FRAXA Grant to Karel Svoboda, PhD — Cold Spring Harbor Laboratory

Karel Svoboda, PhD, at Cold Spring Harbor Laboratory, FRAXA research grantwith Ingrid Bureau, PhD, FRAXA Postdoctoral Fellow (2005)
Adam Oberlander, Technician (2005)

FRAXA Awards:

$40,000 in 2005
$30,000 in 1999

 

Intracortical Circuitry in the Barrel Cortex of FMR1-KO Mice

by Ingrid Bureau, 6/1/2005

One notable feature of Fragile X syndrome are structural abnormalities of dendritic spines in neocortical pyramidal neurons. Dendritic spines are tiny appendages that stud the surface of most neurons in the cerebral cortex. They are the receiving end of most synapses and as a result they play a pivotal role in the communication between neurons.

In patients with Fragile X syndrome, dendritic spines are unusually abundant and tend to have relatively immature forms. This phenotype is also seen in fmr1 knock-out mice, the dominant animal model of Fragile X syndrome. We do not know, however, whether these structural abnormalities in dendritic spines are related to changes in the structure and function of synaptic circuits in the cerebral cortex. More spines may indicate more abundant and promiscuous connections. Ultimately, mental retardation syndromes must have a basis in the neural circuits.

We propose to unravel changes in neocortical circuits in fmr1 knock-out mice (the mouse model of fragile X syndrome). Our studies will be in the barrel cortex, an area in the somatosensory cortex that responds to whisker stimulation. This is a suitable model because FMRP expression is regulated by whisker stimulation. In addition, the barrel cortex is widely used to study the development and plasticity of cortical connectivity. We use laser scanning photo-stimulation to map the pattern of neuronal connectivity in brain slices. This method provides an ‘image’ of functional connectivity impinging onto individual neurons. We will map excitatory synaptic pathways onto neurons from different layers in the barrel cortex of fmr1 knockout mice and compare them to wild-type littermates.

If we discover significant circuit differences in fmr1 knock-out mice, we propose to reintroduce the missing gene in utero directly into specific populations of neurons and determine whether this manipulation is sufficient to correct the defect. Our study may provide a view into circuit mechanisms of Fragile X mental retardation and provide new tools to study fmr1 biology.

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Imaging of Neocortical Dendritic Spine Maturation in FMR1 Knockout Mice Using Two-Photon Laser Scanning Microscopy

by Karel Svoboda, 6/1/1999

One of the most interesting features of the fragile X syndrome is the apparent involvement of dendritic spines. These tiny appendages stud the surface of most neurons in the cerebral cortex, and are the sites where these neurons receive most of their inputs. As a result, they play a pivotal role in the communication between neurons. It is perhaps not surprising, therefore, that in several forms of mental retardation, these structures are somehow altered.

In Fragile X syndrome, they are unusually abundant and tend to have relatively immature forms, similar to what may be seen in the brains of infants. We do not know, however, whether these structural changes in dendritic spines are directly responsible for symptoms of fragile X. This is an especially difficult question to address since dendritic spines can change in size and shape over the course of minutes. Since this motility might itself be a structural manifestation of the biochemical changes that take place with thought processes such as learning and memory, it is important to know how it changes during normal brain development and how it is affected in disorders such as Fragile X syndrome.

The studies we propose are designed to uncover the changing nature of dendritic spine motility as an animal grows from infancy to the equivalent of childhood, and then to discover how this motility during this crucial period is affected in the knockout mouse model of Fragile X syndrome. Finally, if a disorder is detected, we propose to reintroduce the missing gene directly into individual neurons and determine whether this single manipulation is sufficient to correct the earlier-described disorder.

In order to carry out these studies, we will label neurons with a brightly fluorescent protein by infecting them with a harmless virus carrying the gene for the marker. This marker, enhanced green fluorescent protein (EGFP), fills the entire neuron, even the spines, with a bright green label. Using a special custom-designed microscope, neurons filled with this label can be visualized at very high magnification while still in the intact, living animal. The use of this technology (two-photon laser scanning microscopy) offers numerous advantages, especially very minimal damage to the animal and the ability to visualize neurons well below the brain surface. Thus neurons involved in a given pathway (in our case, in the part of the sensory system responsible for sensation using the whiskers) can be observed repeatedly over extended periods of time in normal and fragile X animals at different ages. Finally, the same virus used to introduce the fluorescent marker can be used to introduce, at the same time, the FMRP that the animals lack, rendering bright green only those neurons whose genetic defect has been “corrected.” The effects of reintroduction of the gene can then directly be studied with the same imaging system described above.

We hope that this approach to studying Fragile X syndrome will contribute significantly to our understanding of the way the normal brain develops, as well as to how a single genetic defect can so profoundly affect cognitive function.