- How does activity emerge within immature networks of cortical neurons?
- What are the synaptic and network mechanisms of neuronal plasticity after stroke or during normal aging?
- What are the underlying circuit defects in autism and intellectual dysfunction (e.g., fragile X syndrome)?
We want to answer these and other questions about cortical development and plasticity using 2-photon microscopy of living cortical neurons. Our research employs a variety of techniques ranging from molecular biology to calcium imaging.
Our research is particularly relevant to neuropsychiatric disorders that lack distinct neuropathological features, and could therefore be caused by subtle changes in synapses or neuronal activity that create dysfunctional cortical circuits. These disorders include autism, intellectual disability, epilepsy and schizophrenia. We believe that investigating the mechanisms of synapse formation and axon pruning will identify novel molecular targets that can be exploited for therapeutic purposes in these devastating disorders.
Calcium imaging allows us to study the spatio-temporal dynamics of activity in large ensembles of cortical neurons (see 'movies' below).
We use custom-written code in MATLAB to analyze both spontaneous and sensory-evoked neuronal activity (e.g., extrapolated firing rates, pair-wise correlation coefficients, tuning curves).
After each experiment, the exact location of the imaging within barrel cortex (or visual cortex) is identified using careful reconstructions in fixed tissue. To do so, we match the blood vessel imprint on the surface of the brain to a photograph of the same blood vessels taken through the glass covered cranial window at the time of the experiment.
Our ability to image neurons and other brain structures depends on whether we can introduce fluorescent proteins or dyes into them. One problem for in vivo imaging is that the widely used GFP transgenic mice (thy1) do not express GFP in pyramidal neurons until after the 3rd postnatal week. Because we are interested in the development of dendrites and, in particular, the role of dendritic filopodia, we use in utero electroporation to transfect subsets of Layer 2/3 cortical neuron precursors at embryonic day 16.
Another advantage of in utero electroporation is that we can over-express or knock-down (using siRNA) any gene of interest.
2-photon microscopy allows us to study how dendritic spines or axon boutons are formed during normal cortical development or how they change during plasticity after stroke or with normal aging. Typically we can image synaptic structure in GFP-expressing mice over periods of months, even up to a year.
We are also studying the origin of spine defects in fragile X syndrome (FXS), which is the most commonly inherited form of mental impairment and autism. The disorder is caused by a triplet repeat expansion with the Fmr1 gene, which results in the absence of the fragile X mental retardation protein (FMRP). This protein is important for regulating protein synthesis at the synapse. We found that dendritic spines fail to stabilize properly in an experimental model of FXS during a critical period of brain development.