Camp. Biochem. Physid. Vol. 85C, No. 1, pp. 6146, 1986 Printed in Great Britain

0306~4492/86 $3.00 + 0.00 Pergamon Journals Ltd

A BEHAVIORAL ROLE FOR ENKEPHALINS IN REGULATING LOCOMOTOR ACTIVITY IN THE

INSECT LEUCOPHAEA MADERAE: EVIDENCE FOR HIGH AFFINITY KAPPA-LIKE

OPIOID BINDING SITES

ROBERT FORD,* DUANE M. JACKSON,? LEONA TETRAULT,* JUAN C. TORRES,* PAUL ASSANAH,* JAY HARPER,~ MICHAEL K. LEUNG$

and GEORGE B. STEFANO*~~ *Biological Sciences Program, SUNY/College at Old Westbury, Old Westbury, N.Y. 11568, USA. Telephone: (516) 876-3000, TDepartment of Psychology, Clark College, Atlanta, GA 30310, USA, fPsychology Program, SUNY/College at Old Westbury, Old Westbury, N.Y. 11568, USA and §Chemistry

Program, SUNY/College at Old Westbury, Old Westbury, N.Y. 11568, USA

(Received 10 October 1985)

Abstract-l. D’-Ala-2,met-5-enkephalinamide application to the cerebral ganglia of Leucophaea maderae results in a decrease in locomotor activity. The opiate antagonist, naloxone, can block this effect as well as the depressant effect of morphine on locomotor activity.

2. D-Ala-2,leu-S-enkephalinamide and dynorphin enhance locomotor activity following their topical application to the cerebral ganglia. This effect also can be antagonized by concomitant naloxone treatment.

3. Benzomorphans were the most potent ligands tested in their ability to displace [‘HID’-ala-Z,met-5-enkephalinamide whereas mu and delta ligands were by comparison less potent. These results suggest the presence of kappa-like opioid receptors in Leucophaea cerebral ganglia.

4. The kappa ligands also are potent in enhancing locomotor activity in addition to being weakly antagonized by naloxone. Again, these results indicate the presence of multiple-opiate receptor types in invertebrates.

INTRODUCTION

The known evolutionary relationships among all animals, the presence of many of the same mediators and transmitters in their nervous systems and the recent evidence for the presence of certain opioids in the bivalve Mytilus edulis (Leung and Stefano, 1984;

Stefano and Leung, 1984) make the nervous system of various invertebrates an ideal place to look for opioid functions., Duve et al. (summary, 1986) report the presence of met- and leu-enkephalin-, beta- endorphin- and ACTH-like immunoreactivities in the blowfly, Calliphora oomitoria. Met-enkephalin im- munoreactivity was also found in the cerebral ganglion of Locusta migratoria (Remy and Dubois, 1979). Alpha-endorphin immunoreactivity has also been reported in lepidopteran larva (Thaumetopoea pityocampa; Remy et al., 1978) and met-enkephalin- and beta-endorphin-like activities were found in the midgut of Periplaneta (Nishiitsutsuji-Uwo et al., 1986). In Leucophaea maderae ACTH- and MSH- like compounds have been detected in the corpus cardiacum<orpus allatum complex (Hansen et a/., 1986). Thus, the evidence to date strongly suggests that the insect nervous system may contain a variety of opioid compounds which may resemble those found in mammalian systems.

IjAuthor to whom correspondence should be addressed.

61

Other investigators have demonstrated the pres- ence of opioid binding sites in insect neural tissues. Pert and Taylor (1980) showed that membranes prepared from Drosophila heads bind leu-enkephalin but naloxone does not bind well to these membranes. In the blattarian insect, Leucophaea maderae, D’-ala-2-met-5-enkephalinamide (DAMA) also bound extensively to cerebral ganglion membrane suspensions (Stefano and Scharrer, 1981). In yet another study involving Leucophaea tissues, high- affinity stereospecific opioid binding was demon- strated in midgut preparations (Stefano et al., 1982). These results suggest that these animals possess both opioid substances and receptors and thus the exis- tence of a complex opioid mechanism similar to mammals.

