Michael E. Dailey

Dynamics of microglial activation: A confocal time‐lapse analysis in hippocampal slices

The dynamics of microglial cell activation was studied in freshly prepared rat brain tissue slices. Microglia became activated in the tissue slices, as evidenced by their conversion from a ramified to amoeboid form within several hours in vitro. To define better the cytoarchitectural dynamics underlying microglial activation, we performed direct three‐dimensional time‐lapse confocal imaging of microglial cells in live brain slices. Microglia in tissue slices were stained with a fluorescent lectin conjugate, FITC‐IB4, and stacks of confocal optical sections through the tissue were collected repeatedly at intervals of 2–5 min for several hours at a time. Morphometric analysis of cells from time‐lapse sequences revealed that ramified microglia progress to amoeboid macrophages through a stereotypical sequence of steps. First, in the withdrawal stage, the existing ramified branches of activating microglia do not actively extend or engulf other cells, but instead retract back (mean rate, 0.5–1.5 μm/min) and are completely resorbed into the cell body. Second, in the motility stage, a new set of dynamic protrusions, which can exhibit cycles of rapid extension and retraction (both up to 4 μm/min), abruptly emerges. Sometimes new processes begin to emerge even before the old branches are completely withdrawn. Third, in the locomotory stage, microglia begin translocating within the tissue (up to 118 μm/h) only after the new protrusions emerge. We conclude that the rapid conversion of resting ramified microglia to active amoeboid macrophages is accomplished not by converting quiescent branches to dynamic ones, but rather by replacing existing branches with an entirely new set of highly motile protrusions. This suggests that the ramified branches of resting microglia are normally incapable of rapid morphological dynamics necessary for activated microglial function. More generally, our time‐lapse observations identify changes in the dynamic behavior of activating microglia and thereby help define distinct temporal and functional stages of activation for further investigation. GLIA 33:256–266, 2001.

Rapid formation and remodeling of postsynaptic densities in developing dendrites

The dynamics of postsynaptic density (PSD) formation and remodeling were investigated in live developing hippocampal tissue slices. Time lapse imaging of transfected neurons expressing GFP-tagged PSD95, a prominent PSD protein, revealed that up to 40% of PSDs in developing dendrites are structurally dynamic; they rapidly (<15 min) appear or disappear, but also grow, shrink and move within shafts and spines. New spines containing PSDs were formed by conversion of dynamic filopodia-like spine precursors in which PSDs appeared de novo, or by direct extension of spines or spine precursors carrying preformed PSDs from the shaft. PSDs are therefore highly dynamic structures that can undergo rapid structural alteration within dendrite shafts, spines and spine precursors, permitting rapid formation and remodeling of synaptic connections in developing CNS tissues.

Diverse microglial motility behaviors during clearance of dead cells in hippocampal slices

We used two‐channel three‐dimensional time‐lapse fluorescence confocal imaging in live rat hippocampal slice cultures (1–7 days in vitro) to determine the motility behaviors of activated microglia as they engage dead and dying cells following traumatic brain tissue injury. Live microglia were labeled with a fluorescently conjugated lectin (IB4), and dead neurons were labeled with a membrane‐impermeant fluorescent DNA‐binding dye (Sytox Orange or To‐Pro‐3). Tissue injury during the slicing procedure induced neuronal death and microglial activation, but the density of dead cells diminished ∼ 10‐fold by 7 days in vitro as resident microglia cleared dead cells. In time‐lapse movies (4–20 h long), activated microglia exhibited varying levels of motile and locomotory activity. The motility of microglia could change abruptly following contact by other microglia or death of nearby cells. When neighboring cells died, some microglia rapidly moved toward or extended a process to engulf the dead cell, consistent with a chemotactic signaling response. Dead cell nuclei usually were engulfed and carried along by highly motile and locomoting microglia. The mean time to engulfment was ∼ 5 times faster for newly deceased cells (33 min) than for extant dead cells (160 min), suggesting that the efficacy of microglial phagocytosis in situ might vary with time after cell death or mode of cell death. These observations demonstrate that activated microglia are heterogeneous with respect to motile activity following traumatic tissue injury and further indicate that cell motility in situ is temporally regulated at the single cell level, possibly by direct cell‐cell contact and by diffusible substances emanating from nearby dead cells.

