Effects of dopaminergic drugs on locomotor activity in teleost fish of the genus Oreochromis (Cichlidae): involvement of the telencephalon

Physiology & Behavior, Vol. 64, No. 3, pp. 227–234, 1998 © 1998 Elsevier Science Inc. All rights reserved. Printed in the U.S.A. 0031-9384/98 $19.00 1 .00

PII S0031-9384(98)00038-9

Effects of Dopaminergic Drugs on Locomotor Activity in Teleost Fish of the Genus Oreochromis (Cichlidae): Involvement of the Telencephalon E. Y.-M. MOK AND A. D. MUNRO1 Department of Biological Sciences, The National University of Singapore, Lower Kent Ridge Road, Singapore, 119260 Singapore Received 29 September 1997; Accepted 19 January 1998 MOK, E. Y.-M. AND A. D. MUNRO. Effects of dopaminergic drugs on locomotor activity in teleost fish of the genus Oreochromis (Cichlidae): Involvement of the telencephalon. PHYSIOL BEHAV 64(3) 227–234, 1998.—Single Oreochromis niloticus and O. mossambicus were placed in an unfamiliar white basin for 21 min, and their activity in this open-field situation was recorded from overhead on video. Apomorphine added to the water (2– 8 mg/liter) caused a significant increase in locomotor activity, as assessed by the frequency that a fish swam over a rectilinear array of lines drawn on the base. This effect was attenuated by chlorpromazine (2 mg/liter) and abolished by the D1 antagonist SCH-23390 (1 mg/liter); the D2 antagonist metoclopramide (8 mg/liter) had no effect. Removal of both hemispheres of the telencephalon abolished the response to apomorphine, whereas removal of only one hemisphere or cauterization of the nostrils had no effect. It is concluded that the role of the dopaminergic system in the regulation of locomotor activity is reminiscent of the mammalian mesolimbic, rather than the nigrostriatal, system but that further studies are required to determine the source of the dopaminergic innervation and its likely telencephalic targets. © 1998 Elsevier Science Inc. Dopaminergic drugs

Activity

Teleost fish

Telencephalon

premature to identify homologs of, for example, the tetrapod striatum and the various pallial fields in teleosts. Neuroethological studies on teleosts, mainly involving gross ablation of the telencephalon, have indicated that, apart from a role in processing olfactory information, this area also is important in the regulation of arousal and the modulation of the behavioral outputs of lower brain centers; this has led to comparisons with the functions of limbic components of the mammalian telencephalon (3,10,21,27,48). Flood and Overmier (16) and MacPhail (31) have proposed that the teleost telencephalon may modulate ongoing behavior and learning as a result of the rewarding properties of responses which terminate fearful or frustrating situations; its ablation is thus associated with impairments in avoidance behavior and most types of learning. However, the aim of many studies—to identify an underlying unitary role of the telencephalon in the regulation of teleostean behavior—is probably too simplistic (16). Such neuroethological studies have largely ignored the possible role of lower brain centers in the regulation of telencephalic function in teleosts. In mammals, dopaminergic (DAergic) neurons in the mesencephalon, which are components of the brain’s reward system, and thus important for learning, have been identified as an important source of such an ascending

NEUROANATOMICAL studies indicate that perhaps the most marked difference between the brain of actinopterygian fishes and that of other vertebrates is in the ontogeny, and thus the adult organization, of the telencephalon (37,38). On the one hand, the telencephalon of most vertebrates develops from the anterior neural tube through a process of evagination (or inversion), where the side walls “balloon out” to each surround a lateral ventricle; a primary subdivision into a dorsal pallium and a ventral subpallium has been recognized in the walls of the resultant hollow hemispheres, with subsequent development leading to further compartmentalization of these into more or less discrete nuclear territories. On the other hand, the actinopterygian telencephalon instead arises through the process of eversion, where it is the dorsal portions of the embryonic tube walls which undergo the most lateral displacement, to produce a pair of solid hemispheres with the midline ventricle extending bilaterally under the much expanded roof plate (37). As a result of this atypical mode of development, there is continuing debate about the location of the equivalent of the pallium–subpallium boundary in the actinopterygian telencephalon and thus about the location of field homologs of areas in the telencephalon of mammals and other tetrapods (37,38,47). Thus Nieuwenhuys and Meek (35,37) have argued that it is, at best, 1

