Biomedical subjects

Michael E Dailey

Publications and source records attributed to Michael E Dailey.

9 recordsLinked to original sources

PubMed2006

The P2Y12 receptor regulates microglial activation by extracellular nucleotides.

Microglia are primary immune sentinels of the CNS. Following injury, these cells migrate or extend processes toward sites of tissue damage. CNS injury is accompanied by release of nucleotides, serving as signals for microglial activation or chemotaxis. Microglia express several purinoceptors, including a G(i)-coupled subtype that has been implicated in ATP- and ADP-mediated migration in vitro. Here we show that microglia from mice lacking G(i)-coupled P2Y(12) receptors exhibit normal baseline motility but are unable to polarize, migrate or extend processes toward nucleotides in vitro or in vivo. Microglia in P2ry(12)(-/-) mice show significantly diminished directional branch extension toward sites of cortical damage in the living mouse. Moreover, P2Y(12) expression is robust in the 'resting' state, but dramatically reduced after microglial activation. These results imply that P2Y(12) is a primary site at which nucleotides act to induce microglial chemotaxis at early stages of the response to local CNS injury.

Adenosine Diphosphate↗

PubMed2006

Dendritic arbors of developing retinal ganglion cells are stabilized by beta 1-integrins.

The architecture of dendritic arbors is a defining characteristic of neurons and is established through a sequential but overlapping series of events involving process outgrowth and branching, stabilization of the global pattern, and synapse formation. To investigate the roles of cadherins and beta1-integrins in maintaining the global architecture of the arbor, we used membrane permeable peptides and transfection with dominant-negative constructs to disrupt adhesion molecule function in intact chick neural retina at a stage when the architecture of the ganglion cell (RGC) arbor is established but synapse formation is just beginning. Inactivation of beta1-integrins induces rapid dendrite retraction, with loss of dynamic terminal filopodia followed by resorption of major branches. Disruption of N-cadherin-beta-catenin interactions has no effect; however, dendrites do retract following perturbation of the juxtamembrane region of N-cadherin, which disrupts N-cadherin-mediated adhesion and initiates a beta1-integrin inactivating signal. Thus, developing RGC dendritic arbors are stabilized by beta1-integrin-dependent processes.

Amino Acid Sequence↗

PubMed2006

Early activation, motility, and homing of neonatal microglia to injured neurons does not require protein synthesis.

Neuronal injury in CNS tissues induces a rapid activation and mobilization of resident microglia (MG). It is widely assumed that changes in gene expression drive the morphological transformation of MG and regulate their mobilization during activation. Here, we used acutely excised neonatal rat brain slices to test whether the morphological transformation and homing of MG to injured neurons requires gene expression and de novo protein synthesis. Traumatic injury during excision of live brain tissue slices induces a rapid and transient translocation of a transcription factor, NF-kappaB, to nuclei in MG. This is followed within 4-8 h by an increase in immunolabeling for cell adhesion molecules and lysosomal proteins, accompanied by changes in cell morphology. Application of anisomycin, a protein synthesis inhibitor, prevents the increase in immunolabeling for markers of MG activation but not the morphological transformation. Confocal time-lapse imaging in live tissue slices indicates that MG cell motility (branch extension and retraction) and locomotion are unaffected by anisomycin at early postinjury time-points (<4 h), while at later time-points (4-8 h postinjury) MG locomotion but not motility is inhibited. Thus, activated MG rapidly localize to injured pyramidal neuron cell bodies by 4-h postinjury, even in the presence of anisomycin. Moreover, this early MG activation and homing to injured neurons is unaffected in tissue slices from beta2 integrin deficient mice. These results indicate that gene activation and new protein synthesis coincide with, but are not necessary for, the rapid morphological transformation and early migration-dependent homing of activated MG to injured neurons in CNS tissues.

Animals↗

PubMed2005

Synaptic activity and F-actin coordinately regulate CaMKIIalpha localization to dendritic postsynaptic sites in developing hippocampal slices.

