ILAR 40 (2)

Introduction: Animal Models of Human Vision. ILAR 40 (2): 041.
[Reviewer's Note - Any parts of this introductory article which recapped specific articles published in this ILAR issue were not reviewed as other LABSG members will be summarizing these articles for the LABSG list.] Most of what we learn and remember about the world is based on sight. The visual system is the most complex of the sensory systems. Two million axons in the optic nerves exceed the total number of fibers in all the other sensory nerves. According to the National Eye Institute (NEI), diseases and injury to the cornea are the leading cause of visits to eye specialists. Most blindness and visual disability in the US are caused by diseases and disorders of the light-sensitive retina and the choroid. The retina contains the rod and cone photoreceptors and the complex neuronal network that processes visual signals and relays them to the brain. A large proportion of retinal diseases has a genetic basis. Epidemiological data reveals that myopia is found in 25% of all Americans. About one half of all nearsightedness develops during elementary school and costs more than $1.5 billion per year in eyeglasses alone.
1. What does NEI stand for?
1. NEI - National Eye Institute.

Use of an animal model in studies of bacterial corneal infection. ILAR 40 (2): 043.
The eye is immunologically privileged. Eyelids and bulk tears are thought to be significant in the protection of the cornea from bacterial infections. One of the most common predispositions for bacterial keratitis is contact lens wear, particularly extended wear. P. aeruginosa is one of the commonly isolated pathogens from these cases.
Clinical features of bacterial keratitis include: foreign body sensation, irritation, redness, photophobia, and blurred vision. Focal stromal suppuration is seen early and may progress to ulceration and liquefactive necrosis.
The corneal epithelium is a highly organized and regular, stratified squamous epithelial cell layer that is difficult to maintain in vitro. Cultured corneal epithelial cells are homogeneous, unpolarized, lacking surrounding strutures seen in vivo. These differences can result in altered bacterial interactions.
Bacterial interactions can also be studied by collecting corneal cells from ocular irrigation, but these cells also lack polarity and surrounding structures. The number of cells that can be collected is also limited.
Rabbits and mice are used the most commonly. This paper establishes a new scoring system of 0 to 4 for the area of major opacity, density of the opacity, density and infiltration of the surrounding cornea, and surface irregularity.
1. Give some examples of molecules in tears that provide antibacterial protection to the eye.
2. T F As the corneal stroma is avascular, significant bacterial proliferation may occur prior to immune cell arrival.
1. Lysozyme (cleaves bacterial cell wall peptidoglycan of GP bacteria), lactoferrin (binds iron and inhibits bacterial growth), immunoglobulins. Mucin provides defense by binding bacteria and constant exchange during blinking.
2. True

Transgenic animal models for the study of inherited retinal dystrophies. ILAR 40 (2): 051.
The identification of the causative genes for retinitis pigmentosa and age-related macular degeneration has allowed for the development of animal models of these dystrophies, using transgenic technologies. Describes methodologies. Also describes viral-mediated gene therapy and ribozyme therapy.
1. List some naturally occuring retinal degeneration models that have been used.
2. List the hallmarks of age-related macular degeneration.
3. Why is it frustrating to use transgenic mice to study autosomal dominant diseases?
1. Irish setter, briard dogs, abyssinian cat, rat, and rd and rds mouse models.
2. Cellular accumulations (drusen) in or under the retinal pigmosa epithelium (RPE), irregularities of RPE pigmentation, geographic atrophy, serous detachment of the RPE, and neovascularization.
3. The patient carries one mutant allele and one normal allele. The addition of a transgene does not inactivate or replace the endogenous gene. Therefore, you must knock out the normal gene to study autosomal dominant diseases.

