Barbara Chapman - US grants

DESCRIPTION (provided by applicant): Neuronal activity is important for development of eye-specific segregation and cortical cell orientation specificity in the mammalian visual system. The proposed studies will block or change retinal activity using pharmacological agents or immunotoxins in developing ferrets. Results will show effects of altered patterns of activity on the establishment and maintenance of neuronal connections, of changes in the connection patterns. The first set of experiments (Aim 1) will look quantitatively at the normal time course of segregation of afferents from the two eyes in the lateral geniculate nucleus (LGN), and will determine whether activity blockade during the period of initial segregation actually prevents segregation or causes desegregation or both. The second set of experiments (Aim 2) will determine the effects of removing the normal pattern retinal ganglion cell activity (synchronized bursts of action potentials in neighboring ganglion cells) on the development of receptive field properties in LGN and primary visual cortex. In the third set of experiments (Aim 3), the initial normal segregation of axons from the two eyes will be prevented by activity blockade from PND 1-10, and then the animals will be allowed a period of recovery, resulting in LGNs where the axons from the two eyes are completely segregated, but normal laminae do not form. The effects of this altered retinogeniculate projection on the physiology of single ceils and on functional organization in the primary visual cortex will be studied using electrophysiological recordings, optical imaging, and transneuronal labeling. This will help to determine whether normal lamination of eye-specific inputs in the LGN is necessary for normal visual function. The final series of experiments (Aims 4 and 5) will look at the effects on cortical cell receptive fields of removing ON-center activity during development. Results from these experiments will show how patterns of activity may be involved in developing receptive field properties. The proposed experiments should further our knowledge of the rules of activity-dependent development, and the consequences of disrupting connections; this may have implications for human developmental disorders.

DESCRIPTION (provided by applicant): The overall objective of this research program is to provide a better understanding of the development of retinal ganglion cells (RGCs) and their eye-specific projections to the dorsal lateral geniculate nucleus (dlgn). Five specific aims are proposed designed to fill major gaps in our current knowledge. In specific aim 1 we will employ multiple morphometric measures and quantitative methods to classify mouse RGCs into distinct classes. The mouse offers a powerful model to assess the effects of genetic variations on neuronal development, and for this reason it is vitally important to have a sound basis for assessing the salient properties of RGCs in the normal mouse retina. A cluster analysis of multiple morphometric measures obtained on a preliminary sample of cells suggests that the mouse ganglion cell population is comprised of 14 distinct classes, In specific aim 2 we will test the hypothesis that cholinergic inputs and/or neural circuitry play a key role in the morphological differentiation of mouse RGCs. For this purpose, we will perform the same morphometric measures as in the normal mouse in three different groups of animals: (i) genetically altered animals, lacking the nicotinic acetylcholine receptor;(ii) those in which the cholinergic cells have been depleted by intraocular injection of an immunotoxin targeting these neurons;and (iii) those in which cholinergic synaptic inputs have been chronically blocked during development by pharmacological treatment. In specific aims 3 we will make multi-array recordings from the developing ferret retina, in which neuronal discharges have been perturbed pharmacologically, to define the key features of retinal activity essential for the formation of segregated left and right eye inputs to the dlgn. In specific aim 4 we will define the role of activity in the growth and elaboration of individual retinogeniculate axons during the time that eye-specific projections are normally formed. In specific aim 5 we will test the hypothesis that cholinergic inputs from the basal forebrain to the dlgn are essential for the formation of eye-specific retinogeniculate projections. The completion of the 5 specific aims of this proposal will extend and clarify our understanding of how neuronal activity serves to regulate the development of two key features of mammalian RGCs, their dendritic morphologies and eye-specific projections to the dlgn. This information will be useful in devising empirically based treatments of the myriad ontogenetic disorders than adversely impact the human visual system.

