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Galanin receptors inhibit the spontaneous firing of locus coeruleus neurones and interact with μ-opioid receptors
European Journal of Pharmacology, 230 (1993) 223-230
223
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 52872
Galanin receptors inhibit the spontaneous firing of locus coeruleus neurones and interact with -opioid receptors J a n Sevcik l, E r v i n P. F i n t a a n d P e t e r Illes Department of Pharmacolo,g% Unicersity of Freiburg, W- 7800 Freiburg, Germany
Received 11 September 1992, accepted 27 October 1992
Electrophysiological experiments were performed in a pontine slice preparation of rat brain containing the locus coeruleus (LC). The extracellular part of this study showed that galanin (0.003-0.1 p.mol/I), [Met~]enkephalin (0.01-10 p.mol/l) and noradrenaline (0.1-100 ~mol/l) concentration dependently inhibited the firing rate. Noradrenaline (1 and 3 /zmol/l) had the same effect both before and during the application of galanin (0.001 or 0.01 #mol/l). Similarly, [MetS]enkephalin (0.03 and 0.1 p.mol/l) produced identical inhibition, regardless of the presence or absence of 0.01 p.mol/l galanin. Whereas rauwolscine (1 p.mol/1) potentiated the effect of galanin (0.03 #.tool/I), idazoxan (1 p.mol/1) was inactive. In contrast, both naloxone (0.1 #mol/l) and /3-funaltrexamine (0.1 p.mol/1) facilitated the galanin-induced inhibition. In the intracellular experiments, galanin (0.3 /~mol/l) abolished the spontaneous discharge of action potentials, hyperpolarized the cells and decreased their input resistance. In conclusion, galanin may depress the firing rate by increasing a potassium permeability. Moreover, galanin receptors appear to interact with /x-opioid receptors but not with ~2-adrenoceptors. Galanin; Locus coeruleus neurons; Receptor interaction; Firing rate; Membrane potential
1. Introduction
Galanin, a 29-amino acid residue peptide originally isolated from porcine small intestine (Tatemoto et al., 1983), is widely distributed in the rat central nervous system (Skofitsch and Jacobowitz, 1985; R6kaeus et al., 1987). Galanin has also been found in most noradrenaline-containing neurones of the locus coeruleus (LC) (Skofitsch and Jacobowitz, 1985; Holets et al., 1988). The functional consequences of this co-existence are unclear. It has been demonstrated that, in the LC, both ~2-adrenoceptor and #-opioid receptor agonists increase the same potassium conductance and thereby produce hyperpolarization and depress the spontaneous discharge of action potentials (North and Williams, 1985). LC neurones possess adrenoceptors of the a2-type ( C e d a r b a u m and Aghajanian, 1977; Williams et al., 1985), and opioid receptors of the /x-type only (Williams and North, 1984). It was ob-
Correspondence to: P. Illes, Department of Pharmacology, University of Freiburg. Hermann-Herder-Strasse 5. W-7800 Freiburg, Germany. I On temporary leave from the Institute of Pharmacology CSAV, Albertov 4, 128 00 Prague 2, CSFR.
served that the inhibitory effect of #-agonists is potentiated in the presence of a2-antagonists, suggesting an interaction between the two receptors (llles and N6renberg, 1990; Illes et al., 19901. Furthermore, it was found that neuropeptide Y (NPY) decreases the firing rate by a mechanism similar 1o that for a 2- and #agonists (Illes and Regenold, 1990; Illes et al., 19901. Since NPY potentiated the effect of noradrenaline but not that of [MetS]enkephalin, and the NPY effect was abolished in the presence of the ~2-antagonist, rauwolscine, it was suggested that a selective interaction between NPY receptors and a2-adrenoceptors occurs in the LC (llles and Regenold, 1990; Illes et al., 19901. In addition to the cell somata, the nerve terminals of central noradrenergic neurones also possess inhibitory a 2- (Starke, 1977), /x- (Illes, 1989) and NPY receptors (Martire et al., 1986; Yokoo et al., 1987), which were shown to interact similarly to the respective somatic receptors (Illes, 1989; Illes et al., 1990). It was reported recently that galanin depresses the discharge of spontaneous action potentials in LC neurones (Seutin et al., 1989). However, the extracellular experiments reported gave no information about the ionic mode of action of galanin and the possible interaction of the peptide with noradrenaline. The latter question is especially interesting, since a:-agonists potentiate the inhibitory effect of galanin on noradrenaline re-
224 lease from the terminals of LC neurones, while c%antagonists diminish the galanin effect (Tsuda et al., 1989). In the present extra- and intracellular study, galanin inhibited the spontaneous firing of LC cells by hyperpolarizing the membrane. Moreover, galanin failed to interact with [MetS]enkephalin or noradrenaline, but its inhibitory effect was potentiated by the #-opioid receptor antagonists, naloxone and /3funaltrexamine. Variable results were obtained with two cQ-adrenoceptor antagonists; idazoxan was ineffective, while rauwolscine facilitated the galanin effect.
2.3. Recording techniques
2. Materials and methods
2.3.2. h~tracellular recording
2.1. Brain slice preparation The preparation and maintenance of midpontine slices of rat brain were as previously described (Illes and N6renberg, 1990). In brief, male Wistar rats (150220 g) were anaesthetized with ether and decapitated. Slices of 400-/~m thickness, containing the caudal part of the LC were prepared in oxygenated medium at 1-4°C with a Lancer vibratome. A single slice was transferred to the recording chamber and was superfused at a rate of 2 m l / m i n with medium saturated with 95% 0 2 plus 5% CO x and maintained at 35-36°C. The medium was of the following composition (in retool/l): NaC1 126, KCI 2.5, N a H 2 P O 4 1.2, MgCI 2 1.3, CaCI 2 2.4, N a H C O 3 25, glucose 11, N a , E D T A (I.03 and ascorbic acid 0.3.
2.2. Identification of LC neurones The LC could easily be identified under a binocular microscope (magnification 10-40 times) as a translucent oval area on the ventrolateral border of the fourth ventricle. LC cells fire spontaneously with a constant rate of 0.2-5 Hz.
2.2.1. Extracellular recording The extracellular action potentials are biphasic and longer ( > 2 ms) than those of neighbouring neurones. Since c~2-adrenoceptor agonists depress the firing rate (Cedarbaum and Aghajanian, 1977), we added at the beginning of each experiment a single test concentration of noradrenaline (30 /xmol/l), which produced a complete and reversible inhibition.
2.2.2. lntracellular recording LC cells were distinguished from neighbouring mesencephalic trigeminal neurones by their electrophysiological properties, including spontaneous firing and a marked hyperpolarization in response to noradrenaline (100 /zmol/I).
2. 3.1. Extracellular recording Glass microelectrodes filled with 4 m o l / l NaCI and with a tip resistance of 2 - 4 M,Q were used for recording the firing rate. The electrode signals were passed through a Grass PI6 high-impedance amplifier, filtered and displayed on a Tektronix 5113 oscilloscope. The spikes were gated and counted by means of a WPI 121 window discriminator coupled to an electronic ratemcter and a Watanabe W T R 311 pen recorder. Firing rate was recorded as consecutive 30-s samples.
Recording and current injection were carried out with glass microelectrodes filled with KC1 2 m o l / l (tip resistance 60-1(10 M,O), using a high-impedance preamplifier and a bridge circuit (Axoclamp 2 A). In some experiments LC ceils were constantly hyperpolarized (about 10 mV) by passing through the microelectrode a current just sufficient to prevent the cell from firing spontaneous action potentials. In addition, hyperpolarizing current pulses of constant amplitude and 250-ms duration were delivered at a frequency of 0.5 Hz. The apparent input resistance was calculated from the resuiting peak potential change. The m e m b r a n e potential was determined on withdrawal of the microelectrode from the cell at the end of each experiment.
2.4. Application of drugs and el,aluation of data Various drugs were applied by changing the superfusion medium by means of three-way taps. At the constant flow rate of 2 m l / m i n about 30 s were required until the drug reached the bath.
2.4. 1. Extracellular recording Concentration-response curves for noradrenaline and [MetS]enkephalin were made by applying increasing concentrations of the agonists for 3 rain and the interval between subsequent applications was 10 rain. Concentration-response curves for galanin were made by applying increasing concentrations of the peptide for 5 rain and keeping an interval of at least 15 rain between subsequent applications. Depression of firing rate was measured at its maximum (average of two counting periods), irrespective of drug contact-time, and was expressed as percentage of the average firing rate during the 2 min immediately before addition (average of four counting periods). In every experiment, the ICs0 value, i.e. the concentration that produced 50% inhibition of the spike discharge was estimated graphically. Only one concentration-response curve of one agonist was made on a single cell of a brain slice. The interaction between galanin on the one hand
225 and noradrenaline or [MetS]enkephalin on the other was tested as follows (see fig. 3A). Two concentrations of noradrenaline (1, 3 /zmol/l) or [MetS]enkephalin (0.03, 0.1 /xmol/l) were used, which inhibited the discharge of action potentials by less than 25 and 50%, respectively, as estimated from the concentration-response curves of these agonists. At first, a lower and a higher concentration of the same drug (either noradrenaline or [MetS]enkephalin) were applied consecutively. Both concentrations were then reapplied in the presence of a low concentration of galanin (0.001 or 0.01 p.mol/l). Finally, the first mode of application was repeated. The contact time of all compounds was 3 min, except in the case of galanin, which was applied 5 rain before noradrenaline or [MetS]enkephalin and was kept in the medium for another 3 min together with these agonists. The drug-free intervals were 10-15 rain. Drug effects were expressed as percent inhibition of the average firing rate measured immediately before the addition of each agonist concentration. Furthermore, we tested for a possible interaction between az-adrenoceptor antagonists (idazoxan, rauwolscine) or /x-opioid receptor antagonists (naloxone, /3-funaltrexamine) and galanin. Galanin was applied for 5 min in a concentration (0.03 ixmol/l), which inhibited the firing rate by about 50%. Galanin (0.03 /xmol/1) was reapplied after another 35 rain, either in the absence or the presence of idazoxan (1 ixmol/l), rauwolscine (1 ixmol/1) or naloxone (0.1 /xmol/l). The antagonists were in contact with the tissue for 10 min before and during the application of galanin. The effect of galanin was expressed as percentage inhibition of the average firing rate measured immediately before addition of the peptide. Experiments with /3funaltrexamine were carried out with a slightly modified protocol. /3-Funaltrexamine (0.1 p~mol/l) was applied 10 rain after the washout of galanin (0.03 tzmol/1), and was present for an additional 15 rain. A 10-min superfusion with drug-free medium then followed, before galanin (0.03 txmol/l) was added again for 5 min. When the antagonism between /3-funaltrexamine and [MetS]enkephalin was tested, [MetS]enkephalin (0.3 txmol/l) was applied instead of galanin for 3 rain, both before and after treating the tissue with /3funaltrexamine (0.1 /~mol/1). The effects of most antagonists (idazoxan, rauwolscine, naloxone) on their own were measured 10 min after addition, and were expressed as percent inhibition of the average firing rate determined immediately before drug application. The effect of /3funaltrexamine was determined both 15 min after addition and 10 min after washout.