Animal models of myopia: learning how vision controls the size of the eye. ILAR 40 (2): 059.
Up to 25% of children in the U.S. become myopic (nearsighted) as they grow up and become adults. Most children have little or no refractive error, which is referred to as emmetropic. Some children become hyperopic (farsighted). Myopic parents tend to have myopic children. If both parents are myopic, a child has a 30-40% probability of becoming myopic.
Animal models were developed in the 1970s to study the mechanisms underlying refractive error. Scientists learned that the visual environment exerts a great influence on refractive state by controlling the axial length of the eye during the postnatal developmental period. As the eye matures, the axial length increases. The chick, tree shrew, and macaque monkey are the primary animal models used to study myopia.
Myopia can be due to congenital, physiological (=simple or school myopia) or pathological (=progressive, degenerative myopia) mechanisms. Physiologic myopia develops after age 6 and is primarily a refractive error. Pathological myopia involves an abnormal lengthening of the eye. Elongation of the eye can lead to retinal tears and retinal detachment. Due to an enlarged globe, open-angle glaucoma can also be a risk factor.
Animals raised in a visually restricted environment can develop environmentally induced myopia. Animals reared from near birth with the eyelids of one eye surgically closed to prevent visual images from being focused on the retina become myopic in the visual form-deprived eye. The fellow control eye is normal. Visual form deprivation consistently produces myopia in chicks and tree shrews.
Normal refractive development in most animals appears to parallel human refractive development. Also, changes in the eyes of animals with induced myopia are generally very similar to the changes seen in myopic human eyes. Consequently, studies with animals in visual form deprivation experiments indicate that visual environment plays a critical role in the development of ocular refraction and axial length.
Studies in animals have provided evidence that axial length (vitreous chamber depth) is actively regulated by a visual feedback mechanism. The most important contribution of animal models is that these experiments have revealed the existence of an active emmetropization mechanism that matches the axial length of the eye to its optical power.
These are important factors in selecting an animal model: phylogentic closeness to humans; maturation rate; similarity of ocular structure to humans; similarity of normal development to humans; ability to study the enviroonmental effects during the juvenile period; ability to control the visual environment precisely; presence of myopic changes similar to those in humans; and presence of accommodative mechanisms similar to humans. Although no animal model is ideal, there are definite benefits from studying several different species.
Chicks (Gallus gallus domesticus) have been used to study myopia more than any other animal. Chicks are readily available, relatively inexpensive, and they mature rapidly. Chicks show reliable responses to deprivation and to minus and plus lenses, both in developing induced myopia and in recovery from induced myopia. The greatest concerns about the chick as an animal model are that they are phylogenetically distant from humans and have numerous differences in ocular structure and physiology.
Tree shrews (Tupaia glis belangeri) are small mammals, native to southeast Asia, that are closely related to primates. These mammals are highly dependent on vision, have a cone-dominated retina, and are diurnal. They are easy to house, breed rapidly, and mature quickly.
A big disadvantage of tree shrews is they are not generally available. The government of Thailand has not allowed export of these species since 1980. Since they are not primates, they are not covered by CITES.
Macaque monkeys (Macaca mulatta) are the animal model most closely related to humans. Optics, ocular structure, and eye size are very similar to humans. Additionally, the normal pattern of ocular development and the ocular changes in eyes with induced myopia are very similar to humans. Disadvantages of monkeys are: typically they have single infants that mature slowly, young monkeys are not readily available, and it is difficult and costly to house and maintain large numbers of monkeys.
There is similarity of responses across different animal species. These cross-species similarities suggest that the emmetropization mechanism is fundamentally similar in vertebrate eyes. Other animal species that have been shown to become myopic in response to environmental manipulations include: cat, marmoset, rabbit, kestrel, gray squirrel, and fish.
Future directions seek to learn more about the emmetropization mechanism and the causes of refractive error. A goal is to characterize more fully the specific stimuli on the retina that comprise the signal for increased or decreased axial enlongation rate. Scientists seek to answer questions about the nature of the neural signaling, its timing and duration, and the routes by which circadian and light/dark affect it.
1. What is myopic vs. hyperoptic?
2. What are the three primary animal models of myopia?
3. Excluding congenital myopia, what are the two broad categories of myopia?
4. Name some concerns about the chick as an animal model.
5. What is the concern about the tree shrew as an animal model?
6. Define "CITES." What is the genus and species of the gray squirrel?
7. List 5-6 other species which have been shown to become myopic in response to environmental
1. nearsighted ; farsighted
2. chicken, tree shrew, macaque monkey
3. physiological (=simple or school) myopia; pathological (=progressive or degenerative) myopia
4. Chicks are phylogenetically distant from humans and have numerous differences in ocular structure
and physiology. Chicks have laterally placed eyes and do not have a fovea.
5. Tree shrews are not generally available. A breeding colony is necessary to have access to juvenile
tree shrews.
6. Convention in International Trade in Endangered Species of Wild Flora and Fauna.
Sciurus carolinensis
7. cat, marmoset, rabbit, kestrel, gray squirrel, fish (cichlid)

Nonhuman primate models of visually based cognition. ILAR 40 (2): 078.
Discusses the need to study signal processing inside the brain while the brain produces intelligent behavior and that of the experimental preparations currently available for studying cognitive function in alert animals, the most powerful and versatile is the awake, behaving monkey.

