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In JoVE (1)
Other Publications (20)
- Proceedings of the National Academy of Sciences of the United States of America
- Neuron
- Neuron
- Cerebral Cortex (New York, N.Y. : 1991)
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Trends in Neurosciences
- Cell
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Developmental Neurobiology
- Cerebral Cortex (New York, N.Y. : 1991)
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- PLoS Biology
- Biology Letters
- PloS One
- Communicative & Integrative Biology
- Communicative & Integrative Biology
- PloS One
- Proceedings of the National Academy of Sciences of the United States of America
- Proceedings of the National Academy of Sciences of the United States of America
- Glia
Articles by Ania Majewska in JoVE
JoVE Neuroscience
Preparation of Mouse Brain Tissue for Immunoelectron Microscopy
1Department of Neurobiology and Anatomy, University of Rochester, 2Douglas Mental Health University Institute
We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.
Other articles by Ania Majewska on PubMed
Motility of Dendritic Spines in Visual Cortex in Vivo: Changes During the Critical Period and Effects of Visual Deprivation
Cortical dendritic spines are highly motile postsynaptic structures onto which most excitatory synapses are formed. It has been postulated that spine dynamics might reflect synaptic plasticity of cortical neurons. To test this hypothesis, we have investigated spine dynamics during the critical period in mouse visual cortex in vivo with and without sensory deprivation. The motility of spines on apical dendrites of layer 5 neurons was assayed by time-lapse two-photon microscopy. Spines were motile at the ages examined, postnatal days (P)21-P42, although motility decreased between P21 and P28 and then remained stable through P42. Binocular deprivation from before the time of eye-opening up-regulated spine motility during the peak of the critical period (P28), without affecting average spine length, class distribution, or density. Deprivation at the start of the critical period had no effect on spine motility, whereas continued deprivation through the end of the critical period appeared to reduce spine motility slightly. We conclude that spine motility might be involved in critical-period plasticity and that reduction of activity during the critical period enhances spine dynamics.
Dendritic Spine Dynamics Are Regulated by Monocular Deprivation and Extracellular Matrix Degradation
The mammalian primary visual cortex (V1) is especially susceptible to changes in visual input over a well-defined critical period, during which closing one eye leads to a loss of responsiveness of neurons to the deprived eye and a shift in response toward the open eye. This functional plasticity can occur rapidly, following even a single day of eye closure, although the structural bases of these changes are unknown. Here, we show that rapid structural changes at the level of dendritic spines occur following brief monocular deprivation. These changes are evident in the supra- and infragranular layers of the binocular zone and can be mimicked by degradation of the extracellular matrix with the tPA/plasmin proteolytic cascade. Further, monocular deprivation occludes a subsequent effect of matrix degradation, suggesting that this mechanism is active in vivo to permit structural remodeling during ocular dominance plasticity.
Dendritic Spine Geometry: Functional Implication and Regulation
Dendritic spines are tiny protrusions on dendritic shafts where most excitatory synapses are located. Recent advances in imaging technologies have given us great insight into the function of spines as biochemical compartments. Here we review recent evidence suggesting that the geometry of dendritic spines controls postsynaptic calcium signaling and is bidirectionally regulated during synaptic plasticity.
Effects of Synaptic Activity on Dendritic Spine Motility of Developing Cortical Layer V Pyramidal Neurons
It is increasingly clear that dendritic spines play an important role in compartmentalizing post-synaptic signals and that their dynamic morphological properties have functional consequences. Here, we examine this issue using two-photon microscopy to characterize spine motility on layer V pyramidal neurons in acute slices of the developing mouse cortex. In this system, all spine classes except filopodia become less dynamic as development proceeds. General manipulations of activity (TTX or KCl treatment) do not alter spine dynamics, although increased glutamatergic transmission (AMPA or NMDA treatment) stabilizes developing cortical spines. These effects on spine dynamics do not appear to be related to AMPA or NMDA receptor expression as assessed with immunolabeling, as there is no correlation between spine motility and AMPA (GluR1/2) or NMDA (NR1/NR2B) receptor subunit expression on a spine by spine basis. These results indicate that activity through glutamatergic synapses is important for regulating spine motility in the developing mouse cortex, and that the relative complement of receptors, while different across morphological classifications, cannot account for differences in dynamic structural changes in dendritic spines.