tial. After complete washout of the peptide, the cells were continuously hyperpolarized by passing current through the recording electrode and, in addition, constant hyperpolarizing current pulses were injected at regular intervals. Under these conditions galanin was reapplied for another 4 min, in order to measure the change in input resistance.
2.5. Materials The following drugs were used: galanin (Bachem, Bubendorf, Switzerland); idazoxan hydrochloride, [MetS]enkephalin acetate (Sigma, Deisenhofen, FRG); naloxone hydrochloride (Du Pont, Wilmington, DE, USA); ( - ) - n o r a d r e n a l i n e hydrochloride (Hoechst, Frankfurt am Main, FRG); /3-funaltrexamine hydrochloride (RBI, Natick, MA, USA); rauwolscine hydrochloride (Roth, Karlsruhe, FRG). Stock solutions (1-10 m m o l / l ) of all drugs were prepared with distilled water; only /3-funaltrexamine was dissolved in methanol. Further dilutions were made with medium. Equivalent quantities of the solvents had no effect.
2.6. Statistics Means + S.E.M. are given throughout. The paired Student's t-test was used for comparison of means and for comparison of means with zero. A probability level of 0.05 or less was considered to be statistically significant.
3. Results
3.1. Extracellular recording 3.1.1. Effects of noradrenaline, [MetS]enkephalin and galanin All LC neurones included in this extracellular study fired spontaneously with an average rate of 1.01 + 0.04 Hz (n = 94). Galanin (0.003-0.1 /xmol/l), noradrenaline (0.1-100 /xmol/l) and [MetS]enkephalin (0.01-10 p.mol/l), concentration dependently inhibited the generation of action potentials (fig. 1A and B). The inhibition was completely reversible on washout. The ICs0 values determined from the concentration-response curves were 0.03 + 0.01 tzmol/1 (n = 6; galanin), 0.20 _+ 0.04 tzmol/l (n = 6; [MetS]enkephalin) and 7.4 _+ 3.3 txmol/1 (n = 6; noradrenaline). Hence galanin was the most potent of the agonists tested.
2.4. 2. Intracellular recording
3.1.2. Interaction of galanin with a2-adrenoceptor and I~-opioid receptor agonists
Galanin was applied for 4 min to spontaneously spiking LC neurones at the resting membrane poten-
Based on the concentration-response curves (fig. 1), concentrations of noradrenaline (1, 3 ~mol/1) and
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Fig. I. Inhibitory effects of galanin, [Met~]enkephalin and noradrenaline on the firing rate of LC neurones. Pontine slices of rat brain were prepared and the frequency of spontaneous action potentials was recorded extracellularly as consecutive 30-s samples. (A) Representative experiment from six slices. Galanin was in contact with the tissue as indicated by the horizontal bars. (B) Concentration-response curves for galanin (o), [MetS]enkephalin (el and noradrenaline (zx). Means_+S.E.M. from six slices each. GAL, galanin; ENK, [MetS]enkephalin; NA, noradrenaline. % 6&
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[ M e t S ] e n k e p h a l i n (0.03, 0 . 1 / x m o l / l ) w e r e c h o s e n which i n h i b i t e d t h e f i r i n g rate by less t h a n 25 a n d 5 0 % , respectively. T h e e x p e r i m e n t a l s c h e d u l e is s h o w n in fig. 3A; the a c t i o n s of n o r a d r e n a l i n e (1 a n d 3 p, m o l / l ) w e r e t h e s a m e in t h e p r e s e n c e a n d in the a b s e n c e of 0.001 / x m o l / 1 (fig. 2A) or 0.01 # m o l / l g a l a n i n (fig. 2B). Similarly, [ M e t S ] e n k e p h a l i n (0.03 a n d 0.1 / x m o l / l ) p r o d u c e d i d e n t i c a l i n h i b i t i o n , r e g a r d l e s s of w h e t h e r it was a p p l i e d in the p r e s e n c e or a b s e n c e of 0.01 i x m o l / l g a l a n i n (fig. 3 A a n d B). T h e i n h i b i t o r y effects o f b o t h noradrenaline and [MetS]enkephalin showed a tend e n c y to i n c r e a s e slightly with time; this t r e n d only r e a c h e d statistical s i g n i f i c a n c e in t h e case o f n o r a d r e n a l i n e 3 t x m o l / l in a single series of e x p e r i m e n t s (fig. 2A).
3.1.3. Interaction of galanin with e~2-adrenoceptor and Ix-opioid receptor antagonists T h e effect of g a l a n i n (0.03 # t o o l / l ) did n o t c h a n g e o n r e p e a t e d a p p l i c a t i o n (fig. 4A). I d a z o x a n (1 p, m o l / 1 ) failed to a l t e r t h e g a l a n i n - i n d u c e d i n h i b i t i o n , while r a u w o l s c i n e (1 i z m o l / 1 ) i n c r e a s e d it (fig. 4B). Both n a l o x o n e (0.1 / x m o l / l ) a n d ] 3 - f u n a l t r e x a m i n e (0.1 # t o o l / l ) p o t e n t i a t e d t h e d e c r e a s e in firing c a u s e d b} g a l a n i n ( 0 . 0 3 / x m o l / 1 ) (fig. 4C). I d a z o x a n a n d r a u w o l s c i n e w e r e u s e d in c o n c e n t r a t i o n s (1 / x m o l / 1 ) , w h i c h m a r k e d l y a t t e n u a t e d the effecl of n o r a d r e n a l i n e ( 1 - 1 0 # t o o l / l ) (Illes a n d N 6 r e n b e r g , 1990). T h e c o n c e n t r a t i o n of n a l o x o n e a p p l i e d (0,1 # t o o l / l ) also c o u n t e r a c t e d the effect o f [ M e t S ] e n k e p h a l i n ( 0 . 1 - 1 t x m o l / l ) (Illes a n d N 6 r e n b e r g , 1990).
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Figl 2. [~ack of interaction between galanin and noradrenaline in LC neurones, Extracellular recording. (A) Noradrenaline (1 and 3 p, mol/I) had the same effect both before (first set of two columns) and during (second set of two columns) the application of galanin (0.001 txmol/l). The action of 3 ,amol/l, but not 1 p, mol/l noradrenaline was increased after the washout of galanin (0.001 /xmol/I) (third set of two columns; * P < 0.05). The galanin-induced inhibition was superimposed on the effect of noradrenaline determined in the presence of galanin. (B) Noradrenaline (1 and 3 >tool/l) had the same effect before (first set of two columns), during (second set of two columns) and afte,r (third set of two columns) the application of a higher concentration (0.01 ,amol/I) of galanin. Means+S.E.M. from six slices both in (A) and (B). NA, noradrenaline; GAL. galanin.
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Fig. 3. Lack of interaction between galanin and [MetS]enkephalin in LC neurones. Extracellular recording. (A) Representative experiment from six slices. Drug contact times are indicated by the horizontal bars. (B) [MetS]Enkephalin (1 and 3 #tool/l) had the same effect before (first set of two columns), during (second set of two columns) and after (third set of two columns) the application of galanin ([I.{)l /xmol/l). The galanin-induced inhibition was superimposed on the effect of [MetS]enkephalin determined in the presence of galanin. Means+S.E.M. from six slices. ENK, [MetS]enkephalin; GAL, gahmin.