DESCRIPTION (provided by applicant): Degenerative diseases that result in the loss of photoreceptors, including Age-related Macular Degeneration (AMD) and Retinitis Pigmentosa (RP), are common causes of blindness in the developed world. These diseases largely spare the connections and functions of the retinal ganglion cells that link the retina to central targets in the brain. Therefore, the strategy of rendering retinal ganglion cells directly responsive to light holds a great deal of promise for developing therapies for these diseases. Light-sensitive ganglion cells would bypass the damaged photoreceptors, but take advantage of the normal visual pathways beyond the retina. Other laboratories have previously demonstrated that it is possible to express the light-activated cation channel, channelrhodopsin-2 (ChR2), in retinal cells. Studies have shown that in a rodent model of photoreceptor degeneration, expression of ChR2 can render retinal cells photosensitive. Light responses due to the expression of ChR2 in the retina are transmitted to the visual cortex, and can underlie rudimentary visual behaviors. A great deal of work remains to be done, however, before the use of ChR2 or similar light- activated molecules to treat retinal degenerative diseases could be contemplated in a clinical setting. This R21 proposal addresses two important issues that need to be tackled. Specific aim 1 will determine how best to obtain widespread, stable expression of ChR2 in retinal ganglion cells in a non- rodent model system, and whether such expression leads to visual responses in retinal ganglion cells that are transmitted to lateral geniculate nucleus and primary visual cortex cells. Specific aim 2 will determine whether the vision caused by the expression of ChR2 in retinal ganglion cells can either enhance or interfere with the development and adult function of normal visual pathways. Successful completion of these two aims will bring the field significantly closer to the goal of being able to translate light-activated channel technology into the clinic, and will provide preliminary data needed to apply for funding to determine whether this approach can be used to produce behaviorally useful vision in a retinal detachment model of blindness. PUBLIC HEALTH RELEVANCE: The long-term goal of this research is to develop methods to cure forms of blindness caused by the loss of photoreceptor function including Age-related Macular Degeneration and Retinitis Pigmentosa. Experiments will test whether artificially making retinal cells photosensitive through expression of light- activated ion channels is a promising approach to curing these diseases.

DESCRIPTION (provided by applicant): In the mature mammalian visual system, information from the two eyes is anatomically segregated into layers in the lateral geniculate nucleus, and into ocular dominance columns in primary visual cortex. During the past half century, the development of this segregation has served as a premier model for the development of specific connections in the brain. Groundbreaking work in the 1960s showed that neuronal activity is important for the normal development of ocular dominance columns. If poor vision or no vision is experienced by one eye early in life, then that eye loses anatomical and functional connections to the brain. This results in blindness through the affected eye later in life, even if the cause of the poor vision is fixed so that the eye itself functions normally. Many studies over the ensuing years have looked at the effects of altering or abolishing neuronal activity on the development of eye-specific segregation. Findings from these studies lead to the following conclusions: 1) During development, specific connections in the visual system emerge over time from initially diffuse connections, 2) abolishing retinal activity during development maintains the initial imprecise connections, and 3) temporal correlation in the firing patterns of neurons within each eye, and lack of correlation between eyes is responsible for the development of eye-specific connections. These conclusions formed the dogma of the field of development of sensory systems, and significantly influenced related fields such as neurodevelopmental disorders, adult plasticity in the brain, and learning and memory. However, new experiments over the past decade have questioned each of these conclusions. This proposal will address the controversies by performing the following specific aims. 1) What is the normal pattern of development of ocular dominance columns? Anatomical tracing methods will be used to determine whether afferents serving the two eyes are initially overlapping or segregated in primary visual cortex. 2) What is the effect of complete retinal activity blockade on the development of eye-specific segregation in the LGN and cortex? Pharmacological agents will be used to silence retinal ganglion cell action potentials during development, and the effects on segregation will be examined. 3) What aspects of retinal ganglion cell activity are important for normal development of eye-specificity in the LGN? Manipulations that alter patterns of activity in the retina will be compared in order to determine which aspects of activity patterns are associated with normal segregation versus lack of segregation. 4) What is the role of correlated activity in the development of segregation? Light activatable molecules will be used to control activity patterns to determine whether increased correlation of firing of neighboring cells between the two eyes leads to lack of eye-specific segregation. These experiments will not only help to resolve important basic science controversies, but will also have clinical implications. Understanding the initial state of connections in the brain, the effects of absent or altered activity, and the key aspects of activity patterns that are needed for normal development, will all aid in the understanding and eventual treatment of neurodevelopmental disorders. PUBLIC HEALTH RELEVANCE: Neurodevelopmental disorders, including autism, schizophrenia, fetal alcohol syndrome, seizure disorders, attention deficit / hyperactivity disorder, etc. are devastating to families and costly to society. In order to understand and eventually treat these disorders, it is necessary to understand how the brain develops. The current proposal will answer questions about normal development of connections in the brain, and how connections are altered when neuronal activity is affected. The understanding of basic developmental neurobiology gained from these experiments may someday help in the treatment of neurodevelopmental disorders.