To whom requests for reprints should be addressed. E-mail: [email protected]

227

228 input (e.g., 5,12,14,15,24,29,62,63). Traditionally, two separate DAergic projections have been recognized. Best studied is the mesostriatal one from the substantia nigra to the dorsal striatum, as part of the “extrapyramidal” system for the enactment of motor programs (e.g., 5,62). Disruptions of this system (including with the drug MPTP) result in movements being more slowly initiated and performed, whereas enhanced DAergic transmission is associated with increased performance of stereotyped behaviors (e.g., 5,14,29,62). The second, mesolimbic system involves a DAergic projection from the ventral tegmental area, adjoining the substantia nigra, to the ventromedial striatum, other portions of the forebrain limbic system, and portions of the cortex (5). These DAergic projections have been implicated as playing a permissive role in affective and motivational states through the general, coordinated regulation of their targets. Typically, lesions of the mesolimbic system are reported to result in decreased locomotor activity, whether spontaneous or for the exploration of a novel environment, together with attentional deficits and impairment of some forms of learning, whereas DAergic stimulation has the opposite effect (5,29,54). Mesolimbic targets in mammals are more sensitive than mesostriatal ones to the effects of apomorphine (a nonspecific dopamine agonist at both D1 and D2 receptors) and d-amphetamine (an indirect DAergic agonist): thus low doses of d-amphetamine result in increased general locomotor activity, whereas higher doses also act on mesostriatal projections to induce stereotyped behaviors (14,23). Similarly, there is evidence that certain divisions of the telencephalon receive a DAergic innervation in teleosts (reviewed by Meek (35)). However, the provenance of this DAergic innervation has not been determined experimentally, and it has generally been considered that homologs of the DAergic mesencephalic cell groups are lacking (reviewed by Meek (35); but see Discussion). Nevertheless, DAergic drugs can influence the behavior of various teleost fishes (6,25,26,36; Munro et al., manuscript in preparation). Thus the agonist apomorphine increases locomotor activity in a cichlid (36), with evidence for apparent stereotyped behaviors reminiscent of the effects of this drug on the mammalian mesostriatal system (see above). There is also evidence that d-amphetamine has a hedonic role in goldfish, recalling its rewarding effects in mammals (6). Given the general consensus that teleosts lack a mestelencephalic DAergic projection, it is been suggested that the presence of extensive DAergic projections within motor centers in the brain stem and spinal cord may be important in modulating arousal and locomotion (49), thereby providing a potential alternative to the telencephalon as a site of action for apomorphine and other such drugs. The present experiments, using cichlid teleosts of the genus Oreochromis, aimed to, first, identify the behavioral effects of apomorphine in a novel open field and determine the nature of the receptors involved. Secondly, gross ablation of one or both hemispheres was used as the initial means for addressing the question of whether the telencephalon is indeed involved in mediating these effects. Because drugs were administered by dissolving them in the water of the test tank (36), another experiment was done where the nares of some fish were destroyed by electrocautery, to test the possibility that the drugs may instead be having indirect, olfactory actions. Initial experiments were done with O. niloticus; however, limited supplies of this species meant that we had to switch to O. mossambicus for subsequent experiments.

