Vincenzo De Paola – Wilbrecht Lab

Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window

To understand the cellular and circuit mechanisms of experience-dependent plasticity, neurons and their synapses need to be studied in the intact brain over extended periods of time. Two-photon excitation laser scanning microscopy (2PLSM), together with expression of fluorescent proteins, enables high-resolution imaging of neuronal structure in vivo. In this protocol we describe a chronic cranial window to obtain optical access to the mouse cerebral cortex for long-term imaging. A small bone flap is replaced with a coverglass, which is permanently sealed in place with dental acrylic, providing a clear imaging window with a large field of view (~0.8–12 mm2). The surgical procedure can be completed within ~1 h. The preparation allows imaging over time periods of months with arbitrary imaging intervals. The large size of the imaging window facilitates imaging of ongoing structural plasticity of small neuronal structures in mice, with low densities of labeled neurons. The entire dendritic and axonal arbor of individual neurons can be reconstructed.

Holtmaat A, Bonhoeffer T, Chow, Chuckowree J, De Paola V, Hofer S, Hubener M, Keck T, Lee W-C A, Knott G, Mrsic-Flogel T, Mostany R, Nedivi E, Portera-Cailliau C, Svoboda K, Trachtenberg J, Wilbrecht L. 2009. Long-term, high-resolution imaging in the mouse neocortex through an imaging window. Nature Protocols 4(8):1128-44. (Full Text)

Cell type-specific structural plasticity of axonal branches and boutons in the adult neocortex

We imaged axons in layer (L) 1 of the mouse barrel cortex in vivo. Axons from thalamus and L2/3/5, or L6 pyramidal cells were identified based on their distinct morphologies. Their branching patterns and sizes were stable over times of months. However, axonal branches and boutons displayed cell type-specific rearrangements. Structural plasticity in thalamocortical afferents was mostly due to elongation and retraction of branches (range, 1-150 microm over 4 days; approximately 5% of total axonal length), while the majority of boutons persisted for up to 9 months (persistence over 1 month approximately 85%). In contrast, L6 axon terminaux boutons were highly plastic (persistence over 1 month approximately 40 %), and other intracortical axon boutons showed intermediate levels of plasticity. Retrospective electron microscopy revealed that new boutons make synapses. Our data suggest that structural plasticity of axonal branches and boutons contributes to the remodeling of specific functional circuits.

De Paola V, Holtmaat A, Knott G, Song S, Wilbrecht L, Caroni P, Svoboda K. 2006. Cell type-specific structural plasticity of axonal branches and boutons in the adult neocortex. Neuron. 49(6):861-75 (Full Text)