227 Finally, the a n t a g o n i s m b e t w e e n /~-funaltrexamine (0.1 ~ m o l / 1 ) a n d [ M e t S ] e n k e p h a l i n (0.3 ~ m o l / l ) was tested. W h e r e a s [ M e t S ] e n k e p h a l i n (0.3 ~ m o l / 1 ) completely abolished the firing rate of u n t r e a t e d LC n e u r o n e s (100.0 _+ 0.0%; n = 5), it p r o d u c e d only 16.4 _+ 1.8% i n h i b i t i o n w h e n a 15-min i n c u b a t i o n of the tissue with /3-funaltrexamine (0.1 ~ m o l / 1 ) was followed by a 10rain w a s h o u t period (n = 5; P < 0.01). I n a few experiments, w h e n [ M e t S ] e n k e p h a l i n (0.3 ~ m o l / 1 ) was reapplied after a n o t h e r 35 min, the inhibitory p o t e n c y of the p e p t i d e recovered partially (49.1 _+ 7.4%; n = 3; P < 0.01). Idazoxan (1 ~ m o l / 1 ) ( - 5 . 2 _ + 4 . 6 % ; n = 11), rauwolscine (1 ~ m o l / l ) ( - 2 . 5 + 4.2%; n = 11) a n d naloxo n e (1 p.mol/1) (2.5 + 6.8%; n = 9) all left the firing rate u n c h a n g e d 15 rain after their a d d i t i o n (P > 0.05). In contrast, /3-funaltrexamine (0.1 /~mol/1) d e c r e a s e d firing after a 15-min contact time (28.3 + 4.2%; n = 12; P < 0.01) a n d this i n h i b i t i o n persisted after an additional 10 rain in a drug-free m e d i u m (26.8-+ 4.5%; n = 12; P < 0.01). %
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Fig. 5. Effect of galanin on LC neurones. Intracellular recording. Two original tracings are shown in (A) and (B). (A) Galanin (0.3 p.mol/l) abolished spontaneous firing and hyperpolarized the neurone. The full height of the action potentials was not reproduced by the pen recorder. The interval between the upper and lower traces is shown. Galanin was present for the period indicated by the horizontal bar. (B) Galanin (0.3 ~mol/l) produced hyperpolarization and decreased the input resistance. The membrane potential of the neurone was slightly raised by continuous current injection in order to suppress spontaneous firing. Downward deflections represent electrotonic potentials caused by hyperpolarizing current pulses of constant amplitude. The electrotonic potentials are directly proportional to the input resistance. At the peak of the galanin response, the membrane potential was temporarily restored to its pre-drug value with a depolarizing current. The contact time for galanin is indicated by the horizontal bars. Experiments in (A) and (B) were performed on two different slices.
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Fig. 4. Interaction between galanin receptors, and a2-adrenoceptors or #-opioid receptors in LC neurones. Extracellular recording. Oalanin (0.03 ~mol/l) was applied twice; the second application was either in the absence of a2-adrenoceptor and /~-opioid receptor antagonists (A), or in their presence (B; C). (A) In the absence of antagonists galanin (0.03/,mol/1) caused reproducible inhibition (rq; n = 8). (B) Idazoxan (1 ixmol/l) (ca; n = 11) did not alter the effect of galanin (0.03 p.mol/l), while rauwolscine (1 /~mol/l) ( [] ; n = 11) potentiated it. (C) Both naloxone (0.1 /*tool/l) (®; n = 9 ) and /3-funaltrexamine (0.1 /~mol/l) ([]; n = 7) facilitated the effect of galanin (0.03 #mol/l). GAL, galanin; IDA, idazoxan; RAU, rauwolscine; NAL, naloxone; /3-FNA, /3-funaltrexamine. Asterisks indicate significant differences from the effect of the first application of galanin 0.03/*mol/I (* P < 0.05; ** P < 0.01). Means+ S.E.M. from n slices.
T h e resting m e m b r a n e p o t e n t i a l of the seven LC cells i m p a l e d with i n t r a c e l l u l a r microelectrodes was - 5 4 . 7 + 1.5 mV. G a l a n i n (0.3 p.mol/1) abolished the firing a n d caused a h y p e r p o l a r i z a t i o n of 10.4 + 1.1 m V (n = 7; P < 0.01; fig. 5A). W h e n the m e m b r a n e potential of the cells was increased (about 10 mV) by c u r r e n t injection, the f u r t h e r h y p e r p o l a r i z a t i o n i n d u c e d by g a l a n i n (0.3 ~ m o l / l ) was 9.9 _+ 1.4 m V (n = 7; P < 0.01); at the same time the i n p u t resistance decreased from 136.1 _+ 10.1 to 120.8 _+ 8.4 M.Q by 11.1 _+ 1.3% (n = 7; P < 0.01) (fig. 5B). This decrease persisted w h e n the m e m b r a n e p o t e n t i a l was t e m p o r a r i l y restored to its p r e - d r u g value with a d e p o l a r i z i n g c u r r e n t (fig. 5B).
4. Discussion I n the p r e s e n t e x p e r i m e n t s galanin, a possible cot r a n s m i t t e r of n o r a d r e n a l i n e ( R 6 k a e u s , 1987), depressed the firing rate of LC n e u r o n e s . T h e inhibitory effect of g a l a n i n arises at specific receptors, since it is p r e v e n t e d by a competitive a n t a g o n i s t (Bartfai et al., 1991). T h e g a l a n i n receptors are situated on the som a t a of LC cells a n d not on n e i g h b o u r i n g n e u r o n e s
228 (Seutin et al., 1989). In contrast to an earlier study (Seutin et al., 1989), we observed the peptide effect in all LC cells investigated rather than only in some ceils. It is suspected that this discrepancy may be due to the fact that Seutin et al. (1989) did not test the sensitivity of their supposed LC neurones to noradrenaline (Cedarbaum and Aghajanian, 1977). In our opinion it is absolutely necessary to do this; identification on purely eIectrophysiological grounds, i.e. from the shape of spontaneous action potentials, may not be sufficient. Intracellular recordings in LC cells showed that galanin produces hyperpolarization and a reduction of input resistance, which persists when the membrane potential is manually clamped to its pre-drug value. Input resistance changes that are secondary to hyperpolarization are thereby eliminated (Williams et al., 1984). The ions involved might be potassium and not chloride as the microelectrodes were filled with KCI, and under these conditions an increased permeability of the membrane to C1- depolarizes, rather than hyperpolarizes LC neurones (Cherubini et al., 1988). A recent study, published during the preparation of this manuscript, also demonstrated a galanin-induced hyperpolarization and conductance increase in the LC (Bartfai et al., 1991). Galanin enhances a potassium permeability in myenteric neurones (Palmer et al., 1986; Tamura et al., 1988), postganglionic parasympathetic neurones (Konopka et al., 1989), pheochromocytoma cells (De Weille et al., 1989) and insulinoma cells derived from pancreatic tumours (De Weille et al., 1988). In insulinoma cells (De Weille et al., 1988), galanin opens a class of potassium channels (KATe) , which is sensitive to intracellular ATP (Ashcroft and Ashcroft, 1990). The KAT e channels can be closed by sulphonylurea antidiabetics, such as glibenclamide (Ashcroft and Ashcroft, 1990). Galanin continued to hyperpolarize LC neurones in spite of the presence of glibenclamide (E.P. Finta and P. Illes, unpublished observation); thus, the involvement of KAy p channels can be dismissed. It is quite possible that galanin acts in the LC, at inwardly rectifying potassium channels also opened by a2-adrenoceptor and p,-opioid receptor agonists (Miyake et al., 1989). Galanin is not the only neuropeptide co-localized with noradrenaline in the LC (Skofitsch and Jacobowitz, 1985; Holets et al., 1988); enkephalins (Olpe and Steinmann, 199l) and neuropeptide Y (Everitt et al., 1984; Holets et al., 1988) are also present in the noradrenergic perikarya. Noradrenaline itself may be released from dendrites or recurrent axon collaterals (Aghajanian et al., 1977) and cause hyperpolarization via the activation of somatic az-adrenoceptors (Aghajanian and VanderMaelen, 1982). It may be assumed that these neuropeptides are co-released with noradrenaline and modulate its effect.
In LC neurones, both c~2-adrenoceptors and /~opioid receptors appear to be coupled to potassium channels via a pertussis toxin-sensitive G-protein (G~ or G o) (Aghajanian and Wang, 1986). Coupling of galanin-receptors to a G-protein was demonstrated in binding studies (Fisone et al., 1989). Whether potassium channels are opened via the subsequently inhibited adcnylcyclase/protein kinase A system or by the G-protein itself, awaits further clarification. The present experiments showed that two #-opioid receptor antagonists potentiate the inhibitory effect of galanin on the firing rate of LC neurones. Naloxone causes a reversible blockade of opioid receptors with a slight preference for the ~- over the 8- and K-type, while /3-funaltrexamine is an irreversible antagonist and is highly selective for ~-opioid receptors (Illes et al., 1989). Our findings with /3-funaltrexamine were rather unexpected. Firstly, it caused a long-lasting depression of spontaneous firing when given alone. We did not investigate the mechanism of this effect; a possible explanation is a partial agonistic activation of #-opioid receptors. Secondly, it decreased the effect o~ [MetS]enkephalin (see also Williams and North, 1984) reversibly. However, the inability of both /3funaltrexamine and naloxone to increase the discharge of action potentials excludes the possibility that, under the present in vitro conditions, there is a major tonic control of neuronal activity by spontaneously released opioid peptides. Thus, the potentiation of the galanin effect by the two antagonists was probably not due to competition with endogenous agonists at the #-opioid receptor. The results obtained with two a2-adrenoceptor antagonists were variable. Idazoxan (Doxey et al., 1983) did not alter the galanin effect, while rauwolscine (Weitzell et al., 1979) facilitated it. Rauwolscine was probably active because of its binding to 5-HTIA sites (Broadhurst et al., 1988) which are abundant in the LC (Weismann-Nanopoulos et al., 1985). Functional studies also proved the existence of 5-HTIA receptors on excitatory amino acid (EAA) and y-aminobutyric acid (GABA) terminals ending on LC cells (Bobker and Williams, 1989). Since idazoxan is highly selective for az-adrenoceptors, its inability to potentiate the galanin effect argues against an interaction between galanin receptors and az-adrenoceptors. Noradrenaline and [MetS]enkephalin depressed firing to a similar extent both in the absence and presence of low concentrations of galanin. Thus, galanin receptors appear to interact unidirectionally with #opioid receptors; a similar interaction between galanin receptors and a2-adrenoceptors does not seem to take place. In this context it is interesting to note that somatic a2-adrenoceptors and #-opioid receptors of LC neurones also influence each other; both idazoxan and rauwolscine potentiated the effect of [MetS]en -
229
kephalin, although noradrenaline did not alter it (Illes and N6renberg, 1990). az-Adrenoceptors, NPY receptors and tx-opioid receptors were shown to interact both in the cell somata (Illes and Regenold, 1990; Illes and N6renberg, 1990) and the nerve terminals (Yokoo et al., 1987; Schoffelmeer et al., 1986). Although the second-messenger and effector mechanisms of all three types of receptors seem to be identical (Aghajanian and Wang, 1986; Miyake et al., 1989; Illes and Regenold, 1990), the types of interaction differ. For example rauwolscine inhibits the effect of NPY (Illes and Regenold, 1990), but potentiates the effect of [MetS]enkephalin (Illes and N6renberg, 1990). Hence, the interaction between the receptors may occur in the plasma membrane and not in the common second-messenger system (Illes, 1989; Illes et al., 1990). Whereas presynaptic galanin receptors may be influenced by neighbouring az-adrenoceptors (Tsuda et al., 1989), no such interaction was found between the respective somatic receptors of LC neurones. In contrast, galanin receptors and ix-opioid receptors situated at the perikarya of LC cells were shown to interact unidirectionally. Since [MetS]enkephalin and galanin have a similar mode of action (see above), interference with the common second-messenger system of the occupied receptors might be postulated. However, the inability of galanin to alter the action of [MetS]en kephalin argues against this assumption. Moreover, the potentiation of the galanin effect after the blockade of the non-activated ix-opioid receptors by naloxone locates the site of interaction in the plasma membrane.
Acknowledgements We are grateful to Dr. W. N6renberg for a number of helpful discussions. Dr. J. Sevcik is the recipient of a research fellowship from the Alexander von Humboldt-Stiftung. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 325).
References Aghajanian, G.K. and C.P. VanderMaelen, 1982, a2-Adrenoceptormediated hyperpolarization of locus coeruleus neurons: intracellular studies in vivo, Science 215, 1394. Aghajanian, G.K. and Y.Y. Wang, 1986, Pertussis toxin blocks the outward currents evoked by opiate and a2-agonists in locus coeruleus neurons, Brain Res. 371,390. Aghajanian, G.K., J.M. Cedarbaum and Y.Y. Wang, 1977, Evidence for norepinephrine-mediated collateral inhibition of locus coeruleus neurons, Brain Res. 136, 570. Ashcroft, S.J.H. and F.M. Ashcroft, 1990, Properties and functions of ATP-sensitive K-channels, Cell Sign. 2, 197. Bartfai, T., K. Bedecs, T. Land, LJ. Langel, R. Bertorelli, P. Girotti, S. Consolo, X. Xu, Z. Wiesenfeld-Hallin, S. Nilsson, V.A. Pieribone and T. H6kfelt, 1991, M-15: high-affinity chimeric peptide that blocks the neuronal actions of galanin in the hippocampus,
locus coeruleus, and spinal cord, Proc. Natl. Acad. Sci. U.S.A. 88, 10961. Bobker, D.H. and J.T. Williams, 1989, Serotonin agonists inhibit synaptic potentials in the rat locus coeruleus in vitro via 5-hydroxytryptaminelA and 5-hydroxytryptaminel8 receptors, J. Pharmacol. Exp. Ther. 250, 37. Broadhurst, A.M., B.S. Alexander and M.D. Wood, 1988, Heterogeneous 3H-rauwolscine binding sites in rat cortex: two alpha 2adrenoceptor subtypes or an additional non-adrenergic interaction, Life Sci. 43, 83. Cedarbaum, J.M. and G.K. Aghajanian, 1977, Catecholamine receptors of locus coeruleus neurones: pharmacological characterization, Eur. J. Pharmacol. 44, 375. Cherubini, E., R.A. North and J.T. Williams, 1988, Synaptic potentials in rat locus coeruleus neurones, J. Physiol. 406, 431. De Weille, J., H. Schmid-Antomarchi, M. Fosset and M. Lazdunski, 1988, ATP-sensitive K + channels that are blocked by hypoglycemia-inducing sulfonylureas in insulin-secreting cells are activated by galanin, a hyperglycemia-inducing hormone, Proc. Natl. Acad. Sci. U.S.A. 85, 1312. De Weille, J., M. Fosset, H. Schmid-Antomarchi and M. Lazdunski, 1989, Galanin inhibits dopamine secretion and activates a potassium channel in pheochromocytoma cells, Brain Res. 485, 199. Doxey, J.C., A.G. Roach and C.F.C. Smith, 1983, Studies on RX 781094: a selective, potent and specific antagonist of a2-adrenoceptors, Br. J. Pharmacol. 78, 489. Everitt, B.J., T. H6kfelt, L. Terenius, K. Tatemoto, V. Mutt and M. Goldstein, 1984, Differential co-existence of neuropeptide Y (NPY)-like immunoreactivity with catecholamines in the central nervous system of the rat. Neuroscience 11,443. Fisone, G., l]. Langel, M. Carlquist, T. Bergman, S. Consolo, T. H6kfelt, A. Unden, S. Andell and T. Bartfai, 1989, Galanin receptor and its ligands in the rat hippocampus, Eur. J. Biochem. 181,269. Holets, V.R., T. H6kfelt, A. R6kaeus, L. Terenius and M. Goldstein, 1988, Locus coeruleus neurons in the rat containing neuropeptide Y, tyrosine hydroxylase or galanin and their efferent projections to the spinal cord, cerebral cortex and hypothalamus, Neuroscience 24, 893. llles, P., 1989, Modulation of transmitter and hormone release by multiple neuronal opioid receptors, Rev. Physiol. Biochem. Pharmacol. 112, 139. Illes, P. and W. N6renberg, 1990, Blockade of a2-adrenoceptors increases opioid iz-receptor-mediated inhibition of the firing rate of rat locus coeruleus neurones, Naunyn-Schmiedeb. Arch. Pharmacol. 342, 490. llles, P. and J.T. Regenold, 1990, Interaction between neuropeptide Y and noradrenaline on central catecholamine neurons, Nature 344, 62. Illes, P., H.D. Weber, J. Neuburger, B. Bucher, J.T. Regenold and W. N6renberg, 1990, Receptor interactions at noradrenergic neutones, in: Presynaptic Receptors and the Question of Autoregulation of Neurotransmitter Release eds. S. Kalsner and T.C. Westfall (Ann. N.Y. Acad. Sci., Vol. 604) p. 197. Konopka, L.M., T.W. McKeon and R.L. Parsons, 1989, Galanin-induced hyperpolarization and decreased membrane excitability of neurons in mudpuppy cardiac ganglia, J. Physiol. 410, 107. Martire, M., K. Fuxe, G. Pistritto, P. Preziosi and L.F. Agnati, 1986, Neuropeptide Y enhances the inhibitory effects of clonidine on 3H-noradrenaline release in synaptosomes isolated from the medulla oblongata of the male rat, J. Neural Transm. 67, 113. Miyake, M., M.J. Christie and R.A. North, 1989, Single potassium channels opened by opioids in rat locus coeruleus neurons, Proc. Natl. Acad. Sci. U.S.A. 86, 3419. North, R.A. and J.T. Williams, 1985, On the potassium conductance increased by opioids in rat locus coeruleus neurons, J. Physiol. 364, 265.
230 Olpe, H.R. and M. Steinmann, 1991, Responses of locus coeruleus neurons to neuropeptides, Prog. Brain Res. 88, 241. Palmer, J.M., M. Schemann, K. Tamura and J.D. Wood, 1986, Galanin mimics slow synaptic inhibition in myenteric neurons, Eur. J. Pharmacol. 124, 379. R6kaeus, A., 1987, Galanin: a newly isolated biologically active neuropeptide, Trends Neurosci. 10, 158. Schoffelmeer, A.N.M., J. Putters and A.H. Mulder, 1986, Activation of presynaptic c~2-adrenoceptors attenuates the inhibitory effect of /z-opioid receptor agonists on noradrenaline release from brain slices, Naunyn-Schmiedeb. Arch. Pharmacol. 333, 377. Seutin, V., P. Verbanck, L. Massotte and A. Dresse, 1989, Galanin decreases the activity of locus coeruleus neurons in vitro, Eur. J. Pharmacol. 164, 373. Skofitscb, G. and D.M. Jacobowitz, 1985, Immunohistochemical mapping of galanin-like neurons in the rat central nervous system, Peptides 6, 509. Starke, K., 1977, Regulation of noradrenaline release by presynaptic receptor systems, Rev. Physiol. Biochem. Pharmacol. 77, 1. Tamura, K., J.M. Palmer, C.K. Winkelmann and J.D. Wood, 1988, Mechanism of action of galanin on myenteric neurons, J. Neurophysiol. 60, 966. Tatemoto, K., A. R6kaeus, H. J6rnvall, T.J. McDonald and V. Mutt, 1983, Galanin a novel biologically active peptide from porcine intestine, FEBS Lett. 164, 124.
Tsuda, K., H. Yokoo and M. Goldstein, 1989, Neuropeptide Y and galanin in norepinephrine release in hypothalamic slices, Hypertension 14, 81. Weissmann-Nanopoulos, D., E. Mach, J. Magre, Y. Demassey and J.-F. Pujol, 1985, Evidence for the localization of 5-HTIA binding sites on serotonin containing neurons in the raphe dorsalis and raphe centralis nuclei of the rat brain, Neurochem. Int. 7, 1061. Weitzell, R., T. Tanaka and K. Starke, 1979, Pre- and postsynaptic effects of yohimbine stereoisomers on noradrenaline transmission in the pulmonary artery of the rabbit, Naunyn-Schmiedeb. Arch. Pharmacol. 308, 127. Williams, J.T. and R.A. North, 1984, Opiate-receptor interactions on single locus coeruleus neurones, Mol. Pharmacol. 26, 489. Williams, J.T., R.A. North, S.A. Shefner, S. Nishi and T.M. Egan, 1984, Membrane properties of rat locus coe.ruleus neurones, Neuroscience 13, 137. Williams, J.T., G. Henderson and R.A. North, 1985, Characterization of a2-adrenoceptors which increase potassium conductance in rat locus coeruleus neurones, Neuroscience 14, 95. Yokoo, H., D.H. Schlesinger and M. Goldstein, 1987, The effect of neuropeptide Y (NPY) on stimulation-evoked release of [3H]norepinephrine (NE) from rat hypothalamic and cerebral cortical slices. Eur. J. Pharmacol. 143, 283.
