Synaptic plasticity and gravity: Ultrastructural, biochemical and physico-chemical fundamentals

63—(l)72, 1992 Adv. Space Res. Vol. 12,No. l,pp.(l) Printed in Great Britain. All rights reserved.

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SYNAPTIC PLASTICITY AND GRAVITY: ULTRASTRUCTURAL, BIOCHEMICAL AND PHYSICO-CHEMICAL FUNDAMENTALS H. Rahmann, K. Slenzka, K. H. Kortje and R. Hilbig Zoological Institute, University of Stuttgart-Hohenheim, D-7000 Stuttgart 70 (Hohenheim), Germany

ABSTRACT On the basis of quantitative disturbances of the swimming behaviour of aquatic vertebrates (loop-swimming in fish and frog larvae) following long-term hyper-g-exposure the question was raised whether or not and to what extent changes in the gravitational vector might influence the CNS at the cellular level. Therefore, by means of histological, histochemical and biochemical analyses the effect of 2-4 x g for 9 days on the gross morphology of the fish brain, and on different neuronal enzymes was investigated. In order to enable a more precise analysis in future-ii~-experimentsof any gravity-related effects on the neuronal synapses within the gravity-perceptive integration centers differentiated electron-microscopical and electronspectroscopical techmques have been developed to accomplish an ultrastructural localization of calcium, a high-affinity Ca2+~ATPase,creatine kinase and cytochrome oxidase. In hyper-g animals vs. 1-g controls, a reduction of total brain volume (15 %), a decrease in creatine kinase activity (20 %), a local increase in cytochrome oxidase activity, but no differences in Ca2~/Mg2~-ATPase activities were observed. Ultrastructural peculiarities of synaptic contact formation in gravity-related integration centers (Nucleus magnocellularis) were found. These results are discussed on the basis of a direct effect of hyper-gravity not only on the gravitysensitive neuronal integration centers but possibly also on the physico-chetnical properties of the lipid bilayer ofneuronal membranes in general. INTRODUCTION Since the dawn of life on earth gravity has been a more or less stable factor influencing the nhylogenetic development of all living organisms. Modem technology and gravity-relatedexperiments (space flights, parabolic aircraft flights, clinostat etc.), however, enable researchers to study the ways in which alterations in gravity also influence the early ontogenetic develonment and finally the behaviour of living beings. This kind of investigations was focused so far primarily on the vestibular organs and were inspired by frequent reports by US astronauts and USSR cosmonauts of space-sickness in flight subsequent to changes in gravity /1/. Since, however, significant alterations in the organization of the vestibular organs in response to gravitational changes had been found neither in humans nor in animals, research interest now concentrates on the analysis of the central nervous system (CNS). In this regard, basic neurobiological investigations are urgently needed in order to analyze the effect of gravitational changes on all levels of neuronal activities, from behaviour to any possible functional and structural reactions within the gravity-related, higher neuronal integration centers. Dramatic failures take place concerning behaviour, brain morphology, histochemistry, ultrastructure and biochemistry as a result of long-term environmental disturbances /2-19/. If these disturbances occur during critical developmental phases for relative brief periods only, or if there is any loss ofsensory input, the organism is able to compensate to a certain degree by activating related structures /5,10,14,18,19/. This compensatory phenomenon is referred to as neuronal Dlasticitv. In aquatic vertebrates (fishes and amphibians) changes in the gravitational vector were found to induce significant and long-lasting alterations in the swinuning behaviour (“loop swimming”), /2,4,9,11,12,13,16,20/. It was concluded that gravity is of great importance, especially during the phase of ontogenetic development, for the differentiation not only of the vestibular system within the inner ear but especially also for the differentiation of neuronal networks within the gravity-related integration centers in the CNS. On this background, in preparation of the planned micro-gravity-related STATEX-experiments to be carried Alit

