Chemoreceptor Cells as Concentration Slope Detectors: Preliminary Evidence from the LobsterNoseAuthor(s): Erik Zettler and Jelle AtemaSource: Biological Bulletin, Vol. 197, No. 2, Centennial Issue: October, 1899-1999 (Oct., 1999),pp. 252-253Published by: Marine Biological LaboratoryStable URL: http://www.jstor.org/stable/1542633 .

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REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

Reference: Biol. Bull. 197: 252-253. (October 1999)

Chemoreceptor Cells as Concentration Slope Detectors: Preliminary Evidence from the Lobster Nose Erik Zettlerl and Jelle Atema (Boston University Marine Program, Marine Biological Laboratory, Woods Hole, Massachusetts 02543)

Chemoreceptor cells of the American lobster Homarus ameri- canus distinguish between different chemical compounds, as well as their concentrations (1, 2, 3). After an initial phasic response to an increase in stimulus concentration, chemoreceptor cells quickly adapt to the higher background and, within one to a few seconds, spike frequency reverts to zero or to a low tonic level (2, 3). The chemical signature of an odor source is dispersed through its fluid medium by generally turbulent flow resulting in patches of differ- ent sizes and shapes. As these spatial patches pass by a sensor they appear as concentration peaks over time. The onset slopes of these

peaks depend on the shape and concentration of the individual

patches. Despite the chaotic nature of turbulence there is a spatial gradient of patches within such an odor plume. Patches disperse in

statistically describable patterns, creating "odor landscapes" such that, closer to the source, the average odor peak heights and onset

slopes are greater (1, 4). If an animal could distinguish between shallow and steep odor onset slopes, it could orient itself in a turbulent plume and move towards or away from the odor source.

We have investigated the response of chemoreceptor cells of the lateral antennule of the American lobster Homarus americanus to odor onset slopes generated by computer-controlled piston pumps (Millipore model 510). The odor stimulus (a 0.01% aqueous ex- tract of TetraMarin fish food) was mixed with a dopamine tracer that allowed us to measure the actual stimulus concentration pro- file of each slope with high spatial and temporal resolution; the measurement was made with an IVEC-5 system (In Vivo Electro- Chemistry, Harvard Apparatus). Lateral antennules were excised and inserted, with aesthetasc sensilla facing up, into an--acrylic olfactometer chamber that permitted perfusion with cold oxygen- ated Ringers and use of a suction electrode to record from the

proximally exposed nerve bundle. The distal section bearing the aesthetascs was bathed with a constant flow of 10 ml min-' of filtered seawater into which the food odor and tracer dissolved in seawater could be injected by the piston pumps. Using a micro-

manipulator, the measuring tip of the IVEC electrode was placed within the aesthetasc tuft. Details of this tracer system, the olfac- tometer, and the extracellular recording techniques for this prep- aration are found elsewhere (3).

Four cells were tested with each of two stimulus slopes (Low and Medium). One of these cells was tested with an additional

slope (High) generated by suddenly opening a valve to gravity feed the stimulus to the preparation. This delivery system is similar to one used earlier (3) to achieve steep onset ramps but does not allow for easily controlled slope variation. Because all cells tested showed a similar response, we chose to show results from the cell with the greatest number of treatments (Fig. 1). Average spike frequency, calculated during the first second of stimulus concen- tration rise, was higher for steeper onset slopes, ranging from 3

'Also at Sea Education Association, Woods Hole, Massachusetts.

spikes per second for the Low slope to 60 spikes per second for the High slope. Despite this 20-fold increase in spike frequency in response to steeper slopes, the ratio of spike frequency to stimulus concentration change varied only by a factor of two. Since the length of the slopes and the peak concentrations varied between treatments and the purpose of this study was to investigate the effects of onset slopes, we also compared the spike frequency during the initial rise from background to when the dopamine tracer reached 8 ,IM (the highest concentration reached by the Low slope). Examined in this way, the change in concentration is identical across treatments, so differences in spike frequency

