RESUMO
Within the developing central nervous system, the dynamics of synapse formation and elimination are insufficiently understood. It is ideal to study these processes in vivo, where neurons form synapses within appropriate behavioral and anatomical contexts. In vivo analysis is particularly important for long-range connections, since their development cannot be adequately studied in vitro. The corpus callosum (CC) represents a clinically-relevant long-range connection since several neurodevelopmental diseases involve CC defects. Here, we present a novel strategy for in vivo longitudinal and rapid time-lapse imaging of CC presynaptic terminal development. In postnatal mice, the time-course of CC presynaptic terminal formation and elimination was highly variable between axons or groups of axons. Young presynaptic terminals were remarkably dynamic - moving, dividing to generate more boutons, and merging to consolidate small terminals into large boutons. As synaptic networks matured, presynaptic mobility decreased. These rapid dynamics may be important for establishing initial synaptic contacts with postsynaptic partners, refining connectivity patterns or modifying synapse strength during development. Ultimately, this in vivo imaging approach will facilitate investigation of synapse development in other long-range connections and neurodevelopmental disease models.
Assuntos
Corpo Caloso/fisiologia , Terminações Pré-Sinápticas/fisiologia , Animais , Corpo Caloso/ultraestrutura , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica , Vias Neurais/fisiologia , Terminações Pré-Sinápticas/ultraestruturaRESUMO
Intravital imaging of the immune system is a powerful technique for studying biology of the immune response in the spinal cord using a variety of disease models ranging from traumatic injury to autoimmune disorders. Here, we will discuss specific technical aspects as well as many intriguing biological phenomena that have been revealed with the use of intravital imaging for investigation of the immune system in the spinal cord. We will discuss surgical techniques for exposing and stabilizing the spine that are critical for obtaining images, visualizing immune and CNS cells with genetically expressed fluorescent proteins, fluorescent labeling techniques and briefly discuss some of the challenges of image analysis.
Assuntos
Microscopia Intravital/métodos , Neuroimagem/métodos , Medula Espinal , Animais , HumanosRESUMO
Peripheral immune cells are critical to the pathogenesis of neurodegenerative diseases including multiple sclerosis (MS) (Hendriks et al., 2005; Kasper and Shoemaker, 2010). However, the precise sequence of tissue events during the early asymptomatic induction phase of experimental autoimmune encephalomyelitis (EAE) pathogenesis remains poorly defined. Due to the spatial-temporal constrains of traditional methods used to study this disease, most studies had been performed in the spine during peak clinical disease; thus the debate continues as to whether tissue changes such as vessel disruption represent a cause or a byproduct of EAE pathophysiology in the cortex. Here, we provide dynamic, high-resolution information on the evolving structural and cellular processes within the gray matter of the mouse cortex during the first 12 asymptomatic days of EAE induction. We observed that transient focal vessel disruptions precede microglia activation, followed by infiltration of and directed interaction between circulating dendritic cells and T cells. Histamine antagonist minimizes but not completely ameliorates blood vessel leaks. Histamine H1 receptor blockade prevents early microglia function, resulting in subsequent reduction in immune cell accumulation, disease incidence and clinical severity.
Assuntos
Córtex Cerebral/patologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Espaço Subaracnóideo/patologia , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/patologia , Barreira Hematoencefálica/patologia , Encefalomielite Autoimune Experimental/induzido quimicamente , Substância Cinzenta/patologia , Antagonistas dos Receptores Histamínicos H1/farmacologia , Hidroxizina/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Toxina Pertussis , Linfócitos T/imunologiaRESUMO
NG2 is purportedly one of the most growth-inhibitory chondroitin sulfate proteoglycans (CSPGs) produced after spinal cord injury. Nonetheless, once the severed axon tips dieback from the lesion core into the penumbra they closely associate with NG2+ cells. We asked if proteoglycans play a role in this tight cell-cell interaction and whether overadhesion upon these cells might participate in regeneration failure in rodents. Studies using varying ratios of CSPGs and adhesion molecules along with chondroitinase ABC, as well as purified adult cord-derived NG2 glia, demonstrate that CSPGs are involved in entrapping neurons. Once dystrophic axons become stabilized upon NG2+ cells, they form synaptic-like connections both in vitro and in vivo. In NG2 knock-out mice, sensory axons in the dorsal columns dieback further than their control counterparts. When axons are double conditioned to enhance their growth potential, some traverse the lesion core and express reduced amounts of synaptic proteins. Our studies suggest that proteoglycan-mediated entrapment upon NG2+ cells is an additional obstacle to CNS axon regeneration.
