Kazuyuki Sugino

Electrical properties and fusion dynamics of in vitro membrane vesicles derived from separate parts of the contractile vacuole complex of Paramecium multimicronucleatum.

SUMMARY The contractile vacuole complex of Paramecium multimicronucleatum transforms into membrane-bound vesicles on excision from the cell. The I–V relationship was linear in a voltage range of– 80 to +80 mV in all vesicles, despite being derived from different parts of the contractile vacuole complex. No voltage-gated unit currents were observed in membrane patches from the vesicles. Vesicles derived from the radial arm showed a membrane potential of >10 mV, positive with reference to the cytosol, while those derived from the contractile vacuole showed a residual (<5 mV) membrane potential. The electrogenic V-ATPases in the decorated spongiome are responsible for the positive potential, and Cl– leakage channels are responsible for the residual potential. The specific resistance of the vesicle membrane (∼6 kΩ cm2) increased, while the membrane potential shifted in a negative direction when the vesicle rounded. An increase in the membrane tension (to∼ 5×10–3 N m–1) is assumed to reduce the Cl– leakage conductance. It is concluded that neither voltage- nor mechano-sensitive ion channels are involved in the control of the fluid segregation and membrane dynamics that govern fluid discharge cycles in the contractile vacuole complex. The membrane vesicles shrank when the external osmolarity was increased, and swelled when the osmolarity was decreased, implying that the contractile vacuole complex membrane is water permeable. The water permeability of the membrane was 4–20×10–7 μm s–1 Pa–1. The vesicles containing radial arm membrane swelled after initially shrinking when exposed to higher external osmolarity, implying that the V-ATPases energize osmolyte transport mechanisms that remain functional in the vesicle membrane. The vesicles showed an abrupt (<30 ms), slight, slackening after rounding to the maximum extent. Similar slackening was also observed in the contractile vacuoles in situ before the opening of the contractile vacuole pore. A slight membrane slackening seems to be an indispensable requirement for the contractile vacuole membrane to fuse with the plasma membrane at the pore. The contractile vacuole complex-derived membrane vesicle is a useful tool for understanding not only the biological significance of the contractile vacuole complex but also the molecular mechanisms of V-ATPase activity.

Cell volume control in Paramecium: factors that activate the control mechanisms

SUMMARY A fresh water protozoan Paramecium multimicronucleatum adapted to a given solution was found to swell until the osmotic pressure difference between the cytosol and the solution balanced the cytosolic pressure. The cytosolic pressure was generated as the cell swelled osmotically. When either one or both of these pressures was somehow modified, cell volume would change until a new balance between these pressures was established. A hypothetical osmolyte transport mechanism(s) was presumably activated when the cytosolic pressure exceeded the threshold value of ∼1.5 × 105 Pa as the cell swelled after its subjection to a decreased osmolarity. The cytosolic osmolarity thereby decreased and the volume of the swollen cell resumed its initial value. This corresponds to regulatory volume decrease (RVD). By contrast, another hypothetical osmolyte transport mechanism(s) was activated when the cell shrank after its subjection to an increased osmolarity. The cytosolic osmolarity thereby increased and volume of the shrunken cell resumed its initial value. This corresponds to regulatory volume increase (RVI). The osmolyte transport mechanism responsible for RVD might be activated again when the external osmolarity decreases further, and the cytosolic osmolarity thereby decreases to the next lower level. Similarly, another osmolyte transport mechanism responsible for RVI might be activated again when the external osmolarity increases further, and the cytosolic osmolarity thereby increases to the next higher level. Stepwise changes in the cytosolic osmolarity caused by a gradual change in the adaptation osmolarity found in P. multimicronucleatum is attributable to these osmolyte transport mechanisms. An abrupt change in the amount of fluid discharged from the contractile vacuole seen immediately after changing the external osmolarity reduces an abrupt change in cell volume and thereby protects the cell from the disruption of the plasma membrane by excessive stretch or dehydration during shrinkage.

