Methods for Measuring Phototaxis of Cell Populations and Individual Cells

Two quantitative methods have been devised for studying the phototactic response of Chlamydomonas. In the first procedure, the movement of a cell population is continuously monitored photometrically, yielding a permanent record of the time course of the response. The monitoring system consists of a pair of photovoltaic cells connected in a comparison circuit, and a dim red light which passes through the swimming chamber and strikes the photocells. A stimulus beam enters the chamber at right angles to the monitoring light. The movement of algae towards the stimulus light produces a difference in output between the photocells. With a continuous stimulus, this difference increases in a nearly linear fashion for several ninutes. The slope of the linear portion depends on such factors as stimulus ntensity and wavelength and is used as the index of the response. In the second procedure, a photomicrographic method is used to resolve the population response into its components; i.e., the number of cells responding, the directness of the swimming path, and the rate of swimming. The photographs are taken with a fixed exposure time, during which each cell in the field describes a swimming track on the film. Swimming rate is determined by measuring the length of the photographed track, while directness of path is indicated by the angle between the track and the stimulus light beam. The number of cells going towards the light is calculated from counts made while observing the culture through the microscope. These methods have been used to investigate the dependence of phototaxis on stimulus intensity and wavelength, age of culture, and pre-illumination. This work formed a portion of a doctoral thesis submitted by one of the authors (M. E. Feinleib) to Harvard University, Cambridge, Mass. The work was supported in part by a U.S. Public Health Fellowship, No. FI-GM-17, 305–04, and by a National Science Foundation research grant, No. G14266.     

Encoding of sound duration by neurons in the auditory cortex of the little brown bat, Myotis lucifugus

Responses of 117 single- or multi-units in the auditory cortex (AC) of bats (Myotis lucifugus) to tone bursts of different stimulus durations (1– 400 ms) were studied over a wide range of stimulus intensities to determine how stimulus duration is represented in the AC. 36% of AC neurons responded more strongly to short stimulus durations showing short-pass duration response functions, 31% responded equally to all pulse durations (i.e., all-pass), 18% responded preferentially to stimuli having longer durations (i.e., long-pass), and 15% responded to a narrow range of stimulus durations (i.e., band-pass). Neurons showing long-pass and short-pass duration response functions were narrowly distributed within two horizontal slabs of the cortex, over the rostrocaudal extent of the AC. The effects of stimulus level on duration selectivity were evaluated for 17 AC neurons. For 65% of these units, an increase in stimulus intensity resulted in a progressive decrease in the best duration. In light of the unusual intensity-dependent duration responses of AC neurons, we hypothesized that the response selectivities of AC neurons is different from that in the brainstem. This hypothesis was validated by results of study of the duration response characteristics of single neurons in the inferior colliculus. Accepted: 8 November 1996     

Temperature Effect on the Photo-Accumulation and Phobic Response of Volvox aureus*

SYNOPSIS. The effect of temperature on photoaccumulation and photophobic response of Volvox aureus were studied. The algae exhibited positive photoaccumulation at room temperature and negative at low temperature. When stimulated with light of intermediate intensiy (～ 5 × 10³ lux), the phobic response of the algae consisted of a decrease in the frequency or the cessation of flagellar movement in the anterior cells. At room temperature, an increase in light intensity elicited the phobic response, whereas at low temperature a decrease in light intensity was the effective stimulus. The phobic response lasted only a few seconds. The positive and negative photoaccumulations of the algae could be explained by the brief cessation of flagellar movement in the anterior cells, elicited by an increase of stimulus light at room temperature or a decrease of stimulus at low temperature.     

