The localization and assay of chemical elements by microprobe methods

Hall, T. A.; Gupta, Brij L.

doi:10.1017/s0033583500005114

Cited by 89 publications

(35 citation statements)

References 86 publications

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“…Spectra were recorded at Ϫ160°C or below. Data were processed and quantified by established procedures (Shuman et al, 1976;Hall and Gupta, 1983;K itazawa et al, 1983;Buchanan et al, 1993) (Hall and Gupta, 1983) provides these data directly. Concentrations in millimoles per liter of original wet volume, which are more intuitively usef ul in physiology, can be estimated by multiplying dry weight concentrations by the dry mass fraction of analyzed organelles; typical values are 15, 25, and 40% dry mass for cytoplasm, ER, and mitochondria, respectively (Buchanan et al, 1993).…”

Section: Methodsmentioning

confidence: 99%

“…As a first step in establishing the roles of, as well as the interplay between, each of these mechanisms in dendritic Ca 2ϩ handling, we have determined the amount and location of intracellular C a in proximal dendrites of CA3 pyramidal cells in hippocampal slice cultures, both at rest and at defined times after neuronal activity. These experiments were performed by exploiting the high analytical sensitivity and subcellular resolution of modern (Leapman and Andrews, 1991;Buchanan et al, 1993;Andrews et al, 1994) energy-dispersive x-ray (EDX) microanalysis (Shuman et al, 1976;Hall and Gupta, 1983), a technique that-in distinction to imaging with Ca 2ϩ -sensitive fluoresecent dyes-provides direct and quantitative measurements of the concentration of total C a within identified subcellular compartments.…”

Section: Abstract: Calcium Regulation; Calcium Sequestration; Hippocmentioning

confidence: 99%

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Activity-Dependent Calcium Sequestration in Dendrites of Hippocampal Neurons in Brain Slices

Leapman

et al. 1997

J. Neurosci.

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Synaptic activity-dependent changes in the spatio-temporal distribution of calcium ions regulate important neuronal functions such as dendritic integration and synaptic plasticity, but the processes that terminate the free Ca 2ϩ transients associated with these changes remain unclear. We have characterized at the electron microscopic level the intracellular compartments involved in buffering free Ca 2ϩ transients in dendritic cytoplasm of CA3 neurons by measuring the larger changes in the concentrations of total Ca that persist for several minutes after neuronal activity. Quantitative energy-dispersive x-ray microanalysis of cryosections from hippocampal slice cultures rapidly frozen 3 min after afferent synaptic activity identified a subset of dendritic endoplasmic reticulum (ER) as a highcapacity Ca 2ϩ buffer. Calcium sequestration by cisterns of this subset of ER was graded, reversible, and dependent on a thapsigargin-sensitive Ca 2ϩ -ATPase. Sequestration was so robust that after repetitive high-frequency stimulation the Ca content of responsive ER cisterns increased as much as 20-fold. These results demonstrate that a subpopulation of ER is the major dendritic Ca sequestration compartment in the minutes after neuronal activity.

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Section: Methodsmentioning

confidence: 99%

Section: Abstract: Calcium Regulation; Calcium Sequestration; Hippocmentioning

confidence: 99%

Activity-Dependent Calcium Sequestration in Dendrites of Hippocampal Neurons in Brain Slices

Leapman

et al. 1997

J. Neurosci.

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“…The sections were analyzed either by EPMA, in the spot mode, or by x-ray mapping. The instrumentation and methods for EPMA, including calibration, have been published (20)(21)(22)(23)(24).…”

Section: Methodsmentioning

confidence: 99%

Electron probe microanalysis of calcium release and magnesium uptake by endoplasmic reticulum in bee photoreceptors.

