Joshua T Trachtenberg – 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)

Transient and Persistent Dendritic Spines in the Neocortex In Vivo

Dendritic spines were imaged over days to months in the apical tufts of neocortical pyramidal neurons (layers 5 and 2/3) in vivo. A fraction of thin spines appeared and disappeared over a few days, while most thick spines persisted for months. In the somatosensory cortex, from postnatal day (PND) 16 to PND 25 spine retractions exceeded additions, resulting in a net loss of spines. The fraction of persistent spines (lifetime ≥ 8 days) grew gradually during development and into adulthood (PND 16–25, 35%; PND 35–80, 54%; PND 80–120, 66%; PND 175–225, 73%), providing evidence that synaptic circuits continue to stabilize even in the adult brain, long after the closure of known critical periods. In 6-month-old mice, spines turn over more slowly in visual compared to somatosensory cortex, possibly reflecting differences in the capacity for experience-dependent plasticity in these brain regions.

Holtmaat A, Trachtenberg, JT, Wilbrecht L, Shepherd GM, Zhang XQ, Knott GW, Svoboda K. 2005. Transient and Persistent Dendritic Spines in the Neocortex In Vivo. Neuron. 45(2):279-91 (Full Text)