Geotactic Behavior of Chlamydomonas

SYNOPSIS The normal negative geotaxis (motility oriented against gravity) of Chlamydomonas is an energy-dependent response that requires coordinated flagellar activity. It is evident from quantitative assays that the rate of geotaxis is steady, and slow relative to the average swimming speed.
Geotaxis is inhibited when the horizontal swimming path is less than 200 μm, suggesting that normal geotactic reorientation maneuvers involve long gradual turns. Videomicrographic tracking of cells confirms that such turns are common. In contrast, contact-reorientations generate random cell orientations. When collision frequencies increase, geotaxis in inhibited. The mechanism of normal geotactic orientation, then, depends on long slow reorientation maneuvers (from net downward to net upward vectors) that require hundreds of micrometers of free swimming space. Mechanisms of geotaxis that would require passive reorientation or sedimentation, or rapid active responding, are excluded.
Unusually dense populations sediment with atypical rapidity, probably due to formation of functionally aggregated subpopulations.
Sodium azide causes an inhibition of orientation behavior that is selective relative to its effects on general motility. Evidence presented suggests that active physiologic mechanisms for geotaxis should be reconsidered.     

Kompensation extremer Umwelteinflüsse beiBalanus balanus als Anpassung an die sessile Lebensweise

In the sessile barnacleBalanus balanus L. the relationship between stimulus duration and reaction intensity varies considerably. Investigations on the light reactions (increased cirral activity per unit time) and shadow reactions (decreased cirral activity per unit time) showed that increase of stimulus duration at a temporal range of 1–5 min and 30–360 min causes an increased response intensity, whereas, at a range of 5–30 min, the response intensity decreases. Two adaptive systems are assumed to be responsible for this behaviour. The peculiarities of the light and shadow reactions are interpreted as a result of interactions of central and peripheral components, but the unusual relationship between stimulus duration and reaction intensity is probably based on a greater influence of central components. The biological significance of the light and shadow reactions is discussed in relation to the larval and adult life: By means of the central variation of stimulus threshold in the described protective shadow responsesB. balanus seems to compensate for extreme environmental conditions. The light responses observed in the adults are considered as resulting from metamorphosis of larval swimming activity.     

Effect of larval ontogeny, turbulence and light on prey attack rate and swimming activity in herring larvae

The effects of ontogeny (larval size), light and turbulence on the attack rate and swimming activity (proportion of time swimming and duration of swimming bout) of herring larvae (15-28 mm TL) have been investigated. Emphasis was put on the experimental design in order to create a set-up where the turbulence intensity distribution could be accurately measured as well as controlled in the entire experimental tank.Both larval size (ontogeny) and light had a significant positive effect on prey attack rate. Likewise, an intermediate increase in turbulence had a positive effect on prey attack rate, but this effect was dependent of light intensity and larval size.At low light (1.5 μE m² s⁻¹) intermediate turbulence increased the prey attack rate significantly for larger larvae (26 and 28 mm), while at high light (18 μE m² s⁻¹) intermediate turbulence had only a significant positive effect on the attack rate of smaller larvae 20 and 23 mm.In general, our data show a dome-shaped response of turbulence on attack rate and a U-shaped response of turbulence on swimming activity.For herring larvae >20 mm, the maximum (attack rate) and minimum (swimming activity) response of turbulence were found at intermediate turbulence intensities (energy dissipation rates between 7∗10⁻⁸ and 1∗10⁻⁶ W/kg). The highest turbulence level tested (8∗10⁻⁶ W/kg) showed only negative effects, as attack rates where at the lowest and swimming activity at the highest.Swimming activity increased with larval size or light, and decreased at intermediate turbulence. Compared to turbulent intensities under natural conditions this implies that larger herring larvae at 10 m depth have to be exposed to wind speeds of more than 17 m/s before negative effects on attack rate and swimming activity occurs.     

Swimming of Daphnia galeata x hyalina in response to changing light intensities: Influence of food availability and predator kairomone

Experiments showed that phototactic downward swimming in Daphnia galeata x hyalina as caused by a relative increase in light intensity (stimulus) is influenced by predator kairomone and food availability. The swimming responses at four different combinations of food availability and fish kairomone were analysed. Addition of both food and kairomone led to a significant increase in percentage of animals that responded to the light stimulus, but there was no significant interaction effect.We also found that kairomone and food had significant impact on displacement velocity and on the time between start of the stimulus and onset of the response.     

On the pressure response of Eurydice sp. (Isopoda)

Eurydice responds to increased pressure within the 67 to 790 mb range by an upward swimming, the duration of the response being dependent on the magnitude of the pressure change. Release of pressure is followed by downward swimming or passive sinking.

The responses are orientated primarily to gravity, but with a subsidiary photic influence, and brief pulses of 1–2 sec duration are less effective in eliciting a response.

Adaptation to sustained high pressure appears to involve a shift in the stimulus/response curve with no evident loss of sensitivity.

The pattern of swimming behaviour observed following sinusoidal pressure cycles suggests that, over the range of cycles studied, the parameter most effective in inducing the response is change of pressure, or perhaps pressure itself, rather than the velocity component of the stimulus.

Pressure perception is not affected by surface active substances, nor by changes in sea water concentration, suggesting that the process of transduction does not involve compression of a hydrogen gas film on the body surface.     

The photobehaviour of Daphnia spp. as a model to explain diel vertical migration in zooplankton

Many pelagic animal species in the marine environment and in lakes migrate to deeper water layers before sunrise and return around sunset. The amplitude of these diel vertical migrations (DVM) varies from several hundreds of metres in the oceans to approx. 5–20 m in lakes. DVM can be studied from a proximate and an ultimate point of view. A proximate analysis is intended to reveal the underlying behavioural mechanism and the factors that cause the daily displacements. The ultimate analysis deals with the adaptive significance of DVM and the driving forces that were responsible for the selection of the traits essential to the behavioural mechanism. The freshwater cladoceran Daphnia is the best studied species and results can be used to model migration behaviour in general. Phototaxis in Daphnia spp., which is defined as a light-oriented swimming towards (positive phototaxis) or away (negative phototaxis) from a light source, is considered the most important mechanism basic to DVM. A distinction has been made between primary phototaxis which occurs when light intensity is constant, and secondary phototaxis which is caused by changes in light intensity. Both types of reaction are superimposed on normal swimming. This swimming of Daphnia spp. consists of alternating upwards and downwards displacements over small distances. An internal oscillator seems to be at the base of these alternations. Primary phototaxis is the result of a dominance of either the upwards or the downwards oscillator phase, and the direction depends on internal and external factors: for example, fish-mediated chemicals or kairomones induce a downwards drift. Adverse environmental factors may produce a persistent primary phototaxis. Rare clones of D. magna have been found that show also persistent positive or negative primary phototaxis and interbreeding of the two types produces intermediate progeny: thus a genetic component seems to be involved. Also secondary phototaxis is superimposed on normal swimming: a continuous increase in light intensity amplifies the downwards oscillator phase and decreases the upwards phase. A threshold must be succeeded which depends on the rate and the duration of the relative change in light intensity. The relation between both is given by the stimulus strength versus stimulus duration curve. An absolute threshold or rheobase exists, defined as the minimum rate of change causing a response if continued for an infinitely long time. DVM in a lake takes place during a period of 1-5-2 h when light changes are higher than the rheobase threshold. Accelerations in the rate of relative increase in light intensity strongly enhance downwards swimming in Daphnia spp. and this enhancement increases with increasing fish kairomone and food concentration. This phenomenon may represent a ‘decision-making mechanism’ to realize the adaptive goal of DVM: at high fish predator densities, thus high kairomone concentrations, and sufficiently high food concentrations, DVM is profitable but not so at low concentrations. Body axis orientation in Daphnia spp. is controlled with regard to light-dark boundaries or contrasts. Under water, contrasts are present at the boundaries of the illuminated circular window which results from the maximum angle of refraction at 48–9° with the normal (Snell's window). Contrasts are fixed by the compound eye and appropriate turning of the body axis orients the daphnid in an upwards or an obliquely downwards direction. A predisposition for a positively or negatively phototactic orientation seems to be the result of a disturbed balance of the two oscillators governing normal swimming. Some investigators have tried to study DVM at a laboratory scale during a 24 h cycle. To imitate nature, properties of a natural water column, such as a large temperature gradient, were compressed into a few cm. With appropriate light intensity changes, vertical distributions looking like DVM were obtained. The results can be explained by phototactic reactions and the artificial nature of the compressed environmental factors but do not compare with DVM in the field. A mechanistic model of DVM based on phototaxis is presented. Both, primary and secondary phototaxis is considered an extension of normal swimming. Using the light intensity changes of dawn and the differential enhancement of kairomones and food concentrations, amplitudes of DVM could be simulated comparable to those in a lake. The most important adaptive significance of DVM is avoidance of visual predators such as juvenile fish. However, in the absence of fish kairomones, small-scale DVMs are often present, which were probably evolved for UV-protection, and are realized by not enhanced phototaxis. In addition, the ‘decision-making mechanism’ was probably evolved as based on the enhanced phototactic reaction to accelerations in the rate of relative changes in light intensity and the presence of fish kairomones.     