223
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 52872
Galanin receptors inhibit the spontaneous firing of locus coeruleus neurones and interact with -opioid receptors J a n Sevcik l, E r v i n P. F i n t a a n d P e t e r Illes Department of Pharmacolo,g% Unicersity of Freiburg, W- 7800 Freiburg, Germany
Received 11 September 1992, accepted 27 October 1992
Electrophysiological experiments were performed in a pontine slice preparation of rat brain containing the locus coeruleus (LC). The extracellular part of this study showed that galanin (0.003-0.1 p.mol/I), [Met~]enkephalin (0.01-10 p.mol/l) and noradrenaline (0.1-100 ~mol/l) concentration dependently inhibited the firing rate. Noradrenaline (1 and 3 /zmol/l) had the same effect both before and during the application of galanin (0.001 or 0.01 #mol/l). Similarly, [MetS]enkephalin (0.03 and 0.1 p.mol/l) produced identical inhibition, regardless of the presence or absence of 0.01 p.mol/l galanin. Whereas rauwolscine (1 p.mol/1) potentiated the effect of galanin (0.03 #.tool/I), idazoxan (1 p.mol/1) was inactive. In contrast, both naloxone (0.1 #mol/l) and /3-funaltrexamine (0.1 p.mol/1) facilitated the galanin-induced inhibition. In the intracellular experiments, galanin (0.3 /~mol/l) abolished the spontaneous discharge of action potentials, hyperpolarized the cells and decreased their input resistance. In conclusion, galanin may depress the firing rate by increasing a potassium permeability. Moreover, galanin receptors appear to interact with /x-opioid receptors but not with ~2-adrenoceptors. Galanin; Locus coeruleus neurons; Receptor interaction; Firing rate; Membrane potential
1. Introduction
Galanin, a 29-amino acid residue peptide originally isolated from porcine small intestine (Tatemoto et al., 1983), is widely distributed in the rat central nervous system (Skofitsch and Jacobowitz, 1985; R6kaeus et al., 1987). Galanin has also been found in most noradrenaline-containing neurones of the locus coeruleus (LC) (Skofitsch and Jacobowitz, 1985; Holets et al., 1988). The functional consequences of this co-existence are unclear. It has been demonstrated that, in the LC, both ~2-adrenoceptor and #-opioid receptor agonists increase the same potassium conductance and thereby produce hyperpolarization and depress the spontaneous discharge of action potentials (North and Williams, 1985). LC neurones possess adrenoceptors of the a2-type ( C e d a r b a u m and Aghajanian, 1977; Williams et al., 1985), and opioid receptors of the /x-type only (Williams and North, 1984). It was ob-
Correspondence to: P. Illes, Department of Pharmacology, University of Freiburg. Hermann-Herder-Strasse 5. W-7800 Freiburg, Germany. I On temporary leave from the Institute of Pharmacology CSAV, Albertov 4, 128 00 Prague 2, CSFR.
served that the inhibitory effect of #-agonists is potentiated in the presence of a2-antagonists, suggesting an interaction between the two receptors (llles and N6renberg, 1990; Illes et al., 19901. Furthermore, it was found that neuropeptide Y (NPY) decreases the firing rate by a mechanism similar 1o that for a 2- and #agonists (Illes and Regenold, 1990; Illes et al., 19901. Since NPY potentiated the effect of noradrenaline but not that of [MetS]enkephalin, and the NPY effect was abolished in the presence of the ~2-antagonist, rauwolscine, it was suggested that a selective interaction between NPY receptors and a2-adrenoceptors occurs in the LC (llles and Regenold, 1990; Illes et al., 19901. In addition to the cell somata, the nerve terminals of central noradrenergic neurones also possess inhibitory a 2- (Starke, 1977), /x- (Illes, 1989) and NPY receptors (Martire et al., 1986; Yokoo et al., 1987), which were shown to interact similarly to the respective somatic receptors (Illes, 1989; Illes et al., 1990). It was reported recently that galanin depresses the discharge of spontaneous action potentials in LC neurones (Seutin et al., 1989). However, the extracellular experiments reported gave no information about the ionic mode of action of galanin and the possible interaction of the peptide with noradrenaline. The latter question is especially interesting, since a:-agonists potentiate the inhibitory effect of galanin on noradrenaline re-
224 lease from the terminals of LC neurones, while c%antagonists diminish the galanin effect (Tsuda et al., 1989). In the present extra- and intracellular study, galanin inhibited the spontaneous firing of LC cells by hyperpolarizing the membrane. Moreover, galanin failed to interact with [MetS]enkephalin or noradrenaline, but its inhibitory effect was potentiated by the #-opioid receptor antagonists, naloxone and /3funaltrexamine. Variable results were obtained with two cQ-adrenoceptor antagonists; idazoxan was ineffective, while rauwolscine facilitated the galanin effect.
2.3. Recording techniques
2. Materials and methods
2.3.2. h~tracellular recording
2.1. Brain slice preparation The preparation and maintenance of midpontine slices of rat brain were as previously described (Illes and N6renberg, 1990). In brief, male Wistar rats (150220 g) were anaesthetized with ether and decapitated. Slices of 400-/~m thickness, containing the caudal part of the LC were prepared in oxygenated medium at 1-4°C with a Lancer vibratome. A single slice was transferred to the recording chamber and was superfused at a rate of 2 m l / m i n with medium saturated with 95% 0 2 plus 5% CO x and maintained at 35-36°C. The medium was of the following composition (in retool/l): NaC1 126, KCI 2.5, N a H 2 P O 4 1.2, MgCI 2 1.3, CaCI 2 2.4, N a H C O 3 25, glucose 11, N a , E D T A (I.03 and ascorbic acid 0.3.
2.2. Identification of LC neurones The LC could easily be identified under a binocular microscope (magnification 10-40 times) as a translucent oval area on the ventrolateral border of the fourth ventricle. LC cells fire spontaneously with a constant rate of 0.2-5 Hz.
2.2.1. Extracellular recording The extracellular action potentials are biphasic and longer ( > 2 ms) than those of neighbouring neurones. Since c~2-adrenoceptor agonists depress the firing rate (Cedarbaum and Aghajanian, 1977), we added at the beginning of each experiment a single test concentration of noradrenaline (30 /xmol/l), which produced a complete and reversible inhibition.
2.2.2. lntracellular recording LC cells were distinguished from neighbouring mesencephalic trigeminal neurones by their electrophysiological properties, including spontaneous firing and a marked hyperpolarization in response to noradrenaline (100 /zmol/I).
2. 3.1. Extracellular recording Glass microelectrodes filled with 4 m o l / l NaCI and with a tip resistance of 2 - 4 M,Q were used for recording the firing rate. The electrode signals were passed through a Grass PI6 high-impedance amplifier, filtered and displayed on a Tektronix 5113 oscilloscope. The spikes were gated and counted by means of a WPI 121 window discriminator coupled to an electronic ratemcter and a Watanabe W T R 311 pen recorder. Firing rate was recorded as consecutive 30-s samples.
Recording and current injection were carried out with glass microelectrodes filled with KC1 2 m o l / l (tip resistance 60-1(10 M,O), using a high-impedance preamplifier and a bridge circuit (Axoclamp 2 A). In some experiments LC ceils were constantly hyperpolarized (about 10 mV) by passing through the microelectrode a current just sufficient to prevent the cell from firing spontaneous action potentials. In addition, hyperpolarizing current pulses of constant amplitude and 250-ms duration were delivered at a frequency of 0.5 Hz. The apparent input resistance was calculated from the resuiting peak potential change. The m e m b r a n e potential was determined on withdrawal of the microelectrode from the cell at the end of each experiment.
2.4. Application of drugs and el,aluation of data Various drugs were applied by changing the superfusion medium by means of three-way taps. At the constant flow rate of 2 m l / m i n about 30 s were required until the drug reached the bath.
2.4. 1. Extracellular recording Concentration-response curves for noradrenaline and [MetS]enkephalin were made by applying increasing concentrations of the agonists for 3 rain and the interval between subsequent applications was 10 rain. Concentration-response curves for galanin were made by applying increasing concentrations of the peptide for 5 rain and keeping an interval of at least 15 rain between subsequent applications. Depression of firing rate was measured at its maximum (average of two counting periods), irrespective of drug contact-time, and was expressed as percentage of the average firing rate during the 2 min immediately before addition (average of four counting periods). In every experiment, the ICs0 value, i.e. the concentration that produced 50% inhibition of the spike discharge was estimated graphically. Only one concentration-response curve of one agonist was made on a single cell of a brain slice. The interaction between galanin on the one hand
225 and noradrenaline or [MetS]enkephalin on the other was tested as follows (see fig. 3A). Two concentrations of noradrenaline (1, 3 /zmol/l) or [MetS]enkephalin (0.03, 0.1 /xmol/l) were used, which inhibited the discharge of action potentials by less than 25 and 50%, respectively, as estimated from the concentration-response curves of these agonists. At first, a lower and a higher concentration of the same drug (either noradrenaline or [MetS]enkephalin) were applied consecutively. Both concentrations were then reapplied in the presence of a low concentration of galanin (0.001 or 0.01 p.mol/l). Finally, the first mode of application was repeated. The contact time of all compounds was 3 min, except in the case of galanin, which was applied 5 rain before noradrenaline or [MetS]enkephalin and was kept in the medium for another 3 min together with these agonists. The drug-free intervals were 10-15 rain. Drug effects were expressed as percent inhibition of the average firing rate measured immediately before the addition of each agonist concentration. Furthermore, we tested for a possible interaction between az-adrenoceptor antagonists (idazoxan, rauwolscine) or /x-opioid receptor antagonists (naloxone, /3-funaltrexamine) and galanin. Galanin was applied for 5 min in a concentration (0.03 ixmol/l), which inhibited the firing rate by about 50%. Galanin (0.03 /xmol/1) was reapplied after another 35 rain, either in the absence or the presence of idazoxan (1 ixmol/l), rauwolscine (1 ixmol/1) or naloxone (0.1 /xmol/l). The antagonists were in contact with the tissue for 10 min before and during the application of galanin. The effect of galanin was expressed as percentage inhibition of the average firing rate measured immediately before addition of the peptide. Experiments with /3funaltrexamine were carried out with a slightly modified protocol. /3-Funaltrexamine (0.1 p~mol/l) was applied 10 rain after the washout of galanin (0.03 tzmol/1), and was present for an additional 15 rain. A 10-min superfusion with drug-free medium then followed, before galanin (0.03 txmol/l) was added again for 5 min. When the antagonism between /3-funaltrexamine and [MetS]enkephalin was tested, [MetS]enkephalin (0.3 txmol/l) was applied instead of galanin for 3 rain, both before and after treating the tissue with /3funaltrexamine (0.1 /~mol/1). The effects of most antagonists (idazoxan, rauwolscine, naloxone) on their own were measured 10 min after addition, and were expressed as percent inhibition of the average firing rate determined immediately before drug application. The effect of /3funaltrexamine was determined both 15 min after addition and 10 min after washout.
