Enkephalin-like substance in Aplysia nervous tissue and actions of leu-enkephalin on single neurons

Life Sciences, Vol. 38, pp. 1529-1534 Printed in the U.S.A.

ENKEPHALIN-LIKE

Pergamon Press

SUBSTANCE IN APLYSIA NERVOUS TISSUE AND ACTIONS OF LEU-ENKEPHALIN ON SINGLE NEURONS

Michael K. Leung I Katherine S.-Rozsa 2, Andrew Hall 2 Sunita Ku~uvilla 3', George B. Stefano 3 and David O. ' Carpenter ~ iChemistry Program, SUN~/College at Old Westbury, Old Westbury, N.Y. 11568; LWadsworth Center for Laboratories and Research, N.Y.S. Department of Health, Empire State Plaza, Albany, N.Y. 12201; JBiological Sciences Program, SUNY/ColIege at Old Westbury, Old Westbury, N.Y. 11568. (Received in final form February ii, 1986) SUMMARY Extract from the abdominal ganglia of Aplysia was fractionationed by high performance liquid chromatography. Several large uv absorbing peaks were found. One of these peaks from the ganglionic extract was analyzed by displacement assay with the use of MYtilus edulis membranes. The results revealed the substance in this peak was able to displace 3H-D-AIa2, met5-enkephalinamide. In electrophysiological studies of abdominal and cerebral neurons, depolarizing and hyperpolarizing responses were obtained from some neurons, including neurons in the identified cerebral B cell cluster. A smaller population of cells exhibited biphasic responses. Some of the responses could be depressed by prior naloxone treatment. In conclusion, an endogenous opioid system, using substance related to but distinct from the enkephalins, may exist in Aplysia. In recent years opioid mechanisms in invertebrates have been the subject of numerous intensive investigations (for review, i) . In the land snail, Helix pomatia, a naloxone-reversible action of met-enkephalin an--~ morphine on the activity of single identified neurons has been reported (2). In this study met-enkephalin inhibited the bursting pattern of the Br-type neuron. In a more recent report this inhibition of bursting was a t t r i b u t e d to the prolongation of the interburst period as a resuIt of hyperpolarizing the cell ( 3 ) . Recently, met- and leu-enkephalin have been identified in Mytilus edulis pedal ganglia (4~). Since these opioids were found in a marine mollusc it ~is of interest to determine whether the nervous system of ~plysia c~lifornia, which has been well studied and has many identified cells, also contains opioids and whether there are receptors ~for enkephalin on Aplysia neurons. METHODS Experiments were carried out on A plysia california obtained from Pacific Biomarine Supply Co. (VeniCe, California) and maintained in an aquarium at 16QC. For neurophysiological 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.

1530

Enkephalin-like Substance in

Aplysia

Vol. 38, No. 16, 1986

studies the cerebral ganglia were dissected from the animal pinned to a sylastic base (Slygard 184, Dow Corning) and constantly perfused with artificial sea water as described elsewhere (5). The connective tissue sheath of the ganglia was removed above the B cluster. The cells of the B cluster were identified on the basis of their soma location and response to applied substances. Neuronal somata were penetrated with two independent glass microelectrodes filled with potassium acetate (2 M) having a resistance of 3-8 M ~ and a voltage-clamped with a Dagan Model 8500 amplifie~. Transmembrane current was recorded with a ground current monitor connected to bath via Ag/AgCI2 junction. Both current and voltage clamp mode of recording was used. Membrane resistance was monitored by applying constant current pulses (~0.5 nA) of 0.8 sec duration every 4 sec. Leu-enkephalin was applied by pressure injection from the micropipette of 5 um tip diameter, while the opiate antagonist, naloxone, was added to the bath. The microejection electrode was positioned above cell body so as to maximize the response of the cells. Fast green FCF (.Aldrich) was added to the pressure pipette in order to visualize the pipette ejection and removal. Peptides were all applied at 10-15 min intervals to avoid desensitization of the cells. Acid extraction of Aplysia abdominal ganglia was carried out as previously described ( 4 ) . The ganglia were homogenized with a Brinkman Polytron homogenizer with four 3-s bursts in an extraction solution (25 Pg/ml) containing i M acetic acid, 20 HCI, i ug each of phenylmethylsulfonyl fluoride and pepstatin A per ml, and 1% 2-mercaptoethanol. The homogenate was clarified by centrifugation at 27,000 g for I h. The supernatant was then deproteinized by the addition of 50% (w/v) trichloroacetic acid (TCA) to reach a final concentration of 10% (w/v), followed by centrifugation at 27,000 g for 30 min° The lipids and TCA in the supernatant were removed by extraction 3 times with equal volumes of diethyl ether. The aqueous portion was lyophilized and stored. All of these steps were carried out at 4°C. All steps subsequent to extraction were carried out at ambient temperature. The lyophilized extract was dissolved in an HPLC buffer (i ganglion/ ul) consisting of i0 mM ammonium acetate, pH 4.0. The HPLC system used was a Beckman Model 334 liquid chromatograph equipped with a Beckman/Altex C-RIA integrator. Before injection, the sample was clarified by centrifugation at i0,000 g for i0 min. An aliquot was then subjected to HPLC on a Brownlee RP-300 reverse-phase column (4.6 x 250 ram). The column was eluted at a flow-rate of 2 ml/min with a solvent system consisting of the HPLC buffer and a linear gradient of 5-25% 2-propanol in 30 min. For displacement studies aliquots of pedal ganglia membrane suspension, obtained from freshly collected My tilus edulis, were incubated for 20 min at 22°C with 2 nM of nonradioactive opioid compounds together with opioid standards or fractions from Apylsia ganglionic extract where indicated. 3H-D-AIa2, met5~nkephalinamide (3H-DAMA; I nM) was then added to the mixture and it was incubated for an additional 60 min at 4°C. The incubation buffer and the separation of bound from free 3H-DAMA have been described in detail elsewhere ( 6 ) . One hundred percent binding is defined as bound 3H-DAMA in the presence of I0 uM