Remodeling of Synaptic Structure in Sensory Cortical Areas in Vivo
Although plastic changes are known to occur in developing and adult cortex, it remains unclear whether these changes require remodeling of cortical circuitry whereby synapses are formed and eliminated or whether they rely on changes in the strength of existing synapses. To determine the structural stability of dendritic spines and axon terminals in vivo, we chose two approaches. First, we performed time-lapse two-photon imaging of dendritic spine motility of layer 5 pyramidal neurons in juvenile [postnatal day 28 (P28)] mice in visual, auditory, and somatosensory cortices. We found that there were differences in basal rates of dendritic spine motility of the same neuron type in different cortices, with visual cortex exhibiting the least structural dynamics. Rewiring visual input into the auditory cortex at birth, however, failed to alter dendritic spine motility, suggesting that structural plasticity rates might be intrinsic to the cortical region. Second, we investigated the persistence of both the presynaptic (axon terminals) and postsynaptic (dendritic spine) structures in young adult mice (P40-P61), using chronic in vivo two-photon imaging in different sensory areas. Both terminals and spines were relatively stable, with >80% persisting over a 3 week period in all sensory regions. Axon terminals were more stable than dendritic spines. These data suggest that changes in network function during adult learning and memory might occur through changes in the strength and efficacy of existing synapses as well as some remodeling of connectivity through the loss and gain of synapses.
Plasticity and Specificity of Cortical Processing Networks
The cerebral cortex is subdivided into discrete functional areas that are defined by specific properties, including the presence of different cell types, molecular expression patterns, microcircuitry and long-range connectivity. These properties enable different areas of cortex to carry out distinct functions. Emerging data argue that the particular structure and identity of cortical areas derives not only from specific inputs but also from unique processing networks. The aim of this review is to summarize current information on the interplay of intrinsic molecular cues with activity patterns that are driven by sensory experience and shape cortical networks as they develop, emphasizing synaptic connections in networks that process vision. This review is part of the TINS special issue on The Neural Substrates of Cognition.
In Vivo Two-photon Imaging Reveals a Role of Arc in Enhancing Orientation Specificity in Visual Cortex
Cortical representations of visual information are modified by an animal's visual experience. To investigate the mechanisms in mice, we replaced the coding part of the neural activity-regulated immediate early gene Arc with a GFP gene and repeatedly monitored visual experience-induced GFP expression in adult primary visual cortex by in vivo two-photon microscopy. In Arc-positive GFP heterozygous mice, the pattern of GFP-positive cells exhibited orientation specificity. Daily presentations of the same stimulus led to the reactivation of a progressively smaller population with greater reactivation reliability. This adaptation process was not affected by the lack of Arc in GFP homozygous mice. However, the number of GFP-positive cells with low orientation specificity was greater, and the average spike tuning curve was broader in the adult homozygous compared to heterozygous or wild-type mice. These results suggest a physiological function of Arc in enhancing the overall orientation specificity of visual cortical neurons during the post-eye-opening life of an animal.
Next-generation Optical Technologies for Illuminating Genetically Targeted Brain Circuits
Emerging technologies from optics, genetics, and bioengineering are being combined for studies of intact neural circuits. The rapid progression of such interdisciplinary "optogenetic" approaches has expanded capabilities for optical imaging and genetic targeting of specific cell types. Here we explore key recent advances that unite optical and genetic approaches, focusing on promising techniques that either allow novel studies of neural dynamics and behavior or provide fresh perspectives on classic model systems.
Rapid, Long-term Labeling of Cells in the Developing and Adult Rodent Visual Cortex Using Double-stranded Adeno-associated Viral Vectors
Chronic in vivo imaging studies of the brain require a labeling method that is fast, long-lasting, efficient, nontoxic, and cell-type specific. Over the last decade, adeno-associated virus (AAV) has been used to stably express fluorescent proteins in neurons in vivo. However, AAV's main limitation for many studies (such as those of neuronal development) is the necessity of second-strand DNA synthesis, which delays peak transgene expression. The development of double-stranded AAV (dsAAV) vectors has overcome this limitation, allowing rapid transgene expression. Here, we have injected different serotypes (1, 2, 6, 7, 8, and 9) of a dsAAV vector carrying the green fluorescent protein (GFP) gene into the developing and adult mouse visual cortex and characterized its expression. We observed labeling of both neurons and astrocytes with serotype-specific tropism. dsAAV-GFP labeling showed high levels of neuronal GFP expression as early as 2 days postinjection and as long as a month, surpassing conventional AAV's onset of expression and matching its longevity. Neurons labeled with dsAAV-GFP appeared structurally and electrophysiologically identical to nonlabeled neurons, suggesting that dsAAV-GFP is neither cytotoxic nor alters normal neuronal function. We also demonstrated that dsAAV-labeled cells can be imaged with subcellular resolution in vivo over multiple days. We conclude that dsAAV is an excellent vector for rapid labeling and long-term in vivo imaging studies of astrocytes and neurons on the single cell level within the developing and adult visual cortex.
