David C. Wood

ACTION SPECTRUM AND ELECTROPHYSIOLOGICAL RESPONSES CORRELATED WITH THE PHOTOPHOBIC RESPONSE OF STENTOR COERULEUS

Abstract— The blue‐green ciliate. Stentor coeruleus, is found predominantly in shady places. This concentration occurs because stentor responds when swimming from a shaded area to a lighted area by reversing the direction of its ciliary beat and reorienting its swimming direction until it once again is in the shaded area. A graded receptor potential is recorded from microelectrodes in vacuoles of stentor when the animal is photically stimulated. For all but very weak stimuli this receptor potential is sufficient to elicit a regenerative transmembrane response of variable amplitude in a swimming animal. Suprathreshold electrical stimuli also elicit this regenerative response. In turn the regenerative response is coupled to ciliary reversal. Thus ciliary reversal appears to be produced whenever the photic receptor potential crosses the threshold for elicitation of the regenerative response.

Membrane permeabilities determining resting, action and mechanoreceptor potentials inStentor coeruleus

SummaryIn response to mechanical stimuli the protozoan,Stentor coeruleus, contracts in an all-or-none fashion and simultaneously reverses the direction of its ciliary beat. These behaviors have previously been shown to be correlated with the presence of a mechanoreceptor potential and all-or-none action potential (Wood 1970, 1973a). In the studies reported below the ionic bases of the resting, receptor and action potentials ofStentor were determined by use of intracellular microelectrodes penetrating animals chilled to 8.5–10 °C. The resting potential is most dependent on the extracellular concentration of KCl but some dependence on CaCl2 concentration was also observed. If allowance is made for the large increases in membrane conductance observed in solutions containing 2–8 mM KCl it is found that the resting potential data are well described by a modified form of the Goldman equation wherePCa/PK = 0.068 andPCl/PK = 0.072. The intracellular ionic activities (Ki+ = 13.1 mM, Cli−= 9.9 mM, Cai+ = 0 mM) which provide the best fit of this equation to the resting potential data are in close agreement with the intracellular concentration values measured by flame microspectrophotometry (Ki=12.4 mM, Cli = 9.4 mM) except in the case of Cai where most of the intracellular concentration is presumed to be bound. 65 to 75 mV action potentials are produced by suprathreshold depolarizations but contractions were not generally seen in these chilled animals, only ciliary reversals. The action potential peak varies with CaCl2 concentration with a slope of 12.6 mV/10 fold change but varies only slightly with KCl or Cl− concentration. These peak potentials are well described by assuming that thePCa/Pk = 7.9 andPCl/PK=1.0 at the time of the action potential peak. Depolarizing receptor potentials and brief inward receptor currents were observed for all forms of punctate and gross bodily mechanical stimulation employed. No evidence was found for any form of hyperpolarizing mechanoreceptor potentials as observed in some other ciliates. The reversal potential of the mechanoreceptor current varied with CaCl2 concentration in a manner similar to that of the action potential peak. As in the case of the action potential both thePCa/Pk andpcl/pk ratios appear to increase as a result of mechanical stimulation to 9.3–15 and 1.2–1.95 respectively. Mechanoreceptor currents are voltage dependent being increased when the membrane is depolarized above resting potential and decreased when the membrane is hyperpolarized. In general the electrophysiological characteristics ofStentor appear similar to those ofParamecium andStylonychia, but its resting membrane appears more selectively permeable to K+, it produces only depolarizing receptor potentials when mechanically stimulated and the initial action potential elicited by depolarizing current pulses can be all-or-none even in culture medium.

Partial Cloning of Putative G-Proteins Modulating Mechanotransduction in the Ciliate Stentor

Abstract Signal transduction systems known to utilize G-proteins in higher eukaryotes undoubtedly evolved prior to the development of metazoa. Pharmacological evidence indicates that the ciliates Paramecium, Stentor, and Tetrahymena all utilize signaling systems similar to those found in mammals. However, there has been relatively little direct evidence for the existence of G-proteins in ciliates. Since highly conserved heterotrimeric G-proteins form the basis of receptor-coupled signal transduction systems in a wide variety of metazoa, it is of interest to know if these important signaling molecules were early to evolve and are present and functionally important in a wide variety of unicellular organisms. We have previously shown that mechanotransduction in Stentor is modulated by opiates in a manner that may involve pertussis toxin-sensitive G-proteins. Here we utilize drugs known to interact with G-proteins to further test for the involvement of these important signaling molecules in Stentor mechanotransduction. We present behavioral and electrophysiological data demonstrating that putative G-proteins in Stentor decrease mechanical sensitivity by modulating the mechanotransduction process. In addition, we report the partial cloning of 4 G-protein α-subunits from Stentor. We confirm that these clones are of Stentor origin and are transcribed. Furthermore, we employ antisense oligodeoxynucleotide-mediated knockout to demonstrate that these ciliate G-proteins exert a modulatory influence on Stentor behavior, and that a Gi/Go-like clone mediates the inhibitory action of β-endorphin on mechanotransduction.

Is tube-escape learning by protozoa associative learning?

The ciliate protozoa, Stentor and Paramecium, have been reported to escape from the bottom end of narrow capillary tubes into a larger volume of medium with increasing rapidity over the course of trials. This change in behavior has been considered an apparent example of associative learning. This decrease in escape time is not due to a change in the protozoas environment, their swimming speed, frequency of ciliary reversals, or the proportion of time spent forward or backward swimming. Instead, most of the decrease results from a decrease in the proportion of time spent in upward swimming. However, a similar decrease in upward swimming occurs when the task is altered to require escape from the upper end of the capillary tubes. Because the protozoa exhibit the same change in behavior regardless of the reinforcing stimulus, tube-escape learning is not associative learning.