We examined the timing and mechanisms of CaMKIIalpha recruitment to nascent synapses of developing rat hippocampal pyramidal neurons in slice culture. Time-lapse confocal imaging shows that GFP-CaMKIIalpha in transfected neurons accumulates in spines as they are forming, and loss of CaMKIIalpha coincides with spine destabilization. Immunolabeling shows that endogenous CaMKIIalpha is concentrated at postsynaptic sites in spines under ambient slice culture conditions, and this is not disrupted by short-term (3 h) synaptic activity blockade or Latrunculin-induced F-actin depolymerization. However, the combination of activity blockade and F-actin depolymerization significantly reduces synaptic CaMKIIalpha. Conversely, postsynaptic activation induces synaptic recruitment of CaMKIIalpha even in the presence of F-actin depolymerizing drugs. Thus, synaptic-activity-dependent mechanisms and (synaptic activity-independent) F-actin-based mechanisms are individually sufficient and act in parallel to localize CaMKIIalpha to the dendritic spine compartment. Moreover, the timing of CaMKIIalpha recruitment to developing spines suggests a role for CaMKIIalpha in spine assembly and maintenance.

Actins↗

PubMed2005

Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures.

Protoplasmic astrocytes in mammalian CNS tissues in vivo have a highly complex 3D morphology, but in dissociated cell cultures they often assume a flattened, fibroblast-like morphology bearing only a few, simple processes. By fluorescent labeling and confocal reconstruction we show that many astrocytes in organotypic hippocampal slice cultures exhibit a more native complex cytoarchitecture. Although astrocytes at the surface of slice cultures show a reactive form with several thick glial fibrillary acidic protein (GFAP)-positive processes, astrocytes situated in deeper portions of tissue slices retain a highly complex 3D morphology with many fine spine- or veil-like protrusions. Dozens of astrocytes can be labeled in single slice cultures by gene gun-mediated ballistic delivery of gold or tungsten particles carrying cDNAs (Biolistics), lipophilic dyes (DiOlistics), or fluorescent intracellular calcium indicators (Calistics). Expression of a membrane-targeted form of eGFP (Lck-GFP) is superior to soluble eGFP for resolving fine astrocytic processes. Time-lapse confocal imaging of Lck-GFP transfected astrocytes or "calistically" labeled astrocytes show structural remodeling and calcium transients, respectively. This approach provides an in vitro system for investigating the functional architecture, development and dynamic remodeling of astrocytes and their relationships to neurons and glia in live mammalian brain tissues.

Animals↗

PubMed2005

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.

Animals↗

PubMed2004

Localization of synapsin-I and PSD-95 in developing postnatal rat cerebellar cortex.

The comparative localization of two prominent synaptic proteins, synapsin-I (Syn-I) and PSD-95, was investigated in slices of developing (P3-P21) rat cerebellar cortex using double- or triple-label fluorescence immunohistochemistry and confocal microscopy. During the first postnatal week, Syn-I and PSD-95 immunoreactive (IR) puncta were strongly concentrated in the Purkinje cell layer (PCL) where they circumscribed irregularly shaped PC somata, forming pericellular nests that likely correspond to early climbing fiber synapses. PSD-95 and Syn-I puncta also were found along the shafts and at the tips of growing PC dendrite branches labeled with calbindin. During the second postnatal week, synaptic puncta were lost from the PC layer, while many new puncta were added to the molecular layer (ML). At P10, about half of the PCs were circumscribed by PSD-95 or Syn-I puncta, whereas at P14 no PCs were circumscribed. By P14, PSD-95 and Syn-I became most strongly localized to many small puncta in the ML and to large clusters at mossy fiber rosettes in the glomerular layer (GL) where PSD-95 often encircled Syn-I clusters. Some large clusters in the GL contained only PSD-95 or Syn-I, but not both, suggesting differential growth or remodeling of pre- and post-synaptic structures. No PSD-95 staining of pre-synaptic terminal pinceau was observed during the first 3 weeks of postnatal development. Thus, in relation to PCs, there is a developmental shift in PSD-95 localization whereby, first, it is concentrated on PC cell bodies and short dendrites (P3-P7), then it is lost on PC cell bodies (P7-14) and becomes localized almost exclusively to PC dendrites (P14-P21).

Animals↗

PubMed2004

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 (IB(4)), 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 approximately 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 approximately 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.

Animals↗

PubMed2002

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 microm/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.

Aging↗