Postsynaptic Deregulation in GAP-43 Heterozygous Mouse Barrel Cortex
Formation of whisker-related barrels in primary somatosensory cortex (S1) requires communication between presynaptic thalamocortical afferents (TCAs) and postsynaptic cortical neurons. GAP-43 is crucially involved in targeting TCAs to postsynaptic S1 neurons but its influence on the interactions between these 2 elements has not been explored. Here, we tested the hypothesis that reduced early expression of presynaptic GAP-43 (GAP-43 heterozygous [HZ] mice) alters postsynaptic differentiation of barrel cells. We found a transient increase in cytochrome oxidase staining between P6 and P14 in HZ animals, indicative of increased metabolic activity in barrel cortex during this time. Golgi impregnation and microtubule-associated protein 2 immunohistochemistry showed anomalous dendritic patterning in GAP-43 HZ cortex at P5, with altered dendritic length and branching and abnormal retention of dendrites that extend into developing septa. This deficiency was no longer apparent at P7, suggesting partial recovery of dendritic pruning processes. Finally, we showed early defects in synaptogenesis from P4 to P5 with increased colocalization of NR1 and GluR1 staining in HZ mice. By P7, this colocalization had normalized to wild type levels. Taken together, our findings suggest abnormal postsynaptic differentiation in GAP-43 HZ cortex during early barrel development, followed by adaptive compensation and partial phenotypic rescue.
Structural Dynamics of Synapses in Vivo Correlate with Functional Changes During Experience-dependent Plasticity in Visual Cortex
The impact of activity on neuronal circuitry is complex, involving both functional and structural changes whose interaction is largely unknown. We have used optical imaging of mouse visual cortex responses and two-photon imaging of superficial layer spines on layer 5 neurons to monitor network function and synaptic structural dynamics in the mouse visual cortex in vivo. Total lack of vision due to dark-rearing from birth dampens visual responses and shifts spine dynamics and morphologies toward an immature state. The effects of vision after dark rearing are strongly dependent on the timing of exposure: over a period of days, functional and structural changes are temporally related such that light stabilizes spines while increasing visually driven activity. The effects of long-term light exposure can be partially mimicked by experimentally enhancing inhibitory signaling in the darkness. Brief light exposure, however, results in a rapid, transient, NMDA-dependent increase of cortical responses, accompanied by increased dynamics of dendritic spines. These findings indicate that visual experience induces rapid reorganization of cortical circuitry followed by a period of stabilization, and demonstrate a close relationship between dynamic changes at single synapses and cortical network function.
Microglial Interactions with Synapses Are Modulated by Visual Experience
Microglia are the immune cells of the brain. In the absence of pathological insult, their highly motile processes continually survey the brain parenchyma and transiently contact synaptic elements. Aside from monitoring, their physiological roles at synapses are not known. To gain insight into possible roles of microglia in the modification of synaptic structures, we used immunocytochemical electron microscopy, serial section electron microscopy with three-dimensional reconstructions, and two-photon in vivo imaging to characterize microglial interactions with synapses during normal and altered sensory experience, in the visual cortex of juvenile mice. During normal visual experience, most microglial processes displayed direct apposition with multiple synapse-associated elements, including synaptic clefts. Microglial processes were also distinctively surrounded by pockets of extracellular space. In terms of dynamics, microglial processes localized to the vicinity of small and transiently growing dendritic spines, which were typically lost over 2 d. When experience was manipulated through light deprivation and reexposure, microglial processes changed their morphology, showed altered distributions of extracellular space, displayed phagocytic structures, apposed synaptic clefts more frequently, and enveloped synapse-associated elements more extensively. While light deprivation induced microglia to become less motile and changed their preference of localization to the vicinity of a subset of larger dendritic spines that persistently shrank, light reexposure reversed these behaviors. Taken together, these findings reveal different modalities of microglial interactions with synapses that are subtly altered by sensory experience. These findings suggest that microglia may actively contribute to the experience-dependent modification or elimination of a specific subset of synapses in the healthy brain.
A Trade-off Between Embryonic Development Rate and Immune Function of Avian Offspring is Revealed by Considering Embryonic Temperature
Long embryonic periods are assumed to reflect slower intrinsic development that are thought to trade off to allow enhanced physiological systems, such as immune function. Yet, the relatively rare studies of this trade-off in avian offspring have not found the expected trade-off. Theory and tests have not taken into account the strong extrinsic effects of temperature on embryonic periods of birds. Here, we show that length of the embryonic period did not explain variation in two measures of immune function when temperature was ignored, based on studies of 34 Passerine species in tropical Venezuela (23 species) and north temperate Arizona (11 species). Variation in immune function was explained when embryonic periods were corrected for average embryonic temperature, in order to better estimate intrinsic rates of development. Immune function of offspring trades off with intrinsic rates of embryonic development once the extrinsic effects of embryonic temperatures are taken into account.
The Mouse Primary Visual Cortex is a Site of Production and Sensitivity to Estrogens
The classic female estrogen, 17β-estradiol (E2), has been repeatedly shown to affect the perceptual processing of visual cues. Although gonadal E2 has often been thought to influence these processes, the possibility that central visual processing may be modulated by brain-generated hormone has not been explored. Here we show that estrogen-associated circuits are highly prevalent in the mouse primary visual cortex (V1). Specifically, we cloned aromatase, a marker for estrogen-producing neurons, and the classic estrogen receptors (ERs) ERα and ERβ, as markers for estrogen-responsive neurons, and conducted a detailed expression analysis via in-situ hybridization. We found that both monocular and binocular V1 are highly enriched in aromatase- and ER-positive neurons, indicating that V1 is a site of production and sensitivity to estrogens. Using double-fluorescence in-situ hybridization, we reveal the neurochemical identity of estrogen-producing and -sensitive cells in V1, and demonstrate that they constitute a heterogeneous neuronal population. We further show that visual experience engages a large population of aromatase-positive neurons and, to a lesser extent, ER-expressing neurons, suggesting that E2 levels may be locally regulated by visual input in V1. Interestingly, acute episodes of visual experience do not affect the density or distribution of estrogen-associated circuits. Finally, we show that adult mice dark-reared from birth also exhibit normal distribution of aromatase and ERs throughout V1, suggesting that the implementation and maintenance of estrogen-associated circuits is independent of visual experience. Our findings demonstrate that the adult V1 is a site of production and sensitivity to estrogens, and suggest that locally-produced E2 may shape visual cortical processing.
Experience-dependent Plasticity in Visual Cortex: Dendritic Spines and Visual Responsiveness
To determine the relationship between synaptic structural changes and cortical function, we recently published a study where we imaged dendritic spines using two-photon in vivo microscopy while monitoring network activity in the visual cortex using intrinsic signal imaging. By manipulating cortical activity levels by dark-rearing mice and re-exposing them to light, we found a close inverse correspondence between dendritic spine structural dynamics and visually evoked cortical function on a timescale of days. Light exposure following dark-rearing slowly increased visually evoked cortical processing and stabilized dendritic spine structure, an effect partially mimicked by diazepam injections in dark reared mice suggesting that this slow recovery is mediated by inhibitory signaling. Surprisingly, very brief (2 h) periods of light exposure led to an NMDA-dependent rapid reorganization of cortical networks with an early emergence of visually-evoked cortical activation and enhanced spine dynamics. Here we further explore the relationship between spine morphology and visual function.
A Role for Microglia in Synaptic Plasticity?
Remodeling of brain circuits, including the formation, modification and elimination of synaptic structures, occurs throughout life as animals adapt to their environment. Until very recently, known mechanisms for experience-dependent synaptic plasticity had placed neurons and their structural interactions with astrocytes in the spotlight. However microglia, the immune cells of the brain, are very active even in the absence of pathological insults and their processes periodically contact dendritic spines and axon terminals in vivo.1-3 This intriguing behavior prompted us to explore, using electron microscopy and two-photon in vivo imaging in the primary visual cortex of juvenile mice, a possible role for quiescent microglia in the modification of synaptic structures.4 Our work uncovered subtle changes in the behavior of microglia during manipulations of visual experience including regulation of perisynaptic extracellular spaces, contact with subsets of structurally dynamic and transient dendritic spines, and phagocytic engulfment of intact synapses. Based on these results, here we further discuss three means of synapse modification or elimination that could be mediated by microglia in the context of normal experience-dependent plasticity.