Neuronal activity regulates glutamate transporter dynamics in developing astrocytes

Glutamate transporters (GluTs) maintain a low ambient level of glutamate in the central nervous system (CNS) and shape the activation of glutamate receptors at synapses. Nevertheless, the mechanisms that regulate the trafficking and localization of transporters near sites of glutamate release are poorly understood. Here, we examined the subcellular distribution and dynamic remodeling of the predominant GluT GLT‐1 (excitatory amino acid transporter 2, EAAT2) in developing hippocampal astrocytes. Immunolabeling revealed that endogenous GLT‐1 is concentrated into discrete clusters along branches of developing astrocytes that were apposed preferentially to synapsin‐1 positive synapses. Green fluorescent protein (GFP)‐GLT‐1 fusion proteins expressed in astrocytes also formed distinct clusters that lined the edges of astrocyte processes, as well as the tips of filopodia and spine‐like structures. Time‐lapse three‐dimensional confocal imaging in tissue slices revealed that GFP‐GLT‐1 clusters were dynamically remodeled on a timescale of minutes. Some transporter clusters moved within developing astrocyte branches as filopodia extended and retracted, while others maintained stable positions at the tips of spine‐like structures. Blockade of neuronal activity with tetrodotoxin reduced both the density and perisynaptic localization of GLT‐1 clusters. Conversely, enhancement of neuronal activity increased the size of GLT‐1 clusters and their proximity to synapses. Together, these findings indicate that neuronal activity influences both the organization of GluTs in developing astrocyte membranes and their position relative to synapses.

Purines induce directed migration and rapid homing of microglia to injured pyramidal neurons in developing hippocampus

Traumatic CNS injury activates and mobilizes resident parenchymal microglia (MG), which rapidly accumulate near injured neurons where they transform into phagocytes. The mechanisms underlying this rapid ‘homing’ in situ are unknown. Using time‐lapse confocal imaging in acutely excised neonatal hippocampal slices, we show that rapid accumulation of MG near somata of injured pyramidal neurons in the stratum pyramidale (SP) results from directed migration from tissue regions immediately adjacent to (<200 μm from) the SP. Time‐lapse sequences also reveal a ‘spreading activation wave’ wherein MG situated progressively farther from the SP begin to migrate later and exhibit less directional migration toward the SP. Because purines have been implicated in MG activation and chemotaxis, we tested whether ATP/ADP released from injured pyramidal neurons might account for these patterns of MG behavior. Indeed, application of apyrase, which degrades extracellular ATP/ADP, inhibits MG motility and homing to injured neurons in the SP. Moreover, bath application of exogenous ATP/ADP disrupts MG homing by inducing directional migration toward the slice exterior and away from injured neurons. These results indicate that extracellular ATP/ADP is both necessary and sufficient to induce directional migration and rapid homing of neonatal MG to injured neurons in situ. Rapid, ATP/ADP‐dependent MG homing may promote clearance of dead and dying cells and help limit secondary damage during the critical first few hours after neuronal injury.

Confocal Microscopy of Living Cells

If a picture is worth a thousand words, then a movie may be worth a million words. Microcinematography and, later, video microscopy have provided great insight into biological phenomena. One limitation, however, has been the difficulty of imaging in three dimensions. In many cases, observations have been made on cultured cells that are thin to start with or tissue preparations that have been sectioned.

Juxtavascular microglia migrate along brain microvessels following activation during early postnatal development

Some parenchymal microglia in mammalian brain tissues, termed “juxtavascular microglia,” directly contact the basal lamina of blood vessels; however, the functional consequences of this unique structural relationship are unknown. Here we used a rat brain slice model of traumatic brain injury to investigate the dynamic behavior of juxtavascular microglia following activation. Juxtavascular microglia were identified by confocal 3D reconstruction in tissue slices stained with a fluorescent lectin (FITC‐IB4) that labels both microglia and blood vessel endothelial cells. Immunolabeling confirmed that juxtavascular cells were true parenchymal microglia (OX42+, ED2−) and not perivascular cells or pericytes. Time‐lapse imaging in live tissue slices revealed that activating juxtavascular microglia withdraw most extant branches but often maintain contact with blood vessels, usually moving to the surface of a vessel within 1–4 h. Subsequently, some microglia migrate along the parenchymal surface of vessels, moving at rates up to 40 μm/h. Activated juxtavascular microglia sometimes repeatedly extend veil‐like protrusions into the surrounding tissue, consistent with a role in tissue surveillance. Juxtavascular cells were twice as likely as nonjuxtavascular cells to be locomotory by 10 h in vitro, suggesting an enhanced activation response. Moreover, 38% of all juxtavascular cells migrated along a vessel, whereas this was never observed for a nonjuxtavascular cell. These observations identify a mobile subpopulation (10%–30%) of parenchymal microglia that activate rapidly and are preferentially recruited to the surfaces of blood vessels following brain tissue injury. The dynamic and sustained interaction of microglia with brain microvessels may facilitate signaling between injured brain parenchyma and components of the blood‐brain barrier or circulating immune cells of the blood in vivo. GLIA 37:229–240, 2002.