Macaque monkeys can be trained with operant conditioning techniques to perform a wide variety of simple cognitive tasks and perform these tasks for several hours each day, in exchange for positive rewards, while experimenters monitor the electrical activity of individual nerve cells by means of small microelectrodes positioned at known locations within the brain so an experimenter can pick up electrical signals from one neuron after the other, allowing the experimenter to study the activity of individual cells while the monkey performs a behavioral task of interest. Micro-electrodes in the brain cause the animals no discomfort because the brain lacks primary pain sensors.

The goal is to explore the interplay between neural activity and behavior in an attempt to understand how cognitive functions arise from the coordinated activity of systems of neurons.

Vision is of paramount importance in primate behavior, and the visual system of the macaque is strikingly similar, both anatomically and functionally, to that of humans but, some distinctly human cognitive functions can be studied only in humans.

In some situations, neural activity can be monitored noninvasively from outside the head using electroencephalographic but the spatial resolution of the non-invasive techniques is quite poor and yields relatively little information concerning exactly which brain circuits are active during any particular task. Recording neural activity with a microelectrode, however, permits excellent spatial resolution to the level of the single cell with minimal risk of significant damage to the brain.

The data one actually records with a microelectrode are brief electrical impulses (about 1-msec duration), that are generated by neurons in response to chemical messages from earlier cells in the processing chain.

Visual Attention Most models use awake, behaving monkeys, trained on a simple, well-controlled behavioral scheme that taps the cognitive system of interest and then record neural activity while the animals perform the task. Generally, the primate chair is transported to the laboratory and positioned in front of a television screen on which visual stimuli are displayed. The primate chair is surrounded by a magnetic search coil apparatus that monitors eye position The animal works on a designated task to receive liquid rewards through a lick tube mounted on the primate chair. The monkey receives no water overnight and is therefore thirsty at the beginning of each experiment; the monkey will work diligently for fluid rewards until achieving satiation. When the animal is no longer motivated to work, the experiment is ended and the monkey returned to its home cage.

As an example of a visual attention model- The monkey was required to look directly toward a point of light presented on a television screen and maintain its gaze at this location for a few seconds until the light vanished. While the animal fixated on the point of light in the center of the screen, a second visual stimulus was flashed to the right of the fixation point, in a region of the visual field that is "analyzed" by the single neuron under study. This region is called the neuron's receptive field, and stimuli flashed in the receptive field elicit a brief burst of action potentials from the neuron.. The stimulus was entirely irrelevant to the behavioral task; the monkey was rewarded only for maintaining its gaze on the fixation target at the center of the screen. Nevertheless, the neuron responded weakly to the irrelevant stimulus, consistent with the fact that we can see irrelevant stimuli even though we typically ignore them. A variation on the above experiment-as before, the trial began with the onset of a fixation point, and the monkey was required to direct its gaze toward this location. Then a visual stimulus was flashed to the right of the fixation point within the receptive field of the (same) neuron under study. In the new task, however, the monkey was required to make a quick movement of its eyes (a saccade) to gaze at the new visual stimulus as soon as it appeared on the screen. Only in this manner could the animal obtain a reward. The monkey moved its eyes to the new location about 250 msec after onset of the new stimulus. Note, however, that until the monkey actually executed the eye movement, the sequence of visual stimuli falling on the retina was identical to the experiment above.

A third variation- all events were again identical through the onset of the visual stimulus to the right of the fixation point. In the new experiment, however, the monkey was required to continue maintaining its gaze on the fixation point while moving its hand to touch the visual stimulus to obtain a reward. This manipulation ensured that the visual stimulus would again be relevant to the animal's behavior but removed the necessity for an eye movement. If the enhanced visual response resulted from an eye movement, it should disappear in the new experiment. However, the enhanced response remained intact under the new experimental conditions providing strong evidence that the enhanced visual response actually resulted from visual attention rather than from a specific motor act.

Visual Motion Perception The primary cortical visual area (V1) resides at the back of the brain and is the major recipient of visual information flowing from the retina to the cerebral cortex. In primates, directionally selective neurons first appear in V1 but are substantially more numerous, percentage-wise, in several extrastriate visual areas that receive anatomical projections from V1 such as the middle temporal visual area (MT), in which more than 90% of the neurons are directionally selective. Thus, MT appears to be specialized for processing motion information, and neurons that prefer a particular direction of motion are clustered together into cortical columns. Roughly speaking, a complete set of direction columns exists for each receptive field location represented in MT, so that each direction of motion can be encoded at each point in the visual field. Most V1 neurons, for example, respond selectively to the orientation of the edges of a stimulus, whereas other neurons appear to encode the color of a stimulus or the distance of a stimulus from the animal. Thus, directionally selective neurons are thought to play an important role in motion perception, orientation-selective neurons in form perception, and so on.

