Long-term changes in the activity of Purkinje cells and efferent cerebellar neurons following bilateral destruction of the inferior olive

The spontaneous discharge frequency of Purkinje cells and neurones of the cerebellar nuclei was evaluated in rats after complete bilateral destruction of their inferior olive with 3-acetylpyridine, performed one day to six months before. The deafferentation from the climbing fibers produced an increased inhibitory action of the Purkinje cells on their target neurones, lasting at least for one week. A relative compensation took place progressively during the first month, but the normal activity of the circuit did not recover even after six months.     

A stereotaxic atlas and implantation technique for nuclei of the diencephalon of Atlantic salmon (Salmo salar) parr.

A stereotaxic apparatus and technique for its implantation in diencephalic nuclei of Atlantic salmon parr of 20 to 30 g body weight is described. An atlas of nuclei in the diencephalon is also presented.     

Structure and activity of proteins from pathogenic fungi Phytophthora eliciting necrosis and acquired resistance in tobacco

The phytopathogenic fungi Phytophthora cryptogea and Phytophthora capsici cause systemic leaf necrosis on their non-host tobacco; in culture they release proteins, called cryptogein and capsicein, which elicit similar necrosis. In addition, both proteins protect tobacco against invasion by the pathogen Phytophthora nicotianac, the agent of the tobacco black shank, that is unable to produce such an elicitor. Cryptogein causes visible leaf necrosis starting at about 1 microgram/plant, whereas 50-fold as much capsicein is required for the same reaction. Capsicein induces protection even in near absence of leaf necrosis. The activities of both elicitors are eliminated upon pronase digestion. They are proteins of similar Mr (respectively 10,323 and 10,155) and their complete amino acid sequences were determined. They consist of 98 residues, with some internal repetitions of hexapeptides and heptapeptides. 85% identity was observed between both sequences: only two short terminal regions are heterologous, while the central core is entirely conserved. Secondary structure predictions, hydropathy and flexibility profiles differ only around position 15 and at the C-terminus; these modifications could play a role in the modulation of their biological activities. After a search of the sequence data bases, they appear to be novel proteins.     

Spermiogenesis in the rainbow trout (Salmo gairdneri)

In an ultrastructural study on the spermiogenesis of the rainbow trout (Salmo gairdneri R.) four spermatogenetic stages were identified. In young round spermatids, the nuclear chromatin was first heterogeneous (euchromatin and heterochromatin). Subsequently, it became more homogeneous and started to condense in the form of coarse granules and fibers and then into fibrils associated in ribbon-like elements which eventually partly fused together. During early spermiogenesis, a juxtanuclear vacuole appeared in the area where the nuclear envelope was specialized due to condensation of material between the two envelopes and a slight accumulation of nuclear material. This area was finally located in the anterior part of spermatids and spermatozoa; it probably plays a role during fertilization. A flagellar rootlet appeared early in spermiogenesis; it may play a role in the attachment of the flagellum to the nucleus since it persisted until the centriolar complex was definitively fixed in the implantation fossa. The flagellum did not display a plasma membrane and was first located in the cytoplasm, but when it was later extruded from the cell, it acquired a membrane. The cytoplasm was rich in ribosomes (free or in small groups) but poor in membranous organelles. The few mitochondria polarized around the centriolar complex were finally organized into an annular mid-piece. The spermatids remained connected by intercellular bridges until the end of spermiogenesis. The complexity of trout spermiogenesis is intermediate between that in poecilids and that in carp and pike, which have very simple spermatozoa. The role of the material from the nucleus and the cytoplasm reaching the Sertoli cell in the control of spermatogenesis has been discussed.     