MOK AND MUNRO MATERIALS AND METHODS

Fish Juvenile O. niloticus (standard length 5–7 cm) were obtained from the Primary Productivity Department, Sembawang, and maintained in large stock tanks until use. O. mossambicus of a comparable size were obtained from our laboratory stocks. Each fish was used only once, unless otherwise stated. Operations These were performed under anesthesia with tricaine methanesulfonate (Sigma) and fall within ethical guidelines for National University of Singapore. For brain operations (Experiments 3–5), a flap was cut in the roof of the skull between the eyes and bent back to expose the telencephalon. After swabbing any fatty tissue from the exposed surface of the brain, one (Experiment 4) or both (Experiments 3 and 5) hemispheres of the telencephalon were aspirated in experimental fish by suction with a flamed pipette connected to a water pump. Teleost saline (7) was used to fill the cranial cavity, and the bony flap then replaced and secured with superglue. Controls received the same treatment, except that the brain was left intact. During the subsequent 7-day recovery period, the wounds showed evidence of healing and there were no mortalities in any experiment. In Experiment 6, the olfactory epithelium of eight O. mossambicus was destroyed by electrocautery using a radio-frequency current generator (Erbotom T50B); another eight fish were only anesthetized, as unoperated controls. Fish were individually isolated for 7 days prior to testing. Drugs The hydrochloride salts of apomorphine, chlorpromazine, and metoclopramide were obtained from Sigma; R(1)-SCH-23390 HCl was supplied by Research Biochemicals International. All drugs were administered by dissolving them in the water of the observation tanks immediately before adding the fish. General Test Procedure In Experiments 1–3, the observation tank was a white basin (42 3 28 3 10.5 cm deep) containing 8 liters of water (depth ' 7 cm). Black lines divided the base up into twelve 10.5 3 7 cm rectangles. In Experiments 4 – 6, smaller tanks (28 3 17 3 17 cm deep, lined with white paper) containing 4 liters of water (depth ' 8.5 cm) were used, with the base divided into nine equal-sized zones. The observation tanks were placed on a thick piece of foam rubber to damp out vibrations. Care was taken to ensure that they were evenly illuminated, without any shaded areas. A video camera directly above the tank was used to record the fishes’ activity. Video recordings were analyzed for the number of line crossings over 20-s intervals, starting at 99 50” and 199 50” in all but Experiment 2, where activity was recorded between 149 50” and 159 10”. Histology Brains of operated fish were exposed and fixed in Bouin’s for 1 week. Thereafter they were removed from the floor of the skull, dehydrated, and processed for routine wax histology. Transverse or horizontal sections (7 mm) were stained with the Klu¨ver and Barrera technique. Statistical Analysis This was done using SPSS 41. A preliminary analysis of the data indicated that the frequency of line crossings (x) was zero in

DOPAMINERGIC DRUGS AND ACTIVITY IN FISH

229

TABLE 1

TABLE 2

Effects of Apomorphine and Chlorpromazine on Locomotor Activity in Oreochromis niloticus

Effects of Apomorphine (4 mg/liter) and the D1 Antagonist SCH-23390 (1 mg/liter) on Locomotor Activity in Oreochromis mossambicus

Line Crossings/20 s* Treatment

n

10 min

20 min

Control Chlorpromazine 2 mg/liter 4 mg/liter Apomorphine 2 mg/liter 4 mg/liter 8 mg/liter Apomorphine (4 mg/liter) 1 chlorpromazine (2 mg/liter) Apomorphine (4 mg/liter) 1 metoclopramide (8 mg/liter)

12

0.33 6 0.19a

1.58 6 0.48a

6 6

1.33 6 0.49a 0.50 6 0.34a

0.67 6 0.33a 2.50 6 0.92a

6 6 5

5.33 6 1.56Ab 13.50 6 3.94AB 15.40 6 2.79AB

12.17 6 3.75A 16.00 6 4.19A 17.00 6 2.95A

4

4.00 6 1.87b

3

17.33 6 3.76AB

* Mean 6 standard error of combined vertical and horizontal line crossings. Within each column, A and B are significantly greater than a and b, respectively (p , 0.05).

some replicates and that the variances of various treatments were not homogeneous. In such cases, analyses were performed on the log(x 1 1)-transformed data. RESULTS

Experiment 1 O. niloticus were tested (Table 1) with either untreated water, water treated with either apomorphine (2, 4, and 8 mg/liter) or chlorpromazine (2 and 4 mg/liter), or a combined treatment of apomorphine (4 mg/liter) with either chlorpromazine (2 mg/liter) or metoclopramide (8 mg/liter). Repeated-measures ANOVA of the transformed data indicated there was a significant difference not only between treatments but also between times. Paired t-tests indicated that the latter was attributable to differences between observation periods for controls (p , 0.001) and fish treated with 2 mg/liter of apomorphine (p , 0.02); for 4 mg/liter of chlorpromazine, the difference was almost significant (p , 0.06). Thus the data for each observation period were analyzed separately by one-way ANOVA and least significant difference (LSD) tests (Table 1). The activity of control fish was low but increased with time (Table 1). Fish receiving either dose of chlorpromazine also had low activities (Table 1). Apomorphine was associated with an increase in activity, although the effect of the lowest dose (2 mg/liter) was slower in onset than that of higher doses (Table 1). Based on observations made at 10 min, the effects of apomorphine (4 mg/liter) were partially blocked by chlorpromazine (2 mg/liter) but not by 8 mg/liter of metoclopramide (Table 1). Experiment 2 O. mossambicus were tested with either untreated water or that treated with either apomorphine (4 mg/liter), SCH-23390 (1 mg/liter), or the same doses of both drugs together. There was a highly significant effect on line-crossing activity [F(3, 25) 5 24.48]; p , 0.001), which was attributable to the effects

Treatment

Control Apomorphine SCH-23390 Apomorphine 1 SCH-23390

n

Line Crossings*

5 7 8 6

3.20 6 1.02a 25.57 6 6.31A 1.50 6 1.05a 0.50 6 0.34a

* Mean 6 standard error of combined vertical and horizontal line crossings. Within-column comparisons indicate that A is significantly greater than a (p , 0.05).

of apomorphine on its own: SCH-23390 abolished the response to apomorphine (Table 2). Experiment 3 Sixteen O. niloticus had the whole telencephalon removed, and 16 served as controls. Seven days later, they were tested either with untreated water or with that containing either 8 mg/liter of apomorphine or 4 mg/liter of chlorpromazine (Table 3). Histology confirmed that the telencephalon was removed in experimental fish. The adjoining anterior preoptic area in the telencephalic peduncle (57) was damaged to a variable extent, which could not be related to individual differences in the pattern of response; thus the results for all ablated fish, irrespective of the degree of damage to the preoptic area, are combined in Table 3. Repeated-measures ANOVA of the transformed data indicated that there was a significant difference between treatments [F(5, 26) 5 5.08, p , 0.005] and also between the first and second observation periods [F(1, 26) 5 12.55, p , 0.005]; there was no evidence for any interaction [F(5, 26) 5 0.93, p , 0.40]. Paired t-tests confirmed that there were significant differences between observation times for both sham-operated (p , 0.02) and telencephalon-ablated (p , 0.001) controls and for ablated fish treated with 4 mg/liter of apomorphine (p , 0.03). Thus the data for each observation period were analyzed separately by one-way ANOVA and LSD tests.

TABLE 3 Effects of Bilateral Telencephalon Ablation on the Locomotor Response to Apomorphine (8 mg/liter) and Chlorpromazine (4 mg/liter) in Oreochromis niloticus Line Crossings/20 s* Treatment

Sham-operated controls untreated apomorphine chlorpromazine Telencephalon-ablated untreated apomorphine chlorpromazine

n

10 min

20 min

6 7 3

1.00 6 0.52a 5.33 6 1.26A 0.67 6 0.67a

2.33 6 0.71b 10.00 6 3.14B 6.33 6 4.91

6 7 3

0.67 6 0.67a 1.43 6 0.48a 0.67 6 0.67a

1.00 6 0.82b 3.29 6 0.92b 1.00 6 1.00b

* Mean 6 standard error of combined vertical and horizontal line crossings. Within-column comparisons indicate that A and B are significantly greater than a and b, respectively (p , 0.05).

230

MOK AND MUNRO TABLE 4

Effects of Unilateral Telencephalon Ablation on the Locomotor Response to Apomorphine (8 mg/liter) in Oreochromis niloticus Line Crossings/20 s*

Sham-operated controls (n 5 18) Ablation of left hemisphere (n 5 12) Ablation of right hemisphere (n 5 12)

Untreated

Apomorphine

0.56 6 0.22a 0.67 6 0.36a 1.08 6 0.40a

3.14 6 0.52A 3.83 6 0.44A 3.25 6 0.62A

* Mean 6 standard error of combined vertical and horizontal line crossings for each observation period (n 5 number of observations, with 2 for each fish). A is significantly greater than a (p , 0.05).