tial. After complete washout of the peptide, the cells were continuously hyperpolarized by passing current through the recording electrode and, in addition, constant hyperpolarizing current pulses were injected at regular intervals. Under these conditions galanin was reapplied for another 4 min, in order to measure the change in input resistance.
2.5. Materials The following drugs were used: galanin (Bachem, Bubendorf, Switzerland); idazoxan hydrochloride, [MetS]enkephalin acetate (Sigma, Deisenhofen, FRG); naloxone hydrochloride (Du Pont, Wilmington, DE, USA); ( - ) - n o r a d r e n a l i n e hydrochloride (Hoechst, Frankfurt am Main, FRG); /3-funaltrexamine hydrochloride (RBI, Natick, MA, USA); rauwolscine hydrochloride (Roth, Karlsruhe, FRG). Stock solutions (1-10 m m o l / l ) of all drugs were prepared with distilled water; only /3-funaltrexamine was dissolved in methanol. Further dilutions were made with medium. Equivalent quantities of the solvents had no effect.
2.6. Statistics Means + S.E.M. are given throughout. The paired Student's t-test was used for comparison of means and for comparison of means with zero. A probability level of 0.05 or less was considered to be statistically significant.
3. Results
3.1. Extracellular recording 3.1.1. Effects of noradrenaline, [MetS]enkephalin and galanin All LC neurones included in this extracellular study fired spontaneously with an average rate of 1.01 + 0.04 Hz (n = 94). Galanin (0.003-0.1 /xmol/l), noradrenaline (0.1-100 /xmol/l) and [MetS]enkephalin (0.01-10 p.mol/l), concentration dependently inhibited the generation of action potentials (fig. 1A and B). The inhibition was completely reversible on washout. The ICs0 values determined from the concentration-response curves were 0.03 + 0.01 tzmol/1 (n = 6; galanin), 0.20 _+ 0.04 tzmol/l (n = 6; [MetS]enkephalin) and 7.4 _+ 3.3 txmol/1 (n = 6; noradrenaline). Hence galanin was the most potent of the agonists tested.
2.4. 2. Intracellular recording
3.1.2. Interaction of galanin with a2-adrenoceptor and I~-opioid receptor agonists
Galanin was applied for 4 min to spontaneously spiking LC neurones at the resting membrane poten-
Based on the concentration-response curves (fig. 1), concentrations of noradrenaline (1, 3 ~mol/1) and
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[ M e t S ] e n k e p h a l i n (0.03, 0 . 1 / x m o l / l ) w e r e c h o s e n which i n h i b i t e d t h e f i r i n g rate by less t h a n 25 a n d 5 0 % , respectively. T h e e x p e r i m e n t a l s c h e d u l e is s h o w n in fig. 3A; the a c t i o n s of n o r a d r e n a l i n e (1 a n d 3 p, m o l / l ) w e r e t h e s a m e in t h e p r e s e n c e a n d in the a b s e n c e of 0.001 / x m o l / 1 (fig. 2A) or 0.01 # m o l / l g a l a n i n (fig. 2B). Similarly, [ M e t S ] e n k e p h a l i n (0.03 a n d 0.1 / x m o l / l ) p r o d u c e d i d e n t i c a l i n h i b i t i o n , r e g a r d l e s s of w h e t h e r it was a p p l i e d in the p r e s e n c e or a b s e n c e of 0.01 i x m o l / l g a l a n i n (fig. 3 A a n d B). T h e i n h i b i t o r y effects o f b o t h noradrenaline and [MetS]enkephalin showed a tend e n c y to i n c r e a s e slightly with time; this t r e n d only r e a c h e d statistical s i g n i f i c a n c e in t h e case o f n o r a d r e n a l i n e 3 t x m o l / l in a single series of e x p e r i m e n t s (fig. 2A).
3.1.3. Interaction of galanin with e~2-adrenoceptor and Ix-opioid receptor antagonists T h e effect of g a l a n i n (0.03 # t o o l / l ) did n o t c h a n g e o n r e p e a t e d a p p l i c a t i o n (fig. 4A). I d a z o x a n (1 p, m o l / 1 ) failed to a l t e r t h e g a l a n i n - i n d u c e d i n h i b i t i o n , while r a u w o l s c i n e (1 i z m o l / 1 ) i n c r e a s e d it (fig. 4B). Both n a l o x o n e (0.1 / x m o l / l ) a n d ] 3 - f u n a l t r e x a m i n e (0.1 # t o o l / l ) p o t e n t i a t e d t h e d e c r e a s e in firing c a u s e d b} g a l a n i n ( 0 . 0 3 / x m o l / 1 ) (fig. 4C). I d a z o x a n a n d r a u w o l s c i n e w e r e u s e d in c o n c e n t r a t i o n s (1 / x m o l / 1 ) , w h i c h m a r k e d l y a t t e n u a t e d the effecl of n o r a d r e n a l i n e ( 1 - 1 0 # t o o l / l ) (Illes a n d N 6 r e n b e r g , 1990). T h e c o n c e n t r a t i o n of n a l o x o n e a p p l i e d (0,1 # t o o l / l ) also c o u n t e r a c t e d the effect o f [ M e t S ] e n k e p h a l i n ( 0 . 1 - 1 t x m o l / l ) (Illes a n d N 6 r e n b e r g , 1990).
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Figl 2. [~ack of interaction between galanin and noradrenaline in LC neurones, Extracellular recording. (A) Noradrenaline (1 and 3 p, mol/I) had the same effect both before (first set of two columns) and during (second set of two columns) the application of galanin (0.001 txmol/l). The action of 3 ,amol/l, but not 1 p, mol/l noradrenaline was increased after the washout of galanin (0.001 /xmol/I) (third set of two columns; * P < 0.05). The galanin-induced inhibition was superimposed on the effect of noradrenaline determined in the presence of galanin. (B) Noradrenaline (1 and 3 >tool/l) had the same effect before (first set of two columns), during (second set of two columns) and afte,r (third set of two columns) the application of a higher concentration (0.01 ,amol/I) of galanin. Means+S.E.M. from six slices both in (A) and (B). NA, noradrenaline; GAL. galanin.
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Fig. 3. Lack of interaction between galanin and [MetS]enkephalin in LC neurones. Extracellular recording. (A) Representative experiment from six slices. Drug contact times are indicated by the horizontal bars. (B) [MetS]Enkephalin (1 and 3 #tool/l) had the same effect before (first set of two columns), during (second set of two columns) and after (third set of two columns) the application of galanin ([I.{)l /xmol/l). The galanin-induced inhibition was superimposed on the effect of [MetS]enkephalin determined in the presence of galanin. Means+S.E.M. from six slices. ENK, [MetS]enkephalin; GAL, gahmin.
227 Finally, the a n t a g o n i s m b e t w e e n /~-funaltrexamine (0.1 ~ m o l / 1 ) a n d [ M e t S ] e n k e p h a l i n (0.3 ~ m o l / l ) was tested. W h e r e a s [ M e t S ] e n k e p h a l i n (0.3 ~ m o l / 1 ) completely abolished the firing rate of u n t r e a t e d LC n e u r o n e s (100.0 _+ 0.0%; n = 5), it p r o d u c e d only 16.4 _+ 1.8% i n h i b i t i o n w h e n a 15-min i n c u b a t i o n of the tissue with /3-funaltrexamine (0.1 ~ m o l / 1 ) was followed by a 10rain w a s h o u t period (n = 5; P < 0.01). I n a few experiments, w h e n [ M e t S ] e n k e p h a l i n (0.3 ~ m o l / 1 ) was reapplied after a n o t h e r 35 min, the inhibitory p o t e n c y of the p e p t i d e recovered partially (49.1 _+ 7.4%; n = 3; P < 0.01). Idazoxan (1 ~ m o l / 1 ) ( - 5 . 2 _ + 4 . 6 % ; n = 11), rauwolscine (1 ~ m o l / l ) ( - 2 . 5 + 4.2%; n = 11) a n d naloxo n e (1 p.mol/1) (2.5 + 6.8%; n = 9) all left the firing rate u n c h a n g e d 15 rain after their a d d i t i o n (P > 0.05). In contrast, /3-funaltrexamine (0.1 /~mol/1) d e c r e a s e d firing after a 15-min contact time (28.3 + 4.2%; n = 12; P < 0.01) a n d this i n h i b i t i o n persisted after an additional 10 rain in a drug-free m e d i u m (26.8-+ 4.5%; n = 12; P < 0.01). %
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Fig. 5. Effect of galanin on LC neurones. Intracellular recording. Two original tracings are shown in (A) and (B). (A) Galanin (0.3 p.mol/l) abolished spontaneous firing and hyperpolarized the neurone. The full height of the action potentials was not reproduced by the pen recorder. The interval between the upper and lower traces is shown. Galanin was present for the period indicated by the horizontal bar. (B) Galanin (0.3 ~mol/l) produced hyperpolarization and decreased the input resistance. The membrane potential of the neurone was slightly raised by continuous current injection in order to suppress spontaneous firing. Downward deflections represent electrotonic potentials caused by hyperpolarizing current pulses of constant amplitude. The electrotonic potentials are directly proportional to the input resistance. At the peak of the galanin response, the membrane potential was temporarily restored to its pre-drug value with a depolarizing current. The contact time for galanin is indicated by the horizontal bars. Experiments in (A) and (B) were performed on two different slices.
