fixed and sliced. Individual labeled cells from the interior of a slice are then visualized on an upright fluorescence microscope and impaled with a micropipet containing rhodamine or fluorescein-peroxidase conjugates. The cells are filled by iontophoresis, postfixed, and the peroxidase is oxidized into a permanent, opaque reaction product. The dendritic morphology of the neurons is then reconstructed under camera lucida and quantified.

A simple water-immersion condenser for imaging living brain slices on an inverted microscope.

Due to some physical limitations of conventional condensers, inverted compound microscopes are not optimally suited for imaging living brain slices with transmitted light. Herein is described a simple device that converts an inverted microscope into an effective tool for this application by utilizing an objective as a condenser. The device is mounted on a microscope in place of the condenser, is threaded to accept a water immersion objective, and has a slot for a differential interference contrast (DIC) slider. When combined with infrared video techniques, this device allows an inverted microscope to effectively image living cells within thick brain slices in an open perfusion chamber.

Retinal activity regulates developmental switches in functional properties and ifenprodil sensitivity of NMDA receptors in the lateral geniculate nucleus.

Previous studies have shown that marked changes occur in the kinetic properties of N-methyl-D-aspartate (NMDA) receptors during development of the visual pathways. In the lateral geniculate nucleus (LGN) of the ferret, excitatory postsynaptic currents (EPSCs) induced by activation of NMDA receptors display a very slow decay time during the first postnatal month, then become shorter in duration following eye-opening (around postnatal day 32; P32). In view of the critical role that NMDA receptors play in activity-dependent refinement of visual connections during development, we have examined the mechanisms that underlie these changes and how they are regulated. To examine the role of retinal activity, whole-cell recordings were conducted in the LGN slice preparation obtained from normal ferrets and ferrets treated with continuous intraocular application of tetrodotoxin (TTX) from P25 until the time of recording. Blockade of ganglion cell activity with TTX prevented the changes in decay rate of the postsynaptic current induced by NMDA receptors. Treated animals older than P40 had NMDA-EPSCs markedly longer in duration than normal animals at a similar age, resembling responses present in normal newborn animals. To examine whether changes in subunit composition of the NMDA receptor may contribute to the maturation of its kinetic properties, we used the antagonist ifenprodil, which produces selective inhibition of heteromeric NMDA receptors containing the NR-2B subunit. Ifenprodil induced profound inhibition of NMDA receptor activity in normal young animals and TTX-treated mature animals, but substantially less inhibition in normal mature animals. These findings indicate that retinal activity is required for the developmental switch from a juvenile form of the NMDA receptor to a more mature form, possibly affecting NR2 subunit expression.

Chronic NMDA receptor antagonism during retinotopic map formation depresses CaM kinase II differentiation in rat superior colliculus.

We examined the effects of chronic NMDA receptor antagonism on the normal postnatal differentiation of calcium- and calmodulin-dependent kinase II (CaM kinase II) in the rat superior colliculus. At postnatal day (P) zero, most CaM kinase II protein, as well as CaM kinase II activity, was detected in the soluble fraction. In vitro phosphorylation of P0 superior colliculus revealed several prominent substrates in both the particulate and soluble fractions. At P19 there was more particulate enzyme than soluble enzyme, and CaM kinase II activity in the particulate fraction was higher than in P0 particulate tissue. Additionally, in vitro phosphorylation of P19 superior colliculus revealed many more CaM kinase II substrates. Chronic NMDA receptor antagonism with 2-amino-5-phosphonovalerate (DL-AP5) caused CaM kinase II to retain many of the characteristics of the enzyme found in P0 untreated superior colliculus. In P19 superior colliculus treated with LD-AP5 from birth, most of the protein was in the soluble fraction, CaM kinase II activity was largely restricted to the soluble fraction, and only a few substrates were observed by in vitro phosphorylation. These effects were not observed in tissue treated with the inactive isomer, L-AP5. These results suggest that synaptic maturation is slowed by antagonism of NMDA receptors during retinotopic map formation.

Preservation of spatial learning in fyn tyrosine kinase knockout mice.

Previous work has shown that knockout mice lacking the fyn tyrosine kinase gene (fyn-/-) are impaired in spatial learning. Here, we have re-examined the spatial learning of fyn-/- mutants in an open field water maze. Unlike wild-type mice, fyn-/- knockouts often floated without moving when placed in the water but could swim adequately when their hind feet were mechanically stimulated. Under these conditions, fyn-/- mice showed significant improvement over trials in locating a hidden platform. On a transfer trial, at the end of training, they spent a disproportionate amount of time swimming in the location of the previously hidden platform. These findings suggest that fyn-/- knockouts are capable of spatial learning, but suffer an impairment that compromises their ability to swim normally.

Uses of vision by rats in play fighting and other close-quarter social interactions.

Enucleated juvenile rats were compared to sighted juveniles, and tested over six trials. In some of these trials, the vibrissae were clipped and the test chamber was flooded with white noise. Even though the enucleated rats played, they did so in an atypical manner. They tended to initiate more playful and other social contacts, and were more likely to defend themselves if contacted. When they did defend themselves, they adopted behavior patterns that were more likely to evade the partner's attack. In addition, the enucleated rats were hypersensitive to the partner, being more likely to respond defensively when contacted further from the nape (the main play target). All these changes in play fighting by nonsighted rats suggest that the loss of vision leads to motivational changes in activity and reactivity, and so has an indirect effect on play behavior. In addition, direct evidence is also provided to show that vision is used to orient attacks to the nape. When the vibrissae were closely clipped, the sighted rats continued to make direct attacks on the partner's napes, whereas the nonsighted rats did not. Rather, they first contacted some other part of the partner's body and then oriented to the nape. Another test paradigm was used to determine whether vision is used to trigger defensive responses. The rats were partially food deprived as adults and were filmed in a food wrenching and dodging situation where one rat was given a food pellet and the other allowed to steal it. Measurement of the distance at initiation of the lateral swerve away from the approaching partner (i.e., dodge) showed that when the vibrissae are clipped, the sighted rats continued to initiate dodges at the same distance, whereas the nonsighted rats could not. Therefore, vision appears to have an active role in organizing movement sequences of attack and defense in play fighting and other close-quarter interactions.