The results (Table 3) indicate that apomorphine stimulated activity in sham-operated fish, although to a less marked extent than in Experiment 1. Ablation of the telencephalon abolished this response (Table 3). Those sham-operated fish tested with chlorpromazine (4 mg/ liter) showed some evidence for an increase in activity in the second observation period (Table 3). This was not apparent in the telencephalon-ablated fish. Experiment 4 Six O. niloticus had the left, and six the right, hemisphere removed, and another seven fish were sham-operated controls. The fish were tested, once with 8 mg/liter of apomorphine and once as an untreated control in random sequence, on Days 7 and 8. Histology confirmed that all of the hemisphere in question was removed, whereas the other appeared to be undamaged. There was variable damage to the anterior preoptic area on the aspirated side, which could not be related to the locomotor activity of the fish as a control or in response to apomorphine. Table 4 summarizes the results. Repeated-measures ANOVA of the transformed data indicated that there was a significant difference between treatments [F(5, 34) 5 12.53, p , 0.001] but no significant difference between the first and second observation periods [F(1, 34) 5 0.11, p , 0.70]. Subsequent analysis indicated that there were no differences between groups as controls and that apomorphine uniformly increased the frequency of line crossings in each group (Table 4). A x2 analysis between groups indicated that neither operation nor treatment had a significant effect on the direction (clockwise versus counterclockwise) in which they crossed the lines (data not shown). Experiment 5 Eight O. mossambicus had their telencephalon bilaterally ablated, and eight other fish served as sham-operated controls. The fish were tested on Days 7 and 8 with untreated water and with water treated with 8 mg/liter of apomorphine; the day on which they received apomorphine was randomized. Histology showed that three of the eight telencephalon-ablated fish had the whole of the telencephalon removed; the most anterior portion of the brain remaining was the preoptic area. The remaining five fish still had the olfactory bulbs and the ventralmost portion of the telencephalon intact (the area Vv of Northcutt and Davis (38)). However, the data for all eight fish were pooled in the following analysis, in view of the lack of any evident behavioral difference between the two subgroups. Repeated-measures ANOVA of the transformed data indicated that there was a significant difference between treatments [F(3, 26)

5 48.70, p , 0.001] but not between the first and second observation periods [F(1, 26) 5 2.82, p ' 0.10]; there was, however, a significant interactive effect (F 5 8.36, p , 0.001), attributable to a significant increase in activity between observation periods for telencephalon-ablated fish after apomorphine treatment (Table 5). Comparison between groups for each time indicated that control activity was reduced in ablated fish (p , 0.001 at 10 min and p , 0.02 at 20 min; LSD test). Apomorphine caused a sustained increase in locomotor activity of sham-operated fish (p , 0.001 for each time; paired t-test). The drug had a similar effect on telencephalon-ablated fish (p , 0.01 at 10 min and p , 0.001 at 20 min; paired t-test); however, the response to apomorphine (Table 5) was somewhat delayed, with activity at 20 min being higher than that at 10 min (p , 0.01; paired t-test). The increase in locomotor activity in ablated fish (Table 5) was less than that seen for sham-operated fish (LSD test). Experiment 6 Microscopic examination confirmed the destruction of the olfactory epithelium in cauterized fish. Repeated-measures ANOVA of the transformed data indicated that there was a significant difference between treatments [F(3, 28) 5 19.26, p , 0.001] but not between the first and second observation periods [F(1, 28) 5 0.23, p . 0.60]. Cautery of the nares had no effect on locomotor activity of the fish when untreated and had only a mild moderating effect on the response to apomorphine at 20 min (Table 6). DISCUSSION

General Control fish showed only low levels of activity, especially in the case of O. niloticus. This inactivity may result from the short-term effects of exposure of fish to an unfamiliar open-field situation (18,28,60). Over time, there was a progressive increase in locomotor activity, which may reflect the loss of a “fear response” (61), similar to the habituation seen in mammals (2). On its own, ablation of either or both telencephalic hemispheres had little obvious effect on the activity of either species of tilapia. This is in broad agreement with studies on other teleosts, where telencephalon ablation typically had no effect on general locomotor activity; various workers (see Introduction) have thus suggested that such an operation, by causing quantitative rather than qualitative changes in other behaviors, reflects effects on specific

