Epidemiologic studies demonstrate an association between childhood exposure to anesthesia and an increase in subsequent learning and behavioral disorders. These data are complemented by animal studies showing that rat pups exposed to anesthetics exhibit lasting deficits in learning. Previous investigators have demonstrated that anesthetic agents can increase apoptosis in the developing brain, but little has been done to assess the effects of anesthetics on the development of the neurons that remain. Our preliminary data raise a concern that some commonly used anesthetics presented at clinically relevant doses may interfere with the response of axonal growth cones to the cues that guide them to the correct dendritic targets, thus potentially disrupting the formation of brai circuits critical for higher functions, such as learning. In this proposal we will examine the effets of the major classes of anesthetic agents on axon guidance and circuit formation, with a view towards discovering mechanisms of this deleterious effect and determining how anesthetic plans may be designed to avoid or minimize it.
The Specific Aims are: (1) Test the effects of general anesthetic agents on the response of axonal growth cones to guidance cues. The hypothesis to be tested is that anesthetic agents with activity at GABAA receptors and/or NMDA receptors can substantially interfere with growth cone function, including sensing of and response to guidance cues. Using time-lapse microscopy and quantitative analysis of axon morphology we will determine the effects of the major classes of anesthetic agents on axonal growth cone motility in the absence of guidance cues. Then, using a collapse assay and gradient turning assay, we will test whether anesthetics interfere with the response of the axonal growth cone to repulsive and attractive guidance cues. Finally, we will determine whether the mechanism of action is an effect of anesthetics on GABAA receptors and/or NMDA receptors that leads to alterations in the levels and distribution of growth cone Ca2+, a principal intracellular regulator of the guidance response. (2) Determine whether effects of general anesthetics on axon guidance can lead to errors in circuit formation. The hypothesis to be tested is that anesthetic agents can disrupt axon targeting, thus leading to errors in circuit formation. The trajectory of axons in an organotypic slice culture model of axon guidance that is exposed to anesthetics will be assessed for errors in development to determine which agents may have deleterious effects. Subsequently, the development of the mossy fibers and the Schaffer collaterals, two tracts of the hippocampus that participate in a critical learning circuit, will be assessed after anesthetic exposure to determine which agents, what doses, and what exposure times can disrupt circuit formation. These studies will take place in organotypic brain slice culture using quantitative microscopy and electrophysiologic techniques. Results will be confirmed by microscopic analysis of these tracts in intact mice exposed to the anesthetic agents determined in slice culture to interfere with axon guidance and tract formation.
Each year tens of thousands of children in the United States are anesthetized for medical and surgical procedures, and there is a concern that the anesthetic agents that are routinely administered may increase rates of learning disability and behavioral disorders. Our preliminary data indicate that some commonly used anesthetic agents interfere with the development of the connections between brain cells, a process that creates the circuits of the mature brain. In this proposal we will study the effects of a comprehensive panel of anesthetic agents on the developing mouse brain in order to establish which agents are at fault, determine what properties interfere with the development of brain circuits, and explore how anesthetic plans could be designed to minimize these effects.
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