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)

Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window2009-07-16T14:45:49+00:00

Spine growth precedes synapse formation in the adult neocortex in vivo

Dendritic spines appear and disappear in an experience-dependent manner. Although some new spines have been shown to contain synapses, little is known about the relationship between spine addition and synapse formation, the relative time course of these events, or whether they are coupled to de novo growth of axonal boutons. We imaged dendrites in barrel cortex of adult mice over 1 month, tracking gains and losses of spines. Using serial section electron microscopy, we analyzed the ultrastructure of spines and associated boutons. Spines reconstructed shortly after they appeared often lacked synapses, whereas spines that persisted for 4 d or more always had synapses. New spines had a large surface-to-volume ratio and preferentially contacted boutons with other synapses. In some instances, two new spines contacted the same axon. Our data show that spine growth precedes synapse formation and that new synapses form preferentially onto existing boutons.

Knott GW, Holtmaat A, Wilbrecht L, Welker E, Svoboda K. 2006. Spine growth precedes synapse formation in the adult neocortex in vivo. Nature Neuroscience 9, 1117 – 1124 (2006)
Published online: 6 August 2006 | doi:10.1038/nn1747 (Full Text)

Spine growth precedes synapse formation in the adult neocortex in vivo2006-08-06T15:40:06+00:00

Experience-dependent and cell-type specific spine growth in the neocortex

Functional circuits in the adult neocortex adjust to novel sensory experience, but the underlying synaptic mechanisms remain unknown. Growth and retraction of dendritic spines with synapse formation and elimination could change brain circuits. In the apical tufts of layer 5B (L5B) pyramidal neurons in the mouse barrel cortex, a subset of dendritic spines appear and disappear over days, whereas most spines are persistent for months. Under baseline conditions, new spines are mostly transient and rarely survive for more than a week. Transient spines tend to be small, whereas persistent spines are usually large. Because most excitatory synapses in the cortex occur on spines, and because synapse size10 and the number of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are proportional to spine volume, the excitation of pyramidal neurons is probably driven through synapses on persistent spines. Here we test whether the generation and loss of persistent spines are enhanced by novel sensory experience. We repeatedly imaged dendritic spines for one month after trimming alternate whiskers, a paradigm that induces adaptive functional changes in neocortical circuits. Whisker trimming stabilized new spines and destabilized previously persistent spines. New-persistent spines always formed synapses. They were preferentially added on L5B neurons with complex apical tufts rather than simple tufts. Our data indicate that novel sensory experience drives the stabilization of new spines on subclasses of cortical neurons. These synaptic changes probably underlie experience-dependent remodelling of specific neocortical circuits.

Holtmaat A*, Wilbrecht L*, Knott G, Welker, E, Svoboda K. 2006. Experience-dependent and cell-type specific spine growth in the neocortex. Nature 441, 979-983 (22 June 2006) | doi:10.1038/nature04783 (Full Text)

Experience-dependent and cell-type specific spine growth in the neocortex2006-06-22T15:13:44+00:00

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)

Cell type-specific structural plasticity of axonal branches and boutons in the adult neocortex2006-03-16T15:45:09+00:00

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)

Transient and Persistent Dendritic Spines in the Neocortex In Vivo2005-01-19T15:49:30+00:00

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