TABLE 5 Effects of Bilateral Telencephalon Ablation on the Locomotor Response to Apomorphine (8 mg/liter) in Oreochromis mossambicus Total Number of Line Crossings* Treatment

Sham-operated (n 5 7) untreated apomorphine Telencephalon-ablated (n 5 8) untreated apomorphine

10 min

20 min

4.86 6 1.24Ab 19.43 6 1.34AB

2.43 6 0.84a 20.14 6 2.41AB

0.13 6 0.13ab 4.73 6 1.35Ab

0.13 6 0.13a 10.50 6 1.84Ab

* Mean 6 standard error of combined vertical and horizontal line crossings. Within-column comparisons indicate that A and B are significantly greater than a and b, respectively (p , 0.05).

DOPAMINERGIC DRUGS AND ACTIVITY IN FISH TABLE 6 Effects of Cauterization of the Nares on the Locomotor Response to Apomorphine (8 mg/liter) in Oreochromis mossambicus Total Number of Line Crossings*

Unoperated (N 5 8) untreated apomorphine Nares cauterized (N 5 8) untreated apomorphine

10 min

20 min

3.88 6 0.88a 14.88 6 2.40A

3.00 6 0.68a 16.88 6 2.36AB

2.87 6 1.41a 11.50 6 1.39A

2.25 6 0.41a 12.13 6 1.91Ab

* Mean 6 standard error of combined vertical and horizontal line crossings. Within-column comparisons indicate that A and B are significantly greater than a and b, respectively (p , 0.05).

arousal, reminiscent of the effects of lesions of the limbic system in mammals. Effects of DAergic Drugs Apomorphine enhanced locomotor activity in both oreochromines, in agreement with the findings of Munro (36) for another cichlid (Aequidens pulcher) and Mok et al. (manuscript in preparation) for a cyprinid (Puntius tetrazona). There was evidence that this effect was dose-dependent: the two highest doses had a more rapid action in Experiment 1. On the other hand, in contrast to the results of Munro (36) for the cichlid A. pulcher and Mok et al. (manuscript in preparation) for the cyprinid P. tetrazona, chlorpromazine (a nonspecific D1 and D2 antagonist (8,53)) did not decrease activity in O. niloticus using the present paradigm. This is probably related to the already low levels of activity in control fish as a result of being introduced into a novel environment. However, chlorpromazine was able to at least partially block the response to apomorphine, consistent with the hypothesis that DAergic stimulation may enhance activity. The present experiments provide preliminary information on the likely class of DAergic receptors involved. Although a relatively high dose of the specific D2 blocker metoclopramide ('24 mM) was unable to block the response to apomorphine in O. niloticus, the D1 antagonist SCH-23390 ('3 mM) was able to do so in O. mossambicus. Interpretation of the data is confounded by the use of different species; however, we believe that because the two tilapias are very closely related (they readily hybridize in captivity), it is reasonable to conclude that that apomorphine is acting by way of receptors which are more D1- than D2-like in their properties. Similarly, a greater range of doses of metoclopramide and various other D2 antagonists (haloperidol, sulpiride) did not abolish the response to apomorphine in a cyprinid fish, unlike chlorpromazine and the D1 antagonist SCH 24543 (Mok et al., manuscript in preparation). The Telencephalon as a Target The effect of apomorphine on locomotor activity in Oreochromis species is at least partly mediated by actions which require the presence of at least one telencephalic hemisphere (Experiments 3–5); the reason for the residual responsiveness in O. mossambicus, but not in O. niloticus, remains to be clarified. Such effects cannot be attributed to the telencephalic processing of olfactory responses to this drug in O. mossambicus (Experiment 6). Removal of only one telencephalic hemisphere in O. niloticus