HIV-1 Tat-induced Microgliosis and Synaptic Damage Via Interactions Between Peripheral and Central Myeloid Cells
Despite the ability of combination antiretroviral treatment (cART) to reduce viral burden to nearly undetectable levels in cerebrospinal fluid and serum, HIV-1 associated neurocognitive disorders (HAND) continue to persist in as many as half the patients living with this disease. There is growing consensus that the actual substrate for HAND is destruction of normal synaptic architecture but the sequence of cellular events that leads to this outcome has never been resolved. To address whether central vs. peripheral myeloid lineage cells contribute to synaptic damage during acute neuroinflammation we injected a single dose of the HIV-1 transactivator of transcription protein (Tat) or control vehicle into hippocampus of wild-type or chimeric C57Bl/6 mice genetically marked to distinguish infiltrating and resident immune cells. Between 8-24 hr after injection of Tat, invading CD11b(+) and/or myeloperoxidase-positive leukocytes with granulocyte characteristics were found to engulf both microglia and synaptic structures, and microglia reciprocally engulfed invading leukocytes. By 24 hr, microglial processes were also seen ensheathing dendrites, followed by inclusion of synaptic elements in microglia 7 d after Tat injection, with a durable microgliosis lasting at least 28 d. Thus, central nervous system (CNS) exposure to Tat induces early activation of peripheral myeloid lineage cells with phagocytosis of synaptic elements and reciprocal microglial engulfment of peripheral leukocytes, and enduring microgliosis. Our data suggest that a single exposure to a foreign antigen such as HIV-1 Tat can lead to long-lasting disruption of normal neuroimmune homeostasis with deleterious consequences for synaptic architecture, and further suggest a possible mechanism for enduring neuroinflammation in the absence of productive viral replication in the CNS.
Rapid Experience-dependent Plasticity of Synapse Function and Structure in Ferret Visual Cortex in Vivo
The rules by which visual experience influences neuronal responses and structure in the developing brain are not well understood. To elucidate the relationship between rapid functional changes and dendritic spine remodeling in vivo, we carried out chronic imaging experiments that tracked visual responses and dendritic spines in the ferret visual cortex following brief periods of monocular deprivation. Functional changes, which were largely driven by loss of deprived eye responses, were tightly regulated with structural changes at the level of dendritic spines, and occurred very rapidly (on a timescale of hours). The magnitude of functional changes was correlated with the magnitude of structural changes across the cortex, and both these features reversed when the deprived eye was reopened. A global rule governed how the responses to the two eyes or changes in spines were altered by monocular deprivation: the changes occurred irrespective of regional ocular dominance preference and were independently mediated by each eye, and the loss or gain of responses/spines occurred as a constant proportion of predeprivation drive by the deprived or nondeprived eye, respectively.
Experience-dependent Regulation of CaMKII Activity Within Single Visual Cortex Synapses in Vivo
Unbalanced visual input during development induces persistent alterations in the function and structure of visual cortical neurons. The molecular mechanisms that drive activity-dependent changes await direct visualization of underlying signals at individual synapses in vivo. By using a genetically engineered Förster resonance energy transfer (FRET) probe for the detection of CaMKII activity, and two-photon imaging of single synapses within identified functional domains, we have revealed unexpected and differential mechanisms in specific subsets of synapses in vivo. Brief monocular deprivation leads to activation of CaMKII in most synapses of layer 2/3 pyramidal cells within deprived eye domains, despite reduced visual drive, but not in nondeprived eye domains. Synapses that are eliminated in deprived eye domains have low basal CaMKII activity, implying a protective role for activated CaMKII against synapse elimination.
Effects of Aging and Sensory Loss on Glial Cells in Mouse Visual and Auditory Cortices
Normal aging is often accompanied by a progressive loss of receptor sensitivity in hearing and vision, whose consequences on cellular function in cortical sensory areas have remained largely unknown. By examining the primary auditory (A1) and visual (V1) cortices in two inbred strains of mice undergoing either age-related loss of audition (C57BL/6J) or vision (CBA/CaJ), we were able to describe cellular and subcellular changes that were associated with normal aging (occurring in A1 and V1 of both strains) or specifically with age-related sensory loss (only in A1 of C57BL/6J or V1 of CBA/CaJ), using immunocytochemical electron microscopy and light microscopy. While the changes were subtle in neurons, glial cells and especially microglia were transformed in aged animals. Microglia became more numerous and irregularly distributed, displayed more variable cell body and process morphologies, occupied smaller territories, and accumulated phagocytic inclusions that often displayed ultrastructural features of synaptic elements. Additionally, evidence of myelination defects were observed, and aged oligodendrocytes became more numerous and were more often encountered in contiguous pairs. Most of these effects were profoundly exacerbated by age-related sensory loss. Together, our results suggest that the age-related alteration of glial cells in sensory cortical areas can be accelerated by activity-driven central mechanisms that result from an age-related loss of peripheral sensitivity. In light of our observations, these age-related changes in sensory function should be considered when investigating cellular, cortical, and behavioral functions throughout the lifespan in these commonly used C57BL/6J and CBA/CaJ mouse models. © 2012 Wiley Periodicals, Inc.