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Figure 1. Duplicate odor pulses for the three slopes (top traces in each panel), and the resulting spikes from a single chemoreceptor cell. The concentration of the dopamine tracer (measured at 200 Hz using IVEC-5) is plotted on the left y-axis; the dopamine was mixed in seawater with the odor stimulus and is assumed to disperse in a manner similar to the odor (4). The lower section of each panel represents the occurrence of individ- ual spikes grouped into 100 ms bins (right y-axis). For both the stimulus trace and the spikes, the first run is shown in black, and the second run in gray. Despite some variation in the pump outputs (especially after the peak concentration was reached) agreement during the initial rise in concen- tration was good between replicates. Spikes were digitized and sorted to ensure that they would represent a single receptor cell. Spike frequency increased with increasing odor onset slope.

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CHEMORECEPTION AND BEHAVIOR CHEMORECEPTION AND BEHAVIOR

should be due mainly to the different slopes. The spike frequency (and rise time) during this portion of each slope was 3.1 Hz (1800 ms) for the Low slope, 5.5 Hz (980 ms) for the Medium slope, and 70 Hz (160 ms) for the High slope. Once the concentration stopped rising, cells adapted to the constant background within a couple of seconds. We note that the lack of a response to the second slope rise in the Medium slope (middle panel between 7 and 8 seconds) of Figure 1 may be due to the large fluctuations in the output of the piston pumps preceding this rise. These periodic fluctuations (some of which exceed 5 ILM) would cause cumulative adaptation in the chemoreceptor cell such that the overall response is de- pressed (5). Because spike adaptation can occur rapidly, beginning within 500 ms of the stimulus presentation, a steeper onset slope may minimize adaptation and provide the highest frequency for a

given concentration. We will not discuss here the intracellular signal transduction pathways that may be involved in the excita- tion and adaptation phenomena observed.

These preliminary results demonstrate the feasibility of deliver- ing measured concentration slopes and provide initial evidence that chemoreceptor cells can act as "slope detectors." Odor slope

should be due mainly to the different slopes. The spike frequency (and rise time) during this portion of each slope was 3.1 Hz (1800 ms) for the Low slope, 5.5 Hz (980 ms) for the Medium slope, and 70 Hz (160 ms) for the High slope. Once the concentration stopped rising, cells adapted to the constant background within a couple of seconds. We note that the lack of a response to the second slope rise in the Medium slope (middle panel between 7 and 8 seconds) of Figure 1 may be due to the large fluctuations in the output of the piston pumps preceding this rise. These periodic fluctuations (some of which exceed 5 ILM) would cause cumulative adaptation in the chemoreceptor cell such that the overall response is de- pressed (5). Because spike adaptation can occur rapidly, beginning within 500 ms of the stimulus presentation, a steeper onset slope may minimize adaptation and provide the highest frequency for a

given concentration. We will not discuss here the intracellular signal transduction pathways that may be involved in the excita- tion and adaptation phenomena observed.

These preliminary results demonstrate the feasibility of deliver- ing measured concentration slopes and provide initial evidence that chemoreceptor cells can act as "slope detectors." Odor slope

discrimination could be very useful for orienting and tracking in a fluid environment, because so much physical information about the plume and odor source is present in the distribution of slopes in the eddy field (4). In this study, single cells could discriminate be- tween a range of odor onset slopes with rise times similar to those measured in laboratory studies of jet plumes (1, 4). In general, animals may have different chemoreceptor cells "tuned" to partic- ular ranges of pulse characteristics, such as frequency, height, length, and slope (1, 2, 3).

Thanks to D. Mellon and G. Gomez for helpful discussions. Supported by NSF grant IBN-9723542 to JA and a B.U.M.P. Humes alumni Award to EZ.

