Abstracts of papers by Hans Machemer and coworkers

Abstracts 31 - 60

60
Galvanotaxis: Grundlagen der elektromechanischen Kopplung und Orientierung bei Paramecium.
(Galvanotaxis: basics on electromotor coupling and orientation in Paramecium)
Since the mid-forties three branches of science have cast new light into the delicate organization of ciliates including their cilia: (1) electron microscopy, (2) cellular electrophysiology and (3) biochemistry. Today the understanding of the physiology of ciliates is quite extensive and detailed.  In the present chapter only those basics are to be described and explained, which are indispensable for the causal understanding of galvanotaxis in Paramecium: Morphology - Filament-sliding mechanism of ciliary motion - Ciliary motion as controlled by Ca ions - Role of the surface membrane - The cell as exposed to a voltage gradient - Effects of the voltage gradient on ciliary motion and behaviour - Cell culture - Observations and experiments.

59
Ion channels and behaviour: ciliates as cellular models.
Ciliates are provided with voltage-dependent channels comparable to those occurring in neurons of invertebrates or vertebrates.  Ciliates employ, in addition, receptor channels, as they may be found in cell membranes of sensory organs in metazoans. The perception of stimuli, and their transduction into an appropriate behaviour, are organized at the unicellular level.  Hence, ciliates may be thought of as “swimming sensory nerve cells”. Ion channels play a crucial role in modulating cellular behaviour, both by generating electrical membrane activity for signal integration and by controlling ion fluxes across the cell membrane which eventually serve as second messengers to intracellular organelles.

Contents

1.      Introduction

2.      Mechanoreceptor Channels

2.1      Anterior Channel

2.2      Posterior Channel

3.      Voltage-Dependent Channels

3.1      Calcium channels

3.2      Potassium channels

4.      Behavioural significance of the Ion Channels

5.      Perspectives.


58
Motor control of cilia. In (Görtz HD, ed) Paramecium.
Ciliary activity includes two different aspects: the mechanisms of how cilia are caused to move and how they are caused to change their movements. The present chapter deals, in the first place, with the second aspect, whereas the basic machinery of the cilium is briefly treated.
PERSPECTIVES: The understanding of the ciliary machine and its control in Paramecium has greatly improved after electrophysiological and cell reactivation methods have been systematically applied to this organism. It is now known that a firm link exists between the membrane potential and ciliary function and that Ca ions play a crucial mediating role. Unless the membrane of the viable cell was destroyed, attempts to uncouple the axoneme from the electric membrane signal had been unsuccessful.  The manner of translation of the bipolar and graded membrane signal into an equally fine-tuned ciliary motor response remains still largely unexplained.  Assuming that a Ca concentration transmits the membrane signal to the axoneme, the question arises as to how a uniform Ca concentration at a particular time can act to induce nine pairs of microtubular doublets to perform different sliding programs in sequence, and to perform these sequences differently at different levels of the axonemal cross-section. In addition, we are asking how a change in the intraciliary Ca concentration can reprogram the sliding of the nine sets of doublets such as to maintain the polarity of the cycle, but with the power stroke redirected. These questions illustrate our vast ignorance about the physiological principles of the cilium. In the near future, it will take a lasting and interdisciplinary effort to further unveil the mysteries of this tiny, but most elegant eukaryotic sliding machine.

Contents

1    Introduction

2    Galvanotaxis: A Classic Reviewed

3    Current Methods

4    The Cilium is a Rotary Sliding Machine

      4.1   Microtubular Sliding and Ciliary Gyration

      4.2   Rotation of the Central Complex

5    Parameters of Ciliary Activity

      5.1   Frequency

      5.2   Polarization of the Cycle

      5.3   Inclination, a Non-Cyclic Response

6    Reactivation of Ciliary Axonemes

      6.1   Roles of Ca and Mg

      6.2   Is Ca the Universal Regulator?

7    Depolarization-Induced Ciliary Activity (DCA)

      7.1   Inclination

      7.2   Cyclic Orientational and Frequency Responses

      7.3   The ‘Transient Inactivation’ Phenomenon

      7.4   Sequence in Ciliary Responses Following an Action Potential

8    Hyperpolarization-Induced Ciliary Activity (HCA)

      8.1   Inclination

      8.2   Cyclic Orientational and Frequency Responses

      8.3   Sequence in Ciliary Responses Following a Hyperpolarizing Stimulus Pulse

9    Adaptation

10  Steps in Electromotor Coupling

      10.1 DCA

      10.2 HCA

11  Perspectives.


57
Electrophysiology. In (Görtz HD, ed) Paramecium.
The present survey deals with some passive electrical properties (resistance, capacitance, cable properties) in a section on the resting membrane. Two more sections are devoted to active responses of the membrane following stimuli and shifts in voltage. These three central chapters will be placed between notes characterizing the electrical organization of Paramecium as being simple in design and sophisticated in performance.
CONCLUSION: The electrophysiological organization of Paramecium, as viewed from the present state of the data, is summarized in a diagram including relations between the membrane conductance of particular cell compartments, ions and potentials.  It is seen (Fig. 4) that the membrane potential is regulated in such a way as to swing between the equilibrium potentials of Ca and K ion batteries. External stimuli are transduced in changing the membrane conductance for Ca and/or K.  Signal integration and conduction occurs at the basis of potentials (= shifts in membrane potential).  The Ca ion is central for regulating the Ca and K membrane conductances for homeostasis and the excitable Ca channel of the cilia. All these regulations serve one major function: the guidance of the behaviour of the organism so that it can adequately respond to the stimuli from the outside world. It is clear that the membrane functions of a cell are embedded in a multitude of other provisions of the eukaryotic cell organization, such as structure, morphogenesis, biochemistry and genetics.  The present book, in assembling various aspects of the biology of Paramecium, may serve to provide a more complete view of the relevance of bioelectric control for the daily life of the single-cell organism.

