ISSN:1059-910X    

DOI:10.1002/jemt.1070290406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

ISSN:1059-910X    

DOI:10.1002/jemt.1070290402

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractThe microscopy of biological specimens has traditionally been a two‐dimensional imaging method for analyzing what are in reality three‐dimensional (3‐D) objects. This has been a major limitation of the application of one of science's most widely used tools. Nowhere has this limitation been more acute than in neurobiology, which is dominated by the necessity of understanding both large‐and small‐scale 3‐D anatomy. Fortunately, recent advances in optical instrumentation and computational methods have provided the means for retrieving the third dimension, making full 3‐D microscopic imaging possible. Optical designs have concentrated on the confocal imaging mode while computational methods have made 3‐D imaging possible with wide field microscopes using deconvolution methods. This work presents a brief review of these methods, especially as applied to neurobiology, and data using both approaches. Specimens several hundred micrometers thick can be sampled allowing essentially intact neurons to be imaged. These neurons Image analysis in 3‐D is as important as visualization in 3‐D. Automated methods of cell counting and analysis by nuclear detection as well as tracing of individual neurons are presented. ©

ISSN:1059-910X    

DOI:10.1002/jemt.1070290403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractChanges in the intracellular Ca2+concentration ([Ca2+]i) within CA1 hippocampal pyramidal neurons were imaged using confocal laser scanning microscopy in conjunction with Ca2+‐sensitive fluorescent indicators. The imaging was performed in thick hippocampal brain slices while simultaneously measuring or controlling electrical activity with sharp microelectrodes or whole‐cell patch‐clamp electrodes. The combination of imaging and electrophysiology was essential for interpreting the changes in [Ca2+]i. We compared the increases in [Ca2+]iproduced by either of two methods–direct depolarization of the cell via the somatic electrode or high‐frequency stimulations of synaptic inputs. The increases in [Ca2+]iin the soma and proximal dendrites caused by both methods were of comparable magnitude and they always decayed within seconds in healthy cells. However, the spatial patterns of distal Ca2+increases were different. Separate sets of synaptic inputs to the same cell resulted in different spatial patterns of [Ca2+]itransients. We isolated and observed what appeared to be a voltage‐independent component of the synaptically mediated [Ca2+]itransients. This work demonstrates that the combination of neurophysiology and simultaneous confocal microscopy is well suited for visualizing and analyzing [Ca2+]iwithin neurons throughout the CNS and it raises the possibility of routinely relating subcellular [Ca2+]ichanges to structural and functional modifications. © 1994 Wil

ISSN:1059-910X    

DOI:10.1002/jemt.1070290404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractIn order to study changes in synaptic structure that accompany learning and memory, we have developed optical methods to visualize dendritic spines and presynaptic terminals in living, electrically monitored brain slices maintained in vitro. Focal microapplication of the fluorescent lipophilic dye DiI provides Golgi‐like staining of small numbers of cells and processes that can be resolved clearly using confocal microscoopy; viability of stained cells is established by exclusion of the fluorescent DNA‐binding dye ethidium bromide. Serial optical sections are enhanced by deconvolution and other image processing methods. The resulting high‐resolution images are combined in an automated procedure to generate three‐dimensional reconstructions, in which submicron synaptic structures can be viewed and measured. These unbiased methods allow volume changes in individual, living synaptic structures to be assessed quantitatively over periods of hours or days in development or in response to stimulation, drug application, or other perturbations. © 1994 Wiley

ISSN:1059-910X    

DOI:10.1002/jemt.1070290405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractThree‐dimensional reconstruction protocols in confocal microscopy are typically considered in terms of rendering separate stacks of optical sections. Single stacks, however, include volumes that are often too small to permit descriptions of entire neurons, complete axonal arbors, or complex neural networks. Furthermore, traditional tissue preparation protocols generally yield specimens too limited to permit reconstructions of complex neural systems. For 3‐D analyses of extensive networks such as the autonomic nervous system projections within the viscera, it is critical to incorporate appropriate tissue techniques, including suitable tracer protocols, into the reconstruction strategy. This report summarizes complementary technologies, including whole mount procedures, tracer techniques for identifying single fibers in situ, and methods of examining stacks of optical images, which make it practical to describe the complete terminal field of an individual axon in the gastrointestinal tract. Such methods establish that vagal motor axons travel long distances within their target organs, collateralize frequently, and ramify extensively. Vagal afferents have extensive, complex, and, in some cases, polytopic arbors within target tissues. © 1994 Wiley‐Lis

ISSN:1059-910X    

DOI:10.1002/jemt.1070290407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractStudies were undertaken to develop microscopic methods and imaging procedures that would permit identification of sites of intradendritic microelectrode recordings from pyramidal cells in hippocampal slice preparations. Intradendritic recording were obtained with sharp microelectrodes filled with the dye lucifer yellow. Following a recording session a neuron was iontophoretically injected with the dye and imaged by fluorescence videomicroscopy. Images were stored on videotape for later analysis. They provided a record of the location of the microelectrode recording site. After withdrawal of the microelectrode, slices were processed histologically and imaged a second time with a Bio‐Rad 600 confocal attachment on an Olympus BH‐2 microscope. Confocal images provided detailed anatomical information in three dimensions. In most instances, a clear identification of the recording site was achieved by comparing video images containing the recording electrode and confocal images.Neurophysiological recordings obtained from proximal and distal apical dendrites were markedly different. Proximal dendritic recordings were similar to those obtained from pyramidal cell soma. However, distal dendrites were not electroresponsive when depolarized by intracellular current injection. The techniques described here, or variations that employ patch electrodes, could provide valuable information that should further an understanding of the properties of dendrites in the central nervous system. © 1994 Wiley‐Lis

ISSN:1059-910X    

DOI:10.1002/jemt.1070290408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractStellation is the process by which astrocytes change from epithelial‐like to process‐bearing cells. Stellation occurs following activation of either cyclic AMP‐dependent protein kinase or protein kinase C. This process occurs through tubulin‐dependent rearrangement of the cytoskeleton. We have evaluated the ability of phorbol, 12‐myristate, 13‐acetate (PMA) to induce astrocyte stellation. Astrocytes from five brain regions (cerebellum, cerebral cortex, hippocampus, diencephalon, and brain‐stem) were examined to determine if all astrocytes would exhibit similar responses to this activator of protein kinase C. Stellation was evaluated following cell fixation by either phase optics using conventional light microscop, or scanning laser confocal light microscopy of cultures prepared using immunocytochemistry for tubulin and glial fibrillary acidic protein. Both the number of cells responding to PMA and the sensitivity to PMA varied for astrocytes from each brain region. PMA‐induced stellation was most robust in cerebellar and brainstem astrocytes, with greater than 70% responding. Less than 40% of hippocampal and diencephalic astrocytes responded to PMA at the maximum does (10−5M). PMA also induced different numbers of processes or branching patterns of processes on astrocytes from different brain regions. The protein kinase C induced stellation response in astrocytes supports the hypothesis that astrocytes contribute to neural plasticity. © 19

ISSN:1059-910X    

DOI:10.1002/jemt.1070290409

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

ISSN:1059-910X    

DOI:10.1002/jemt.1070290401

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY