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The rate and retention of the habituation of the shadow reflex in Balanus improvisus (Cirripedia)
Anim. Behav., 1970,18, 61 6-620
THE RATE AND RETENTION OF THE HABITUATION OF THE SHADOW REFLEX IN BALANUS IMPRO VISUS (CIRRIPEDIA) BY
KARL Y. H. LAGERSPETZ & LIISA KIVIVUORI
Laboratory of Zoophysiology, Department of Zoology, University of Turku, Finland
Habituation is generally considered to be the simplest type of learning (Thorpe 1956, p . 54). The characteristics of habituation have been summarized by Thompson & Spencer (1966) in their extensive review of the subject . In spite of its wide occurrence among representatives of probably all animal phyla, only a few studies have been concerned with the mechanisms of habituation and with the mechanisms of the recovery of the habituated response . Bruner & Tauc (1966) studied the compound and simple excitatory post-synaptic potentials in the left pleural ganglion of the sea hare Aplysia during habituation of the tentacular contraction response to mechanical and electrical stimulation . A series of stimuli caused a progressive diminution of the post-synaptic potentials of interneurones . Using series of successive stimuli, repeated at intervals, Bruner & Tauc (1966) found that the effect lasted for some time after the end of the train of stimuli . It was, however, completely abolished by other types of stimuli . An extensive study of the effects of various factors on the rate of habituation of the startle reflex in the polychaete worm Nereis was made by Clark (1960a, b) using mechanical and photic stimuli. Clark also paid attention to the retention of habituation, i .e . on the variables affecting the recovery of the habituated response . Massed photic stimuli (light on 2 s with an interval of 30 s between successive stimuli) resulted in a more rapid habituation than spaced stimuli (light on for 12 s with an interval of 180 s between successive stimuli) . In his group experiments, complete habituation (all animals failed to react) was achieved in forty-seven trials in massed conditions, and in sixty-four trials in spaced conditions. The corresponding figures after a pause of 1 hr were twenty-two and thirtytwo trials, and after a pause of 5 hr, fifty-two and forty-two trials . The results indicate a somewhat better retention after a slower rate of habituation . However, in group experiments the number of stimuli presented to each animal in the group was the same, irrespective of its individual rate of habituation . Thus the amount of over-training varied, and was not controlled .
Gwilliam (1963, 1966) studied the mechanism of the shadow reflex in barnacles (Balanus). The habituation of this reflex was first described by Pi6ron (1910, p 121) . Through electrophysiological recording from different parts of the reflex arc, Gwilliam (1963, 1966) was able to localize the site of habituation to the path from the supreoesophageal ganglion to the motor neurones of the ventral ganglion . The aim of the present experiments was to study the factors affecting the retention of habituation, or conversely, the recovery of the habituated response, in a simple and well-known reflex system . The rate of habituation was thought to be one of the main variables affecting retention . Thus the rate of habituation of the shadow reflex in the barnacle Balanus improvisus (Crustacea : Cirripedia) was varied either by using different individuals or by using different temperatures . Retention was measured by repeated exposures of the animals to series of stimuli . Overtraining was avoided. Another factor which was thought to affect retention, was the externally applied concentration of the assumed neurotransmitter of the reflex system . Methods The test animals were collected during summer months from sea outside the city of Turku (salinity of the Baltic sea water six parts per thousand) together with pieces of the substratum (mostly Fucus) on which they were attached . The animals were stored in aerated sea water at -}-10°C. For the experiment, a piece of substratum with the attached animal was transferred to a glass dish containing sea water . The dish was placed in a translucent water bath . The bath was partially surrounded by black cardboard, which left only one side open . A microscope lamp placed at a fixed distance from the animal dish was used as the light source . The light beam from the lamp to the animal dish could be periodically interrupted by a black circular cardboard disc, from which sectors of appropriate size had been cut out . The disc was attached on the axis of an electrically driven 616
LAGERSPETZ & KIVIVUORI : HABITUATION OF THE SHADOW REFLEX IN BARNACLES
kymograph . In most experiments, the stimulus was a shadow lasting 4 s and the interval between stimuli lasted for 8 s. In all experiments, the animals were allowed to adapt for 30 min to the light conditions in the animal dish before the beginning of the experiment . The temperature of the water in the animal dish was measured with a thermocouple . It could be varied by changing the temperature of the water bath surrounding the animal dish . The response of the animals to the shadow consisted in the cessation of the beat of the cirri, the withdrawal of cirri, and the closure of the shell . The first stimuli usually produced a long retraction, while the animal responded to the later stimuli with a retraction lasting for a few seconds only, and finally, no retraction was elicited . Stimuli were presented at the same rate even when the barnacle was retracted . When the animal responded to only one out of five successive stimuli, it was considered to be habituated . The number of stimuli needed for this degree of habituation was used as the inverse measure of the rate of habituation . Retention was tested by repeating the series of stimuli after 2 . 5, 5 or 10 min . During this interval the animals were in light in the animal dish. The second series of stimuli was also interrupted when the criterion for habituation was reached. This procedure was again repeated after a second similar interval . Each animal was used for one experiment only . The numbers of stimuli needed for habituation in the three successive series were designated as h 1 , h 2 and h 3 . Retention (r) was calculated as the percentage of decrease in the number of stimuli needed for habituation : h1 h2
x 100,
h2-h3 r2=h2h 2
x 100 .