Contents

1    Introduction

2    A Historical Note

3    Ion Batteries and Membrane Channels

      3.1    The Ca-K Membrane Circuit

      3.2    Other Ion Batteries

      3.3    The Membrane Channels

4    Properties of the Resting Membrane

      4.1    Membrane Potential

      4.2    Surface Potentials and the ‘Ca-Paradox’

      4.3    Leakage Conductance

      4.4    Resistance

      4.5    Capacitance and Membrane Area

      4.6    Cable Properties

5    Responses to Stimuli

      5.1    Sensory Transduction

      5.2    Receptor Potentials

      5.3    Anterior Mechanoreceptor Response

      5.4    Posterior Mechanoreceptor Response

      5.5    Role of Cilia in Mechanosensory Transduction

      5.6    Responses to Temperature

      5.7    Responses to Light

6    Voltage-Dependent Responses

      6.l     Channels for Excitation and Homeostasis

      6.2    Action Potential

      6.3    Membrane Rectification

7    Topology of Ion Channels

      7.l     Channels for Cell Homeostasis Restricted to the Soma

      7.2    Mechanoreceptor Channels Restricted to the Soma

      7.3    Voltage-Dependent Ca Channel Restricted to Cilia

8    Conclusion.


56
Das Experiment: Können Einzeller lernen? Prüfung klassischen Konditionierungsexperiment.
(The experiment: Do unicells learn? Tests applying classical conditioning)
The evolution of learning in animals has been tracked down to simple metazoans, but it was always a controversial question, whether or not unicellular organisms have a capacity for learning.  In the present experiment using Paramecium caudatum we asked the question what kind of learning should be tested.  We used the classical conditioning scheme which aims at establishing a “conditioned reflex”.  The results show that neither an increase in the unconditional stimulus (an electric voltage gradient) nor doubling of the time of training induced a modification of the response to the conditional stimulus (illumination). This result applies to training of single cells as well as to mass cell experiments.  The present results agree with the general conclusion that unicellular organisms cannot be conditioned.

55
Übungen zur Elektrophysiologie tierischer Zellen und Gewebe.
(Exercises in electrophysiology of animal cells and tissue)
INTRODUCTION (by Erwin Neher, Göttingen)
Electronic devices for data acquisition should ideally establish an unequivocal relationship between the object for measurement and the measured value. This is commonly achieved without major problems in the technical realm. In the case of biological preparations, however, this postulate is not at all trivial.  Biological sources of signals often have a high internal electric resistance so that many kind of disturbances have to be taken into consideration.  The environment plays a role.  The intersection between the biological preparation and the preamplifier, the microelectrode, has its own fallacies. Therefore, it is difficult to understand a signal at the oscilloscope and to interpret it correctly.  It requires, beyond understanding the specifications of instruments, a good deal of experience in handling preparations and electrodes. This experience may be achieved in a troublesome way doing large numbers of failing experiments.  Alternatively, the student may simulate biological measurements using electrical circuit diagrams as representing thoroughly defined objects. According to general experience, this is the more efficient way at least during a period of basic learning.
This book is a valuable guide to this goal.  It specifies the tools, proposes experiments, which largely cover the problems of electrophysiological measurement, and poses more advancing questions. The latter should not be neglected in order to go beyond the circuit diagrams.  An Appendix on basic physics and many insertions on general techniques of measurement make this book, apart from serving as a guide for exercises, a valuable textbook.

54
From structure to behaviour: Stylonychia as a model system for cellular physiology.
The ciliate Stylonychia is a versatile unicellular organism with a variety of outstanding structural and physiological properties. The present review seeks to assemble some of the building blocks which eventually lead to a complex pattern of cellular behaviour. In the first part, the cell morphology is described with emphasis on the ciliary apparatus. The behaviour of Stylonychia is documented, including modifications that occur in response to stimuli.
The second part analyzes the physiological and behavioural functions. These include the electrical properties of the membrane at rest and during stimulation. The basis of electrical excitation in Stylonychia, a two-peak action potential, is investigated by studying ion channels of the cell membrane. The control of ciliary organelles, the behaviour of which is recorded with high-speed cinematography, is discussed as a function of membrane potential, extracellular concentrations of ions, and ion-currents flowing across the membrane. Due to the accumulation of pertinent data, Stylonychia is an attractive system for the study of cellular electromechanical coupling and ciliary motor performance. The results lead to some conclusions about how behavioural control is achieved. The present account emphasizes experimental approaches. Techniques used are described briefly, where appropriate. We seek a synthesis of structure, motility, and physiological data. The integrating function of the cell membrane, with its regional differences of excitation and motility, is one of the central issues of this review. Frequent reference is made to comparable work in the fields of protistology and physiology. It is the aim of the authors to show that an inherently multidisciplinary approach of research in unicellular eukaryotes can contribute to the resolution of major questions of cell physiology.

