Biomedical subjects

J C Fiala

Publications and source records attributed to J C Fiala.

7 recordsLinked to original sources

PubMed2005

Reconstruct: a free editor for serial section microscopy.

Many microscopy studies require reconstruction from serial sections, a method of analysis that is sometimes difficult and time-consuming. When each section is cut, mounted and imaged separately, section images must be montaged and realigned to accurately analyse and visualize the three-dimensional (3D) structure. Reconstruct is a free editor designed to facilitate montaging, alignment, analysis and visualization of serial sections. The methods used by Reconstruct for organizing, transforming and displaying data enable the analysis of series with large numbers of sections and images over a large range of magnifications by making efficient use of computer memory. Alignments can correct for some types of non-linear deformations, including cracks and folds, as often encountered in serial electron microscopy. A large number of different structures can be easily traced and placed together in a single 3D scene that can be animated or saved. As a flexible editor, Reconstruct can reduce the time and resources expended for serial section studies and allows a larger tissue volume to be analysed more quickly.

Animals↗

PubMed2004

Dendritic spines disappear with chilling but proliferate excessively upon rewarming of mature hippocampus.

More dendritic spine synapses occur on mature neurons in hippocampal slices by 2 h of incubation in vitro, than in perfusion-fixed hippocampus. What conditions initiate this spinogenesis and how rapidly do the spines begin to proliferate on mature neurons? To address these questions, CA1 field of the hippocampus neurons expressing green fluorescent protein in living slices from mature mice were imaged with two-photon microscopy. Spines disappeared and dendrites were varicose immediately after slice preparation in ice-cold artificial cerebrospinal fluid (ACSF). Electron microscopy (EM) revealed disrupted dendritic cytoplasm, enlarged or free-floating postsynaptic densities, and excessive axonal endocytosis. Upon warming dendritic varicosities shrank and spines rapidly reappeared within a few minutes illustrating the remarkable resilience of mature hippocampal neurons in slices. When membrane impermeant sucrose was substituted for NaCl in ACSF dendrites remained spiny at ice-cold temperatures and EM revealed less disruption. Nevertheless, spine number and length increased within 30 min in warm ACSF even when the extracellular calcium concentration was zero and synaptic transmission was blocked. When slices were first recovered for several hours and then chilled in 6 degrees C ACSF many spines disappeared and the dendrites became varicose. Upon re-warming varicosities shrank and spines reemerged in the same position from which they disappeared. In addition, new spines formed and spines were longer suggesting that chilling, not the initial injury from slicing, caused the spines to disappear while re-warming triggered the spine proliferation on mature neurons. The new spines might be a substrate for neuronal recovery of function, when neurons have been chilled or exposed to other traumatic conditions that disrupt ionic homeostasis.

Animals↗

PubMed2001

Cylindrical diameters method for calibrating section thickness in serial electron microscopy.

UNLABELLED: An estimate of section thickness is required for measuring structures in serial section microscopy. Mean section thickness is estimated reliably by averaging the ratios of the diameters of cylindrical objects, such as mitochondria, to the number of sections they span. This cylindrical diameters method improves the accuracy of section thickness as inferred from the colour of sections floating in water. The cylindrical diameters method gives the same answer as that obtained by the minimal folds METHOD: It is preferable because it can be done in a series that has no folds that can distort and obscure the objects that are being measured.

Animals↗

PubMed1998

Synaptogenesis via dendritic filopodia in developing hippocampal area CA1.

To determine the role of dendritic filopodia in the genesis of excitatory synaptic contacts and dendritic spines in hippocampal area CA1, serial section electron microscopy and three-dimensional analysis of 16 volumes of neuropil from nine male rat pups, aged postnatal day 1 (P1) through P12, were performed. The analysis revealed that numerous dendritic filopodia formed asymmetric synaptic contacts with axons and with filopodia extending from axons, especially during the first postnatal week. At P1, 22 +/- 5.5% of synapses occurred on dendritic filopodia, with 19 +/- 5.9% on filopodia at P4, 20 +/- 8.0% at P6, decreasing to 7.2 +/- 4.7% at P12 (p < 0.02). Synapses were found at the base and along the entire length of filopodia, with many filopodia exhibiting multiple synaptic contacts. In all, 162 completely traceable dendritic filopodia received 255 asymmetric synaptic contacts. These synapses were found at all parts of filopodia with equal frequency, usually occurring on fusiform swellings of the diameter. Most synaptic contacts (53 +/- 11%) occurred directly on dendritic shafts during the first postnatal week. A smaller but still substantial portion (32 +/- 12%) of synapses were on shafts at P12 (p < 0.036). There was a highly significant (p < 0.0002) increase in the proportion of dendritic spine synapses with age, rising from just 4.9 +/- 4.3% at P1 to 37 +/- 14% at P12. The concurrence of primarily shaft and filopodial synapses in the first postnatal week suggests that filopodia recruit shaft synapses that later give rise to spines through a process of outgrowth.

