Colloque franco-belge de M.E., mai 1988

COMPARATIVE LOCALIZATION AND QUANTIFICATION OF ELECTROLYTES IN CARDIAC CELLS AFTER CHEMICAL OR RAPID FREEZE FIXATION. A SECONDARY ION MASS SPECTROMETRY STUDY (SIMS) IN CONTROL AND LI-TREATED MICE Marie-C~cile MONY, Josiane AIOUN, Eliane LARRAS-REGARD. INSERM U66 Institut Gustave Roussy 94800 ViIle- ju i f , France et Laboratoire de Biologie Vertebras, Universit~ Paris-Sud, 91400 Orsay, France.

Localization of fundamental electrolytes: Na, Mg, K, Ca has been studied in heart tissue after either chemical or rapid freeze fixation according to Escaig (1). After chemical procedure images were mainly artefactual due to ion diffusion and translocation: intensity of K images was very weak, Ca pictures were also diffused, Na images expressed translocation of Na from Na-cacodyiate buffer and physiological or supraphysiological Li due to chronic treatment was not detected. In order to avoid loss of diffusible ions, the tissue was submitted to rapid freeze fixation and freeze substitution prior to resin embedding. In order to validate the quality of the freeze fixation the tissue was obser- ved by means of electron microscope. The preservation of the structures was good in the f i r s t 10 Pm near the surface of freezing; then small ice crystals appeared, but their size was compatible with the I Wm spatial resolution of the secondary ion mass spectrometry (SIMS) instrument. The multielemental analysis was performed in an even area of tissue sections. Elemental distributions were either super- imposed or complementary at nuclear and cytoplasmic levels. Relative quantification evidenced two popu- lations of cells according to their Ca concentration. Moreover the method allowed imaging of Li in treated animals. NapMg, K, Ca distributions were not affected by the treatment. Measurements demonstra- ted undesired suspected effects of Li on electrolyte balance: a significant decrease of Na and K con- centrations as well as an increase of Ca concentration were observed in heart fibers whereas Mg concen- tration was not modified. Disorders of electrolyte concentrations in myocytes after lithium chronic treatment are in agreement with secondary effects of the metal reported in cardiology. SIMS applied to rapid frozen and freeze substituted tissues offers a new appraisal for understanding therapeutic and secondary effects of drugs on cellular and subcellular targets. (1) ESCAIG J., J. Microsc. 126, 221-229 (1982)

X-RAY ~ICROANALYSIS OF CALCIUM-CONTAINING GLIAL GRANULES AND ESTIMATION OF THE EXTRACELLULAR SPACE IN APLYSIA FREEZE-SUBSTITUTED GANGLIA. Erick KEICHER and Ghislain NICAISE. Laboratoire de Cytologie Exp~riment~le, Universit~ de Nice, Pare V~Irose, 0605# Nice (France).

This study concerns the granule-containing glial cells surrounding the giant somata (R2 and LPII) of Ap~ysla. Such granules are more often found in marine than in freshwater mollusks and may represent a calcium store used to compensate for the excess Na + in the extracellular milieu of marine species (see 1). To test this hypothesis, the calcium content of Aplysia californica glial granules was measured by X-ray microanalysis, usi~; 9 the Hall method, in a Camebax-TEM system equipped with a wavelength-dispersive spectrometer and an energy-dispersive detector Tracor TN 2000, after rapid freezing in liquid propane, cryosubstitution in presence of oxalic acid, and resin embedding (2). The results show that in glial granules there is an effective calcium store, which varies from low calcium concentrations (3 mmoles/kgof epon-~mbedded tissue) to more than 90 mM. This may be indicative of different loading ~tates of the glia] granules. In addition to Ca, glial granule analysis shows the presence of phosphorus, sulphur, and psrticularly chlorine.

The extracellular space was estimated by a point latt ice method, using electron micrographs of conventionally stained thin sections. In freeze-substituted ganglia, i t ~epresents 40 % (± 2, s.e.m., n = 33) of the perlneuronal volume This value can be compared to that obtoined with chemically fixed ganglia (26 % ~ 0.7, n = l l l i ; the shrinking of the extraceIlular spa~ is reminiscent of that observed in Vertebrates and could be due to glia] oedema during fixation.

I t can be speculated that the calcium content of the glia] granules could raise the concentration of the extracellular space by l mM.

(1) Julllard A.K. and Nicaise G., Neurosclence 13, 1387-1396 (1984). (2) Blalneau S., Julllard A.K., Amsellem J. and Nicaise G., Histochem. 87, 545-555 (1987).

QUANTITATIVE X-RAY MICROANALYSIS OF CALCIUM IN SEA URCHIN EGGS AFTER QUICK-FREEZING AND CRYOSUBSTITUTION : VALIDITY OF THE METHOD. Isabel]e GILLOT (1), Brigitte CIAPA (2), Guy DE RENZlS (2), Patrick PAYAN (2), Christian SARDET (1) and Ghislain NICAISE (3). (I) Biologie Cellulaire Marine, CEROV, 06230 Villefranche 8or Met (France), (2) Physiologie Cellu~aire et Comparde, and (3) Cytologie Exp~rimenta~e, Universit~de Nice, 06054 Nice (France).

Several authors have used freeze-substitution to study the distribution of calcium, not only in plant (see I) but also in animal cells (I-4). However, the validity of the technique has been tested on only a few cell types or organelles.

We tested the fate of both total and exchangeable calcium on the sea urchin (Paracentrotus lividus) egg after the various treatments needed for freeze-substitution. We compared the total Ca content of the egg by X-ray microana]ysis of epon-embedded sections of freeze-substituted eggs (6.2 ± 0.7 mmoles/ kg of epon-embedded tissue) and by destructive spectrophotometric analysis of fresh eggs (32.3 ~ 1.3 nmoles/mg of protein), taking into account a water content of 53 %. Eggs stuck on aluminium foil were frozen in liquid propane (see 4) and freeze-substituted at -g2°c for 4 days in ethanol containing molecular sieve and IO to 20 mM oxalic acid. After progressive warming, the eggs were embedded in epon. X-ray microanalysis was performed with the Hall method in a Philips CMI2 transmission micros- cope equipped with a Tracor TN 5400 analyzer ; a probe of 5 ~m was used on 150 nm thick sections collected rapidly on water with formvar-coated copper grids. After loading with 45Ca, the activity of the eggs and of the substitution f luid were measured at the different steps of the method.

The exchangeable 45Ca as well as the total Ca appear practically unaffected by the treatment ; although a possible redistribution of Ca inside the egg was not directly tested, the preparative technique can be considered appropriate for our material.

(1) Harvey D.M.R., J. Microsc. (London) 127, 209-221 (]982). (2) Geyer G., HalbhUber K.J. and Benser A., Acta histochem. 48, 257-26] (]974). (3) Ornberg R.L. and Reese T.S., Fed. Proc. 39, 2802-2808 (]980). (4) B1aineau S., Julliard A.K., Amsellem J. and Nicaise G., Histochem. 87. 545-555 (1987).