Contents

I.       Introduction

II.      Cell Structure

          A. Phenotype

          B. Cytoarchitecture

          C. Motor Organelles

          D. Cytometry

              1. Soma

              2. Membranelles

              3. Cirri

              4. Paroral undulating membranes

III.     Ciliary Motor Functions

          A. Marginal Cirri

              1. Noncyclic nonpolar activity

              2. Cyclic nonpolar activity

              3. Cyclic polar activity

              4. Noncyclic polar activity

              5. Residual oscillation

          B. Cirri of the Ventral Surface

          C. Membranelles

IV.     The Daily Life of the ‘Walking Neuron’

V.      Conditioning Experiments

VI.     Some Methods and Techniques

          A. Cell Culture and Cell Preparation

          B. Membrane Potential Recording and Current Injection

          C. Voltage-clamp Recording

          D. Mechanical Stimulation

VII.   Electrical Properties of the Resting Membrane

          A. Membrane Potential

          B. Resistance and Capacitance

          C. Length Constant

VIII.  Stimulus Reception

          A. Mechanosensory Responses

              1. Bipolarity of responses

              2. Sensitive structures

              3. Specificity of responses

              4. Conductance gradients

          B. Characteristics of Mechanosensory Ion Channels

              1. Ion selectivity of the anterior response

              2. Ion selectivity of the posterior response

              3. Time course of receptor responses

              4. Voltage sensitivity of receptor currents

IX.     Electrical Excitation

          A. The Composite Action Potential

          B. Temperature-dependence.


53
Axial-view recording: An approach to assess the third dimension of the ciliary cycle.
High-frequency ciné data of the frontal cirri of Stylonychia viewed parallel to the ciliary base (“axial view”) have been geometrically analyzed so as to isolate a steady inclination of the proximal shaft during the ciliary cycle. Correction for this inclination allows assessment of the pathway of the shaft during the cycle and thereby parameters such as the directional change rate (time derivative of the xy-projected direction of the proximal ciliary shaft), the angular velocity (absolute measure of the ciliary displacement rate), and the rate of reorientation movement of the cilium corresponding to the translocation rate of the most active among microtubular doublets. The sliding velocity results from the product of angular velocity and the distance between two neighbouring doublets. A model is proposed which shows the implications of cyclic ciliary reorientations for the circular translocation of the activation of active sliding of axonemal doublets.

52
Electromotor coupling in cilia. In (Lüttgau HC, ed) Membrane control of cellular activity.
Ciliary movement bears interesting similarities to muscular function: mechanical forces are generated by an intracellular sliding-filament mechanism, which works in a unidirectional manner.  The ciliary membrane includes voltage-sensitive Ca channels, and ionic Ca is an intracellular messenger of the electromotor coupling process.  The present review focuses on basic mechanisms in electromotor coupling of cilia.  Accounts of the structure and function of protozoan and metazoan cilia have been given elsewhere. Therefore, only principles of the ciliary machine will be recalled here.  Emphasis is given to topics of controversy and so far unanswered questions.

Contents

1. Introduction

2. Principles of the ciliary machine

2.1  Elementary building blocks

2.2  Dynein cycle

2.3  Sliding and bending

2.4  Axonemal twisting

2.5  Sliding transfer and gyration

2.6  Planar beating

2.7  Core of axoneme

2.8  Rotation of the central complex

2.9  Role of the central complex

3. Ciliary responses in demembranated preparations

3.1 The two-machine hypothesis

3.2 Ca-dependent regulation of frequency and direction of beating

4. Ciliary responses under membrane voltage control

4.1 Ciliary frequency profile

4.2 Inactivation

4.3 Ciliary inclination

4.4 Orientation of the power stroke

4.5 Temporal polarization of the cycle

4.6 Spatial polarization of the cycle

5. Is the cilium a good cable?

6. Compartmentation of ciliary and somatic spaces

7. Pathways of electromotor coupling

7.1 Depolarization controls Ca-mediated regulation of cyclic ciliary activity

7.2 Depolarization controls Ca-mediated regulation of orientational ciliary responses

7.3 Is hyperpolarization-dependent ciliary activation mediated by calcium?

8. Perspectives.


51
Parameters of the ciliary cycle under membrane voltage control.
Axial views of depolarization- and hyperpolarization-dependent activation of the frontal cirri of Stylonychia were cinematically recorded at high rate (250 frames/s) under voltage-clamp. Images of a cirrus performing the cycle were processed by using computer assistance. In responding to the polarity and amplitude of the voltage signal, a cirrus inclines proximally with a particular angle and orientation.  The ciliary cycle - always counterclockwise - is superimposing upon steady inclination.  Correction for inclination allowed the assessment of the directional change rate and, after inclusion of the amplitude data, the determination of the ciliary angular velocity during the cycle.  The method serves to isolate a new ciliary parameter: inclination, and to register precisely parameters of the cycle which may be meaningful for the understanding of the microtubule sliding mechanism.