The present study provides additional evidence for the presence of endogenous opioid mechanisms in insects, specifically Leucophaea. The results indicate that the endogenous opioid system interacts with locomotor regulatory mechanisms and may involve a kappa-like receptor population.

MATERIAL AND METHODS

Adult L.eucophaea were selected from stock colonies maintained for many years under controlled conditions on a diet of dog chow, apples and water. At Clark College they were maintained on a diet of water and “roach food” purchased from Carolina Biological Supply Company. The

62 ROBERT FORD et al.

animals were maintained on a natural light cycle. Twenty- four hours prior to testing the animals were placed in individual wire cages (ht. 9cm, diam. 8cm) and provided with food.

For the in DI’L.O application of drugs or vehicle the animal was held stationary. A 1 ml tuberculin syringe was posi- tioned by hand and the needle forced into the anteriordorsal surface of the head in the proximity of the central suture, and above the antenna1 suture just above and between the compound eyes. The injection volume was 100 ~1 given over 1 min. The barrel of the needle was sealed in warm wax which was then pressed firmly on the head after the needle was withdrawn, so as to prevent any backflow of solution. Dye injections confirmed that backflow was negligible since the cerebral ganglia was heavily stained.

Immediately after the injection the animal was placed in a Entomex 1001 behavior monitoring chamber (Columbus Instruments, Columbus, Ohio) for a 15 min observation period divided into five observation periods. The Entomex uses a light sensitive grid floor to acquire the activity count. The Entomex is interfaced with an Apple II computer. In addition to this method of data collection organisms were placed in boxes that had checkered bottoms (0.5 cm squares)

and individuals counted the number of boxes stepped into during the same observation periods. In the last procedure the individuals counting were blind as to treatments. Experi- ments were also performed utilizing the Videomex (Col- umbus Instruments) behavioral monitoring equipment in conjunction with an enhanced Apple IIe computer. This last method divides a surface into small cm regions and the surface is constantly being evaluated for the occupation of new space. The computer program then analyzes and com- pares space occupancy which is translated into locomotion. Each dose of pharmacalogical agent was given to at least six animals. Statistical analysis was by way of a one tailed Student’s r-test.

For binding studies, the dissection of the adult male cerebral ganglia was carried out under 4°C saline as pre- viously described (Stefano and Scharrer, 1981). The ganglia were homogenized, suspensions prepared and binding experiments carried out as previously described (Stefano and Scharrer, 1981). The potent synthetic enkephalin analog DAMA ([3H]DAMA; 15.1 Ci/mmol) was used as the ex- ogenous opioid ligand. Ahquots of suspension (0.2 ml, 0.12 mg protein) were incubated in triplicate at 4°C for

60 24 hour Act~wty of Lec~phaeO MockrOe

gt$ IO 2 $.F'F' IO 2 6W

24 hour cycle

Fig. I, The group activity of 40 animals was monitored by the Videomex-Apple IIe system for 3 days. The activity numbers represent the mean number of grids the animals enter during the observation period. Each point represents the mean reading for that hour k SEM. The difference between the 9:00 a.m. reading and the 10:00 p.m. reading is

significant (P < 0.01).

90 min with the radiolabelled ligand in the presence of dextrorphan (1OpM) or levorphanol (10pM) in IOmM Tris-HCI buffer, pH 7.4, containing 0.1% BSA and 150 mM KCI. Separation of free ligand from membrane- bound ligand was by filtration under reduced pressure through GF/B glass filters previously soaked, for 45 min at 4°C in buffer containing 0.5% BSA. Filters were then counted in a Packard 460 CD liquid scintillation counter (57% efficiency). Stereospecific binding is defined as binding in the presence of 10 PM levorphanol. Protein concentration was determined by the method of Lowry et al. (1951).

For displacement analysis aliquots of adult male cerebral ganglia membrane suspension were incubated with non- radioactive opioid compounds at seven concentrations for IO min at 22°C and then with [ZH]DAMA (I nM) for 60 min at 4°C. One hundred per cent binding is bound [‘HIDAMA in the presence of IOpM dextrorphan minus bound [‘HIDAMA in the presence of IOpM levorphanol. IC,, is defined as the concentration of drug which elicits half- maximal inhibition of specific [‘HIDAMA binding. The mean + SE for three experiments is reported for each com- pound tested.

The peptides employed in this study were puchased from Peninsula Laboratory. Naloxone was a gift from Endo Laboratories.

RESULTS

Adult Leucophaea used in the present study were more active during the light than during the dark (Fig. 1). A probable explanation for this reversal of normal activity is that animals were routinely fed early in the mornings and thus were forced to leave their paper dwellings to feed. Control organisms and those treated with vehicle all tend to have high activity in the first period of observation (Fig. 2). This is probably associated with the trauma of being handled.

Leucophow Locomotor Actwty

0 control o vehde

25

1

I 2 3 4 5

Achwty Perds (3 ml” 1

Fig. 2. Recorded by both Entomex and Videomex systems. Each point represents the mean + SEM of five individual

determinations.

Kappa binding site in cockroach 63

40

35

30

25

za .%> 20

‘C P

15

IO

5

Effects of DAMA on Locomotor Activity

Cl *hide 0 .5/M DAMA l I +4 DAMA b 5j~M a4MA . 10j~t.I MMA

I 2 3 4 5

Activity Periods (3rmnj

Fig. 3. Recorded and monitored by the Videomex-Apple IIe system. Each point represents the mean of five determinations f SEM. At a dose of 1, 5 and 10pM DAMA the change or depression of locomotor activity is statistically significant when compared to vehicle treatment (Fig. 2). Dose 1 PM at period 4, P < 0.01; 5 PM periods 2 and 3, P < 0.01, periods 4 and 5, P < 0.005; 10 PM periods

2-5, P < 0.005.

Injection of DAMA and morphine results in a complete and prolonged decrease in activity (Figs 3 and 4). The decrease in activity is dose-dependent and can be antagonized by concomitant naloxone injec- tion (Fig. 5). Taken together the results appear to indicate that both DAMA and morphine exert a potent effect on locomotor control. In addition, DAMA appears to be more potent than morphine in

30

25

Effects of Morphine

0 vehicle 0 5pM Morphme

l 50pM Morphine

A IOOP Maphine

I 2 3 4 5

Actdy FWWIS (3mln )

Fig. 4. As noted in Fig. 3, 50 PM morphine periods 4 and 5, P i 0.05; periods 4 and 5, P < 0.005.

this regard. The dose of naloxone required to antag- onize this affect is rather large and will be discussed later. Injections of met-enkephalin (10-8-10-4 M, data not shown) do not alter the organism’s loco- motor activity. This lack of effect may be explained by the presence of endogenous enkephalinases or nonspecific digestion. The relatively high doses em- ployed may be due to the nature of the drug applica- tion, namely a restricted whole body injection.

Interestingly, administration of o-ala-2-leu- 5enkephalinamide (DALA; lOA M), dynorphin A (2 x 10e6 M) and the benzomorphan, cyclazocine (10e6 M), to the vicinity of the cerebral ganglia results in an increase in locomotor activity (P < 0.05) by 36, 41 and 34%, respectively. Concomitant naloxone and DALA injection prevents the increase in activity. Naloxone, when injected alone (Fig. 5) causes a nonsignificant increase in activity. Also, given the apparent affinity of kappa ligands (in the next sec- tion), it was of interest to try dynorphin A (l-13). The effects of both dynorphin A and cyclazocine are naloxone reversible (5 x 10m5 M).

In addition to the previously described activity alterations following opioid administration we noted that certain individuals exhibited other abnormal activities. A few of the DALA-treated individuals would continue to have high activity levels by run-

ning in a circle for up to 24 hr. Interestingly, follow- ing opioid treatments the organisms would not eat for up to a day.