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Fig. 4. Interaction between galanin receptors, and a2-adrenoceptors or #-opioid receptors in LC neurones. Extracellular recording. Oalanin (0.03 ~mol/l) was applied twice; the second application was either in the absence of a2-adrenoceptor and /~-opioid receptor antagonists (A), or in their presence (B; C). (A) In the absence of antagonists galanin (0.03/,mol/1) caused reproducible inhibition (rq; n = 8). (B) Idazoxan (1 ixmol/l) (ca; n = 11) did not alter the effect of galanin (0.03 p.mol/l), while rauwolscine (1 /~mol/l) ( [] ; n = 11) potentiated it. (C) Both naloxone (0.1 /*tool/l) (®; n = 9 ) and /3-funaltrexamine (0.1 /~mol/l) ([]; n = 7) facilitated the effect of galanin (0.03 #mol/l). GAL, galanin; IDA, idazoxan; RAU, rauwolscine; NAL, naloxone; /3-FNA, /3-funaltrexamine. Asterisks indicate significant differences from the effect of the first application of galanin 0.03/*mol/I (* P < 0.05; ** P < 0.01). Means+ S.E.M. from n slices.
T h e resting m e m b r a n e p o t e n t i a l of the seven LC cells i m p a l e d with i n t r a c e l l u l a r microelectrodes was - 5 4 . 7 + 1.5 mV. G a l a n i n (0.3 p.mol/1) abolished the firing a n d caused a h y p e r p o l a r i z a t i o n of 10.4 + 1.1 m V (n = 7; P < 0.01; fig. 5A). W h e n the m e m b r a n e potential of the cells was increased (about 10 mV) by c u r r e n t injection, the f u r t h e r h y p e r p o l a r i z a t i o n i n d u c e d by g a l a n i n (0.3 ~ m o l / l ) was 9.9 _+ 1.4 m V (n = 7; P < 0.01); at the same time the i n p u t resistance decreased from 136.1 _+ 10.1 to 120.8 _+ 8.4 M.Q by 11.1 _+ 1.3% (n = 7; P < 0.01) (fig. 5B). This decrease persisted w h e n the m e m b r a n e p o t e n t i a l was t e m p o r a r i l y restored to its p r e - d r u g value with a d e p o l a r i z i n g c u r r e n t (fig. 5B).
4. Discussion I n the p r e s e n t e x p e r i m e n t s galanin, a possible cot r a n s m i t t e r of n o r a d r e n a l i n e ( R 6 k a e u s , 1987), depressed the firing rate of LC n e u r o n e s . T h e inhibitory effect of g a l a n i n arises at specific receptors, since it is p r e v e n t e d by a competitive a n t a g o n i s t (Bartfai et al., 1991). T h e g a l a n i n receptors are situated on the som a t a of LC cells a n d not on n e i g h b o u r i n g n e u r o n e s
228 (Seutin et al., 1989). In contrast to an earlier study (Seutin et al., 1989), we observed the peptide effect in all LC cells investigated rather than only in some ceils. It is suspected that this discrepancy may be due to the fact that Seutin et al. (1989) did not test the sensitivity of their supposed LC neurones to noradrenaline (Cedarbaum and Aghajanian, 1977). In our opinion it is absolutely necessary to do this; identification on purely eIectrophysiological grounds, i.e. from the shape of spontaneous action potentials, may not be sufficient. Intracellular recordings in LC cells showed that galanin produces hyperpolarization and a reduction of input resistance, which persists when the membrane potential is manually clamped to its pre-drug value. Input resistance changes that are secondary to hyperpolarization are thereby eliminated (Williams et al., 1984). The ions involved might be potassium and not chloride as the microelectrodes were filled with KCI, and under these conditions an increased permeability of the membrane to C1- depolarizes, rather than hyperpolarizes LC neurones (Cherubini et al., 1988). A recent study, published during the preparation of this manuscript, also demonstrated a galanin-induced hyperpolarization and conductance increase in the LC (Bartfai et al., 1991). Galanin enhances a potassium permeability in myenteric neurones (Palmer et al., 1986; Tamura et al., 1988), postganglionic parasympathetic neurones (Konopka et al., 1989), pheochromocytoma cells (De Weille et al., 1989) and insulinoma cells derived from pancreatic tumours (De Weille et al., 1988). In insulinoma cells (De Weille et al., 1988), galanin opens a class of potassium channels (KATe) , which is sensitive to intracellular ATP (Ashcroft and Ashcroft, 1990). The KAT e channels can be closed by sulphonylurea antidiabetics, such as glibenclamide (Ashcroft and Ashcroft, 1990). Galanin continued to hyperpolarize LC neurones in spite of the presence of glibenclamide (E.P. Finta and P. Illes, unpublished observation); thus, the involvement of KAy p channels can be dismissed. It is quite possible that galanin acts in the LC, at inwardly rectifying potassium channels also opened by a2-adrenoceptor and p,-opioid receptor agonists (Miyake et al., 1989). Galanin is not the only neuropeptide co-localized with noradrenaline in the LC (Skofitsch and Jacobowitz, 1985; Holets et al., 1988); enkephalins (Olpe and Steinmann, 199l) and neuropeptide Y (Everitt et al., 1984; Holets et al., 1988) are also present in the noradrenergic perikarya. Noradrenaline itself may be released from dendrites or recurrent axon collaterals (Aghajanian et al., 1977) and cause hyperpolarization via the activation of somatic az-adrenoceptors (Aghajanian and VanderMaelen, 1982). It may be assumed that these neuropeptides are co-released with noradrenaline and modulate its effect.
In LC neurones, both c~2-adrenoceptors and /~opioid receptors appear to be coupled to potassium channels via a pertussis toxin-sensitive G-protein (G~ or G o) (Aghajanian and Wang, 1986). Coupling of galanin-receptors to a G-protein was demonstrated in binding studies (Fisone et al., 1989). Whether potassium channels are opened via the subsequently inhibited adcnylcyclase/protein kinase A system or by the G-protein itself, awaits further clarification. The present experiments showed that two #-opioid receptor antagonists potentiate the inhibitory effect of galanin on the firing rate of LC neurones. Naloxone causes a reversible blockade of opioid receptors with a slight preference for the ~- over the 8- and K-type, while /3-funaltrexamine is an irreversible antagonist and is highly selective for ~-opioid receptors (Illes et al., 1989). Our findings with /3-funaltrexamine were rather unexpected. Firstly, it caused a long-lasting depression of spontaneous firing when given alone. We did not investigate the mechanism of this effect; a possible explanation is a partial agonistic activation of #-opioid receptors. Secondly, it decreased the effect o~ [MetS]enkephalin (see also Williams and North, 1984) reversibly. However, the inability of both /3funaltrexamine and naloxone to increase the discharge of action potentials excludes the possibility that, under the present in vitro conditions, there is a major tonic control of neuronal activity by spontaneously released opioid peptides. Thus, the potentiation of the galanin effect by the two antagonists was probably not due to competition with endogenous agonists at the #-opioid receptor. The results obtained with two a2-adrenoceptor antagonists were variable. Idazoxan (Doxey et al., 1983) did not alter the galanin effect, while rauwolscine (Weitzell et al., 1979) facilitated it. Rauwolscine was probably active because of its binding to 5-HTIA sites (Broadhurst et al., 1988) which are abundant in the LC (Weismann-Nanopoulos et al., 1985). Functional studies also proved the existence of 5-HTIA receptors on excitatory amino acid (EAA) and y-aminobutyric acid (GABA) terminals ending on LC cells (Bobker and Williams, 1989). Since idazoxan is highly selective for az-adrenoceptors, its inability to potentiate the galanin effect argues against an interaction between galanin receptors and az-adrenoceptors. Noradrenaline and [MetS]enkephalin depressed firing to a similar extent both in the absence and presence of low concentrations of galanin. Thus, galanin receptors appear to interact unidirectionally with #opioid receptors; a similar interaction between galanin receptors and a2-adrenoceptors does not seem to take place. In this context it is interesting to note that somatic a2-adrenoceptors and #-opioid receptors of LC neurones also influence each other; both idazoxan and rauwolscine potentiated the effect of [MetS]en -
229
kephalin, although noradrenaline did not alter it (Illes and N6renberg, 1990). az-Adrenoceptors, NPY receptors and tx-opioid receptors were shown to interact both in the cell somata (Illes and Regenold, 1990; Illes and N6renberg, 1990) and the nerve terminals (Yokoo et al., 1987; Schoffelmeer et al., 1986). Although the second-messenger and effector mechanisms of all three types of receptors seem to be identical (Aghajanian and Wang, 1986; Miyake et al., 1989; Illes and Regenold, 1990), the types of interaction differ. For example rauwolscine inhibits the effect of NPY (Illes and Regenold, 1990), but potentiates the effect of [MetS]enkephalin (Illes and N6renberg, 1990). Hence, the interaction between the receptors may occur in the plasma membrane and not in the common second-messenger system (Illes, 1989; Illes et al., 1990). Whereas presynaptic galanin receptors may be influenced by neighbouring az-adrenoceptors (Tsuda et al., 1989), no such interaction was found between the respective somatic receptors of LC neurones. In contrast, galanin receptors and ix-opioid receptors situated at the perikarya of LC cells were shown to interact unidirectionally. Since [MetS]enkephalin and galanin have a similar mode of action (see above), interference with the common second-messenger system of the occupied receptors might be postulated. However, the inability of galanin to alter the action of [MetS]en kephalin argues against this assumption. Moreover, the potentiation of the galanin effect after the blockade of the non-activated ix-opioid receptors by naloxone locates the site of interaction in the plasma membrane.