231 had no effect on the response to apomorphine; moreover, there was no evidence for increased turning in one particular direction, whether the fish were treated with apomorphine or not. This lack of any turning bias contrasts with data for homoiotherms: in mammals, unilateral injections of dopamine agonists (e.g., apomorphine) into the striatum induce contraversive movements (44), consistent with evidence for increased dopamine release in the contralateral striatum during turning in the rat (17). Conversely, such injections of D2 antagonists in mammals (44), like unilateral lesions of the mesostriatal system in both birds (45) and mammals (14,44), lead to an ipsiversive biasing of postures and turning movements. It would seem, therefore, that the telencephalon of O. niloticus contains bilateral center(s) which receive a DAergic input and which respond to dopamine agonists by inducing a generalized, rather than side-directed, increase in locomotor activity. The location of such center(s) remains to be determined; immunohistochemical studies on other teleosts indicate that the DAergic innervation of the telencephalon is primarily to certain areas of the ventral (subpallial?) subdivision of the telencephalon as well as to ventrolateral and posterior portions of the dorsal (pallial?) subdivision (35). Preliminary studies indicate that aspiration of the medial and/or the lateral dorsal subdivisions (including much of the field Dc) had no effect on the response to apomorphine (Mok and Munro, unpublished), but more refined studies are clearly required to localize the likely areas involved. The absence of any effects comparable with manipulations of the mesostriatal DAergic system in amniotes is consistent with the proposal of Nieuwenhuys and Meek (35,37) that any homolog of the striatum in teleosts, if present, may show fundamental functional differences. Source of Endogenous DAergic Control There is no experimental information on the location of the cells responsible for the DAergic innervation of different portions of the telencephalon in teleosts (35). As noted in the introduction, neuroanatomical studies have generally been interpreted as indicating that actinopterygians lack homologs of the DAergic mesencephalic groups of mammals; in this respect, they resemble agnathans (but see below) and holocephalans (reviewed by Meek (35) and Smeets and Reiner (55)). In contrast, there is evidence for such mesostriatal and mesolimbic projections in elasmobranchs and tetrapods (reviewed by Smeets and Reiner (55)), with the mesostriatal projections of sauropsids considered to function as components of the basal ganglia in a manner basically similar to that of mammals (20,46). However, recent studies suggest that it may be premature to deny the existence of a homolog of mammalian mesencephalic DAergic systems in teleosts and other actinopterygians. A segmental analysis of the ontogeny of the “mesencephalic” DAergic neurons in the chick indicates that the “ventral tegmental area” includes a diencephalic contribution (43). Subsequent studies on amphibians indicate that in young tadpoles the equivalents of the mesostriatal and mesolimbic systems arise from DAergic neurons in the posterior tuberculum, in the dimesencephalic transition zone; thereafter, further DAergic cells appear in the adjoining ventral mesencephalon, behind (32–34). It was concluded that, as a whole, the DAergic cells involved are homologs of those in the amniote “mesencephalon” (33,34). Similarly, although DAergic neurons have not been described in the agnathan mesencephalon, the extensive DAergic innervation of the presumed striatal homolog by a small number of neurons in the posterior tuberculum in lampreys (42) suggests that an ascending DAergic control of at least some portions of the telencephalon by homologous neurons in the rostroventral brain stem may be plesiomorphic for verte-