r1 = 1
and, respectively :
Results 1. Activity and Habituation Rate Habituation to a shadow lasting for 4 s and
617
presented at intervals of 8 s was studied in 110 animals, the maximum diameter of the base plate (shell length) of which varied from 2 to 10 mm. The animals were not taken from the storage containers at random, but the most active specimens were selected . Before the experiments, the animals had been stored at room temperature (19 to 23°C) for 1 day . Seven test animals responded to the shadow by such a long retraction that habituation could not be produced . From the remaining 103 animals, seventy-six were classified as active. Such animals were regarded as active, in which the longest retraction (except the first one) lasted for less than 1 min (corresponding to five stimulus-interval cycles) . In the twenty-seven passive animals, the maximum retraction time was longer. The number of stimuli needed for the attainment of the criterion for habituation (h) varied from fourteen to 150 . The distribution of the active and passive animals according to the value of h appears in Table I . It is obvious from the direct inspection of the data that the distributions are not different. Since the passive animals had their shell closed for a longer time than the active ones, the result seems to indicate that the habituation process continues also when stimuli are presented during the closure of the shell. Before each experiment, the cirral beating frequency was measured using a stopwatch . Newell & Northcroft (1965) showed that in Balanus balanoides the cirral beating frequency was an exponential function of the body weight. In this study, the correlation coefficient between the shell length and the cirral beating frequency was -0 .360. The correlation was statistically significant at the level of P<0 .001 . The inverse relationship found between the body size and the cirral beating frequency in Balanus balanoides by Newell & Northcroft (1965) is thus also found in Balanus improvisus. The correlation coefficient calculated between the shell length and the number of stimuli needed for habituation (h) was 0 .094 and the correlation between cirral beating frequency and the number of stimuli needed for habituation was 0 .144 . Neither of
Table I . Distribution of the Active and Passive Animals According to the Number of Stimuli Needed for Habituation (h) h
14-30
31-60
61-90
91-120
121-150
No. of active animals
15(20%)
38 (50Y.)
13 (17 %)
7 (19 %)
3 (4 %)
No . of passive animals
6 (22%)
15 (55%)
5(19%)
1( 4 %)
618
ANIMAL BEHAVIOUR, 18, 4
Table IL Numbers of Stimuli Needed for Habituation at Intervals of 5 min (ht, h2, h3) and the Corresponding Retention Values (r1, r2) at Different Experimental Temperatures (± SE) Experimental temperature
h1
r1
h2
r2
12°C
22 . 2±1 .9
30
15 . 6±1 . 4
22°C
50 . 5±5 . 1
49
25. 9±2 .4
these correlation coefficients indicates a statistically significant correlation . These results show that there is a large variation in the rate of habituation of the shadow reflex, measured as the number of stimuli needed for habituation . In addition, this variation cannot be accounted for by the variation in the body size or in the activity of the animals . 2 . Habituation Rate and Retention after Different Recovery Times Altogether, twenty animals were habituated twice at a recovery interval of 2 . 5 min, forty animals using an interval of 5 min, and nineteen with an interval of 10 min . From the values of h 1 and h 2 the retention r 1 was calculated . The correlation coefficient was calculated between the values of h l and r 1 in each group. The correlation coefficients were as follows Recovery interval 2 . 5 min 0 . 656 (P<0 . 01) Recovery interval 5 min 0 . 383 (P<0 . 05) Recovery interval 10 min 0 . 228 NS The correlation coefficients for the two first groups were statistically significant . The re gression equations for these groups were the following : r 1 = - 7 .683 + 40 . 382 x h 1 and r 1 = 7 . 086 + 27 . 005 x h l , respectively. Thus, when a series of stimuli is presented to the animals shortly after they have been habituated, retention of the habituation is proportional to the number of stimuli originally needed to produce the habituation . In other words, the recovery of the response which has occurred is proportional to the original rate of habituation . However, this relationship disappears as the recovery proceeds. The time-course of the recovery for the animals with different habituation rates is presented in Fig . 1 . It is evident that the recovery of the habituated response is most rapid in the group with the most rapidly habituating animals . 3 . Temperature, Habituation Rate and Retention In another series of experiments, twenty-nine animals were habituated three times at 9 ° to 15 °C
h3
N
13
13 . 6±1 . 2
29
25
19 .5±1 .7
36
and thirty-six treated similarly at 19° to 23°C . The mean temperatures were 12° and 22°C, respectively . The numbers of stimuli needed for habituation at each occasion h, as well as the retention values r are given in Table II . The time 100
0 5 :S h, :5 30
• 31<_h I- 60 A61<_h 1 <90 v91<_h,<120
47
0
I I
2 .5
5
I 10
min
Fig . 1 . Retention of habituation as function of recovery time (rest interval in minutes between stimulus periods) and of the original rate of habituation (h 1) .
