With few exceptions biological tissues strongly scatter light, making high-resolution deep imaging impossible for traditional⎯including confocal⎯fluorescence microscopy. Nonlinear optical microscopy, in particular two photon–excited fluorescence microscopy, has overcome this limitation, providing large depth penetration mainly because even multiply scattered signal photons can be assigned to their origin as the result of localized nonlinear signal generation. Two-photon microscopy thus allows cellular imaging several hundred microns deep in various organs of living animals. Here we review fundamental concepts of nonlinear microscopy and discuss conditions relevant for achieving large imaging depths in intact tissue.

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Deep tissue two-photon microscopy

Mounting evidence indicates that microglial activation contributes to neuronal damage in neurodegenerative diseases. Recent studies show that in response to certain environmental toxins and endogenous proteins, microglia can enter an overactivated state and release reactive oxygen species (ROS) that cause neurotoxicity. Pattern recognition receptors expressed on the microglial surface seem to be one of the primary, common pathways by which diverse toxin signals are transduced into ROS production. Overactivated microglia can be detected using imaging techniques and therefore this knowledge offers an opportunity not only for early diagnosis but, importantly, for the development of targeted anti-inflammatory therapies that might slow or halt the progression of neurodegenerative disease.

Microglia-mediated neurotoxicity: uncovering the molecular mechanisms

The organization of behavior: A neuropsychological theory

Microglial cells constitute the resident macrophage population of the CNS. Recent in vivo studies have shown that microglia carry out active tissue scanning, which challenges the traditional notion of 'resting' microglia in the normal brain. Transformation of microglia to reactive states in response to pathology has been known for decades as microglial activation, but seems to be more diverse and dynamic than ever anticipated—in both transcriptional and nontranscriptional features and functional consequences. This may help to explain why engagement of microglia can be either neuroprotective or neurotoxic, resulting in containment or aggravation of disease progression. Moreover, little is known about the heterogeneity of microglial responses in different pathologic contexts that results from regional adaptations or from the progression of a disease. In this review, we focus on several key observations that illustrate the multi-faceted activities of microglia in the normal and pathologic brain.

Microglia: active sensor and versatile effector cells in the normal and pathologic brain

Microglia are highly motile phagocytic cells that infiltrate and take up residence in the developing brain, where they are thought to provide a surveillance and scavenging function. However, although microglia have been shown to engulf and clear damaged cellular debris after brain insult, it remains less clear what role microglia play in the uninjured brain. Here, we show that microglia actively engulf synaptic material and play a major role in synaptic pruning during postnatal development in mice. These findings link microglia surveillance to synaptic maturation and suggest that deficits in microglia function may contribute to synaptic abnormalities seen in some neurodevelopmental disorders.

http://science.sciencemag.org/content/sci/333/6048/1456.full.pdf

Synaptic Pruning by Microglia Is Necessary for Normal Brain Development

Parenchymal microglia are the principal immune cells of the brain. Time-lapse two-photon imaging of GFP-labeled microglia demonstrates that the fine termini of microglial processes are highly dynamic in the intact mouse cortex. Upon traumatic brain injury, microglial processes rapidly and autonomously converge on the site of injury without cell body movement, establishing a potential barrier between the healthy and injured tissue. This rapid chemotactic response can be mimicked by local injection of ATP and can be inhibited by the ATP-hydrolyzing enzyme apyrase or by blockers of G protein-coupled purinergic receptors and connexin channels, which are highly expressed in astrocytes. The baseline motility of microglial processes is also reduced significantly in the presence of apyrase and connexin channel inhibitors. Thus, extracellular ATP regulates microglial branch dynamics in the intact brain, and its release from the damaged tissue and surrounding astrocytes mediates a rapid microglial response towards injury.

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ATP mediates rapid microglial response to local brain injury in vivo

Long-term memories for motor skill tasks are associated with enhanced synaptic efficacy in the motor cortex Here, rapid structural responses in individual neurons are revealed to potentially underlie motor learning skill retention In experiments in which mice were trained to perform a reaching task, new neuronal spines were selectively stabilized within hours, with different spines/putative synapse sets encoding distinct learned motor skills These stabilized morphological changes are proposed to act as a motor memory substrate The learning of novel motor skills through repetitive practice is associated with enhanced synaptic efficacy in the motor cortex However, how motor learning affects neuronal circuitry at the level of individual synapses and how long-lasting memory is structurally encoded in the intact brain remain unknown Synaptic connections in the living mouse brain are now shown to respond to motor-skill learning and permanently rewire; this could be the foundation of durable motor memory Novel motor skills are learned through repetitive practice and, once acquired, persist long after training stops1,2 Earlier studies have shown that such learning induces an increase in the efficacy of synapses in the primary motor cortex, the persistence of which is associated with retention of the task3,4,5 However, how motor learning affects neuronal circuitry at the level of individual synapses and how long-lasting memory is structurally encoded in the intact brain remain unknown Here we show that synaptic connections in the living mouse brain rapidly respond to motor-skill learning and permanently rewire Training in a forelimb reaching task leads to rapid (within an hour) formation of postsynaptic dendritic spines on the output pyramidal neurons in the contralateral motor cortex Although selective elimination of spines that existed before training gradually returns the overall spine density back to the original level, the new spines induced during learning are preferentially stabilized during subsequent training and endure long after training stops Furthermore, we show that different motor skills are encoded by different sets of synapses Practice of novel, but not previously learned, tasks further promotes dendritic spine formation in adulthood Our findings reveal that rapid, but long-lasting, synaptic reorganization is closely associated with motor learning The data also suggest that stabilized neuronal connections are the foundation of durable motor memory