50
Electrical properties and membrane currents in the ciliate Didinium.
l. The electrical properties of the ciliate Didinium nasutum, a Paramecium predator, have been investigated using techniques of constant current injection, voltage-clamp, exchange of solutions, and nonlethal deciliation. The electrical observations have been related to structural characteristics (Fig. 1).
2. The resting potential was primarily K-dependent. K concentrations beyond 1mmol/l depolarized the cell and reduced the input resistance. The resting conductance for Ca was low. Partial substitution of Ca by Mg changed the membrane potential very little and had no effect on the input resistance. Chloride did not influence the membrane properties.
3. The late V/I-relationship was characterized by pronounced outward and minimal inward rectification. It was very little modified by Ca concentrations ranging between 0.125 mmol/1 and 8 mmol/1. Raising the K concentration between 0.063 and 16 mmol/1 did not affect membrane rectification.
4. Membrane depolarization triggered a stimulus-graded action potential, which was Ca-dependent. The action potential rose with rates of up to 6V/s; its overall duration was 100 to 500ms. Repolarization occurred along a fast and a slow (shoulder) component and was followed by pronounced after-hyperpolarization.
5. Complete deciliation of Didinium eliminated the regenerative depolarization of the membrane. The resting potential, input resistance and late current/voltage relationship were not modified by the removal of ciliary membranes. Under voltage-clamp deciliation removed the early inward current and the hump component of the late outward current.
6. Depolarizing voltage-clamp pulses of up to +20 mV revealed a residual inward current following the early transient. The transient included two decay time constants (3.3 ms; 31.8 ms). An 0.8nA persistent inward current was isolated using the difference current of ciliated and deciliated cells. A late outward current rose with depolarizations beyond 20mV including an upward inflection (hump) about 50 ms after step onset.
7. A decreasing Ca/Ba mole-fraction increased the frequency and duration of spontaneous action potentials, and depressed the amplitudes of both, early inward and late outward current under voltage-clamp. In Ca-free Ba solution the unclamped membrane fired repetitively; the depolarizations included shoulders of 10s or more. Inactivation of inward Ba currents was slow under depolarizing voltage-clamp; the late outward current was strongly depressed. In deciliated cells an early inward current was missing in Ca-free Ba solution, and the late I/V relation resembled that of deciliated cells in Ba-free Ca solution.
8. Tail currents following the late outward current of ciliated cells in Ca-solution and of deciliated cells in Ca- or Ba-solutions decayed with similar time constants.
9. In conclusion, the voltage-activated Ca conductance of the ciliary membrane of Didinium compares well with data from ciliates so far studied. Unique is (i) a persistent inward current isolated using minor step depolarization or the difference current of ciliated and deciliated cells, (ii) a slow activating voltage-dependent K conductance, and (iii) a late, potentially Ca-dependent, outward current, which may be related to spatial separation of the cilia from the majority of the soma membrane. Ba interferes with the Ca channel which favours a previous two-binding site model. Ba can inhibit the K conductance from inside the cell after passing the ciliary Ca channel.

49
Was bewegt einen Einzeller?
(What moves the ciliate?)
1. In this talk, the physiological bases of the complex motile behaviour of ciliated unicellular organisms are addressed.
2. Motile single and compound cilia are often specialized for certain functions. They are the structural basis for various forms of movements.
3. Ciliates are not bilaterally organized, and they commonly swim along helical pathways which are interrupted by reversals. They use orthokinesis and klinokinesis for temporal stimulus discrimination.
4. Predominant viscous interaction between the cell and its fluid environment determines the properties of ciliary activity and ciliary metachronism.
5. The cilium gyrates during its beating cycle along a pathway which is polarized in time and in space. The effective stroke may be gradually reoriented, together with the complete bending program of the cycle, within a wide angular range. In addition, the ciliary beating frequency is physiologically regulated.
6. Changes in orientation of ciliary beating are due to a reprogramming of the cyclic microtubule-sliding mechanism; they are not based on ciliary rotation. Active sliding between peripheral microtubular doublets bears some analogies to the sliding-filament mechanism in muscle.
7. Ciliary activity is under control of the membrane potential. Two types of ciliary frequency and directional responses occur: depolarization-induced and hyperpolarization-induced. Depolarizations activate voltage-sensitive ciliary Ca channels so that ionic intraciliary Ca transmits the signal to the axoneme.
8. The electric cable properties of the cell including the cilia are presumably suited for passive spread of potentials without major losses in amplitude.
9. Application of mechanical stimuli to various sites of the surface membrane may elicit receptor potentials of different amplitude and polarity followed by different ciliary responses. This is due to a gradient-type distribution of two species of mechanoreceptor channels in the somatic membrane.
10. Positive and negative receptor potentials passively spread to the ciliary membrane; here, a depolarization elicits a regenerative Ca influx accompanied by an action potential.
11. Separation in space between somatic sensors and the ciliary motor has a functional basis.
12. Topological modification of sensory and motor structures and temporal changes in electric membrane properties of the ciliate cell give ample space for a complex behaviour of the organism. The bases of this behaviour are still insufficiently understood.

48
Mechanoreception in ciliates.
 CONTENTS.
1. Introduction. – 2. Behavioural Responses to Mechanical Stimulation. – 3. Bipolar Nature of Receptor Responses. – 4. Effects of Different Modes of Stimulation. – 5. Ionic selectivity of Receptor Channels. – 5.1 Anterior Mechanoreceptor Response. – 5.2 Posterior Mechanoreceptor Response. – 6. Time characteristics of Receptor Response. – 7. Voltage Sensitivity of Receptor Conductances. – 8. Distribution of Mechanoreceptor Channels. – 9. Are Cilia Mechanosensitive? – 10. Sensory-Motor Coupling. – 11. Concluding Remarks.