interval between the successive stimulus sequences was 5 min . The values of h for successive stimulus sequences are plotted in Fig . 2 . The habituation was significantly more rapid at 12°C than at 22°C . Less retention was found after 5 min at 12°C than at 22 °C . Again, at higher rate of habituation, a more rapid recovery occurred . 4. Acetylcholine, Habituation Rate and Retention The effect of acetylcholine (ACh) on the habituation rate and retention was studied in fourteen animals . Half of these were habituated three times in sea water, then transferred to sea water with 1 . 67 x 10- 5 g per ml ACh chloride
619
LAGERSPETZ & KIVIVUORI : HABITUATION OF THE SHADOW REFLEX IN BARNACLES
The experiment was repeated with 10 - 4 g per ml ACh chloride using eight animals . As these animals were collected in wintertime, the control level is not directly comparable with those presented above . The mean habituation value without ACh was 25 . 3 + 3 . 1, and with ACh 45 . 1 ± 4. 9, the difference being statistically significant (P<0 . 01) .
60-
50
40
30
20
10
0 III I II Fig. 2 . Rate of habituation measured as the number of stimuli needed to habituation (h) in three subsequent experiments, performed at intervals of 5 min . The average habituation rates for the groups at 22° and 12°C show the effect of temperature, the average habituation rates for the groups marked ACh and no ACh show the effect of 1 . 67 x 10 - 5 g per ml ACh chloride on habituation. 1 l I
for I hr and, subsequently habituated in the ACh solution for three times . The other half of the animals were first kept and habituated in 1 . 67 x 10 - 5 g per ml ACh chloride, then allowed to recover in sea water, and again habituated in sea water . As the results were similar in both cases, they were combined and are presented in Table III and in Fig. 2 . Acetylcholine apparently slightly reduced the number of stimuli needed for the first habituation (h 1 ) . However, it affected the subsequent habituations in the opposite way, so that the retention values (r 1 and r 2) were definitely decreased . The difference between the last habituation values (h3) is, when evaluated with the t-test, statistically significant (P<0 .001) .
Discussion Horn (1967) showed that many characteristics of habituation could be accounted for on the hypothesis that a self-generated depression of neuronal sensitivity occurs at one or more points of the system to which a stimulus is repeatedly applied . Horn used the term self-generated depression to include such phenomena as after hyperpolarization, conduction block due to extracellular potassium accumulation around an active fibre, and synaptic depression representing an imbalance between the neurotransmitter mobilization and utilization . Bruner & Tauc (1966) consider the last of these, the transmitter depletion at synaptic terminals as the most likely mechanism of the neuronal habituation in Aplysia. If habituation is considered to be the outcome of transmitter depletion caused by frequent release of the transmitter, then the recovery of the habituated response may be interpreted as a result of synthesis, uptake or axonal transport of the transmitter. This `transmitter depletion hypothesis' of habituation may be used to explain the present data . The observed differences in the rate of habituation could be caused either by differences in the amount of transmitter originally present in the appropriate synaptic terminals, or by differences in the release in response to stimulation . The present results show that the rate of recovery is in its first stages proportional to the rate of the preceding habituation . It is difficult to conceive that the recovery process would be related to the amount of transmitter present before the habituation . It seems more plausible to assume
Table III. Effect of Acetylcholine (ACh) on the Numbers of Stimuli Needed for Habituation at Intervals of 5 min and the Corresponding Retention Values (r1, r2) (± SE) h2
r2
h3
(h1, h 2, h 3)
N
h1
r1
Control
52 .4±7 . 1
58
22 . 1±3 . 9
24
16 .9±2 . 0
14
ACh
40 . 1±5 . 3
27
29 . 5±3 . 7
-
29 . 8±3 . 3
14
620
ANIMAL BEHAVIOUR, 18, 4
that the observed differences in the rate of habituation would be caused by differences in the release of the transmitter, and that the rates of synthesis, uptake and transport are in balance with the rates of transmitter release and breakdown . This would account for the relationship observed between the rate and retention of habituation . Low temperatures certainly depress the metabolism and release of the transmitter substances . Since a proportionality was found between the first habituation and retention values these could be used to yield some information about the temperature characteristics of the processes of transmitter release, respectively transmitter synthesis, uptake and transport . The Q 10 value for h 1 from Table II is 2 .27 and for r 1 1 .63 . This indicates a higher temperature dependence for the transmitter release than for the transmitter recovery. Low temperature thus depressed the transmitter release, and habituation occurred rapidly . On the other hand, the recovery was not affected to an equivalent degree, and the released small amount of transmitter was soon replaeed . In the experiments of Waldes (1938), acetylcholine and atropine strongly affected the shadow reflex in Balanus. This supports the view that cholinergic transmission is involved in the mechanism of the shadow reflex . The results of Waldes (1938) also show that externally applied acetylcholine probably enters the nervous system of barnacles . In the present experiments it was found that externally applied acetylcholine increased the number of stimuli needed for habituation and decreased retention, i .e . enhanced the recovery of the habituated response . In the terms of the transmitter hypothesis, the uptake of acetylcholine to the pre-synaptic terminals, or the post-synaptic depolarization was enhanced . This presumably also happens when dishabituation is produced by stimulating the habituated animal with a different type of stimulus which elicits the same response . Summary 1 . There is large variation in the rate of
habituation of the shadow reflex in the barnacle Balanus improvisus. This variation cannot be accounted for by the variation in the body size or in the activity of the animals . 2. When only a short recovery time is allowed, the rate of the recovery of the habituated response is correlated with the rate of the preceding habituation . The same result is obtained when the habituation rate is varied using different animals or different temperatures . 3. Habituation is more rapid at 12°C than at 22°C. 4. Acetylcholine chloride (1 . 67 x 10- 5 g per ml) enhances the recovery of the habituated response. 5. The results are in agreement with the transmitter depletion hypothesis of habituation . REFERENCES Bruner, J. & Tauc, L . (1966) . Habituation at the synaptic level in Aplysia. Nature, Lond., 210, 37-39 . Clark, R . B. (1960a) . Habituation of the polychaete Nereis to sudden stimuli. I. General properties of the habituation process. Anim. Behav ., 8, 82-91 . Clark, R . B . (1960b) . Habituation of the polychaete Nereis to sudden stimuli . II. Biological significance of habituation . Anim . Behav., 8, 92-103 . Gwilliam, G . F. (1963) . The mechanism of the shadow reflex in Cirripedia . I . Electrical activity in the supraoesophageal ganglion and ocellar nerve . Biol. Bull., 125, 470-485 . Gwilliam, G . F. (1966) The mechanism of the shadow reflex in Cirripedia. II. Photoreceptor cell response, second-order responses, and motor cell output. Biol. Bull., 131, 244-256 . Horn, G. (1967) . Neuronal mechanisms of habituation . Nature, Lond., 215, 707-711 . Newell, R . C . & Northcroft, H. R . (1965). The relationship between cirral activity and oxygen uptake in Balanus balanoides. J. Mar. Biol. Ass. U. K., 45, 387-403 . Pi6ron, H. (1910) . L'Evolution de la Memo ire, 360 pp . Paris : Flammarion. Thompson, R . F. & Spencer, W . A . (1966). Habituation : a model phenomenon for the study of neuronal substrates of behavior. Psychol. Rev., 73, 16-43 . Thorpe, W. H . (1956) . Learning and Instinct in Animals, 493 pp. London : Methuen. Waldes, Vera (1938). Ueber die chemische Beeinflussung des Rhythmus and der Retraktionsdauer der Cirren von Balanus perforatus. Z. vergl. Physiol., 26, 347-361 . (Received 16 January 1970 ; revised 9 April 1970 ; MS. number : 941)