Rapid formation and selective stabilization of synapses for enduring motor memories

https://www.cell.com/neuron/pdf/S0896-6273(05)00309-0.pdf

Development of Long-Term Dendritic Spine Stability in Diverse Regions of Cerebral Cortex

In the cerebral cortex of humans and other mammals, rapid synapse formation during early postnatal life is followed by a substantial loss of synapses through adolescence and into adulthood. The mechanisms that underlie this phenomenon are unknown. It is generally believed that experience leads to an increase in the number of synapses and that's certainly true in early postnatal life but a study in young adolescent mice paints a very different picture. Long-term sensory deprivation increased dendritic spine numbers (hence synaptic links) by reducing the rate of spine elimination. At distinct stages of life it seems that the more experience one has, the more synapses will be lost in the brain. These results emphasize the importance of childhood experience in sculpting neuronal connectivity while the brain is up to the task. A substantial decrease in the number of synapses occurs in the mammalian brain from the late postnatal period until the end of life1,2,3,4,5. Although experience plays an important role in modifying synaptic connectivity6,7,8,9,10,11,12,13,14,15,16,17, its effect on this nearly lifelong synapse loss remains unknown. Here we used transcranial two-photon microscopy to visualize postsynaptic dendritic spines in layer I of the barrel cortex in transgenic mice expressing yellow fluorescent protein. We show that in young adolescent mice, long-term sensory deprivation through whisker trimming prevents net spine loss by preferentially reducing the rate of ongoing spine elimination, not by increasing the rate of spine formation. This effect of deprivation diminishes as animals mature but still persists in adulthood. Restoring sensory experience after adolescent deprivation accelerates spine elimination. Similar to sensory manipulation, the rate of spine elimination decreases after chronic blockade of NMDA (N-methyl-d-aspartate) receptors with the antagonist MK801, and accelerates after drug withdrawal. These studies of spine dynamics in the primary somatosensory cortex suggest that experience plays an important role in the net loss of synapses over most of an animal's lifespan, particularly during adolescence.

Long-term sensory deprivation prevents dendritic spine loss in primary somatosensory cortex

Many lines of evidence suggest that memory in the mammalian brain is stored with distinct spatiotemporal patterns. Despite recent progresses in identifying neuronal populations involved in memory coding, the synapse-level mechanism is still poorly understood. Computational models and electrophysiological data have shown that functional clustering of synapses along dendritic branches leads to nonlinear summation of synaptic inputs and greatly expands the computing power of a neural network. However, whether neighbouring synapses are involved in encoding similar memory and how task-specific cortical networks develop during learning remain elusive. Using transcranial two-photon microscopy, we followed apical dendrites of layer 5 pyramidal neurons in the motor cortex while mice practised novel forelimb skills. Here we show that a third of new dendritic spines (postsynaptic structures of most excitatory synapses) formed during the acquisition phase of learning emerge in clusters, and that most such clusters are neighbouring spine pairs. These clustered new spines are more likely to persist throughout prolonged learning sessions, and even long after training stops, than non-clustered counterparts. Moreover, formation of new spine clusters requires repetition of the same motor task, and the emergence of succedent new spine(s) accompanies the strengthening of the first new spine in the cluster. We also show that under control conditions new spines appear to avoid existing stable spines, rather than being uniformly added along dendrites. However, succedent new spines in clusters overcome such a spatial constraint and form in close vicinity to neighbouring stable spines. Our findings suggest that clustering of new synapses along dendrites is induced by repetitive activation of the cortical circuitry during learning, providing a structural basis for spatial coding of motor memory in the mammalian brain.

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Yi Zuo

Papers

ATP mediates rapid microglial response to local brain injury in vivo

Rapid formation and selective stabilization of synapses for enduring motor memories

Development of Long-Term Dendritic Spine Stability in Diverse Regions of Cerebral Cortex

Long-term sensory deprivation prevents dendritic spine loss in primary somatosensory cortex

Repetitive motor learning induces coordinated formation of clustered dendritic spines in vivo.