Portera-Cailliau Laboratory - 2-Photon Imaging of Neuronal Structure

2-Photon Imaging of Neuronal Structure

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 (see in utero electroporation section).

2-photon microscopy allows us to study how dendritic spines are formed during cortical development, and in particular the role of dendritic filopodia in synaptogenesis.

We can also study the growth and pruning of axons during early postnatal development.

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.

Time-lapse imaging of immature dendritic protrusions of Layer 5 neurons in GFP-M mice. (P = postnatal day)

We can also fill neurons in acute brain slices with fluorescent dyes and then use 2-photon microscopy to image their dendrites and axons. This allows us to do experiments with pharmacology, and test different molecular pathways involved in the regulation of dendritic protrusions, or the elaboration of axons.

Bolus loading of fluorescent dyes into pyramidal neurons in acute brain slices.

We are also studying the origin of spine defects in Fragile X syndrome, which is the most comming inherited form of mental retardation and autism. The disorder is caused by a mutation that results in the absence of the fragile X mental retardation protein, which is important for RNA trafficking in neurons.

For example, we have found that glutamate application to dendrites results in the elongation of dendritic filopodia. This may be a mechanism whereaby nearby axons recruot filopodia to establish early synapses during development. We are currently investigating the molecular pathways involved in this process.

Glutamate recruits dendritic filopodia. The same dendrite before (top) and after (bottom) application of 100 microM glutamate. Note that shaft filopodia (but not growth filopodia) elongate in response to glutamate.