THE RATE AND RETENTION OF THE HABITUATION OF THE SHADOW REFLEX IN BALANUS IMPRO VISUS (CIRRIPEDIA) BY
KARL Y. H. LAGERSPETZ & LIISA KIVIVUORI
Laboratory of Zoophysiology, Department of Zoology, University of Turku, Finland
Habituation is generally considered to be the simplest type of learning (Thorpe 1956, p . 54). The characteristics of habituation have been summarized by Thompson & Spencer (1966) in their extensive review of the subject . In spite of its wide occurrence among representatives of probably all animal phyla, only a few studies have been concerned with the mechanisms of habituation and with the mechanisms of the recovery of the habituated response . Bruner & Tauc (1966) studied the compound and simple excitatory post-synaptic potentials in the left pleural ganglion of the sea hare Aplysia during habituation of the tentacular contraction response to mechanical and electrical stimulation . A series of stimuli caused a progressive diminution of the post-synaptic potentials of interneurones . Using series of successive stimuli, repeated at intervals, Bruner & Tauc (1966) found that the effect lasted for some time after the end of the train of stimuli . It was, however, completely abolished by other types of stimuli . An extensive study of the effects of various factors on the rate of habituation of the startle reflex in the polychaete worm Nereis was made by Clark (1960a, b) using mechanical and photic stimuli. Clark also paid attention to the retention of habituation, i .e . on the variables affecting the recovery of the habituated response . Massed photic stimuli (light on 2 s with an interval of 30 s between successive stimuli) resulted in a more rapid habituation than spaced stimuli (light on for 12 s with an interval of 180 s between successive stimuli) . In his group experiments, complete habituation (all animals failed to react) was achieved in forty-seven trials in massed conditions, and in sixty-four trials in spaced conditions. The corresponding figures after a pause of 1 hr were twenty-two and thirtytwo trials, and after a pause of 5 hr, fifty-two and forty-two trials . The results indicate a somewhat better retention after a slower rate of habituation . However, in group experiments the number of stimuli presented to each animal in the group was the same, irrespective of its individual rate of habituation . Thus the amount of over-training varied, and was not controlled .
Gwilliam (1963, 1966) studied the mechanism of the shadow reflex in barnacles (Balanus). The habituation of this reflex was first described by Pi6ron (1910, p 121) . Through electrophysiological recording from different parts of the reflex arc, Gwilliam (1963, 1966) was able to localize the site of habituation to the path from the supreoesophageal ganglion to the motor neurones of the ventral ganglion . The aim of the present experiments was to study the factors affecting the retention of habituation, or conversely, the recovery of the habituated response, in a simple and well-known reflex system . The rate of habituation was thought to be one of the main variables affecting retention . Thus the rate of habituation of the shadow reflex in the barnacle Balanus improvisus (Crustacea : Cirripedia) was varied either by using different individuals or by using different temperatures . Retention was measured by repeated exposures of the animals to series of stimuli . Overtraining was avoided. Another factor which was thought to affect retention, was the externally applied concentration of the assumed neurotransmitter of the reflex system . Methods The test animals were collected during summer months from sea outside the city of Turku (salinity of the Baltic sea water six parts per thousand) together with pieces of the substratum (mostly Fucus) on which they were attached . The animals were stored in aerated sea water at -}-10°C. For the experiment, a piece of substratum with the attached animal was transferred to a glass dish containing sea water . The dish was placed in a translucent water bath . The bath was partially surrounded by black cardboard, which left only one side open . A microscope lamp placed at a fixed distance from the animal dish was used as the light source . The light beam from the lamp to the animal dish could be periodically interrupted by a black circular cardboard disc, from which sectors of appropriate size had been cut out . The disc was attached on the axis of an electrically driven 616
LAGERSPETZ & KIVIVUORI : HABITUATION OF THE SHADOW REFLEX IN BARNACLES
kymograph . In most experiments, the stimulus was a shadow lasting 4 s and the interval between stimuli lasted for 8 s. In all experiments, the animals were allowed to adapt for 30 min to the light conditions in the animal dish before the beginning of the experiment . The temperature of the water in the animal dish was measured with a thermocouple . It could be varied by changing the temperature of the water bath surrounding the animal dish . The response of the animals to the shadow consisted in the cessation of the beat of the cirri, the withdrawal of cirri, and the closure of the shell . The first stimuli usually produced a long retraction, while the animal responded to the later stimuli with a retraction lasting for a few seconds only, and finally, no retraction was elicited . Stimuli were presented at the same rate even when the barnacle was retracted . When the animal responded to only one out of five successive stimuli, it was considered to be habituated . The number of stimuli needed for this degree of habituation was used as the inverse measure of the rate of habituation . Retention was tested by repeating the series of stimuli after 2 . 5, 5 or 10 min . During this interval the animals were in light in the animal dish. The second series of stimuli was also interrupted when the criterion for habituation was reached. This procedure was again repeated after a second similar interval . Each animal was used for one experiment only . The numbers of stimuli needed for habituation in the three successive series were designated as h 1 , h 2 and h 3 . Retention (r) was calculated as the percentage of decrease in the number of stimuli needed for habituation : h1 h2
x 100,
h2-h3 r2=h2h 2
x 100 .