Assuntos
Antígenos/fisiologia , Axônios/fisiologia , Comunicação Celular/fisiologia , Regeneração Nervosa/fisiologia , Proteoglicanas/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Sinapses/fisiologia , Animais , Antígenos/genética , Axônios/ultraestrutura , Rastreamento de Células , Células Cultivadas , Proteoglicanas de Sulfatos de Condroitina/fisiologia , Fibronectinas/fisiologia , Gânglios Espinais/fisiopatologia , Gânglios Espinais/ultraestrutura , Integrina beta1/fisiologia , Laminina/fisiologia , Camundongos , Camundongos Knockout , Degeneração Neural/fisiopatologia , Proteoglicanas/genéticaRESUMO
Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.
Assuntos
Axônios/patologia , Comunicação Celular/fisiologia , Microscopia Intravital/métodos , Macrófagos/patologia , Microglia/patologia , Traumatismos da Medula Espinal/patologia , Animais , Modelos Animais de Doenças , Inflamação/patologia , CamundongosRESUMO
After traumatic spinal cord injury, functional deficits increase as axons die back from the center of the lesion and the glial scar forms. Axonal dieback occurs in two phases: an initial axon intrinsic stage that occurs over the first several hours and a secondary phase which takes place over the first few weeks after injury. Here, we examine the secondary phase, which is marked by infiltration of macrophages. Using powerful time-lapse multi-photon imaging, we captured images of interactions between Cx3cr1(+/GFP) macrophages and microglia and Thy-1(YFP) axons in a mouse dorsal column crush spinal cord injury model. Over the first few weeks after injury, axonal retraction bulbs within the lesion are static except when axonal fragments are lost by a blebbing mechanism in response to physical contact followed by phagocytosis by mobile Cx3Cr1(+/GFP) cells. Utilizing a radiation chimera model to distinguish marrow-derived cells from radio-resistant CNS-resident microglia, we determined that the vast majority of accumulated cells in the lesion are derived from the blood and only these are associated with axonal damage. Interestingly, CNS-resident Cx3Cr1(+/GFP) microglia did not increasingly accumulate nor participate in neuronal destruction in the lesion during this time period. Additionally, we found that the blood-derived cells consisted mainly of singly labeled Ccr2(+/RFP) macrophages, singly labeled Cx3Cr1(+/GFP) macrophages and a small population of double-labeled cells. Since all axon destructive events were seen in contact with a Cx3Cr1(+/GFP) cell, we infer that the CCR2 single positive subset is likely not robustly involved in axonal dieback. Finally, in our model, deletion of CCR2, a chemokine receptor, did not alter the position of axons after dieback. Understanding the in vivo cellular interactions involved in secondary axonal injury may lead to clinical treatment candidates involving modulation of destructive infiltrating blood monocytes.
Assuntos
Axônios/patologia , Macrófagos/patologia , Microglia/patologia , Traumatismos da Medula Espinal/patologia , Animais , Transplante de Medula Óssea , Receptor 1 de Quimiocina CX3C , Comunicação Celular/imunologia , Feminino , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/imunologia , Microscopia/métodos , Monócitos/imunologia , Monócitos/patologia , Compressão Nervosa , Fagocitose/imunologia , Receptores CCR2/genética , Receptores de Quimiocinas/genética , Nervo Isquiático/patologia , Traumatismos da Medula Espinal/imunologia , Quimeras de TransplanteRESUMO
Fluorescent imaging coupled with high-resolution femto-second pulsed infrared lasers allows for interrogation of cellular interactions deeper in living tissues than ever imagined. Intra-vital imaging of the central nervous system (CNS) has provided insights into neuronal development, synaptic transmission, and even immune interactions. In this review we will discuss the two most common intravital approaches for studying the cerebral cortex in the live mouse brain for pre-clinical studies, the thinned skull and cranial window techniques, and focus on the advantages and drawbacks of each approach. In addition, we will discuss the use of neuronal physiologic parameters as determinants of successful surgical and imaging preparation.
RESUMO
Within the central nervous system (CNS), antigen-presenting cells (APCs) play a critical role in orchestrating inflammatory responses where they present CNS-derived antigens to immune cells that are recruited from the circulation to the cerebrospinal fluid, parenchyma, and perivascular space. Available data indicate that APCs do so indirectly from outside of CNS vessels without direct access to luminal contents. Here, we applied high-resolution, dynamic intravital two-photon laser scanning microscopy to directly visualize extravascular CX3CR1+ APC behavior deep within undisrupted CNS tissues in two distinct anatomical sites under three different inflammatory stimuli. Surprisingly, we observed that CNS-resident APCs dynamically extend their cellular processes across an intact vessel wall into the vascular lumen with preservation of vessel integrity. While only a small number of APCs displayed intravascular extensions in intact, noninflamed vessels in the brain and the spinal cord, the frequency of projections increased over days in an experimental autoimmune encephalomyelitis model, whereas the number of projections remained stable compared to baseline days after tissue injury such as CNS tumor infiltration and aseptic spinal cord trauma. Our observation of this unique behavior by parenchyma CX3CR1+ cells in the CNS argues for further exploration into their functional role in antigen sampling and immune cell recruitment.