Aging↗

PubMed1998

Critical assessment of the involvement of perforations, spinules, and spine branching in hippocampal synapse formation.

Several studies propose that long-term enhancement of synaptic transmission between neurons results from the enlargement, perforation, and splitting of synapses and dendritic spines. Unbiased analyses through serial electron microscopy were used to assess the morphological basis for synapse spilitting in hippocampal area CA1. Few perforated synapses and almost no split (i.e., branched) spines occurred at postnatal day 15, an age of high synaptogenesis; thus, synapse splitting is unlikely to be important during development. The synapse splitting hypothesis predicts an intermediate stage of branched spines with both heads sharing the same presynaptic bouton. Ninety-one branched dendritic spines were traced through serial sections, and the different branches never synapsed with the same presynaptic bouton. Projections from spines, called "spinules," have been thought to extend from perforations in the postsynaptic density (PSD), thereby dividing the presynaptic bouton. Forty-six spinules were traced, and only 13% emerged from perforations in the PSD. Most spinules emerged from the edges of nonperforated PSDs, or from spine necks, where they extended into boutons that were not presynaptic to the spine. In summary, these morphological characteristics are inconsistent with synapse and spine splitting. An alternative is discussed whereby perforated synapses and spinules are transient components of synaptic activation, and branched spines appear from synapses forming in close proximity to one another.

Animals↗

PubMed1996

Metabotropic glutamate receptor activation in cerebellar Purkinje cells as substrate for adaptive timing of the classically conditioned eye-blink response.

To understand how the cerebellum adaptively times the classically conditioned nictitating membrane response (NMR), a model of the metabotropic glutamate receptor (mGluR) second messenger system in cerebellar Purkinje cells is constructed. In the model, slow responses, generated postsynaptically by mGluR-mediated phosphoinositide hydrolysis and calcium release from intracellular stores, bridge the interstimulus interval (ISI) between the onset of parallel fiber activity associated with the conditioned stimulus (CS) and climbing fiber activity associated with unconditioned stimulus (US) onset. Temporal correlation of metabotropic responses and climbing fiber signals produces persistent phosphorylation of both AMPA receptors and Ca(2+)-dependent K+ channels. This is responsible for long-term depression (LTD) of AMPA receptors. The phosphorylation of Ca(2+)-dependent K+ channels leads to a reduction in baseline membrane potential and a reduction of Purkinje cell population firing during the CS-US interval. The Purkinje cell firing decrease disinhibits cerebellar nuclear cells, which then produce an excitatory response corresponding to the learned movement. Purkinje cell learning times the response, whereas nuclear cell learning can calibrate it. The model reproduces key features of the conditioned rabbit NMR: Purkinje cell population response is timed properly; delay conditioning occurs for ISIs of up to 4 sec, whereas trace conditioning occurs only at shorter ISIs; mixed training at two different ISIs produces a double-peaked response; and ISIs of 200-400 msec produce maximal responding. Biochemical similarities between timed cerebellar learning and photoreceptor transduction, and circuit similarities between the timed cerebellar circuit and a timed dentate-CA3 hippocampal circuit, are noted.

Animals↗

PubMedDate not supplied

Extending unbiased stereology of brain ultrastructure to three-dimensional volumes.

OBJECTIVE: Analysis of brain ultrastructure is needed to reveal how neurons communicate with one another via synapses and how disease processes alter this communication. In the past, such analyses have usually been based on single or paired sections obtained by electron microscopy. Reconstruction from multiple serial sections provides a much needed, richer representation of the three-dimensional organization of the brain. This paper introduces a new reconstruction system and new methods for analyzing in three dimensions the location and ultrastructure of neuronal components, such as synapses, which are distributed non-randomly throughout the brain. DESIGN AND MEASUREMENTS: Volumes are reconstructed by defining transformations that align the entire area of adjacent sections. Whole-field alignment requires rotation, translation, skew, scaling, and second-order nonlinear deformations. Such transformations are implemented by a linear combination of bivariate polynomials. Computer software for generating transformations based on user input is described. Stereological techniques for assessing structural distributions in reconstructed volumes are the unbiased bricking, disector, unbiased ratio, and per-length counting techniques. A new general method, the fractional counter, is also described. This unbiased technique relies on the counting of fractions of objects contained in a test volume. A volume of brain tissue from stratum radiatum of hippocampal area CA1 is reconstructed and analyzed for synaptic density to demonstrate and compare the techniques. RESULTS AND CONCLUSIONS: Reconstruction makes practicable volume-oriented analysis of ultrastructure using such techniques as the unbiased bricking and fractional counter methods. These analysis methods are less sensitive to the section-to-section variations in counts and section thickness, factors that contribute to the inaccuracy of other stereological methods. In addition, volume reconstruction facilitates visualization and modeling of structures and analysis of three-dimensional relationships such as synaptic connectivity.

Algorithms↗