Behavioural Stress Responses Predict Environmental Perception in European Sea Bass (Dicentrarchus labrax)

Individual variation in the response to environmental challenges depends partly on innate reaction norms, partly on experience-based cognitive/emotional evaluations that individuals make of the situation. The goal of this study was to investigate whether pre-existing differences in behaviour predict the outcome of such assessment of environmental cues, using a conditioned place preference/avoidance (CPP/CPA) paradigm. A comparative vertebrate model (European sea bass, Dicentrarchus labrax) was used, and ninety juvenile individuals were initially screened for behavioural reactivity using a net restraining test. Thereafter each individual was tested in a choice tank using net chasing as aversive stimulus or exposure to familiar conspecifics as appetitive stimulus in the preferred or non preferred side respectively (called hereafter stimulation side). Locomotor behaviour (i.e. time spent, distance travelled and swimming speed in each tank side) of each individual was recorded and analysed with video software. The results showed that fish which were previously exposed to appetitive stimulus increased significantly the time spent on the stimulation side, while aversive stimulus led to a strong decrease in time spent on the stimulation side. Moreover, this study showed clearly that proactive fish were characterised by a stronger preference for the social stimulus and when placed in a putative aversive environment showed a lower physiological stress responses than reactive fish. In conclusion, this study showed for the first time in sea bass, that the CPP/CPA paradigm can be used to assess the valence (positive vs. negative) that fish attribute to different stimuli and that individual behavioural traits is predictive of how stimuli are perceived and thus of the magnitude of preference or avoidance behaviour.     

Sensitization and habituation of the swimming behavior in ascidian larvae to light

Ascidian larvae of Ciona intestinalis change their photic behavior during the course of development. Newly hatched larvae show no response to a light stimulus at any intensity. At 4 hr after hatching, larvae were induced to start to swimming upon the cessation of illumination, and to stop swimming upon the onset of illumination. At a weaker light intensity (5.0 x 10(-3) J/m (2).s), the larvae showed similar responses to either a single stimulus or repeated stimuli of onset and cessation of light until 10 hr after hatching. At a stronger light intensity (3.2 x 10(-1) J/m(2).s), when the stimulus was repeated, they showed sensitization and habituation of the swimming response. At 3 hr after hatching the larvae failed to show any response to an initial stimulus at any intensity of light, but after several repeated stimuli (sensitization) they showed a swimming response at light intensities above 4.0 x 10(-2) J/m (2).s. At 5 hr and with intensity above 1.0 x 10 (-2) J/m(2).s, the larvae showed photoresponses to the first stimulus, but after several repetitions the larvae failed to stop swimming upon the onset of light (habituation). A repeated series of stimuli at stronger intensities of light caused greater habituation; this habituation was retained for about 1 min. Since the larval central nervous system in Ciona is comprised of only about 100 neurons, learning behavior in ascidian larvae should provide insights for a minimal mechanism of memory in vertebrates.     