CONCLUDING REMARKS. Mechanoreception is an important sensory function of ciliated protozoa, helping them to deal with their physical environment.  Unlike metazoan organs for mechanoreception, which are often anatomically complex and difficult to investigate at the cellular level, ciliates, as “swimming mechanoreceptors”, are comparatively simple structures.  They permit experiments employing direct stimulation and electrical recording.  The ciliary motor output, in addition, is a most sensitive and reliable indicator of the state of excitation of the cell.  As unicellular organisms, ciliates are surrounded by the experimental solution, which appears to pose no problems for the diffusion of ions to and from the cell membrane.  Furthermore, ciliates are easily cloned, mass-cultured and genetically manipulated (Kung 1979).
Mechano-electrical transduction processes can thus be studied at the single-cell level.  The ions channels involved in mechanoreception have been identified and their distribution in the somatic membrane has been grossly assayed.  The channels have been characterized, although still incompletely, by their voltage-sensitivity, time course and ion selectivity.  The roles of the cilia in the transduction process have gained more attention.  New approaches, such as single-channel recording and biochemical and molecular techniques, are needed to further elucidate the process of mechano-electrical transduction.  The formation, turnover rate of the mechanosensitive ion channels during the relatively short cell cycle may give new insights into the properties and functions of ion channels in general.  Sensory-motor coupling in ciliates provides a unique mechanism for the study of behaviour at the cellular level.  Thus ciliated protozoa may in future also serve as promising model systems for investigating stimulus-response coupling in animals.

47
Injected cyclic AMP increases ciliary beat frequency in conjunction with membrane hyperpolarization.
Injections of cyclic AMP (cAMP) and 8-Br-cAMP into Paramecium and external application of isobutylmethylxanthine (IBMX), an inhibitor of cAMP breakdown, to these cells increased the frequency of ciliary beating and hyperpolarized the membrane potential.  When the membrane potential was held equal to the resting potential under voltage clamp, the same experimental conditions which serve to increase intracellular cAMP did not raise the ciliary frequency.  We conclude that cAMP is presumably not the direct mediator of the hyperpolarization-induced ciliary activation, although it may be associated with this motor response.

46
Mechanoresponses in protozoa.
 This lecture deals with mechanoresponses in ciliates only. In fact, insights into the regulating mechanisms of protozoan sensory-motor coupling have been limited to those few species for which electrophysiological and other recording techniques were established. I shall use the term “mechanoresponses” in a way as to include the behavioural events. Such a definition is useful for comparison with bacterial cellular behaviour. - Conclusions.  Mechanoresponses in the ciliate protozoa involve a number of physiological steps which are now emerging to be comparable to similar processes in tissue for stimulus reception, excitation and generation of a motor response. Universality in cellular functions, being indispensable for the unicellular organism, may have been an evolutionary basis of metazoan tissue differentiation.
Some of the properties of sensory-motor coupling are unique to the ciliate, such as the topographical difference in cellular mechanosensitivity and the ability of the ciliate to respond to membrane hyperpolarization as well as depolarization. While ciliates are highly advanced in these respects, there are other properties, such as long response latency of mechanical stimulation and membrane excitation using voltage-sensitive Ca channels, which are undoubtedly archaic properties.  It is the position of protozoans between the worlds of prokaryotic life and metazoan organization which will continue to define their roles as model systems for stimulus-response coupling in animals.

45
Mechanical and electric correlates of mechanoreceptor activation of the ciliated tail in Paramecium.
1. Single mechanical pulses were applied by micro-needle to the sensitive posterior soma membrane of Paramecium which bears a bundle of immobile and elongated tail cilia. The time sequence of events of stimulation and response was recorded using membrane voltage clamp and microphotography. Deciliation techniques were used to determine the role of cilia in receptor current generation.
2. The minimal latency of receptor current was between 2 and 2,5ms. Prior to the rise of the current the soma membrane had been deformed in W-shaped manner, the cell end had widened and shifted anteriorly (Fig. 1).
3. With small stimulus amplitudes first signs of the receptor current included an additional delay of about 5ms. Abrupt transition to the normal response latency occurred upon raising the amplitude of the stimulus.
4. The stimulus amplitude determined the rate and amplitude of the receptor current.  Decreasing rates of the stimulus increased the response latency.
5. The receptor current continued to flow during somatic membrane rebound from indentation. At this time the tail cilia were passively deflected.
6. Cell deciliation using 5% ethanol and mechanical agitation did not affect the minimal response latency, but decreased the sensitivity of the tail area.  Ethanol-treated ciliated cells were more sensitive than normal cells. These tests suggest that stimulus conduction via the cilia can enhance the receptor response.
7. In conclusion, the latency of the receptor current appears to be limited by a transduction mechanism located in the soma. The tail cilia serve to transmit the stimulus including, presumably, spatial and temporal summing of non-local mechanical input.