r1 = 1
and, respectively :
Results 1. Activity and Habituation Rate Habituation to a shadow lasting for 4 s and
617
presented at intervals of 8 s was studied in 110 animals, the maximum diameter of the base plate (shell length) of which varied from 2 to 10 mm. The animals were not taken from the storage containers at random, but the most active specimens were selected . Before the experiments, the animals had been stored at room temperature (19 to 23°C) for 1 day . Seven test animals responded to the shadow by such a long retraction that habituation could not be produced . From the remaining 103 animals, seventy-six were classified as active. Such animals were regarded as active, in which the longest retraction (except the first one) lasted for less than 1 min (corresponding to five stimulus-interval cycles) . In the twenty-seven passive animals, the maximum retraction time was longer. The number of stimuli needed for the attainment of the criterion for habituation (h) varied from fourteen to 150 . The distribution of the active and passive animals according to the value of h appears in Table I . It is obvious from the direct inspection of the data that the distributions are not different. Since the passive animals had their shell closed for a longer time than the active ones, the result seems to indicate that the habituation process continues also when stimuli are presented during the closure of the shell. Before each experiment, the cirral beating frequency was measured using a stopwatch . Newell & Northcroft (1965) showed that in Balanus balanoides the cirral beating frequency was an exponential function of the body weight. In this study, the correlation coefficient between the shell length and the cirral beating frequency was -0 .360. The correlation was statistically significant at the level of P<0 .001 . The inverse relationship found between the body size and the cirral beating frequency in Balanus balanoides by Newell & Northcroft (1965) is thus also found in Balanus improvisus. The correlation coefficient calculated between the shell length and the number of stimuli needed for habituation (h) was 0 .094 and the correlation between cirral beating frequency and the number of stimuli needed for habituation was 0 .144 . Neither of
Table I . Distribution of the Active and Passive Animals According to the Number of Stimuli Needed for Habituation (h) h
14-30
31-60
61-90
91-120
121-150
No. of active animals
15(20%)
38 (50Y.)
13 (17 %)
7 (19 %)
3 (4 %)
No . of passive animals
6 (22%)
15 (55%)
5(19%)
1( 4 %)
618
ANIMAL BEHAVIOUR, 18, 4
Table IL Numbers of Stimuli Needed for Habituation at Intervals of 5 min (ht, h2, h3) and the Corresponding Retention Values (r1, r2) at Different Experimental Temperatures (± SE) Experimental temperature
h1
r1
h2
r2
12°C
22 . 2±1 .9
30
15 . 6±1 . 4
22°C
50 . 5±5 . 1
49
25. 9±2 .4
these correlation coefficients indicates a statistically significant correlation . These results show that there is a large variation in the rate of habituation of the shadow reflex, measured as the number of stimuli needed for habituation . In addition, this variation cannot be accounted for by the variation in the body size or in the activity of the animals . 2 . Habituation Rate and Retention after Different Recovery Times Altogether, twenty animals were habituated twice at a recovery interval of 2 . 5 min, forty animals using an interval of 5 min, and nineteen with an interval of 10 min . From the values of h 1 and h 2 the retention r 1 was calculated . The correlation coefficient was calculated between the values of h l and r 1 in each group. The correlation coefficients were as follows Recovery interval 2 . 5 min 0 . 656 (P<0 . 01) Recovery interval 5 min 0 . 383 (P<0 . 05) Recovery interval 10 min 0 . 228 NS The correlation coefficients for the two first groups were statistically significant . The re gression equations for these groups were the following : r 1 = - 7 .683 + 40 . 382 x h 1 and r 1 = 7 . 086 + 27 . 005 x h l , respectively. Thus, when a series of stimuli is presented to the animals shortly after they have been habituated, retention of the habituation is proportional to the number of stimuli originally needed to produce the habituation . In other words, the recovery of the response which has occurred is proportional to the original rate of habituation . However, this relationship disappears as the recovery proceeds. The time-course of the recovery for the animals with different habituation rates is presented in Fig . 1 . It is evident that the recovery of the habituated response is most rapid in the group with the most rapidly habituating animals . 3 . Temperature, Habituation Rate and Retention In another series of experiments, twenty-nine animals were habituated three times at 9 ° to 15 °C
h3
N
13
13 . 6±1 . 2
29
25
19 .5±1 .7
36
and thirty-six treated similarly at 19° to 23°C . The mean temperatures were 12° and 22°C, respectively . The numbers of stimuli needed for habituation at each occasion h, as well as the retention values r are given in Table II . The time 100
0 5 :S h, :5 30
• 31<_h I- 60 A61<_h 1 <90 v91<_h,<120
47
0
I I
2 .5
5
I 10
min
Fig . 1 . Retention of habituation as function of recovery time (rest interval in minutes between stimulus periods) and of the original rate of habituation (h 1) .
