Lawrence C. Katz - US grants

The primary visual cortex is a model system for understanding the integration and processing of information at a cortical level. The cellular basis of this processing network consists of an elaborate network of intrinsic connections within and between the six cortical layers. In the first few months of life, the complexity of this circuitry increases tremendously, paralleling the emergence of distinct visual response properties in each of the layers. The primary goals of the proposed research are to understand the sequence of events underlying the development of these connections and the emergence of receptive field properties in the cat visual cortex, and to assess the extent to which these connections can be modified by visual experience. This work will utilize anterograde staining with PHA-L to visualized the differentiation of individual geniculo-cortical axons in order to determine if geniculate afferents originating from different laminae develop sequentially or in parallel. Intracellular injections of individual neurons in brain slices will then be used to assess the differentiation of dendrites of layer 4 spiny stellate cells, to determine whether the appearance of complex receptive properties in layer 3 is related to the emergence of connections between layers 4 and 2\3. The development of vertical and horizontal connections of layer 2\3 pyramidal cells will be studied to determine how the laminar specificity of vertical connections emerges, and when the system of clustered horizontal connections within layer 2/3 differentiates. This will be accomplished by identifying a particular class of layer 2/3 cells-those projecting to area 18- by combining a fluorescent retrograde tracer with intracellular dye injections in slices. The possible contribution of the layer 6 to 4 connection in generating end-inhibition will be evaluated by examining when this connection develops relative to the known physiological emergence of end-inhibition. Metabolic labelling with 2-deoxyglucose will be used to examine the overall pattern of orientation column development relative to afferent input and intrinsic circuit development. Finally, the question of whether intrinsic circuits can be altered by visual experience will be examined, by studying the effects of monocular deprivation and induced strabismus on the patterns of intrinsic circuits. These studies will use a combination of anterogradely transported and intracellularly injected fluorescent dyes to visualize the relationship between ocular dominance columns and intrinsic circuits. While previous work has emphasized the changes in afferent distribution resulting from deprivation or misalignment of the eyes, the experiments proposed here will provide the first glimpse of the involvement of intrinsic cortical circuits in these conditions, which cause profound visual dysfunction in humans, and which affect a significant number of young children. These investigations may bring to light previously unobservable consequences of these alterations in visual input, and suggest new therapies for treating or preventing cortical deficits.

The mammalian primary visual cortex is a model system for understanding cortical integration and processing of information. This network consists of an elaborate system of intrinsic connections within and between the six cortical layers. In the first few months of life, the complexity of this circuitry increases tremendously, paralleling the emergence of distinct visual response properties in each layer. The primary goals of the proposed research are to define the roles of activity-independent factors, spontaneous neuronal activity, and patterned visual experience in structuring and modifying horizontal and vertical connections. To determine the mechanisms generating specific vertical connections, the modifiability of the laminar specificity of layer 2/3 pyramidal neurons will be examined. The effects of blocking either patterned visual experience, by binocular lid suture, or of spontaneous neural activity, by intraocular tetrodotoxin injections, will be assessed with single-cell labelling techniques in brain slices. Reconstructions of individual axon arbors will determine whether the normal laminar specificity, in which collaterals are absent from layer 4, breaks down after manipulations of activity levels. To further characterize the signals involved in generating laminar specificity, slices of immature cortex will be maintained in tissue culture in order to differentiate between activity-dependent and activity-independent mechanisms. Manipulations of visual experience will also be used to examine the mechanisms underlying the emergence and refinement of clustered horizontal connections in layer 2/3. Binocular tetrodotoxin injections, and in vitro cultures of tangential brain slices, will reveal whether the emergence of the initial crude pattern of horizontal connections requires spontaneous neural activity. Varying periods of binocular deprivation, followed by restoration of normal vision, will be used to assess the "critical period" during which these intrinsic horizontal connections can be modified by visual experience. Animals will also be reared with induced strabismus to parse out the effects of the amount of visual experience versus the pattern of visual experience on the refinement of clustered connections. While previous investigators have emphasized the relationships between ocular dominance columns and visual activity, the experiments proposed here will provide the first analysis of the involvement of activity-dependent and activity-independent mechanisms in the differentiation of visual cortical circuits. The conditions of cataract-induced binocular deprivation and strabismus cause profound visual dysfunction in humans, and affect a considerable number of young children. The proposed experiments will provide insight into the consequences of these deficits on local circuits in the striate cortex. They will furthermore help determine when intervention can rescue deprivation-induced deficits. Understanding the basic mechanisms should also provide a rationale for new therapies for treating or preventing the cortical consequences of abnormal visual input. Finally, uncovering activity-independent cues involved in circuitry formation may lead to the discovery of novel factors critical to normal brain differentiation.