Literature Cited

1. Atema, J. 1996. Biol. Bull. 191: 129-138. 2. Borroni, P., and J. Atema. 1988. J. Comp. Physiol. A. 164: 67-74. 3. Gomez, G., and J. Atema. 1996. J. Exp. Biol. 199: 1771-1779. 4. Moore, P., and J. Atema. 1991. Biol. Bull. 181: 408-418. 5. Voigt, R., and J. Atema. 1990. J. Comp. Physiol. A. 166: 865-874.

discrimination could be very useful for orienting and tracking in a fluid environment, because so much physical information about the plume and odor source is present in the distribution of slopes in the eddy field (4). In this study, single cells could discriminate be- tween a range of odor onset slopes with rise times similar to those measured in laboratory studies of jet plumes (1, 4). In general, animals may have different chemoreceptor cells "tuned" to partic- ular ranges of pulse characteristics, such as frequency, height, length, and slope (1, 2, 3).

Thanks to D. Mellon and G. Gomez for helpful discussions. Supported by NSF grant IBN-9723542 to JA and a B.U.M.P. Humes alumni Award to EZ.

Literature Cited

1. Atema, J. 1996. Biol. Bull. 191: 129-138. 2. Borroni, P., and J. Atema. 1988. J. Comp. Physiol. A. 164: 67-74. 3. Gomez, G., and J. Atema. 1996. J. Exp. Biol. 199: 1771-1779. 4. Moore, P., and J. Atema. 1991. Biol. Bull. 181: 408-418. 5. Voigt, R., and J. Atema. 1990. J. Comp. Physiol. A. 166: 865-874.

Reference: Biol. Bull. 197: 253-254. (October 1999)

Individual Recognition and Memory in Homarus americanus Male-Female Interactions Cristin Berkey1 and Jelle Atema (Boston University Marine Program, Woods Hole, Massachusetts 02543)

Reference: Biol. Bull. 197: 253-254. (October 1999)

Individual Recognition and Memory in Homarus americanus Male-Female Interactions Cristin Berkey1 and Jelle Atema (Boston University Marine Program, Woods Hole, Massachusetts 02543)

Individual recognition and memory have been studied in Ho- marus americanus, the American lobster. It has previously been demonstrated, through lobster boxing matches, that males can

recognize individuals. When a male encounters the same male it lost to previously, it avoids a new fight. The same animal will fight on subsequent occasions if presented with an unfamiliar opponent (1). The ability to recognize a previous opponent lasts for up to a week, and it is based on olfactory recollection of the opponents' urine (2). The same results have been obtained in female-female encounters (3). We therefore hypothesize that male-female pairs of Homarus americanus can also recognize individuals, as demon- strated by shorter periods of fighting on the second encounter with familiar individuals.

Males and females were paired by carapace length, with no pair differing by more than 3 mm. It was also ensured that in all matched pairs, the animals had not previously met. Animals were

kept in isolation for 24 h before their first boxing match and for the 24 h between the first and second matches. All boxing matches took place in a 240-1 glass aquarium and were videotaped. One male and one female were placed in the tank and separated by a removable, opaque divider. The lobsters were allowed to acclimate for 10 min before the divider was removed, and then allowed to interact for 20 min. There were two sets of fights, group A and

group B. In group A, the same pair of animals fought twice. In

group B, the animals were rotated so that each animal met a new

opponent in the second fight. In both sets, the first and second

fights were separated by 24 h. Any pair of animals that did not

Individual recognition and memory have been studied in Ho- marus americanus, the American lobster. It has previously been demonstrated, through lobster boxing matches, that males can

recognize individuals. When a male encounters the same male it lost to previously, it avoids a new fight. The same animal will fight on subsequent occasions if presented with an unfamiliar opponent (1). The ability to recognize a previous opponent lasts for up to a week, and it is based on olfactory recollection of the opponents' urine (2). The same results have been obtained in female-female encounters (3). We therefore hypothesize that male-female pairs of Homarus americanus can also recognize individuals, as demon- strated by shorter periods of fighting on the second encounter with familiar individuals.

Males and females were paired by carapace length, with no pair differing by more than 3 mm. It was also ensured that in all matched pairs, the animals had not previously met. Animals were

kept in isolation for 24 h before their first boxing match and for the 24 h between the first and second matches. All boxing matches took place in a 240-1 glass aquarium and were videotaped. One male and one female were placed in the tank and separated by a removable, opaque divider. The lobsters were allowed to acclimate for 10 min before the divider was removed, and then allowed to interact for 20 min. There were two sets of fights, group A and

group B. In group A, the same pair of animals fought twice. In

group B, the animals were rotated so that each animal met a new

opponent in the second fight. In both sets, the first and second

fights were separated by 24 h. Any pair of animals that did not

1 Tufts University, Medford, Massachusetts. 1 Tufts University, Medford, Massachusetts.

show a definite loser and winner after the first 20-min period was

disqualified and the fight was not included in the data analysis. The fights were scored using a pre-established scale of agonistic

levels (1) with some additions made for differences that arose as a result of the fights being between males and females. An agonistic level was assigned to each animal every 5 s. The levels ranged from -2 to 5, with -2 demonstrating fleeing behavior and 5

demonstrating claw snapping or claw ripping at the opponent. An overall agonistic scale was calculated for each animal in each fight by summing all of the agonistic values. A fight was determined to be over when one animal ceased to show an agonistic level above one. In this way, the duration of each fight was scored. To prevent bias during analysis of the behavioral tapes, the scorer was blind to whether the tapes were from a first or second fight.

In group A, 10 pairs of fights were carried out. Significantly, in 9 out of 10 instances, no fighting occurred on the second encoun- ter; fighting did occur in the tenth case. In group B, 13 pairs of

fights were carried out. Two sets were disqualified because there was no clear winner after the first fight. In 3 out of 11 instances, no

fighting occurred on the second encounter. In 8 instances, fighting occurred. The fraction of times in which fighting occurred on the second encounter is significantly different between the two groups (P < 0.005 x2 = 8.416 df = 1).

When a fight was between familiar opponents, the aggression level of the loser was significantly lower in the second boxing match than in the first boxing match (P < 0.05 paired t test). However, for unfamiliar opponents, the aggression level of the loser was not significantly lower in the second boxing match than in the first (paired t test). The aggression level of the winner was

show a definite loser and winner after the first 20-min period was

disqualified and the fight was not included in the data analysis. The fights were scored using a pre-established scale of agonistic

levels (1) with some additions made for differences that arose as a result of the fights being between males and females. An agonistic level was assigned to each animal every 5 s. The levels ranged from -2 to 5, with -2 demonstrating fleeing behavior and 5

demonstrating claw snapping or claw ripping at the opponent. An overall agonistic scale was calculated for each animal in each fight by summing all of the agonistic values. A fight was determined to be over when one animal ceased to show an agonistic level above one. In this way, the duration of each fight was scored. To prevent bias during analysis of the behavioral tapes, the scorer was blind to whether the tapes were from a first or second fight.

In group A, 10 pairs of fights were carried out. Significantly, in 9 out of 10 instances, no fighting occurred on the second encoun- ter; fighting did occur in the tenth case. In group B, 13 pairs of

fights were carried out. Two sets were disqualified because there was no clear winner after the first fight. In 3 out of 11 instances, no

fighting occurred on the second encounter. In 8 instances, fighting occurred. The fraction of times in which fighting occurred on the second encounter is significantly different between the two groups (P < 0.005 x2 = 8.416 df = 1).

When a fight was between familiar opponents, the aggression level of the loser was significantly lower in the second boxing match than in the first boxing match (P < 0.05 paired t test). However, for unfamiliar opponents, the aggression level of the loser was not significantly lower in the second boxing match than in the first (paired t test). The aggression level of the winner was

253 253

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