β-endorphin modulates a mechanoreceptor channel in the protozoan Stentor

A series of opiate compounds was tested for their ability to depress the probability that the protozoan Stentor coeruleus would contract in response to mechanical stimulation. Of these β-endorphin proved to be the most effective. The depressive effect of β-endorphin is concentration-dependent with an approximate E.C.50 of 3.0 μM and time-dependent with the maximum depression occurring 15 min after drug exposure. The effect of β-endorphin is blocked by the opiate antagonist naloxone and pertussis toxin suggesting that it is mediated by a G-protein coupled opiate receptor. β-endorphin does not alter responding to photic or electrical stimuli indicating its action is specific to the mechanical stimulus transduction mechanism. In agreement with this conclusion, electrophysiological studies reveal that β-endorphin decreases the amplitude of the mechanoreceptor potential without altering other membrane properties. Voltage clamp analysis shows that β-endorphin acts by decreasing inward currents through the mechanoreceptor channel at transmembrane potentials between -70 and + 20 mV without affecting the outward currents observed at more depolarized voltages. The fact that a mammalian neuromodulatory peptide is capable of producing a specific modulation of an ion channel in a unicellular eukaryote indicates that mechanisms of signal transduction and neuromodulation originated at an early stage in evolution.

Electrophysiology and Photomovement of Stentor

As first reported by Jennings (1906) and Mast (1906) Stentor coeruleus swims away from light sources (photodispersal) and collects in the more shaded areas of an unevenly illuminated container. Stentor are sensitive to light because they contain a pigment called stentorin (Moller, 1962; Walker et al., 1979), which releases H+ ions as a result of light absorption (Walker et al., 1981). This rapid proton release is thought to be an initial step in a series of steps which eventuate in photodispersal (Song et al., 1983). This paper summarizes the available evidence describing some of these intervening processes and the physiological mechanisms producing them.

Generalization of habituation between different receptor surfaces of Stentor

Abstract The protozoan, Stentor coeruleus , was repeatedly stimulated with a reproducible mechanical stimulus applied to a small portion of its cell surface. The animals probability of contracting in response to the stimulus decreased with repeated stimulation. After this habituation had occurred, the site of stimulation was moved to another area of the cell surface, but the decrement in responsiveness had generalized to that area also. Such generalization of habituation was not resultant from the contractions observed during habituation, since contractions induced by electrical stimuli did not depress response probabilities. Contractions in Stentor were associated with diphasic potentials which appeared asynchronously at different sites on the cell surface. These potentials appear to be propagated away from the site of mechanical stimulation at a rate sufficient to explain the speed with which the animal contracts. The directions of propagation observed require that the cell have at least two different receptor surfaces capable of independently initiating action potentials. Generalization of habituation may result from a diffusion process occurring between these receptor surfaces.

DOUBLET Stentor DO NOT DISPLAY PHOTODISPERSAL

Normal Stentor, called singlets since they have a single membranellar band and oral groove surrounding their frontal field, swim away from light sources and collect in the darker areas of an unevenly illuminated container (photodispersal). Phenotypic variants, called doublets since they have 2 membranellar bands and 2 oral grooves, do not exhibit this behavior. Doublets produce photophobic responses and contractions when illuminated at the same fluence rates which produce those responses in singlets, hence their sensitivity to light is normal. Illumination of the frontal field of doublets produces a photophobic response at lower fluence rates than does illumination of their side or posterior. This directional sensitivity is quantitatively similar to that observed in singlets. However, doublets do not reorient their swimming direction after a phobic response as extensively as do singlets. This failure in reorientation is the probable reason that doublets fail to show photodispersal. These results imply that the mechanism producing photodispersal in singlets depends on photophobic responses or some other, presently undescribed, response which requires an asymmetric frontal field.

Protozoa as Models of Stimulus Transduction

To the casual observer one of the most interesting behavioral characteristics of protozoa is their sensitivity to stimuli. At the beginning of this century Jennings (1906) and Mast (1906) described in detail some responses of protozoa to stimuli: the “avoiding reaction” of swimming ciliates when they bump into objects, the swimming behavior which causes flagellates to collect in lighted areas and the responses of amoeba to mechanical stimuli and food particles. These observations leave no doubt that protozoa can sense events in their external environment and therefore must contain receptor mechanisms.

Behaviors Producing Photodispersal in Stentor coeruleus

Abstract The ciliate Stentor coeruleus exhibits photodispersal, that is, these cells swim away from light sources and collect in dimly lighted areas. We imaged and reconstructed the tracks of 48 Stentor to determine which swimming behaviors produced their photodispersal. We observed that their photodispersal is not due to a change in their swimming speed but rather to a change in the frequency with which they reorient their swimming direction. Therefore, their photodispersal must be due to either (1) a gradual reorientation of the organisms swimming direction determined by the direction of the light beam (phototaxis) or (2) multiple randomly directed reorientations in swimming direction that occur less frequently when the cell is swimming away from the light source (biased random walk). Sixteen (19%) of the 83 observed forward swimming tracks lasting three or more seconds exhibited a gradual bending away from the light source consistent with a phototaxis. However, most tracks were interrupted repeatedly by abrupt reorientations resulting from ciliary reversals and “smooth turns” that caused cells to reorient through 5.4 times as many degrees as were needed to direct them away from the light source. When cells were swimming away from the light source, their probability of reorienting was reduced and photodispersal resulted.