Baumann

Walz

Somlyo

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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Honey bee photoreceptors contain large sacs of endoplasmic reticulum (ER) that can be located unequivocally in freeze-dried cryosections. The elemental composition of the ER was determined by electron probe x-ray microanalysis and was visualized in high-resolution x-ray maps. In the ER of dark-adapted photoreceptors, the Ca concentration was 47.5 ± 1.1 mmol/kg (dry weight) (mean ± SEM). During a 3-sec nonsaturating light stimulus, -50% of the Ca content was released from the ER. Light stimulation also caused a highly significant increase in the Mg content of the ER; the ratio ofMg uptake to Ca released was -0.7. Our results show unambiguously that the ER is the source of Ca2+ release during cell stimulation and suggest that Mg2+ can nearly balance the charge movement of Ca2+.The mobilization of Ca2" from an intracellular store by a reaction cascade comprising a cell-surface receptor, a guanine nucleotide binding protein, a phospholipase C, and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] is an important mechanism of signal transduction (1). A great deal of evidence indicates that the endoplasmic reticulum (ER) is the target of Ins(1,4,5)P3 action (2-5) and the organelle involved in the physiological regulation of cytoplasmic Ca2" in nonmuscle cells (6-8), although some authors proposed that the Ins(1,4,5)P3-sensitive Ca2" pool is located in a separate organelle ("calciosome") (9,10). Direct measurements ofthe Ca2+ content and of Ca2+ changes in the ER (or the calciosomes) have been difficult to obtain. Consequently, little is known about the ion movements that must occur during Ca2+ release, to maintain charge balance across the ER membrane. The large sacs of ER in bee photoreceptors (11) are uniquely suitable for addressing these questions because the size makes them clearly identifiable in ultrathin freeze-dried cryosections and allows unambiguous experiments in which the analyzed area is completely within the ER cisternae. Therefore, we determined by electron probe x-ray microanalysis (EPMA) the elemental composition of the ER in situ, in the dark and during light stimulation, that, in invertebrates, is known to be followed by Ins(1,4,5)P3-mediated (12, 13) intracellular Ca2' release (14,15). We quantitated the amount of Ca2' released from the ER during photoresponse and demonstrate an associated uptake of Mg2' into the ER, suggesting that Mg2+ movements make a major contribution toward maintaining electroneutrality during Ca2' release from the ER in bee photoreceptors. Preliminary reports of these findings have been published (16,17). MATERIALS AND METHODSHoney bee drones (Apis mellifera) were kept in the dark at 32°C to 36°C for up to 10 days and fed a concentrated sugar solution. Decapitation and all subsequent manipulations were done under red light (A > 600 nm), which elicits no electrical response of the photoreceptors (18). Thick slices (600-1000 Am) ofthe drone head (19) remained in oxygenated physiological saline containing 170 mM NaCl, 10 mM KCI, 10 mM MgCl2, 1.6 mM CaC12, 10 mM Tris-HCl (pH ...

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“…The theory, instrumentation, and data reduction methods have been described, both in general (21,26,27) and as practiced in our laboratories (22,28).…”

Section: Methodsmentioning

confidence: 99%

Activity-dependent accumulation of calcium in Purkinje cell dendritic spines.

Andrews

Leapman

Landis

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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The calcium content of synapses of parallel fibers on Purkinje cell dendritic spines was determined by electron probe x-ray microanalysis of freeze-dried cryosections from directly frozen slices of mouse cerebellar cortex. In fresh slices frozen within 20-30 sec of excision, calcium concentrations ranging from 0.8 to 18.6 mmol/kg of dry weight were measured in cisterns of smooth endoplasmic reticulum within Purkinje cell dendritic spines. The average calcium content of spine cisterns in rapidly excised slices (6.7 ± 0.6 mmol/kg of dry weight + SEM) was higher than the average calcium content of spine cisterns in brain slices incubated without stimulation for 1-2 hr before direct freezing (2.5 ± 0.4 mmol/kg of dry weight). Depolarization of incubated cerebellar slices by isotonic 55 mM KCI resulted in the accumulation within spine cisterns of very high amounts of calcium or isotonically substituted strontium, both derived from the extracellular fluid. These results suggest that one function of spine cisterns is to sequester free calcium that enters the spine through ligand-gated or voltage-gated channels during synaptic transmission.Dendritic spines are characteristic features of many classes of neurons in the central nervous system. Although the function of dendritic spines is still uncertain (1-4), several lines of evidence suggest that changes in the amount and distribution of intracellular calcium may accompany synaptic activity at spines (5). Thus, recent studies on hippocampal, striatal, and spinal cord neurons show that the N-methyl-D-aspartate (NMDA)-sensitive subtype of glutamate receptor (6-10)-a receptor thought to be present on several kinds of spines (11)-includes a calcium channel (12)(13)(14) and that ligand binding to such receptors results in calcium influx and an increase in free cytoplasmic calcium (15). Significant changes in the calcium concentration of dendritic spines might also be expected on the basis of voltage-dependent calcium channel activity (16).Because spiny neurons often exhibit long-term modulation of synaptic efficacy (17), it has been suggested that spines are the anatomical locus of long-term modulation and that these effects are mediated by calcium ions (5, 14). In the cerebellum, parallel fiber synapses on Purkinje cell dendritic spines are subject to use-dependent effects specifically mediated by glutamate receptors (18) that, in this instance, are quisqualate-selective and have a conductance state similar to the large (40-50 pS), calcium-permeable state of the hippocampal receptor (14,19). Moreover, cerebellar parallel fiber synapses are well-suited to structural studies because they can be directly frozen, undamaged and intact, without prior fixation (20). The accessibility of intact synapses to direct freezing also makes this preparation available to quantitative electron probe x-ray microanalysis (21), a method that can accurately measure the content of calcium and other elements in unfixed, directly frozen tissue at high spatial resolution.The results of such e...

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The localization and assay of chemical elements by microprobe methods

Cited by 89 publications

References 86 publications

Activity-Dependent Calcium Sequestration in Dendrites of Hippocampal Neurons in Brain Slices

Activity-Dependent Calcium Sequestration in Dendrites of Hippocampal Neurons in Brain Slices

Electron probe microanalysis of calcium release and magnesium uptake by endoplasmic reticulum in bee photoreceptors.

Activity-dependent accumulation of calcium in Purkinje cell dendritic spines.

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