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Current Research

Overview


Inside the Portera-Cailliau Lab in early 2015.

How are cortical circuits assembled during development?

- How do cortical neurons recognize each other as appropriate synaptic partners? 
- 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.

In Vivo 2-Photon Calcium Imaging 

Cartoon of bolus loading of calcium indicators for in vivo calcium imaging.
To study how neuronal activity emerges within networks of immature cortical neurons, we use 2-photon calcium imaging with the genetically encoded indicator GCaM P6, as well as in vivo patch-clamp electrophysiology.

Calcium imaging allows us to study the spatio-temporal dynamics of activity in large ensembles of cortical neurons (see 'movies' below). 

Visualizing mouse barrel cortex.

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.
 
In vivo calcium imaging of GCAMP6s-expressing Layer 2/3 neurons in adult visual cortex (30 fps, Unprocessed "movie").

In Utero Electroporation

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.

Left: Layer 2/3 neurons transfected with GFP at E16 using in utero electroporation (epifluorescence; coronal brain slice).  
Right:  Higher magnification of Layer 2/3 neurons at postnatal day 14 (2-photon, coronal brain slice, fixed tissue).

2-Photon Imaging of Neuronal Structure

 
Time-lapse imaging of immature dendritic protrusions of Layer 5 neurons in GFP-M mice. (P = postnatal day)
We use high resolution long-term 2-photon microscopy in vivo and in acute brain slices to examine the development and plasticity of neuronal structure in neocortex. We image neurons that express fluorescent proteins like GFP in transgenic mice or in electroporated mice.
In vivo imaging of Layer 5 pyramidal neuron dendrites in adult GFP-M mice. A: top view (maximum intensity z-projection of 150 slices, 5 micrometers apart), B: Side view x-z projection. C-E: high-resolution images of dendritic segments at various depths below the dura.

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.


Intrinsic optical signal imaging

 
We can also record brain activity non-invasively with intrinsic optical signal imaging, which allows us to map the areas of the cerebral cortex that respond to sensory stimuli.