A Novel Effect in Phycomyces Phototropism : Positive Bending and Compensation Spectrum in Far UV

A novel effect—positive phototropic bending under far UV irradiation (between 260 and 305 nanometers) at low intensities—is reported. Natural compensation points (intensities which cause no bending under unilateral irradiation) have been determined for different wavelengths. The curve connecting these points, the compensation spectrum, divides the intensity-wavelength plane into areas of negative and positive tropism. It is further shown that a highly asymmetrical pattern of light stimulus within the sporangiophore underlies the symmetrical growth response at each compensation point. This suggests that some unknown additional factor is involved in perceiving a UV stimulus at the level of the photoreceptor. It is also demonstrated here that positive tropism in the UV range is due to a lens effect. We conclude that the hypothesis of optical attenuation of the stimulus (considered until now as the most plausible explanation of negative tropism in the UV spectral range) must be dismissed. The results presented here represent the first application of our quantitative theoretical consideration of spatial factors in phototropism heretofore neglected by others.     

Brain-stem auditory evoked potentials in the common marmoset (Callithrix jacchus)

Brain-stem auditory evoked potentials (BAEPs) were recorded in 10 common marmosets (Callithrix jacchus) to investigate the effects of recording electrode configurations, stimulus rate, and stimulus frequency on BAEP wave forms and peak latencies. Tone burst stimulations were used to evaluate the effects of pure tone on BAEP wave forms. Five positive peaks superimposed on positive and negative slow potentials were identified in the BAEP recorded at the linkage between the vertex and the dorsal base of the ear ipsilateral to a monaural stimulus. When the reference electrode was placed at the ipsilateral mastoid or the neck, the amplitudes of positive and negative slow potentials and the incidence of wave I increased. There were no significant changes in peak latencies of BAEP waves with changes in stimulus rate from 5 to 20/s. It was possible to record the BAEPs in response to tone burst stimulations at frequencies extending from 0.5 to 99 kHz. Wave I appeared apparently at high stimulus frequencies; while waves III to V, at low frequencies. Wave II was recorded at frequencies ranging from 0.5 to 99 kHz and comprised a superposition of 2 or 3 potentials.     

Intensity Characteristics of the Noctuid Acoustic Receptor

Spiking activity of the more sensitive acoustic receptor is described as a function of stimulus intensity. The form of the intensity characteristic depends strongly on stimulus duration. For very brief stimuli, the integral of stimulus power over stimulus duration determines the effectiveness. No response saturation is observed. With longer stimuli (50 msec), a steady firing rate is elicited. The response extends from the spontaneous rate of 20–40 spikes/sec to a saturated firing rate of nearly 700 spikes/sec. The characteristic is monotonic over more than 50 db in stimulus intensity. With very long stimuli (10 sec), the characteristics are nonmonotonic. Firing rates late in the stimulus decrease in response to an increase in stimulus intensity. The non-monotonic characteristics are attributed to intensity-related changes in response adaptation.     

Mechanism of Photoaccumulation in Paramecium bursaria

Responses of Paramecium bursaria to light intensity changes were investigated. The resting paramecia show a direction changing response (photophobic response) to a sudden decrease of light intensity, whereas no response was shown to an increase in intensity. The critical intensity decrease dI_c necessary to show the response was measured at various values of initial light intensity, and the ratio dI_c/I was found to be equal to ～0.15. The swimming paramecia show different behavior depending on their swimming direction in the spatial gradient of light intensity. Paramecia show direction change more frequently when they are swimming down the gradient than in the opposite direction. This difference in the rate of direction changing is 13–17%. These results may offer an explanation for the mechanism of photoaccumulation.     

What goes down must come up: symmetry in light-induced migration behaviour of Daphnia

