Within the last decade, major advances have been made in imaging the nervous system with great resolution and minimal invasiveness. This has led to a leap in our understanding of changes in neuronal networks, single cells and subcellular compartments during behaviorally relevant manipulations such as sensation and learning. Macroscopic imaging modalities such as positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) have allowed the read out of both structural and functional aspects of brain function non-invasively in humans and animal models, with the disadvantage of integration of signal over very large numbers of neurons and glia.1 Optical imaging, however, offers the ability to image over a large range of resolutions (Fig. 1) but requires more invasive procedures to deliver the light to the brain surface. Of great interest to in vivo brain imaging are two optical techniques. The first is intrinsic signal imaging, which relies on hemodynamic changes to track the function of neurons, but necessarily integrates over many neurons due to the nature of the hemodynamic signal.2 The other, two photon laser scanning microscopy (TPLSM), has been an important technique in the optical imaging arsenal due to its microscopic resolution and good depth penetration even in highly scattering organs such as the brain.3 TPLSM is a fluorescence-based technique and therefore, depending on the choice of fluorescent probe, can report on structural as well as functional aspects of cells with subcellular resolution. Thus TPLSM has provided unexpected insights in to the dynamics of neurons and glia in vivo.
We have recently used TPLSM to show that the structure of visual cortical neurons is surprisingly plastic in response to manipulation of the visual environment during developmental periods. Dendritic spines are the postsynaptic structures of the overwhelming majority of excitatory synapses in the central nervous system.4 They have a peculiar “lollipop” shape whereby the spherical head of the spine is connected to the parent dendrite through a thin neck.5 Why excitatory synapses maintain these postsynaptic compartments is not yet well understood but the heterogeneity of dendritic spine morphology led Ramon y Cajal who first described these structures over 100 years ago to postulate that their shape related to learning.5 With the recent application of TPLSM to the intact brain it has been possible to examine the structural dynamics of dendritic spines in the same animals before and after manipulations of the visual environment.6
One of the first surprises was that dendritic spines, even in relatively adult animals, were not static but were constantly and rapidly changing their morphologies on a second to minute timescale.7 This spine motility was sensitive to vision as both binocular7 and monocular deprivation,8 as well as dark rearing,9 increased rapid spine dynamics. Surprisingly, dendritic spines could also be completely structurally dismantled and made de novo very rapidly in response to visual input.9
Mice reared in the dark were imaged in the dark and then exposed to light for as little as two hours. The same dendrites were then re-identified and imaged after light exposure (Fig. 2). During the light exposure new protrusions grew at a rate 4 times that of control mice that were kept in complete darkness or in light throughout both imaging sessions. Interestingly, this time scale correlated well with functional imaging experiments using intrinsic signal imaging, which showed profound dampening of the cortical response to vision in dark reared mice compared with light reared controls, and a rapid gain the visually-driven cortical response after two hours of light exposure. This functional regulation of the cortical response to vision may be caused in part by the rapid establishment of new intracortical connections mediated by newly formed dendritic protrusions. This experiment is just one example of many recent studies that have implicated structural changes at dendritic spines as important contributors to functional plasticity.6,10
While neurons are traditionally studied as the mediators of plastic changes in the brain, glia are starting to be recognized for the important roles they play in nervous system function and disease.11 Astrocytes have established roles at synapses, where they contribute to synaptic transmission and plasticity. More recently, a role for microglia, the immune cells of the brain, in normal brain function has been uncovered.12–14 TPLSM studies have shown that these highly ramified cells dynamically sample the brain environment even in the absence of pathological changes15,16 and frequently interact with synaptic elements.12 Such dynamic interactions may lead to alterations in synaptic function, including remodeling of synaptic structure as well as phagocytosis and removal of synaptic elements, and are sensitive to sensory activity. Thus it appears that microglia may be important mediators of rapid synaptic changes in response to vision.
How cortical changes influence the progression of glaucoma is currently being debated17 but rapid vision-mediated changes in cortical circuits could contribute to visual deficits in early glaucoma and limit recovery even if retinal ganglion cell function and connectivity to the brain could be re-established. TPLSM in combination with intrinsic signal imaging could be used to track changes in cortical visual function and structural and molecular level plasticity in dendrites, axons and glia in animal models of glaucoma. Such cortical changes could then be related to retinal deficits to decipher the interplay between retinal and cortical alterations. Recent improvements in endoscopic TPLSM could also allow the examination of similar changes in the lateral geniculate nucleus, the first synapse of retinal ganglion cell axons.18 Information obtained from such imaging studies would be invaluable for understanding cortical contribution to glaucoma.
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