44
Receptor current following controlled stimulation of immobile tail cilia in Paramecium caudatum.
1. Directionally and amplitude-controlled mechanical pulses were applied to identified sites of the tail cilia and the posterior soma membrane of Paramecium under voltage-clamp.
2. Lateral stimuli directed toward more distal sections of the cilia deflected the ciliary bundle as a unit.  No deformations of the soma membrane were detected upon stimulus application to the ciliary shafts (Fig. 3).
3. Outward receptor currents following somatic stimulation arose 3ms after the onset of the electric driving pulse (= response time). The response times after ciliary stimulation were at least 6ms.
4. The response times rose from 6 to beyond 10ms after shifts of the stimulus probe from proximal to more distal sections of the tail cilia.
5. Maximal currents (up to 17nA) were recorded upon stimulation of the apical posterior soma in the centre of the tail. Ciliary stimulation elicited currents which had more irregular time courses of rise and decay.
6. In most cells tested the current amplitudes were reduced with shifts from somatic to ciliary stimulation; the amplitudes even more decreased with stimulus displacement toward the ciliary tips. A fraction of the cells was hypersensitive in that the responses to somatic and ciliary stimulation were enhanced as compared to normal cells.
7. The tail cilia showed no intrinsic directional sensitivity. Minor differences in responsiveness to topographically identified lateral stimulation of the cilia are presumably related to a dorso-ventral differentiation of somatic sensitivity.
8. We conclude that neither the ciliary membrane nor the axoneme and basal body play an active role in mechanotransduction. A local ciliary stimulus appears to be “defocused” so that it affects an extended area of the sensitive soma membrane.

43
Effects of varied culturing and experimental temperature on electrical membrane properties in Paramecium.
Effects of temperature on the electrical properties of the Paramecium membrane were investigated under constant current and voltage-clamp stimulation.  With cells cultured at 18°C, the resting potential was largely stable, when the experimental temperatures varied over the range of 10 to 25°C; on the other hand, the action potential amplitude and membrane input resistance were inversely related to experimental temperature increases.  During voltage clamp, the early calcium current was increased, the time-to-peak decreased, and the early conductance increased with temperature. Similar modifications of the culturing temperature did not affect the resting potential, input resistance and stimulus-response relationships of the action potential, but the early conductance was reduced with increase in temperature. Possible effects of long- and short-term temperature changes upon intraciliary calcium concentration are discussed.

42
Motor control in three types of ciliary organelles in the ciliate Stylonychia.
1. The motor activity of three types of ciliary organelles (compound cilia): membranelles, frontal cirri and marginal cirri, of the hypotrich ciliate Stylonychia mytilus was analyzed using high-speed cinematography (250 images/s).  The cell membrane was voltage-clamped, and step or ramp voltage pulses were applied to study the relationship between membrane polarization and motor performance in the three ciliary organelles simultaneously.
2. At zero current (= resting) potential the membranelles beat at a frequency of around 40 to 45 Hz, whereas frontal and marginal cirri were quiescent.  Positive or negative voltage pulses activated the frontal and marginal cirri, but did not significantly alter the beating frequency of membranelles.
3. Hyperpolarization of the membrane induced beating of the frontal and marginal cirri with the power stroke directed towards the cell posterior (“hyperpolarizing ciliary activation”), whereas depolarization of the membrane induced beating of the frontal and marginal cirri in the reversed direction (“depolarizing ciliary activation”).
4. The threshold for both hyperpolarizing and depolarizing ciliary activation was higher, and the latency was larger, for the frontal cirri than for the marginal cirri.  The maximum frequency attained was smaller in the frontal cirri than in the marginal cirri during hyperpolarization (around 25 Hz versus 35 Hz) as well as during depolarization (around 35 Hz versus 45 Hz).
5. Voltage ramps from –20mV to +20mV with respect to the holding (= resting) potential, rising at rates of 20mV/s caused very small - if any - transient changes in the beating frequency of the membranelles. Responses to voltage ramps exhibited similar frequency-membrane potential relationships as step pulses: both frontal and marginal cirri had much the same frequency-voltage pattern, although different absolute beating frequencies (see above).
6. The performance of the three types of ciliary organelles is discussed in relation to membrane voltage and current. It is concluded that the membranelles have an unidentified mechanism of motor control, whereas the activity of both frontal and marginal cirri is coupled to the membrane potential, as also suggested in Paramecium.

41
Threshold activation and dynamic response range of cilia following low rates of membrane polarization under voltage-clamp.
1. We have applied slow positive and negative voltage ramps to the ciliate Stylonychia under membrane voltage-clamp to study the properties of activation of the cilia within ciliary aggregates (marginal cirri). These cilia are quiescent in the resting state of the membrane.
2. When the rate of polarization of the membrane is sufficiently reduces (£ 3mV/s), a reorientation of the cilia occurs prior to the onset of cyclic activity together with the changes in potential.  Because this response is unrelated to periodic beating of the cilia, we call it “silent reorientation”.  The range of voltage stimuli for this ciliary response is typically 3 to 5mV.
3. Small membrane depolarizations cause silent reorientations of the cell anterior; small hyperpolarizations induce a similar reorientation toward the posterior cell end.
4. With rising membrane hyperpolarization the cyclic activity of the cilia begins abruptly. The observed amplitude of the 3-dimensional beating is larger than 150°, and the starting frequency is at least 20 Hz.
5. Ciliary beating toward the anterior cell end following membrane depolarization rises from zero to maximum within a stimulus range of £ 2mV starting at depolarizations of about +5mV.  The voltage range for ciliary beating toward the cell posterior following hyperpolarization of the membrane is at least 5mV between threshold (approximately -6mV) and saturation.
6. The dynamic response range of individual cirri varies within one cell.  Threshold voltages for both hyperpolarizing and depolarizing activation of the cilia rise along the antero-posterior cell axis.
7. Averaging of the motor responses of several members of the marginal row of cirri illustrates that an integration of the motor responses of many cilia eventually leads to a motor behaviour of the cell which is fine-tuned to small gradations of the voltage stimulus.
8. The application of voltage ramps for the study of ciliary electromechanical coupling implies time-dependent effects on the membrane properties (accommodation) and the ciliary response (cycle time, relaxation time).  The frequency/voltage relationship appears to be adequately represented applying slow ramps (5 to 10mV/s) with moderate amplitudes (± 20mV).