Different memory types for generating saccades at different stages of learning

It has been proposed that positional memories encoded in different types of reference frame are used for the reaching hand movement in different stages of learning. However, the types of reference frame employed for generating behavior at each stage of learning remain unclear, particularly for saccades. To examine the types of reference frame for target positions, we analyzed the saccade of monkeys performing an oculomotor task. The task required the animal to make a learning-based saccade to one of the eight landmark positions specified by the color of a fixation point. According to the color of the fixation point, the target landmark position was fixed in all experimental blocks (FIX trial) or altered to other landmark positions block by block (ALTER trial). Although the monkeys learned the target landmark position in both the FIX and the ALTER trials, once the landmarks became invisible, the success rate remained high only in the FIX trials. These results suggest that the target position was learned on the basis of the landmark positions in the early stage of learning. However, the memory of the target position in space was formed after sufficient training. When the fixation point was shifted horizontally by 5 degrees and the landmarks were invisible, the saccades in the ALTER trials were made to the normal target landmark position whereas those in the FIX trials were made to the point approximately 5 degrees shifted horizontally from the normal target landmark position. These results suggest that the target position in space was initially represented in the head-centered or world-centered reference frame and then in the eye-centered reference frame. Analysis of saccade end-points indicated that a kinematically similar saccade was generated for each FIX trial. These results showed that memories encoded by different reference frames were formed to generate a saccade while the saccade toward the same target was repeatedly executed.

Effects of muscimol injection into the caudate nucleus on performance of cognitive eye movement task in the cat

Intracranial self-stimulation (ICSS) of the medial forebrain bundle (MFB) activates, in the first place, the descending myelinated fibers, synapsing to cholinergic neurons of pedunculopontine tegmental nucleus which project on dopaminergic neurons of the ventral tegmental area (VTA). Thus, ascending dopaminergic pathway is transsynaptically activated to release dopamine in its terminals such as the nucleus accumbens (NAC) and medial prefrontal cortex (mPFC), both of which also receive cholinergic innervations, In the present study, microdialysts was used to examine changes of extracellular acetylcholine (ACh) levels in the rat VTA, NAC and mPFC following ICSS of the MFB. Baseline levels of ACh were 10.2 fmol/M ~1 in the mPFC and undetectable in the VTA and NAC. In the mPFC, ACh efflux increased to a maximum of 222% of baseline during ICSS and returned to baseline soon after ICSS was terminated. On the other hand, in the presence of an acetylcholinesterase inhibitor, ACh efflux increased to a maximum of 334% of baseline in the VTA and 184% m the NAC immediately after the start of ICSS and remained elevated during the experiment. The result suggests a diffuse activation of central cholinergic systems during ICSS of the MFB.

1655 The activity of cat caudate neurons associated with memory-guided eye movement task

To identify brainstem neurons involved in the central rhythm generation of ingestive movements of mammals, we have applied sulphorhodamine during NMDA-induced sucking-like activity of the hypoglossal nerve (XII) in an in vitro brainstemspinal cord preparation of O-4 day-old rats. Neural activities were monitored from the XII as well as the ventral roots of the fourth or fifth cervical spinal cord with suction electrodes. Sulphorhodamine was administrated to the recording chamber through perfusing system after appearance of rhythmical XII activity evoked by the application of N-methyl-Daspartate (NMDA) (group N). NMDA has been shown to induce rhythmical XII activities representing sucking-like tongue movements. Controls were exposed to the dye in the absence of NMDA (group C), in which spontaneous inspiratory activity was observed. Transverse sections of the brainstem were screened for rhodamine emission using an epifluorescence microscope. In group C, labelled cells were found in the hypoglossal nucleus, the ambiguus nucleus, the dorsal motor nucleus of the vagus, the solitary truct nucleus, the lateral reticular nucleus and the dorsal medullary reticular nucleus. In addition to these, labelled cells were found in the facial nucleus and the ventral part of the medial pontobulbar reticular formation in group N.

An Approach to Quantitative Analysis and Modelling of Three-Dimensional Ciliary Motion

Computer simulation of three-dimensional ciliary motion suggests that mechanisms controlling the activity of ciliary beating are localized at the basal region of cilia. An analysis of photographic data of the proximal shaft of a beating cilium, which had been recorded under membrane potential control, allows us to quantitatively describe motion at the level of axonemal functions. There are three parameters of ciliary beating: sliding velocity of outer doublets, transfer rate of sliding activity around the axoneme, and the steady inclination of the axis of beating.