Using standard operant conditioning techniques, the authors trained several monkeys to discriminate the direction of motion in a family of motion stimuli. The visual stimuli are flickering random dot patterns designed specifically to activate direction-selective neurons in the brain. The random dot display can take several forms in which the strength of the motion signal in the display is varied. The difficulty of the discrimination can be smoothly varied by manipulating the percentage of dots in coherent motion. After sufficient training, monkeys typically perform as well as humans at discriminating the direction of motion in these displays. On each trial, the monkey was required to fixate on a small point of light while viewing the random dot display for 1 sec. The coherent motion signal could occur in any of eight directions equally spaced around the clock at 45° intervals. At the end of the 1-sec viewing interval, the monkey reported the direction of the coherent motion signal by moving its eyes from the fixation point to one of eight small targets that corresponded to the eight possible directions of motion. Thus, correct answers could be detected by the computer and reinforced with a liquid reward. The reward contingencies were the same whether or not microstimulation was applied on a given trial. . Microstimulation was applied on half of the trials for each condition, and the stimulating pulses began and ended simultaneously with the onset and offset of the random dot display attempting to influence the monkey's perceptual judgments by activating a specific direction column artificially.

The results from several types of experiments all point toward the conclusion that the direction columns in MT actually provide the signals used by the monkey in performing the direction discrimination task and that microstimulation caused the monkey to increase dramatically its decisions favoring motion up and to the left but caused little or no increase in favor of any other direction. The direction of increased behavioral choices is matched perfectly to the direction of motion encoded by the stimulated column. Artificial activation of a column with a particular preferred direction causes an increase in behavioral choices toward that direction. Thus, the microstimulation signal is interpreted in a meaningful fashion by the monkey as it performs the discrimination task and this result establishes a causal link between the activity of direction-selective neurons in the cortex and a monkey's perceptual decisions on a direction-discrimination task.

Practical Issues Surgical Preparation of the Animals Each animal must be prepared surgically to have (1) a fine wire search coil implanted around the eye, which enables measurement of eye position; (2) a stainless steel post attached to the skull, which allows the head to be stabilized during recording experiments; and (3) a craniotomy, which is then covered by a stainless steel recording cylinder and plastic cap, allowing microelectrodes to be introduced into the brain for electrophysiological experiments. These three procedures may be performed during a single surgery, but because of the potential for infection and regrowth of bone, it is unwise to implant a recording cylinder that will not be used during several months of training.

Training and Daily Routine Awake animals are generally handled with a pole and collar technique, and chaired. After becoming comfortable with the chairing process, the chaired animal is moved to the laboratory each day for extended training on specific behavioral tasks. This phase of the training requires as little as 2 to 3 wk for the simplest tasks but can take the better part of a year for sophisticated tasks involving perceptual threshold measurements or intricate manipulations of attention and memory. Intense effort is expended on each animal. Daily training sessions generally last 1 to 3 hr; session length is typically determined by the monkey's willingness to perform the task. When the monkey ceases working, the session ends and the animal is returned to its home cage.

Controlled Water Intake To ensure adequate motivation for each session, the animal is maintained on a carefully controlled fluid intake schedule, established in consultation with veterinary staff and with prior approval of the institutional animal care and use committee. The desired outcome, of course, is to provide the animal with a sufficient volume of fluid each day to maintain good health, while establishing proper motivation for a few hours of task performance. Some monkeys receive their entire daily allotment of fluid during experimental sessions, and others receive supplemental fluids to bring the total up to the required allotment. The authors have never found a particular formula that works well for all, or even a substantial majority, of monkeys. The primary health concern associated with the controlled water intake paradigm is the potential for dehydration or, in some cases, for the animal to eat less solid food when water intake is being controlled. The controlled water intake paradigm (or controlled food intake paradigm) has been criticized in some circles as unnecessary or inhumane. The general arguments are that monkeys will continue to work for highly desired treats while receiving food and water ad libitum, and that any form of controlled intake causes unacceptable suffering of the animal. The authors state with "Over 15 yr of behavioral work with monkeys, our experience is unequivocal: The vast majority of monkeys will not work at challenging tasks like those described in earlier sections of this paper without the incentive supplied by controlled water intake." and believe that controlled water intake is both scientifically necessary and possible to apply humanely within the laboratory

Psychological Well-being and safety issues and the role of the veterinarian were summarized along with the need for all of the above to be performed according to high professional standards. Current imaging techniques, are severely limited in their spatial and temporal resolution and are unlikely to replace microelectrode recording at any time in the foreseeable future.
No questions