40
Simultaneous recording of responses of membranelles and cirri in Stylonychia under membrane voltage-clamp.
We have studied to motor responses of various compound cilia in Stylonychia, using membrane voltage-clamp, high-frequency cinematography and data processing techniques. The membranelles beat at high frequency, whereas the frontal and marginal cirri are quiescent at the membrane resting potential. Hyperpolarizations and depolarizations specifically activate the cirri, but leave the membranellar frequency unchanged.

39
Tail cilia of Paramecium passively transmit mechanical stimuli to the cell soma.
Calibrated stimuli were applied to ciliary and somatic sites of the tail of Paramecium caudatum under voltage-clamp. Response times were minimal after somatic stimulation and rose in the distal ciliary direction. Receptor current amplitudes were maximal after posterior somatic stimulation. The data suggest a passive role of ciliary shafts and basal bodies in mechanotransduction.

38
Electromechanical coupling in cilia I. Effects of depolarizing voltage steps.
We have studied quantitative aspects of ciliary motor responses to membrane depolarization in the ciliate Stylonychia using voltage-clamp and high-speed cinematography techniques and employing computer-processing methods for evaluation. Depolarizations beyond 4mV activate the cirri (compound cilia) which are at rest in the absence of a stimulus. The power stroke of activated cirri is oriented toward the cell anterior. The frequency and duration of beating increase with rising depolarization. With very large positive stimuli (> 150 mV) activation of the response is delayed until the end of the voltage step (“off-response”). The peak frequency is essentially unaltered during sustained depolarization. The frequency drops exponentially following repolarization of the membrane. The time constant of the decay in ciliary activity rises with the amplitude, not with the duration of the depolarization. The ciliary motor response is most adequately represented by the number of evoked ciliary cycles (ciliary work), and appears to be related to the amplitude of the depolarization.

37
Electromechanical coupling in cilia II. Effects of hyperpolarizing voltage steps.
We have studied the motor responses to membrane hyperpolarization of the marginal cirri in Stylonychia using voltage-clamp, high-speed cinematography, and computer-processing techniques. The cirri started beating when voltage step amplitudes rose beyond 5 mV, and the power stroke was oriented toward the posterior cell end (“hyperpolarizing motor activation”).  The frequency rose slightly during a voltage step, and decreased with similar rates for 100ms following the step end.  Amplitude and duration of the step tended to increase the motor response of the cirri.  The late response declined exponentially.  The time constant of the decay rose with the step amplitude. Among three response parameters tested (frequency, duration, number of cycles), the number of evoked ciliary cycles was best correlated with the amplitude of the hyperpolarization. Comparisons with the responses to depolarizing voltage steps reveal similarities in the relaxation of ciliary activity which appears to be uncoupled, in part, from the electric membrane events during the voltage stimulus.

36
Osmotic tolerance of Ca-dependent excitability in the marine ciliate Paramecium calkinsi.
The electrical membrane properties of the marine (brackish water) ciliate Paramecium calkinsi were investigated under constant-current and voltage-clamp conditions, using two intracellular microelectrodes. The action potential and membrane currents were extremely tolerant to changes in the salinity of the bathing medium. Current-voltage relationships exhibited a moderate inward-going rectification of the membrane upon hyperpolarization, and a prominent outward-going rectification upon depolarization of the membrane. Ion substitution experiments showed that the electrically excitable response is a graded Ca2+-action potential, similar to that found in freshwater ciliates.

35
Mechanoreception and signal transmission in the lateral ciliated cells of the gill of Mytilus.
1. The lateral ciliated cells of the gill epithelium of Mytilus edulis were mechanically stimulated with a fine-tipped glass stylus.  The electrical responses were recorded intracellularly from the ciliated cells, and the ciliary responses were photographed.
2. Mechanical stimuli to the cilia and to the cell surface evoked a depolarizing membrane response (receptor potential) which increased with the rate of rise and the steady-state amplitude of the electric pulse driving the piëzo-transducer.  The effective parameter of the stimulus was the velocity of the stylus.
3. Stimulation of the cilia in various directions (4 parallel and 1 perpendicular to the cell surface), including direct stimulation of the lateral cells, led to similar depolarizing receptor potentials. The “perpendicular” stimulation was among the most effective.
4. The same electric responses were elicited from stimuli applied to any of the 4 rows of lateral ciliated cells.
5. The mechanically elicited depolarizing receptor potentials triggered action potentials.  Such regenerative depolarizations were often missing in the isolated filament preparation, presumably due to subthreshold receptor potentials.
6. The electric membrane responses and arrest responses of the cilia following membrane depolarization were propagated with decrement along several cells.  The area of the ciliary arrest responses increased with stimulus intensity extending to a maximum of 70µm in any direction.
7. Exposure to Ca-free artificial sea water suppressed both the depolarizing receptor potential and the ciliary arrest response. Action potentials with long-lasting plateaus occurred spontaneously in the Ca-free solution.
8. Total substitution of Na with choline in artificial sea water hyperpolarized the membrane, but did not interfere with the generation of receptor potentials.  Spontaneous and mechanically induced action potentials were suppressed in this solution.  Electrical responses were not transmitted to the neighbouring cells, and no ciliary arrest response was seen in the absence of external Na.
9. We conclude that mechanical stimuli impinging on the lateral cells via the cilia generate a Ca-dependent depolarizing receptor potential which elicits a Na-dependent regenerative process. The graded depolarization spreads transcellularly with decrement, and regulates the local arrest response of the cilia, which may play a role in the feeding behaviour of the mussel.