Vol. 38, No. 16, 1 9 8 6

Enkephalin-like Substance in

Aplysia

1531

dextrorphan minus bound 3H-DAMA in the presence of I0 uM levorphanol. RESULTS The extract from 40 Aplysia abdominal ganglia was i n j e c t e d into the HPLC. Aside from t h e p o l a r materials which appeared at the beginning of the chromatogram, several major peaks were observed. No major peak was observed with retention time (Rt) of met-enkephalin. A small peak was present in this area but it was not subjected to further study in this present investigation. However, a very large peak was observed to migrate with the same Rt as leu-enkephalin (data not shown). The HPLC fractions of this peak were lyophilized. The lyophilized material was redissolved in HPLC buffer and was rechromatographed under isocratic conditions with the use of 8% 2-propanol. Duplicated runs under these conditions have consistently shown this material to migrate 0.5 min ahead of the authentic leu-enkephalin standard. As the deviation in Rt of duplicated runs of the standard or the sample by itself was less than 0.09 min we consider this difference of 0.5 min to be significant and the material in the peak is not identical to leu-enkephalin. In order to further evaluate the material, the fraction from this peak was assayed for displacement activities. The displacement studies utilizing 3H-DAMA has been described elsewhere (6)° In this study we did not use Aplysia ganglia membrane suspensions because we did not have an adequate amount. Mytilus pedal ganglia membrane suspensions were employed as a substitute since information exists concerning their binding properties (1,5). As noted in Table I the HPLC fraction does possess displacement activity. However, this activity is weak relative to that of the authentic enkephalins. TABLE I Displacement of 3H-DAMA from M. edulis Pedal Ganglia Membrane Suspension--~y p ~ f i c Aplysia HPLC Fraction 0pioid Peptides

Conc.

DAMA met - enkephalin leu- enkephalin Purified Abdominal Fraction

2 nM 2 nM 2 nM

Substance P

50 ul* I00 ul* i uM

* concentrations unknown. Values from redissolved fractions.

Displacement % of Control i00 94 91 21 44 0 indicate volumes used

In parallel experiments electrophysiological studies were performed in order to determine whether the enkephalins can elicit changes in potential and/or conductance on Aplysia neurons. The techniques used were similar to those described by Slater et al. ( 7 ) . Neurons from various ganglia were studied under either current or voltage clamp with two intracellular electrodes and an extracellular electrode filled with i to i0 mM