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respectively, which are significant for future analysis and interpretation of gravitational effects on the altered behaviour of vertebrates. METHODOLOGY Animals Cichlid-fish larvae (Oreochromis mossambicus; 2 days after hatching = stage 14 at 20°C)and clawed toad larvae (Xenopus laevis; stage 14 = neurula stage) were kept as animal models for gravity research in miniaquaria (Petriperm cell culture vessels, Haereus, FRG), 5 animals in each. Sibling larvae were and randomly separated into control and hyper-g groups. The hyper-g group was centrifuged at a 2.4 x g level for 9 days, while control animals were kept under identical conditions within the centrifuge but at normal 1-g force. Analysis of Swimming Behaviour Immediately after each session of centrifugation and later on during habituation to 1-g each day, the animals were transferred to the motility registration system for quantitative analysis of swimming behaviour. The registration system consisted of a computer-based video analyzer (Videotrack 512 II, Lyonaise Electronique Intern., France) with a frame sample rate of 20 msec. The distance and duration of swimming movements were registered in test periods of 5 minutes each. Three velocity ranges were defined by setting individual thresholds: fast swimming = about 15 mm/sec.; slow swimming = between 15 and 0,2 mm/sec.; and inactivity = < 0,2 mm/sec. Additionally the spatial distribution of the animals in the mini-aquaria (center vs. wall-contact) were observed and registered quantitatively by window computing. The data obtained were statistically evaluated by Student’s t-test and the average and standard deviations were calculated. Histological-Histochemical Preoaration Immediately following each centrifugation session the animals were decapitated, the brains were quickly removed and divided for histological, electron microscopic and biochemical analyses. For histological studies entire larvae were fixed in Bouin’s fixative, dehydrated with ethanol, embedded in methaciylate resin (Historesin, LKB/Bromma, Sweden), cut into 3 ~.imsections and stained with toluidine blue. In control animals afferent nerve fibers from the inner ear were labelled with the enzyme horseradish peroxidase (HRP) after sectioning of the octaval nerve /21/ in order to precisely localize the ~gravity-re1atedregion of the brainstem.For histochemical analyses brains were deep-frozen (isopentan -80’~C)ciyocut into 12 ~&m sections and stained for measurements of cytochrome oxidase activity according to Wong-Riley /22/.Quantitative analyses were performed planimetrically using a computer-based image analysis system (Gesotec, FRG) and the volume of the entire brain and brain regions was calculated. Electron Microsconic Procedures Brains were fixed in paraformaldehyde/glutaraldehyde/Sorensen phosphate-buffer and prepared for stanwas performed dard analyses. Localization of calcium of ausing high-affinity Ca2~-A1Pase using ultrastructural the newly developed procedures of Körtje et al. and /23,24/ energy-filtering electronmicroscopy in combination with energy-loss spectroscopy (Zeiss CEM 902, FRG). Ultracytochemical detection of cytochrome oxidase and creatine kinase was performed as per Wong-Riley /22/ and Biermans et al. /25/. Biochemical Analysis Brains were transferred immediately after dissection to a 10 mM Tris/HC1-buffer, pH 7,4, ultrasonically homogenized and deep-frozen at -80°C.Ca2+~ATPase~assay was adapted for brain tissue as described by Slenzka et al./26/. A creatine kinase assay was adapted for brain tissue as per Oliver /27/ and Nielsen and Ludvigsen /28/. Phvsico-Chemical Procedures Monolayers of various membrane lipids (PC-phosphatidylcholine, PS-phosphatidylserine, G~ 1~-tri-sialoganglioside, etc.) were formed at the air/water interface of a Teflon trough. The surface pressure was measured by a Wilhelmi balance, and the surface potentials were measured with a vibrating plate condensor as previously described /29/ RESULTS Influence of Hvoer-g on the Swimming Behaviour of Fish and Toad Larvae By means of video track imaging a significant altered swimming pattern can be registered immediately after a 15 day-old cichlid fish larvae had been transferred to 1-g conditions following a centrifugation period of 9

Synaptic Plasticity and Gravity

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Fig. 1. Influence of hyper-gravity on the swimming behaviour of 15 days old cichlid fish (A) and clawed toad larvae (B) in comparison to the respective 1-g controls (A resp. B). Representative motility patterns were registered during a 5 mm interval. locally at the wall of the mini-aquaria. In contrast, the hyper-g animals swam and looped nearly randomly throughout the aquarium, however, gliding for some time slowly along the wall of the aquarium. The quantitative evaluation of the swimming behaviour of the hyper-g (9 days) vs. 1-g controls revealed significant differences in duration of movements, in swimming distances and, thus resulting in velocity (Fig. 2 a,b). This was due to all three different speed classes (fast, slow, inactive). In both fish and toad larvae, the swimming distance per minute in hyper-g animals was increased significantly. In contrast, the duration of movements was influenced considerably only in the fish larvae, in which the time spent swimming fast and slowly was increased relative to the intervals ofinactivity (Fig. 2a). A. Duration of Movements / mm

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