Ionic regulation of the plasma membrane potential of rainbow trout (Salmo gairdneri) spermatozoa: role in the initiation of sperm motility

The ionic dependence of the trout sperm plasma membrane potential was analysed by measuring the accumulation of the lipophilic ions 3H-tetraphenylphosphonium (TPP) and 14C-thiocyanate (SCN) following dilution in artificial media isotonic to the seminal fluid. Our data showed that the trout sperm plasma membrane has a mixed conductance: the plasma membrane potential is sensitive upon the transmembrane gradients of K+, Na+, and H+. This potential is negative (less than -40 mV) in a 125 mM choline chloride media (ChM) at pH 8.5. Replacement of choline by sodium has a small depolarizing effect. The membrane potential is about -15 mV in a 125 mM potassium chloride and falls near zero mV only if valinomycin is added. In ChM changing the external pH (pHe) greatly affects the membrane potential: its value rises from less than -40 mV at pHe 9.0 to -17 mV at pHe 5.0. This pH effect is observed also in presence of sodium or potassium. A decrease in the transmembrane proton gradient produced by increasing internal pH without changing pHe induces also a depolarisation of the plasma membrane. In the different media in which trout sperm remain immotile after dilution (media with [K+] greater than 20-40 mM or a pH less than 7.5) the plasma membrane is more depolarized than in media allowing motility, suggesting a relationship between the state of membrane polarization and the intracellular effectors of the axonemal movement.     

Darkness improves growth and delays necrosis in a nonchlorophyllous habituated sugarbeet callus: Biochemical changes

The transfer of light-cultured green normal (N) and white habituated (HNO) sugarbeet callus to darkness reduced the growth of N callus and improved growth and delayed necrosis in the HNO callus. The decrease of dry matter of N callus under darkness was accompanied by a reduced content of carotenoids and by decreased CO₂ fixation, which was compensated by an increased dependency on externally supplied sucrose. The levels of some organic nitrogen compounds such as glutamate, proline, and free polyamines were not affected by transfer to darkness of N or HNO callus. Darkness decreased ethylene emissions in both callus types. In the HNO callus, the sucrose growth dependency and the CO₂ fixation were unaffected by darkness. Chlorophylls were absent both in light and darkness, whereas some carotenoids were accumulated in the HNO callus only in dark conditions. In another connection, a significant increase of peroxidase activity, which did not occur in the N callus, was induced by darkness in the HNO callus. A decreased content of thio-barbituric acid (TBA)-reactive substances was measured in the HNO callus transferred to darkness, whereas an increase was noticed in the N callus placed in the same conditions. These metabolic changes and the reduction of cellular damage in darkness revealed light-induced stress reactions leading to necrosis and to reduced growth of HNO callus. It appeared that darkness allowed the HNO callus to avoid the photooxidation stress. Therefore, the favorable effect of darkness on HNO growth might be explained by the suppression of photooxidative damage due to the absence of carotenoids. The higher peroxidase activity in the HNO callus maintained in darkness raised the problem of heme synthesis in this heterotrophic callus.     

Disturbed nitrogen metabolism associated with the hyperhydric status of fully habituated callus of sugarbeet

The content of polyamines and proline was much lower in a normal (N) callus of Beta vulgaris L. than in a fully habituated hyperhydric (H) callus. The H callus also contained more glutamate and had a higher glutamate dehydrogenase activity. The excess of glutamate, in this chlorophyll-deficient callus, was linked to accumulation of proline and polyamines. Experiments with α-difluoromethylornithine (DFMO) and α-difluoromethylarginine (DFMA) showed that both ornithine decarboxylase and arginine decarboxylase participated in the synthesis of polyamines (especially spermidine and putrescine) and removal of ammonia. It is hypothesized that the H callus was subjected to ammonia stress from the start of the culture. Experiments with gabaculine, an inhibitor of ornithine aminotransferase, showed that this enzyme linked proline degradation to polyamine synthesis through the production of ornithine. This disturbed nitrogen metabolism appeared to be characteristic of the fully habituated callus and might explain the low growth of this hyperhydric tissue.     

Sperm motility in turbot,Scophthalmus marimus: initiation of movement and changes with time of swimming characteristics

Synopsis Turbot sperm motility is observed using dark field microscopy and stroboscopic illumination combined with video recording. Sperm motility is triggered by dilution of spermatozoa in sea water or in non ionic media (glucose or saccharose), presenting osmotic pressure ranging from 300 to 2100 mOsmol. The percentage of motile spermatozoa reaches 100% under conditions of osmotic pressure of 300 to 1100 mOsmol and pH close to 8.0. In full sea water, glucose or saccharose solutions an agglutination of spermatozoa is observed; this is prevented by addition of bovine serum albumin (5 mg ml^–1). Immediately after transfer in activation solutions, 100% spermatozoa are motile in most samples freshly stripped. This percentage drops suddenly between 15 and 30% after 70 to 100 sec. The beat frequency remains at a constant value of 50 Hz during 40 s post activation and then drops suddenly between 15 and 30 Hz. The spermatozoa velocity is about 200 micrometers s^–1 during 30 to 40 s and then declines to a stable value of 100 micrometers s^–1 at 50 s post activation. After 1.20 mn, more and more spermatozoa become motionless. The minimum calculated and averaged distance covered during 1.20 min, is about 12 mm. The high performances of turbot spermatozoa motility are interpreted as a compensatory mechanism for the low sperm production.     