1532

Enkephalin-like Substance in Aplys~a

Vol. 38, No. 16, 1986

leu-enkephalin plus 3 mg/ml fast green in seawater. The extracellular electrode was positioned over the cell being recorded and the leu-enkephalin was applied by pressure using 8 to i0 psi pulses of 0.4 to 1.2 sec duration, with visual inspection of the dye passage as proof that the electrode was functional. Responses to application of leu-enkephalin were obtained from neurons in both abdominal and 6erebral ganglia. In both ganglia some neurons were excited and others were inhibited by leu-enkephalin. One cluster of large identified neurons, the B neurons of the cerebral ganglia, was found to contain a relative~ high proportion of neurons respond£ng to leu-enkephalin and these neurons were studied more completely. The responses on B neurons did not appear to differ from those seen on unidentified abdominal neurons, however. The B neurons, approximately 20 in each bilateral cluster, are known to be involved in movement, and to constitute a more or less homogeneous group of cells (8,9). In the isolated ganglia these neurons are silent at rest. Approximately 50% of more than i00 B neurons studied were affected by leu-enkephalin, with two different ionic responses seen. Approximately half of the affected neurons were depolarized and excited while another 25% showed a slow hyperpolarizing response. Some neurons had biphasic responses, one example of which is shown in Fig. I. Under current clamp (A) the response to application of leu-enkephalin was a brief depolarization followed by a more prolonged hyperpolarization. While both polarities of response were frequently associated with an increase in membrane conductance, in this case it is small, suggesting that the receptors ate in the neuropile distant from the cell body. Under voltage clamp (B-G) the large outward current underlying the late hyperpolarizing response reversed at about -75mV, near the equilibrium potential for K+. From the conductance changes and the voltage dependency of the currents we conclude that on these neurons leu-enkephalin causes an increase in conductance to Na+ for depolarizing the K+ for hyperpolarizing responses. Both responses were depressed to less than 50% of control by naloxone, but only at relatively high bath concentrations (5 raM). DISCUSSION The abdominal ganglia of Aplysia does not contain detectable level of authentic leu-enkephalin with the HPLC procedure used but there is a large peak present which contains material capable of displacing 3H-DAMA in Mytilus ganglionic membrane suspension. Furthermore, identified neurons respond to leu-enkephalin with a variety of types of naloxone-sensitive voltage and conductance changes. Together these observations suggest the presence of an endogenous system using substance related to but distinct from leu-enkephalin. The weak naloxone sensitivity is also consistent with this suggestion. Lukowiak et al. (review i0) has reported that met-enkephalin superfusion of Aplysi a abdominal ganglion caused a decrease in gill withdrawal r e ~ ! e x amplitude and increased its rate of habituation. It was concluded that the peptide was acting presynaptically. He also reported that sexual activity in Aplysia leads to suppression of the gill withdrawal reflex, a phenomena

Vol. 38, No. 16, 1 9 8 6

Enkephalin-likeSubstance in

Aplysia

1533

reversed by naloxone, These observations are also consistent with there being an endogenous opioid system in Aplysia which which influences behavior.

CBR14 MP-56

A

-30 B

....

C ~'~

~

+

-40

-56

E

-75

F

-85

-90

G 2 nA 20 sec

Figure I. Current (A) and voltage (B-G) clamp recordings of the response of a cerebral neuron (cell CBRI4) to pressure application of i0 mM leu-enkephalin. Record A was recorded at the resting membrane potential of -56mV. The deflections are voltage shifts resulting from a i nA current pulse. Leu-enkephalin was applied at the arrow in each record Recurds B-G show voltage clamp traces at different holding potentials, indicated at the right of each trace

1534

Enkephalin-likeSubstance in Aplysia

Vol. 38, No. 16, 1986

ACKNOWLEDGEMENTS This work was supported by NIH Grant RR08180 (M.K.L. and G.B.S.) and NS18435 (KoS.R., A.H., D.O.C.) and ADAMHA-MARC 17138 (G.B.S. and M.K.L.). Ms° S. Kuruvilla is an ADAMHA-MARC Fellow. REFERENCES i. 2. 3. 4. 5.

G.Bo STEFANO, Cel. Mol0 Neurobiol. 2:167-178 (1982). G.B. STEFANO, I. VADASZ and L. HIRIPI, Experientia 36:666667 (1980). J. SALANKI, A. VEHOVSZKY and G.B. STEFANO, Compo Biochem. Physiol., 75c:387-390 (1983). M.K. LEUNG and G.B. STEFANO, Proc. Natl. Acado Sci., USA 81:955-958 (1984) T.Co PELLMAR and D.O. CARPENTER, J. Neurophysiol. 44:423-439

(1980). 6. 7. 8. 9. I0.

R.M. KREAM, R.S. ZUKIN, and G.B. STEFANO, J. Biol. Chem., 255:9218-9224 (.1980). N.T. SLATER, D.O. CARPENTER, J.E. FREEDMAN, and S.H. SNYDER, Brain Res. , 278:266-270 (1983). S.M. FREDMAN, and B0 JAHAN-PARWAR, J. Neurophysiol. 40:608-615 (1977). B. JAHAN-PARWAR, and S°M. FREDMAN, J. Neurophysiolo 49:1481-1503 (1983). Ko LUKOWIAK, Handbook of Comparative Opioid and Related Neuropeptide Mechanisms. Ed. GoB. STEFANO, CRC Press, Boca Raton, FL. (1986).