Assuntos
Sistema Nervoso Central/imunologia , Células Dendríticas/imunologia , Receptores de Quimiocinas/análise , Animais , Receptor 1 de Quimiocina CX3C , Modelos Animais de Doenças , Encefalomielite/imunologia , Encefalomielite/patologia , Camundongos , Microscopia ConfocalRESUMO
Aberrant neuronal re-entry into the cell cycle is emerging as a potential pathological mechanism in Alzheimer disease (AD). However, while cyclins, cyclin dependent kinases (CDKs), and other mitotic factors are ectopically expressed in neurons, many of these proteins are also involved in other pathological and physiological processes, generating continued debate on whether such markers are truly indicative of a bona fide cell cycle process. To address this issue, here we analyzed one of the minichromosome maintenance (Mcm) proteins that plays a role in DNA replication and becomes phosphorylated by the S-phase promoting CDKs and Cdc7 during DNA synthesis. We found phosphorylated Mcm2 (pMcm2) markedly associated with neurofibrillary tangles, neuropil threads, and dystrophic neurites in AD but not in aged-matched controls. These data not only provide further evidence for cell cycle aberrations in AD, but the cytoplasmic, rather than nuclear, localization of pMcm2 suggests an abnormal cellular distribution of this important replication factor in AD that may explain resultant cell cycle stasis and consequent neuronal degeneration.
Assuntos
Doença de Alzheimer/metabolismo , Neurônios/fisiologia , Fase S/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Componente 2 do Complexo de Manutenção de Minicromossomo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Placa Amiloide , Transporte ProteicoRESUMO
Conventional kinesin (Kinesin-1), the founding member of the kinesin family, was discovered in the squid giant axon, where it is thought to move organelles on microtubules. In this study, we identify a second squid kinesin by searching an expressed sequence tag database derived from the ganglia that give rise to the axon. The full-length open reading frame encodes a 1753 amino acid sequence that classifies this protein as a Kinesin-3. Immunoblots demonstrate that this kinesin, unlike Kinesin-1, is highly enriched in chaotropically stripped axoplasmic organelles, and immunogold electron microscopy (EM) demonstrates that Kinesin-3 is tightly bound to the surfaces of these organelles. Video microscopy shows that movements of purified organelles on microtubules are blocked, but organelles remain attached, in the presence Kinesin-3 antibody. Immunogold EM of axoplasmic spreads with antibody to Kinesin-3 decorates discrete sites on many, but not all, free organelles and localizes Kinesin-3 to organelle/microtubule interfaces. In contrast, label for Kinesin-1 decorates microtubules but not organelles. The presence of Kinesin-3 on purified organelles, the ability of an antibody to block their movements along microtubules, the tight association of Kinesin-3 with motile organelles and its distribution at the interface between native organelles and microtubules suggest that Kinesin-3 is a dominant motor in the axon for unidirectional movement of organelles along microtubules.
Assuntos
Axônios , Cinesinas/fisiologia , Organelas/fisiologia , Animais , Decapodiformes , Microscopia Eletrônica , Especificidade da EspécieRESUMO
In Alzheimer disease, neuronal degeneration and the presence of neurofibrillary tangles correlate with the severity of cognitive decline. Neurofibrillary tangles contain the antigenic profile of many cell cycle markers, reflecting a re-entry into the cell cycle by affected neurons. However, while such a cell cycle re-entry phenotype is an early and consistent feature of Alzheimer disease, the mechanisms responsible for neuronal cell cycle are unclear. In this regard, given that a dysregulated cell cycle is a characteristic of cancer, we speculated that alterations in oncogenic proteins may play a role in neurodegeneration. To this end, in this study, we examined brain tissue from cases of Alzheimer disease for the presence of BRCA1, a known regulator of cell cycle, and found intense and specific localization of BRCA1 to neurofibrillary tangles, a hallmark lesion of the disease. Analysis of clinically normal aged brain tissue revealed systematically less BRCA1, and surprisingly in many cases with apparent phosphorylated tau-positive neurofibrillary tangles, BRCA1 was absent, yet BRCA1 was present in all cases of Alzheimer disease. These findings not only further define the cell cycle reentry phenotype in Alzheimer disease but also indicate that the neurofibrillary tangles which define Alzheimer disease may have a different genesis from the neurofibrillary tangles of normal aging.