Microglia: Key Elements in Neural Development, Plasticity, and Pathology

A century after Cajal identified a “third element” of the nervous system, many issues have been clarified about the identity and function of one of its major components, the microglia. Here, we review recent findings by microgliologists, highlighting results from imaging studies that are helping provide new views of microglial behavior and function. In vivo imaging in the intact adult rodent CNS has revolutionized our understanding of microglial behaviors in situ and has raised speculation about their function in the uninjured adult brain. Imaging studies in ex vivo mammalian tissue preparations and in intact model organisms including zebrafish are providing insights into microglial behaviors during brain development. These data suggest that microglia play important developmental roles in synapse remodeling, developmental apoptosis, phagocytic clearance, and angiogenesis. Because microglia also contribute to pathology, including neurodevelopmental and neurobehavioral disorders, ischemic injury, and neuropathic pain, promising new results raise the possibility of leveraging microglia for therapeutic roles. Finally, exciting recent work is addressing unanswered questions regarding the nature of microglial-neuronal communication. While it is now apparent that microglia play diverse roles in neural development, behavior, and pathology, future research using neuroimaging techniques will be essential to more fully exploit these intriguing cellular targets for effective therapeutic intervention applied to a variety of conditions.

Regulation of hippocampal synapse remodeling by epileptiform activity

We examined the regulation of dendritic spines and synapses by epileptiform activity (EA) in rat hippocampal slice cultures. EA, which was induced by a GABA(A) receptor inhibitor, gabazine, reduced pyramidal neuron spine density by approximately 50% after 48 h and also caused an increase in the average length of remaining spines. To directly determine the effects of EA on synapses, we used fluorescent protein-tagged PSD95, which marks postsynaptic densities. EA induced a net loss of synapses on spines but not shafts; conversely, activity blockade (TTX) induced a loss of shaft synapses. Time-lapse confocal imaging in live tissue slices revealed that EA (1) shifts the balance of synapse gain and loss in dendrites leading to a net loss of spine synapses and (2) induces the formation of new filopodia-like dendritic structures having abnormally slow motility. These results identify EA-induced changes in the density and distribution of synaptic structures on dendrites.

Differentiation of substantia gelatinosa-like regions in intraspinal and intracerebral transplants of embryonic spinal cord tissue in the rat

The differentiation of intracerebral and intraspinal transplants of fetal (E14-E15) rat spinal cord was studied to determine the extent to which myelin-free zones in these embryonic grafts exhibit cytological features and immunocytochemical characteristics of the substantia gelatinosa (SG) of the normal spinal cord. Immunocytochemical staining with antiserum to myelin basic protein (MBP) revealed myelin-free areas of varying proportions within fetal spinal cord grafts. These regions were identified in both newborn and adult recipients regardless of whether donor tissue was grafted to heterotopic (intracerebral) or homotopic (intraspinal) sites. As in the SG of the intact spinal cord, the myelin-free regions consisted mainly of small (7-15 microns) diameter neurons. At the ultrastructural level, these cells were surrounded by a neuropil composed of numerous small caliber, unmyelinated axons and intermediate-sized dendrites. Synaptic terminals in these areas were primarily characterized by the presence of clear, round vesicles, although granular vesicles were occasionally found within these terminals. Immunocytochemical staining demonstrated met- and leu-enkephalin-, neurotensin-, substance P-, and somatostatin-like immunoreactive elements within these myelin-free areas. Thus, regions within embryonic spinal cord grafts undergo some topographical differentiation which parallels that of the normal superficial dorsal horn. The presence of SG-like regions illustrates the potential capacity of fetal spinal cord transplants for replacing some intraspinal neuronal populations at the site of a spinal cord injury in neonatal and adult animals. These graft regions may serve as a source of intersegmental projection neurons or establish an extensive intrinsic circuitry similar to that seen in the normal SG. In addition, the definition of these areas provides a useful model to study the innervation patterns of host axons that typically project to the substantia gelatinosa of the normal spinal cord.