232

MOK AND MUNRO

brates. Thus, there is a need to reevaluate whether such ascending DAergic projections are indeed absent in actinopterygians. For example, tyrosine hydroxylase-positive (TH-positive), presumed DAergic, neurons are present in the periventricular nucleus of the posterior tuberculum in Polypterus (47), and TH-positive DAergic cells are present in a comparable area in teleosts (reviewed by Meek (35)), as components of the paraventricular organ (PVO). Although Parent (39,40) suggested that the PVO—which also includes other DAergic neurons which are apparently TH-negative and thus may instead sequester this monoamine from the cerebrospinal fluid (30,35)—is the main source of the ascending DAergic innervation of the telencephalon in teleosts, subsequent experimental studies have failed to support this proposition (35). Clearly, further experimental studies are required to determine which, if any, PVO cells are the source of a DAergic innervation of the teleost telencephalon. The telencephalon itself is another potential source of DAergic control: as in other nonmammalian vertebrates, there is evidence that portions of the telencephalon and olfactory bulb of teleosts include DAergic neurons (reviewed by Meek (35) and Smeets and Reiner (55)). Consistent with a possible wholly intratelencephalic level of action, Goping et al. (19) reported that the transient bradykinesia induced by the drug MPTP in goldfish was associated with short-term effects on DAergic neurons in the dorsal telencephalic area “Dc” (possibly equivalent to the ventral area “Vi” of Meek (35)). Goping et al. (19) thus suggested that the area “Dc”/ “Vi” was the functional homolog of the substantia nigra. In a similar vein, Sas et al. (52) speculated that intrinsic neurons in the ventral telencephalon were responsible for the DAergic innervation of a potential striatal homolog in the basal telencephalon of another teleost. Clearly, again, further studies are required to explore this possibility. Apart from the foregoing, TH-positive DAergic neurons have also been described in the preoptic area and the tuberal area of the hypothalamus and in various portions of the thalamus and the adjoining pretectal area (reviewed by Meek (35)). It is not known whether these contribute to the dopaminergic innervation of the telencephalon and thus whether they could play a role in the normal DAergic regulation of locomotor activity. Functional Significance of DAergic Control Systems Munro (36) reported that apomorphine induced a highly characteristic behavior, “Fluttering,” in the cichlid A. pulcher: fish, each kept on their own in a familiar tank, swam repeatedly up and down the front of the aquarium (the only transparent wall), especially when exposed to a mirror or a model of a conspecific at one side of the tank. It was suggested that this behavior may represent a stereotypic activity (36), reminiscent of activation of the mammalian mesostriatal system (see Introduction). However, there was no evidence that the increased locomotor activity observed here in tilapia, using a different test procedure (a novel, open-field arena), was associated with any obvious stereotypy: the fish swam about at random. A study using the same paradigm as that used here with the cyprinid Puntius tetrazona also suggests that there is no evidence of such a response over a range of doses of apomorphine

where the highest had clear toxic side effects (Mok et al., manuscript in preparation; Ng and Munro, manuscript in preparation). Thus the original interpretation of the characteristic “Fluttering” observed in the cichlid A. pulcher with apomorphine is too simplistic. Fluttering has been described in other cichlids without any drug treatment in circumstances which suggested it was a withdrawal response to a threatening situation (51,59); a similar behavior, escape, with a similar function has been described in an anabantoid (9,58). Furthermore, an apparently identical behavior has been used as an index of migratory tendency in three-spined sticklebacks (Gasterosteus aculeatus (4)). Thus, an alternative interpretation of the effects of apomorphine in A. pulcher and other teleosts is that it stimulates behaviors associated with seeking out more “suitable” (e.g., less threatening) conditions; its actions on the telencephalon would thus be reminiscent of MacPhail’s (31) tentative conclusion that the telencephalon may be principally involved in mediating fear in teleosts and evidence that the DAergic projections of the mesolimbic system mediate escape behaviors and other responses to stress in mammals (1,11,13,22,41,50,56). CONCLUSIONS

It would seem from the present results that apomorphine may act at the level of the telencephalon in a manner more comparable with activation of the mesolimbic (rather than the mesostriatal) system in mammals: there was no evidence for stereotypic behavior, including turning bias in the case of fish with only one telencephalic hemisphere. The increase in activity would seem to be mediated by receptors which are more like mammalian D1 than D2 receptors. This would seem to be the first demonstration of a role for dopamine acting at the level of the telencephalon in any anamniote and the receptors involved, and it would appear to refute the suggestion of Roberts et al. (49) that this catecholamine may instead have locomotor actions at lower levels of the neuraxis. Further neuroanatomical studies are required to determine whether the posterior tuberculum may indeed be the source of the DAergic projection implicated and to localize the telencephalic targets involved. The present finding that DAergic drugs act at the level of the telencephalon to influence behavior, combined with the literature on the gross effects of telencephalic ablation, gives some tentative insights into the possible role(s) for dopamine in the regulation of teleost behavior. Thus it was suggested above that DAergic neurons may mediate responses to fearful stimuli, which can be reconciled with a synthesis of other data based on telencephalic ablation (16,31). Clearly, more sophisticated experiments are required to better define the role of the presumed ascending DAergic projections in teleosts, especially in view of the proposal that such projections are important components of the reward system in mammals (see Introduction) and apparently also in goldfish (6). ACKNOWLEDGEMENTS

We are grateful to the National University for financial support (Grant RP 123/84 to A.D.M.) and to two anonymous referees for their instructive comments.

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