interval between the successive stimulus sequences was 5 min . The values of h for successive stimulus sequences are plotted in Fig . 2 . The habituation was significantly more rapid at 12°C than at 22°C . Less retention was found after 5 min at 12°C than at 22 °C . Again, at higher rate of habituation, a more rapid recovery occurred . 4. Acetylcholine, Habituation Rate and Retention The effect of acetylcholine (ACh) on the habituation rate and retention was studied in fourteen animals . Half of these were habituated three times in sea water, then transferred to sea water with 1 . 67 x 10- 5 g per ml ACh chloride
619
LAGERSPETZ & KIVIVUORI : HABITUATION OF THE SHADOW REFLEX IN BARNACLES
The experiment was repeated with 10 - 4 g per ml ACh chloride using eight animals . As these animals were collected in wintertime, the control level is not directly comparable with those presented above . The mean habituation value without ACh was 25 . 3 + 3 . 1, and with ACh 45 . 1 ± 4. 9, the difference being statistically significant (P<0 . 01) .
60-
50
40
30
20
10
0 III I II Fig. 2 . Rate of habituation measured as the number of stimuli needed to habituation (h) in three subsequent experiments, performed at intervals of 5 min . The average habituation rates for the groups at 22° and 12°C show the effect of temperature, the average habituation rates for the groups marked ACh and no ACh show the effect of 1 . 67 x 10 - 5 g per ml ACh chloride on habituation. 1 l I
for I hr and, subsequently habituated in the ACh solution for three times . The other half of the animals were first kept and habituated in 1 . 67 x 10 - 5 g per ml ACh chloride, then allowed to recover in sea water, and again habituated in sea water . As the results were similar in both cases, they were combined and are presented in Table III and in Fig. 2 . Acetylcholine apparently slightly reduced the number of stimuli needed for the first habituation (h 1 ) . However, it affected the subsequent habituations in the opposite way, so that the retention values (r 1 and r 2) were definitely decreased . The difference between the last habituation values (h3) is, when evaluated with the t-test, statistically significant (P<0 .001) .
