Mouse genetic approaches to studying functionally defined neural circuits and neurodevelopmental disorders [electronic resource]

Over the past several decades, the study of neurobiology has been revolutionized by the development of increasingly sophisticated genetic methods in diverse model organisms. The mouse is the most easily genetically manipulated mammal and has become a favorite model organism for neurobiologists. In two independent projects, I have developed a new method—Targeted Recombination in Active Populations (TRAP)—to facilitate studies of neural circuits in genetically modified mice and have developed and characterized new mouse models of a human neurodevelopmental condition, Smith-Magenis Syndrome. In many brain regions, functionally distinct populations of neurons are intermingled and genetically indistinguishable using current methods. The ability to selectively genetically manipulate such functionally distinct neuronal populations would facilitate experiments to address many outstanding questions in neurobiology. To enable selective genetic access to functionally defined neural circuits, I developed TRAP. This novel method utilizes mice in which the tamoxifen-dependent Cre recombinase CreER has been knocked-in to the loci of the immediate early genes (IEGs) Arc and Fos. Because IEGs are expressed in response to neuronal or synaptic activity, CreER is expressed in an activity-dependent manner in these mice. In the presence of tamoxifen, active neurons that express CreER can undergo a CreER-catalyzed recombination event, while inactive neurons, which do not express CreER, do not undergo recombination; in the absence of tamoxifen, CreER remains inactive. By combining the Arc-CreER and Fos-CreER alleles with transgenes that allow Cre-dependent expression of genetically encoded tools, TRAP enables the selective genetic manipulation of neurons activated during a time window surrounding tamoxifen injection. I characterized this novel method and demonstrate its utility in multiple neural systems—including in the somatosensory, visual, auditory, and hippocampal space representation systems. In a separate project, I began to dissect the function of a gene—Retinoic acid-induced 1 (Rai1)—involved in a rare human neurodevelopmental disorder, Smith-Magenis Syndrome (SMS). Human genetics studies have shown that SMS, which is characterized by general cognitive, motor, and developmental delay in addition to autistic features, behavioral problems, and numerous other multisystemic abnormalities, is caused by haploinsufficiency of RAI1. However, the function of Rai1 in the developing and adult brain and how its disruption leads to the symptoms of SMS are unclear. I found that Rai1 is expressed widely but not ubiquitously throughout the brain in both neurons and astrocytes. To better understand the function of Rai1 and the pathogenic mechanism of SMS, I generated an allele of Rai1 that can be conditionally inactivated in the presence of Cre recombinase. Using this allele, I generated mice in which Rai1 was selectively deleted in different cell types in the brain. I found that global homozygous deletion of Rai1 (using Nestin-Cre) results in lethality in early adulthood, fear learning and motor deficits, and obesity. The fear learning deficit was also apparent in mice lacking Rai1 specifically in inhibitory neurons (using GAD2-Cre). Loss of Rai1 from forebrain excitatory neurons (using Emx1-Cre) and from astrocytes and postnatally born neurons (using mGFAP-Cre) did not produce any phenotypes detectable in our behavioral assays. Using Mosaic Analysis with Double Markers (MADM), I generated mice in which Rai1 was deleted in only a subpopulation of distinctly labeled cells in the brains of otherwise Rai1 heterozygous mice. In Rai1 MADM mice, I observed that Rai1-null Bergmann glia—a specialized cerebellar astrocyte type—are present in fewer numbers than Rai1-wild-type Bergmann glia, suggesting a role for Rai1 in the production or survival of this cell type. Although much more work is needed to understand the function of Rai1 and how its loss leads to SMS, these data suggest some useful starting points for future studies.

Advisor: Luo Liqun
Heller H.
Raymond Jennifer L. advisor. Thesis advisor
Südhof Thomas C. advisor. Thesis advisor