Binding to cerebral ganglia membrane suspensions is monophasic, saturable with respect to radioligand concentration and stereospecific. Scatchard analysis (data not shown) of these data revealed a single class of high-affinity sites with a Kd of 8.0 nM, and a B,,,,, of 39 pmol/g protein as previously noted in detail (Stefano and Scharrer, 1981).

Antoqanlsm of Opote Depreswon Of Locomotor Actndy

0 vchlcle

0 50pM Noloxcne (N) o N + IOOpM Mxphme

A N* l0j~MDAhtA

I 2 3 4 5

Activity Rriods (3mln.J

Fig. 5. As noted in Fig. 3, 50pM naloxone and 100pM morphine at periods 3,4 and 5, P < 0.01 when compared to agent without naloxone. 50pM naloxone and 10pM DAMA at periods 3, 4 and 5, P < 0.01 when compared to

agent without naloxone.

64 ROBERT FORD et al.

Table I, Effect of various ions and trypsin on binding of DAMA by adult male cerebral ganglia

Stereospecifically bound ligand

Reagent Concentration (fmol/mg protein)

[‘H]DAMA 8nm

20.3 f 1.2 KC1 1.5 x IO-’ M 26.8 + 1.8 NaCl 1.5 x IO-’ M 3.6 + I.0 NaCl and M”& 1.5 x IO-‘, IO-‘M 19.7 + 1.6 Trypsin I mg/ml 1.2 * 1.0 Trypsin and trypsin

inhibitor I mg/ml, 2 mg/ml 17.9 + 1.2

Described in Material and Methods section.

Tests on the highly specific and differential effects of various ions on DAMA binding gave the results in Table 1. The binding of [3H]DAMA is reduced by sodium, an effect that is reversed by manganese. The binding of DAMA is abolished by trypsin (Table 1). Pretreatment with soybean trypsin inhibitor reversed the inhibition of opiate binding by trypsin and confirmed that the binding site is proteinaceous.

Displacement of [‘H]DAMA by nonradioactive DAMA was monophasic as a function of non- radioactive ligand concentration. The ability of a variety of other opiates to displace specifically bound [3H]DAMA was investigated (Table 2). The opioid peptides [dynorphin A (l-13) > FK 33 824 >/?- endorphin > DAMA] were the most potent of the ligands tested. The benzomorphans (cyclazocine > (-)-ketocyclazocine) exhibited relatively high poten- cies as a group and the opiate narcotics as a group (etorphine > naltrexone > morphine > nalo- xone) were the least potent. Surprisingly, kappa ligands appear to be quite potent in this regard.

DISCUSSION

The present demonstration of behavioral effects of opioid applications to Leucophaea cerebra1 ganglia is in agreement with previous studies. DAMA was previously shown to bind with high affinity and stereoselectivity to cerebral ganglia membrane sus- pensions (Stefano and Scharrer, 1981) and this was

Table 2. Relative potencies of opiates in reduc- ing [‘HIDAMA (1 nM) binding to L. maderae

cerebral ganglia membrane suspensions

Opioid compounds

Peptides DAMA FK 33824 Dynorphin (I-13) B-Endorphin

;Zl)

8.3 i 0.20 5.6 k 0.50 2.1 + 0.10 7.1 * 0.30

Alkaloids Etorphine Morphine Naltrexone Naloxone

5.9 * 0.20 10.4 + 1.10 6.3 rt 0.50

11.9+ 1.5

Benzomorphans Cyclazocine 1.6 + 0.30 (-)-Ketocyclazocine 2.0 * 0.10

Described in Material and Methods section.

verified in the present study. Specific binding could not be detected in membrane suspensions of the organism’s optic lobes, which lie right next to the cerebral ganglia. The earlier study suggests that in this insect opioid binding sites are confined to specific areas of the organism’s nervous system. In support of our finding Remy and Dubois (1979) demonstrated the presence of met-enkephalin-like material in the cerebral ganglia of Locusta and that the optic lobe region was devoid of immunoreactivity. Thus, in different species the results complement each other. As a result of this occurrence it was of interest to determine if opioid administration would in some way alter some aspect of the organisms behavioral profile.