DESCRIPTION: This project will characterize the appearance of gap junction coupling in developing visual cortex in ferrets and evaluate its role in the establishment of columnar organization. Local cell assemblies display coherent changes in intracellular [Ca] measured with fura-2 due to gap junction communication. In the first specific aim, Fura-2 imaging as well as injection of low molecular weight dyes that cross gap junctions will be used to characterize the timing of the appearance of these "neuronal domains" in ferret visual cortex during early development when functional cortical inputs, architecture, and response organization are becoming established. The cortical layers involved and orientation of domains will be characterized, and time lapse measurements of fura will show the dynamics of formation and dissipation of domains. In the second specific aim, the persistence of domains through early cortical development will be followed by studying slices from late embryonic, neonatal, and postnatal animals up to two months old. The third specific aim focuses on the initiation and propagation of domains. High speed video methods will be used to determine the speed of activation of domains, and this, along with whole cell patch recording of electrical activity of cells within domains and pharmacological blockade of sodium spikes, calcium spikes, and transmitter receptors, will be used to determine the mechanisms for propagation of Ca waves between cells. The influence of neurotransmitters on domains will be studied to determine the likely effects of synaptic inputs as they are established. Finally, in the last specific aim, chronic blocking of gap junction coupling using osmotic minipumps to deliver octanol, halothane, and 16-doxyl stearic acid will be used to test the hypothesis that electrical coupling within domains plays a role in the establishment of the functional columnar organization of cortex and its physiological responses to visual stimuli.

The mammalian primary visual cortex is a model system for understanding cortical integration and processing of information. This network consists of an elaborate system of intrinsic connections within and between the six cortical layers. In the first few months of life, the complexity of this circuitry increases tremendously, paralleling the emergence of distinct visual response properties in each layer. The primary goals of the proposed research are to define the roles of activity-independent factors, spontaneous neuronal activity, and patterned visual experience in structuring and modifying horizontal and vertical connections. To determine the mechanisms generating specific vertical connections, the modifiability of the laminar specificity of layer 2/3 pyramidal neurons will be examined. The effects of blocking either patterned visual experience, by binocular lid suture, or of spontaneous neural activity, by intraocular tetrodotoxin injections, will be assessed with single-cell labelling techniques in brain slices. Reconstructions of individual axon arbors will determine whether the normal laminar specificity, in which collaterals are absent from layer 4, breaks down after manipulations of activity levels. To further characterize the signals involved in generating laminar specificity, slices of immature cortex will be maintained in tissue culture in order to differentiate between activity-dependent and activity-independent mechanisms. Manipulations of visual experience will also be used to examine the mechanisms underlying the emergence and refinement of clustered horizontal connections in layer 2/3. Binocular tetrodotoxin injections, and in vitro cultures of tangential brain slices, will reveal whether the emergence of the initial crude pattern of horizontal connections requires spontaneous neural activity. Varying periods of binocular deprivation, followed by restoration of normal vision, will be used to assess the "critical period" during which these intrinsic horizontal connections can be modified by visual experience. Animals will also be reared with induced strabismus to parse out the effects of the amount of visual experience versus the pattern of visual experience on the refinement of clustered connections. While previous investigators have emphasized the relationships between ocular dominance columns and visual activity, the experiments proposed here will provide the first analysis of the involvement of activity-dependent and activity-independent mechanisms in the differentiation of visual cortical circuits. The conditions of cataract-induced binocular deprivation and strabismus cause profound visual dysfunction in humans, and affect a considerable number of young children. The proposed experiments will provide insight into the consequences of these deficits on local circuits in the striate cortex. They will furthermore help determine when intervention can rescue deprivation-induced deficits. Understanding the basic mechanisms should also provide a rationale for new therapies for treating or preventing the cortical consequences of abnormal visual input. Finally, uncovering activity-independent cues involved in circuitry formation may lead to the discovery of novel factors critical to normal brain differentiation.

[unreadable] DESCRIPTION (provided by applicant): A long-standing hypothesis holds that memory formation involves structural changes in synapses. Indirect evidence shows that dendrites, dendritic spines, and axons in the central nervous system can grow and retract under a variety of different conditions. Recent advances in imaging technology, such as multiphoton microscopy, have made it possible to directly image morphological changes in vitro and in vivo, but it has proven difficult to link these changes to the actual process of learning. This is due to the inaccessibility of brain areas involved in the learning process (such as the hippocampus) and to difficulties in identifying the synapses that are involved in learning. The overall aim of this proposal is to use the mouse olfactory system to determine whether changes in dendritic architecture accompany the formation of long-term memories. The formation of olfactory memories requires the olfactory bulb, whose location makes it very accessible for high-resolution imaging studies. Using genetically engineered mice in which specific neuronal populations are labeled with fluorescent markers, multiphoton imaging of neuronal dendrites over hours, days, and weeks will be used to determine the stability of dendrites in the adult brain. Animals will then learn an odorant discrimination task, and imaging will be used to investigate the extent to which learning the task results in dendritic alterations. To begin to address possible mechanisms involved in dendritic remodeling, a preparation will be developed in which learning can be induced while animals are anesthetized, which will allow real-time observations and manipulations of circuits in the bulb. These experiments will determine whether structural changes are a requisite for long-term memories. Understanding the cellular changes that accompany the formation of memories is critical for understanding how normal memory formation takes place, and in uncovering mechanisms that can be targeted for intervention in the many disorders of memory, both in normal aging and in pathological conditions such as Alzheimer's disease. [unreadable] [unreadable]