Acknowledgements We are grateful to Dr. W. N6renberg for a number of helpful discussions. Dr. J. Sevcik is the recipient of a research fellowship from the Alexander von Humboldt-Stiftung. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 325).
References Aghajanian, G.K. and C.P. VanderMaelen, 1982, a2-Adrenoceptormediated hyperpolarization of locus coeruleus neurons: intracellular studies in vivo, Science 215, 1394. Aghajanian, G.K. and Y.Y. Wang, 1986, Pertussis toxin blocks the outward currents evoked by opiate and a2-agonists in locus coeruleus neurons, Brain Res. 371,390. Aghajanian, G.K., J.M. Cedarbaum and Y.Y. Wang, 1977, Evidence for norepinephrine-mediated collateral inhibition of locus coeruleus neurons, Brain Res. 136, 570. Ashcroft, S.J.H. and F.M. Ashcroft, 1990, Properties and functions of ATP-sensitive K-channels, Cell Sign. 2, 197. Bartfai, T., K. Bedecs, T. Land, LJ. Langel, R. Bertorelli, P. Girotti, S. Consolo, X. Xu, Z. Wiesenfeld-Hallin, S. Nilsson, V.A. Pieribone and T. H6kfelt, 1991, M-15: high-affinity chimeric peptide that blocks the neuronal actions of galanin in the hippocampus,
locus coeruleus, and spinal cord, Proc. Natl. Acad. Sci. U.S.A. 88, 10961. Bobker, D.H. and J.T. Williams, 1989, Serotonin agonists inhibit synaptic potentials in the rat locus coeruleus in vitro via 5-hydroxytryptaminelA and 5-hydroxytryptaminel8 receptors, J. Pharmacol. Exp. Ther. 250, 37. Broadhurst, A.M., B.S. Alexander and M.D. Wood, 1988, Heterogeneous 3H-rauwolscine binding sites in rat cortex: two alpha 2adrenoceptor subtypes or an additional non-adrenergic interaction, Life Sci. 43, 83. Cedarbaum, J.M. and G.K. Aghajanian, 1977, Catecholamine receptors of locus coeruleus neurones: pharmacological characterization, Eur. J. Pharmacol. 44, 375. Cherubini, E., R.A. North and J.T. Williams, 1988, Synaptic potentials in rat locus coeruleus neurones, J. Physiol. 406, 431. De Weille, J., H. Schmid-Antomarchi, M. Fosset and M. Lazdunski, 1988, ATP-sensitive K + channels that are blocked by hypoglycemia-inducing sulfonylureas in insulin-secreting cells are activated by galanin, a hyperglycemia-inducing hormone, Proc. Natl. Acad. Sci. U.S.A. 85, 1312. De Weille, J., M. Fosset, H. Schmid-Antomarchi and M. Lazdunski, 1989, Galanin inhibits dopamine secretion and activates a potassium channel in pheochromocytoma cells, Brain Res. 485, 199. Doxey, J.C., A.G. Roach and C.F.C. Smith, 1983, Studies on RX 781094: a selective, potent and specific antagonist of a2-adrenoceptors, Br. J. Pharmacol. 78, 489. Everitt, B.J., T. H6kfelt, L. Terenius, K. Tatemoto, V. Mutt and M. Goldstein, 1984, Differential co-existence of neuropeptide Y (NPY)-like immunoreactivity with catecholamines in the central nervous system of the rat. Neuroscience 11,443. Fisone, G., l]. Langel, M. Carlquist, T. Bergman, S. Consolo, T. H6kfelt, A. Unden, S. Andell and T. Bartfai, 1989, Galanin receptor and its ligands in the rat hippocampus, Eur. J. Biochem. 181,269. Holets, V.R., T. H6kfelt, A. R6kaeus, L. Terenius and M. Goldstein, 1988, Locus coeruleus neurons in the rat containing neuropeptide Y, tyrosine hydroxylase or galanin and their efferent projections to the spinal cord, cerebral cortex and hypothalamus, Neuroscience 24, 893. llles, P., 1989, Modulation of transmitter and hormone release by multiple neuronal opioid receptors, Rev. Physiol. Biochem. Pharmacol. 112, 139. Illes, P. and W. N6renberg, 1990, Blockade of a2-adrenoceptors increases opioid iz-receptor-mediated inhibition of the firing rate of rat locus coeruleus neurones, Naunyn-Schmiedeb. Arch. Pharmacol. 342, 490. llles, P. and J.T. Regenold, 1990, Interaction between neuropeptide Y and noradrenaline on central catecholamine neurons, Nature 344, 62. Illes, P., H.D. Weber, J. Neuburger, B. Bucher, J.T. Regenold and W. N6renberg, 1990, Receptor interactions at noradrenergic neutones, in: Presynaptic Receptors and the Question of Autoregulation of Neurotransmitter Release eds. S. Kalsner and T.C. Westfall (Ann. N.Y. Acad. Sci., Vol. 604) p. 197. Konopka, L.M., T.W. McKeon and R.L. Parsons, 1989, Galanin-induced hyperpolarization and decreased membrane excitability of neurons in mudpuppy cardiac ganglia, J. Physiol. 410, 107. Martire, M., K. Fuxe, G. Pistritto, P. Preziosi and L.F. Agnati, 1986, Neuropeptide Y enhances the inhibitory effects of clonidine on 3H-noradrenaline release in synaptosomes isolated from the medulla oblongata of the male rat, J. Neural Transm. 67, 113. Miyake, M., M.J. Christie and R.A. North, 1989, Single potassium channels opened by opioids in rat locus coeruleus neurons, Proc. Natl. Acad. Sci. U.S.A. 86, 3419. North, R.A. and J.T. Williams, 1985, On the potassium conductance increased by opioids in rat locus coeruleus neurons, J. Physiol. 364, 265.
230 Olpe, H.R. and M. Steinmann, 1991, Responses of locus coeruleus neurons to neuropeptides, Prog. Brain Res. 88, 241. Palmer, J.M., M. Schemann, K. Tamura and J.D. Wood, 1986, Galanin mimics slow synaptic inhibition in myenteric neurons, Eur. J. Pharmacol. 124, 379. R6kaeus, A., 1987, Galanin: a newly isolated biologically active neuropeptide, Trends Neurosci. 10, 158. Schoffelmeer, A.N.M., J. Putters and A.H. Mulder, 1986, Activation of presynaptic c~2-adrenoceptors attenuates the inhibitory effect of /z-opioid receptor agonists on noradrenaline release from brain slices, Naunyn-Schmiedeb. Arch. Pharmacol. 333, 377. Seutin, V., P. Verbanck, L. Massotte and A. Dresse, 1989, Galanin decreases the activity of locus coeruleus neurons in vitro, Eur. J. Pharmacol. 164, 373. Skofitscb, G. and D.M. Jacobowitz, 1985, Immunohistochemical mapping of galanin-like neurons in the rat central nervous system, Peptides 6, 509. Starke, K., 1977, Regulation of noradrenaline release by presynaptic receptor systems, Rev. Physiol. Biochem. Pharmacol. 77, 1. Tamura, K., J.M. Palmer, C.K. Winkelmann and J.D. Wood, 1988, Mechanism of action of galanin on myenteric neurons, J. Neurophysiol. 60, 966. Tatemoto, K., A. R6kaeus, H. J6rnvall, T.J. McDonald and V. Mutt, 1983, Galanin a novel biologically active peptide from porcine intestine, FEBS Lett. 164, 124.
Tsuda, K., H. Yokoo and M. Goldstein, 1989, Neuropeptide Y and galanin in norepinephrine release in hypothalamic slices, Hypertension 14, 81. Weissmann-Nanopoulos, D., E. Mach, J. Magre, Y. Demassey and J.-F. Pujol, 1985, Evidence for the localization of 5-HTIA binding sites on serotonin containing neurons in the raphe dorsalis and raphe centralis nuclei of the rat brain, Neurochem. Int. 7, 1061. Weitzell, R., T. Tanaka and K. Starke, 1979, Pre- and postsynaptic effects of yohimbine stereoisomers on noradrenaline transmission in the pulmonary artery of the rabbit, Naunyn-Schmiedeb. Arch. Pharmacol. 308, 127. Williams, J.T. and R.A. North, 1984, Opiate-receptor interactions on single locus coeruleus neurones, Mol. Pharmacol. 26, 489. Williams, J.T., R.A. North, S.A. Shefner, S. Nishi and T.M. Egan, 1984, Membrane properties of rat locus coe.ruleus neurones, Neuroscience 13, 137. Williams, J.T., G. Henderson and R.A. North, 1985, Characterization of a2-adrenoceptors which increase potassium conductance in rat locus coeruleus neurones, Neuroscience 14, 95. Yokoo, H., D.H. Schlesinger and M. Goldstein, 1987, The effect of neuropeptide Y (NPY) on stimulation-evoked release of [3H]norepinephrine (NE) from rat hypothalamic and cerebral cortical slices. Eur. J. Pharmacol. 143, 283.