Synchronous triggering of trout sperm is followed by an invariable set sequence of movement parameters whatever the incubation medium.

The movement of live trout spermatozoa is very brief (25 sec at 20 degrees C) and conditions have been developed to get synchronous initiation of sperm motility which allowed quantification of the major parameters of sperm movement during the motility phase. Recorded flagellar beat frequencies decreased steadily from values of 55 Hz at the beginning to 20 Hz at the end of the motility phase. Sperm forward velocities followed a similar pattern from 250 to 20 microns.sec-1 in the same conditions and the diameters of sperm trajectories were reduced from 370 to 40 microns. Thus none of the characteristics of sperm movement was constant during the motile phase which ended abruptly by a straightening of the flagella. The decrease in flagellar beat frequencies and sperm velocities are much greater than what could be extrapolated from the decrease of intracellular ATP (Christen R. et al: Eur. J. Biochem, 166: 667-671, 1987) or from measurements of ATP-dependence of reactivated sperm velocities (Okuno M. and Morisawa N.: In Biological Functions of Microtubules and Related Structures. New York: Academic Press, pp. 151-162, 1982). Therefore, the cessation of flagellar beating at 25 sec is not directly the result of the low concentration of intracellular ATP. The decrease in the diameters of sperm trajectories which occurred during the first part of the motility phase was correlated with [Ca]i measurements (Cosson M.P. et al, Cell Motil. Cytoskeleton, 14:424-434, 1989). The effect of Ca2+ at the axonemal level does not indicates that Ca2+ influx is previous to flagellar beating but rather suggests a classical Ca2+ regulation of the flagellar assymetry. The short duration of the motility phase and the characteristics of sperm movement were very similar in various conditions (high external K+, low pH media) where increased external Ca2+ or divalent ions were shown to overcome K+ and H+ inhibition of sperm motility, both conditions which have been shown to depolarize the plasma membrane potential (Gatti J.L. et al: J. Cell Physiol., 143:546-554, 1990). The present study of the parameters of sperm movement suggests that once motility is initiated, a defined set of axonemal events will take place whatever the external conditions.     

Cytokinin treated microcalli of Phelipanche ramosa: an efficient model for studying haustorium formation in holoparasitic plants

The ability of Orobanchaceae to establish a parasitic relationship is based on the development of a specific organ called haustorium. Previous studies in Phelipanche aegyptiaca and P. ramosa have underlined the interest of producing calli from germinated seeds as an efficient genetic transformation system, but they also pointed the need to improve the attachment rate of infectious calli to the host plant as well as the seed yield of the regenerated plants following attachment. It was previously shown that haustorium formation in P. ramosa is triggered by cytokinins. The present work demonstrates that one month-old microcalli produced from germinated seeds displayed a response to c/tZ similar to germinated seeds, as shown by the temporal expression profile of markers genes of cytokinin-related haustorium initiation, including PrRR5, PrCKX2, PrCKX4, PrTRN2 and PrZFP6. In addition, a 48 h treatment of microcalli with c/tZ (10^?7 M) before infestation triggered a 4-fold increase in the attachment rate of microcalli to tomato roots after 18 days in minirhizotrons when compared to untreated microcalli. Finally, it also outlines that increasing the aggressiveness of microcalli with a c/tZ treatment followed by the transfer of parasitized tomato plants into pots significantly improved seed yield of regenerated P. ramosa plants. Indeed, high amounts of viable seeds which germinated at more than 90% in response to GR24 were harvested after a 10 weeks-co-cultivation period. According to these results, cytokinin treated P. ramosa microcalli thus appear to be a good tool for further functional studies in holoparasitic plants, especially on haustorium formation.