34
Analysis of ciliary beating frequency under voltage-clamp control of the membrane.
 In order to demonstrate the correlations between the membrane potential and ciliary activity slow voltage ramps have been applied under voltage clamp in Paramecium starting with -12mV hyperpolarization and ending with +18mV depolarization.  The ciliary frequency profile (i.e. frequency-voltage relationship) includes those responses of the cilia, where the overall direction of the power stroke is toward the cell anterior (“reversed beating”) and cilia beating in the posterior direction (“normal beating”). The experiments demonstrate that the effects of depolarization and hyperpolarization on the ciliary motor responses are subtractive upon each other.  We hypothesize that hyperpolarization antagonizes the effects of depolarization by decreasing [Ca2+]i by one or several mechanisms such as raised Ca pumping, Ca binding, passive cilio-somatic Ca diffusion and/or reduced Ca conductance of the ciliary membrane (all-Ca hypothesis).

33
Sind Cilien wirklich Fühler? Mechanorezeption bei Ciliaten als Modell.
(Mechanoreception in ciliates is no model system for ciliated sensory cells.)
Mechanical stimuli applied to the somatic surface of Paramecium and Stylonychia produce receptor potentials of specific amplitude and polarity depending on the intensity and site of stimulation. The receptor potentials passively spread from the stimulated area to the cilia where they interfere with Ca membrane conductances and other systems which modify the intraciliary Ca concentration.  This concentration is a sufficient signal for the cilia to regulate their motor response. Artificial deciliation of Paramecium removes the Ca action potential, but does not interfere with mechanoreception.  In the ciliate protozoa the cilia are exclusively motor organelles which are covered with an excitable membrane.

32
Distribution of mechanoreceptor channels in the Paramecium surface membrane.
Different sites of the Paramecium surface were mechanically stimulated with a fine-tipped glass stylus.  Ciliated and deciliated specimens showed a decrease in depolarizing mechanosensitivity and a subsequent increase in hyperpolarizing mechanosensitivity, when the site of stimulation was shifted from the anterior to the posterior end of the cell.  Maximal depolarizing mechanosensitivity was observed slightly posterior to the front end of the cell.  Specimens without cilia failed to produce action potentials, but no differences in mechanosensitivity were detected.  Stimuli given to dorsal surfaces produced larger hyperpolarizations, or smaller depolarizations, than stimuli applied to ventral surfaces at the same latitude.  In voltage-clamped cells posterior stimulation elicited outward receptor currents with 5ms half-time of decay.  Inward receptor currents following anterior stimulation decayed with half-times between 20 and 70 ms.  Reversal potentials of the receptor currents became more positive, when more anterior surfaces were stimulated.  Measurements of the reversal potentials in solutions with varied K and Ca concentrations showed that the posterior receptor currents are exclusively carried by K, whereas the anterior receptor currents are the sum of K and Ca currents. Ca and K mechanoreceptor currents cancel out in the mid-posterior region of the cell.  Quantitative evaluations of the data suggest that the receptor potentials in Paramecium occur due to activated Ca and K mechanoreceptor channels distributing over the somatic cell surface in the manner of overlapping gradients.

31
Ionic conductances of membranes in ciliated and deciliated Paramecium.
1. Paramecium caudatum was deciliated with ethanol.  The ionic conductance of the membrane was investigated with constant current, voltage clamp and mechanical stimuli.
2. The resting potential was not modified by the removal of the cilia.  The dependence of the resting potential on the extracellular concentrations of Ca and K was the same in deciliated and control cells.
3. The input resistance in deciliated and ciliated cells increased after the ethanol treatment.
4. The membrane capacitance decreased to 48% after deciliation, suggesting that the ciliary surface area is equal to the somatic surface area.
5. Deciliation completely removed the regenerative response (graded action potential) elicited by depolarizing current pulses or mechanical stimuli.
6. Deciliated cells retained the depolarizing and hyperpolarizing mechanoreceptor responses.
7. Voltage-clamp experiments demonstrated the loss of the early inward current in deciliated cells; it was restored during ciliary regeneration.  Steady-state current-voltage relationships were unchanged by deciliation.
8. The time courses of the recovery of the membrane capacitance and of the early inward current were similar, suggesting that the number of voltage-sensitive Ca-channels is proportional to the ciliary membrane area.
9. We conclude that the voltage-sensitive Ca channels reside in the ciliary membrane (in confirmation of Dunlap, 1976; Ogura & Takahashi, 1976), while mechanoreceptor channels, rectifier channels and resting conductances are localized in the somatic membrane.