Discussion Horn (1967) showed that many characteristics of habituation could be accounted for on the hypothesis that a self-generated depression of neuronal sensitivity occurs at one or more points of the system to which a stimulus is repeatedly applied . Horn used the term self-generated depression to include such phenomena as after hyperpolarization, conduction block due to extracellular potassium accumulation around an active fibre, and synaptic depression representing an imbalance between the neurotransmitter mobilization and utilization . Bruner & Tauc (1966) consider the last of these, the transmitter depletion at synaptic terminals as the most likely mechanism of the neuronal habituation in Aplysia. If habituation is considered to be the outcome of transmitter depletion caused by frequent release of the transmitter, then the recovery of the habituated response may be interpreted as a result of synthesis, uptake or axonal transport of the transmitter. This `transmitter depletion hypothesis' of habituation may be used to explain the present data . The observed differences in the rate of habituation could be caused either by differences in the amount of transmitter originally present in the appropriate synaptic terminals, or by differences in the release in response to stimulation . The present results show that the rate of recovery is in its first stages proportional to the rate of the preceding habituation . It is difficult to conceive that the recovery process would be related to the amount of transmitter present before the habituation . It seems more plausible to assume
Table III. Effect of Acetylcholine (ACh) on the Numbers of Stimuli Needed for Habituation at Intervals of 5 min and the Corresponding Retention Values (r1, r2) (± SE) h2
r2
h3
(h1, h 2, h 3)
N
h1
r1
Control
52 .4±7 . 1
58
22 . 1±3 . 9
24
16 .9±2 . 0
14
ACh
40 . 1±5 . 3
27
29 . 5±3 . 7
-
29 . 8±3 . 3
14
620
ANIMAL BEHAVIOUR, 18, 4
that the observed differences in the rate of habituation would be caused by differences in the release of the transmitter, and that the rates of synthesis, uptake and transport are in balance with the rates of transmitter release and breakdown . This would account for the relationship observed between the rate and retention of habituation . Low temperatures certainly depress the metabolism and release of the transmitter substances . Since a proportionality was found between the first habituation and retention values these could be used to yield some information about the temperature characteristics of the processes of transmitter release, respectively transmitter synthesis, uptake and transport . The Q 10 value for h 1 from Table II is 2 .27 and for r 1 1 .63 . This indicates a higher temperature dependence for the transmitter release than for the transmitter recovery. Low temperature thus depressed the transmitter release, and habituation occurred rapidly . On the other hand, the recovery was not affected to an equivalent degree, and the released small amount of transmitter was soon replaeed . In the experiments of Waldes (1938), acetylcholine and atropine strongly affected the shadow reflex in Balanus. This supports the view that cholinergic transmission is involved in the mechanism of the shadow reflex . The results of Waldes (1938) also show that externally applied acetylcholine probably enters the nervous system of barnacles . In the present experiments it was found that externally applied acetylcholine increased the number of stimuli needed for habituation and decreased retention, i .e . enhanced the recovery of the habituated response . In the terms of the transmitter hypothesis, the uptake of acetylcholine to the pre-synaptic terminals, or the post-synaptic depolarization was enhanced . This presumably also happens when dishabituation is produced by stimulating the habituated animal with a different type of stimulus which elicits the same response . Summary 1 . There is large variation in the rate of
habituation of the shadow reflex in the barnacle Balanus improvisus. This variation cannot be accounted for by the variation in the body size or in the activity of the animals . 2. When only a short recovery time is allowed, the rate of the recovery of the habituated response is correlated with the rate of the preceding habituation . The same result is obtained when the habituation rate is varied using different animals or different temperatures . 3. Habituation is more rapid at 12°C than at 22°C. 4. Acetylcholine chloride (1 . 67 x 10- 5 g per ml) enhances the recovery of the habituated response. 5. The results are in agreement with the transmitter depletion hypothesis of habituation . REFERENCES Bruner, J. & Tauc, L . (1966) . Habituation at the synaptic level in Aplysia. Nature, Lond., 210, 37-39 . Clark, R . B. (1960a) . Habituation of the polychaete Nereis to sudden stimuli. I. General properties of the habituation process. Anim. Behav ., 8, 82-91 . Clark, R . B . (1960b) . Habituation of the polychaete Nereis to sudden stimuli . II. Biological significance of habituation . Anim . Behav., 8, 92-103 . Gwilliam, G . F. (1963) . The mechanism of the shadow reflex in Cirripedia . I . Electrical activity in the supraoesophageal ganglion and ocellar nerve . Biol. Bull., 125, 470-485 . Gwilliam, G . F. (1966) The mechanism of the shadow reflex in Cirripedia. II. Photoreceptor cell response, second-order responses, and motor cell output. Biol. Bull., 131, 244-256 . Horn, G. (1967) . Neuronal mechanisms of habituation . Nature, Lond., 215, 707-711 . Newell, R . C . & Northcroft, H. R . (1965). The relationship between cirral activity and oxygen uptake in Balanus balanoides. J. Mar. Biol. Ass. U. K., 45, 387-403 . Pi6ron, H. (1910) . L'Evolution de la Memo ire, 360 pp . Paris : Flammarion. Thompson, R . F. & Spencer, W . A . (1966). Habituation : a model phenomenon for the study of neuronal substrates of behavior. Psychol. Rev., 73, 16-43 . Thorpe, W. H . (1956) . Learning and Instinct in Animals, 493 pp. London : Methuen. Waldes, Vera (1938). Ueber die chemische Beeinflussung des Rhythmus and der Retraktionsdauer der Cirren von Balanus perforatus. Z. vergl. Physiol., 26, 347-361 . (Received 16 January 1970 ; revised 9 April 1970 ; MS. number : 941)