During a short period of the year, Daphnia may perform a phenotypically induced diel vertical migration. For this to happen, light-induced swimming reactions must be enhanced both at dawn and at dusk. Enhanced swimming in response to light intensity increase can be elicited by fish-associated kairomone in the laboratory, if food is sufficiently available. However, during the light change at dusk the Daphnia are still in the hypolimnion, where no fish kairomone is present and both temperature and food availability is low. Still, what goes down must come up. This raises questions about how Daphnia tunes its light-induced swimming behaviour to prevailing conditions such that a normal diel vertical migration can be performed. We investigated the symmetry in behavioural mechanism underlying these diel vertical migrations in the hybrid Daphnia galeata × hyalina (Cladocera; Crustacea), with special interest for the environmental cues that are known to affect swimming in response to light increase. That is, we tested whether fish- associated kairomone, food availability, and temperature affected both swimming in response to light intensity increase and decrease similarly. We quantified swimming behaviour during a sequentially increased rate of light change. Vertical displacement velocity was measured and proved to be linearly related to the rate of the light change. The slope (PC) of the function depends on the value of the factors kairomone concentration, food availability, and temperature. The changes of the PC with kairomone concentration and with temperature were similar both at light intensity increases and decreases. The PC also increased with food concentration, although during light increases in a different way from during light intensity decreases. Low food availability inhibited swimming in response to light intensity increase, but enhanced swimming in response to light intensity decrease. Hence, ascent from the deep water layers with low food concentration at dusk is facilitated. These causal relations are part of a proximate decision-making mechanism which may help the individual Daphnia to tune migration to predation intensity and food availability.     

Photoresponse and Learning Behavior of Ascidian Larvae, a Primitive Chordate, to Repeated Stimuli of Step-Up and Step-Down of Light

Ascidians are lower chordates and their simple tadpole-like larvae share a basic body plan with vertebrates. Newly hatched larvae show no response to a stimulus of light. 4 h after hatching, the larvae were induced to swim upon a step-down of light and stop swimming upon a step-up of light. At weaker intensity of light, the larvae show the same response to a stimulus after presentation of repeated stimuli. When intensity of actinic light was increased, the larvae show sensitization and habituation of the swimming response to a stimulus after repeated stimuli of step-down and step-up of the light. Between 2 h 20 min and 3 h 40 min after hatching the larvae did not show any response to the first stimulus, but after several repeatedstimuli they show swimming response to a step-down of light. A repeated series of stimulus cause sensitization. Between 4 h and 7 h after hatching, the larvae show photoresponse to the first stimulus, but after several repetition of the stimuli, the larvae could not stop swimming to a stimulus of a step-up of the actinic light. A repeated series of stimulus cause greaterhabituation. Both sensitization and habituation depend upon intensity ofactinic light.     

Dependence of unit responses of the catlateral geniculate body on photic stimulus contrast

Unit responses in the lateral geniculate body of cats to photic stimuli of different contrast were investigated. The number of spikes in the initial phase of the responses (the first 30–45 msec) was found not to change at first, but then to decrease with an increase in the intensity of background illumination. The background intensity starting from which the response diminishes was shown to increase with an increase in the intensity of the test stimulus. The unit response is a linear function of the logarithm of stimulus contrast if the contrast is changed through variation of the intensity of the test stimulus. If contrast increases on account of a decrease in the intensity of background illumination the responses first increase and then remain unchanged. The range of contrasts within which the response is a linear function is narrowed if the intensity of the test stimulus is reduced. Counting the number of spikes in different time intervals of the response (t) showed that the greater the value of t (within the first 70–90 msec of the response) the steeper the curve of the number of spikes as a function of contrast. The Weber-Fechner law applies in the receptive field of the lateral geniculate body. The results are compared with those of some psychophysiological experiments.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 9, No. 3, pp. 267–274, May–June, 1977.     

The effect of accelerations in light increase on the phototactic downward swimming of Daphnia and the relevance to diel vertical migration

The effect of relative increases in light intensity on photobehaviourwas studied in the hybrid Daphnia galeata x hyalina. We firstcarried out a series of experiments to study the influence offish kairomone on several response variables of light-inducedswimming. With fish kairomone present, an increase in the percentageof reacting daphnids to 100% was found at almost all ecologicallyoccurring relative light change rates that were above threshold.The relationship between the relative increase in light intensity(stimulus) and the time expiring between the onset of the stimulusand the start of the downward swimming response was not influencedby fish kairomone, nor did kairomone alter the functional relationshipbetween stimulus strength and downward displacement velocity,although velocity increased. During the previous experiments,various light change rates were applied, but per test run theserates were constant. The natural relative light increase inthe early morning consists of continuously increasing relativelight change rates, turning into decreasing rates after themaximum is reached