In mammals low doses of opiates may stimulate locomotor activity (10m6 M) whereas higher doses may result in depressed activity (for a review see Cooper et al., 1978). Slightly higher doses (lo-‘M) produce enhanced activity which may result in stereo- typed behavior. Following extremely high doses of opiates, after the cataleptic state is observed, the organisms enter into a state of prolonged activity. These behavioral effects of opiates in mammals may have counterparts in various invertebrates. In the land snail Helix pomatia morphine induces a state of immobilization and muscle rigidity (3 PM), resulting in a loss of the organism’s righting reflex. These effects are reversible by naloxone (10 PM; Stefano et al., 1980; Burrowes et al., 1983). This behavioral effect of morphine is reduced as treatments progress for 4 days and reoccurs if a higher dose of morphine is given on the fifth day (10 p M), thus demonstrating that tolerance has occurred. In Plunariu morphine also reduces locomotor activity (Venturini et al., 1981). Other studies regarding opiate activities in invertebrates also suggest a regulatory motor role (Kavaliers et al., 1983).

The present study demonstrates that the endo- geneous opioid system, which apparently exists in this organism, is involved with regulating locomotor activity. It was, however, quite surprising to find that DALA stimulated whereas DAMA inhibited loco- motor activity. This interesting phenomenon may be explained by suggesting that separate opiate mech- anisms exist and that separate ligands modulate separate functions. In a recent study, the only small peptide enkephalin-like material found in the acid extract of cerebral ganglia fractionated by HPLC in Leucophaea was met-enkephalin-like (Chapman et

Kappa binding site in cockroach 65

al., 1984). This may indicate that larger opioid mol- ecules may be present in this tissue.

However, the “picture” may be more complex given the binding data presented in this report. The doyen among agonists at K-receptors is a non- peptide, ketocyclazocine (Martin et al., 1976). It has also been demonstrated that the peptide dynorphin (Goldstein et al., 1979, 1981) also is, a potent kappa agonist in various assays. These studies note that naloxone is rather weak in displacing kappa agonists. The data presented in the present report suggest that kappa-like receptors may be present in the cerebral ganglia of Leucophaea since dynorphin and the ben- zomorphans are potent binding ligands whereas na- loxone is a weak one. In considering the displacement study in Mytilus edulis, peptides with more mu and delta affinities were found to be potent in reducing FK 33 824 binding and the kappa agonists were the least potent (Kream et al., 1980). In ~e~co~h~e~ the benzomorphans, as a group, are highly potent in reducing DAMA binding, whereas mu and delta ligands were relatively weaker. These results tend to suggest the presence of multiple opioid receptor subtypes in invertebrates. Clearly, more detailed studies must be performed to evaluate these results. Simultaneously with the preparation of this report Santoro et al. (1985) reported that Drosophila tissues appear to contain kappa-like opioid receptors. Thus, in two insects the subpopulations of opioid receptors appear to be quite similar in regard to their prefer- ence for binding ligands.

In addition, opioid substances appear to be in- volved with regulating locomotor activity. It is of interest to note that the enkephalin analogs DAMA and DALA can cause opposite effects in the same tissue. This may indicate the existence of different functional roles. Also, the benzomorphanes were the most potent compounds tested for their ability to increase activity. Naloxone alone was able to enhance locomotor activity suggesting a tonic-opioid influence. This study demonstrates that the opioid mechanisms present in this organism may be ex- tremely complex and highly diversified.

Acknowledgemenrs-This work was partially supported by ADAMHA MARC Grants MH 16573 and 17138 and NIH Grant MBRS 08180. Robert Ford and Juan C. Torres are MBRS research assistants. Paul Assanah and Leona Tet- rault are ADAMHA-MARC Fellows. The work also was partially supported by a Grant from the Long Island Community Foundation, Inc. (G.B.S.).

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