Depression by neuropeptide Y of noradrenergic inhibitory postsynaptic potentials of locus coeruleus neurones

Naunyn-Schmiedeberg's Arch Pharmacol (1992) 346:472- 474

Naunyn-Schmiedeberg's

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Pharmacology © Springer-Verlag 1992

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Depression by neuropeptide Y of noradrenergic inhibitory postsynaptic potentials of locus coeruleus neurones Ervin P. Finta, Jtirgen T. Regenold, and Peter llles Department of Pharmacology, University of Freiburg, Hermann-Herder-Strasse 5, W-7800 Freiburg, Federal Republic of Germany Received April 28, 1992/Accepted June 10, 1992

Summary. I n t r a c e l l u l a r recordings were p e r f o r m e d in a p o n t i n e slice p r e p a r a t i o n o f the rat b r a i n c o n t a i n i n g the locus coeruleus (LC). T h e s p o n t a n e o u s firing o f a c t i o n p o t e n t i a l s was prevented by passing c o n t i n u o u s hyperp o l a r i z i n g current v i a the r e c o r d i n g electrode. F o c a l electrical s t i m u l a t i o n evoked a s y n a p t i c d e p o l a r i z a t i o n ( P S P ) followed by a h y p e r p o l a r i z a t i o n (IPSP). N e u r o p e p t i d e Y (NPY; 0.1 txmol/1) i n h i b i t e d the I P S P only. Pressure eject i o n o f n o r a d r e n a l i n e p r o d u c e d h y p e r p o l a r i z a t i o n which was p o t e n t i a t e d in the presence o f N P Y (0.1 Ixmol/1). Hence, N P Y a p p e a r s to inhibit the release o f n o r a d r e n a line f r o m d e n d r i t e s o r recurrent a x o n collaterals o f LC neurones.

Key words: N e u r o p e p t i d e Y - L o c u s coeruleus - N o r a d r e n a l i n e release Postsynaptic potentiation

Presynaptic

inhibition

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Introduction I n the central nervous system a m a j o r g r o u p o f n o r a d r e n e r g i c n e u r o n e s is s i t u a t e d in the nucleus locus c o e r u l e u s (LC) (DahlstrOm a n d Fuxe 1964; F o o t e et al. 1983). N e u r o p e p t i d e Y (NPY), a m e m b e r o f the p a n c r e a t ic p o l y p e p t i d e f a m i l y is c o - l o c a l i z e d with n o r a d r e n a l i n e in a s u b p o p u l a t i o n o f LC cells, which p r o j e c t m a i n l y to t h e h y p o t h a l a m u s ( H o l e t s et al. 1988). N P Y depresses the electrically-evoked release o f [3H]noradrenaline f r o m hyp o t h a l a m i c , b u t n o t f r o m cerebral cortical slices o f rats, i n d i c a t i n g the presence o f NPY-receptors at t h e t e r m i n a l s o f s o m e LC n e u r o n e s (Yokoo et al. 1987). F o c a l electrical s t i m u l a t i o n o f b r a i n slices c o n t a i n i n g t h e LC evokes a s y n a p t i c d e p o l a r i z a t i o n ( P S P ) followed b y a h y p e r p o l a r i z a t i o n (IPSP; E g a n et al. 1983; W i l l i a m s et al. 1991). T h e I P S P is a b o l i s h e d in the presence o f

Correspondence to: P. Illes at the above address

a 2 - a d r e n o c e p t o r a n t a g o n i s t s , proving the involvement o f a c a t e c h o l a m i n e in the s y n a p t i c response. It was suggested t h a t n o r a d r e n a l i n e released f r o m dendritres o r recurrent a x o n collaterals o f LC n e u r o n e s p r o d u c e s this c h a n g e in m e m b r a n e p o t e n t i a l ( W i l l i a m s et al. 1991). T h e present experiments were designed to find o u t w h e t h e r N P Y inhibits the I P S P by a p r e s y n a p t i c m e c h a n i s m .

Materials and methods Slices of the rat pons (thickness, 300-400 p.m) containing the caudal part of the LC, were prepared as described (Regenold and Illes 1990). Slices were submerged in a continuously flowing (2 ml/min) superfusion medium of the following composition (in mmol/1): NaC1, 126; KC1,2.5; NaH2PO 4, 1.2; MgC12, 1.3; CAC12,2.4; NaHCO3,25; glucose, 11; ascorbic acid, 0.3 and Na2EDTA, 0.03. The medium was saturated with 95°7002 plus 5°70CO 2 and maintained at 35-36°C. The LC was visually identified under a binocular microscope. LC cells were distinguished from neighbouring mesencephalic trigeminal neurones by their electrophysiological properties including spontaneous firing at a frequency of 0.2-5 Hz, and by hyperpolarization to noradrenaline (Williams et al. 1985). Recording and current injection was carried out with glass microelectrodes filled with KC1 (2 mol/1; tip resistance, 50- 80 Mf~) using a high impedance pre-amplifier and a bridge circuit (Axoclamp 2A). LC cells were constantly hyperpolarized (about 10 mV) by passing current through the microelectrode; thereby the generation of spontaneous action potentials was prevented. Synaptic potentials were evoked by single square wave pulses (0.8-1.5 ms duration, 40-50 V intensity, 0.1 Hz frequency) applied to bipolar tungsten electrodes, insulated except their tips and inserted about 50 Ixm into the slice near to the site of recording. The stimulation parameters were chosen so as to obtain an IPSP of about 4 inV. Four synaptic potentials were averaged immediately before and 5 min after the application of NPY (0.1 Ixmol/1). Noradrenaline (10 mmol/1; dissolved in medium) was pressure ejected every 1-2 min from a micropipette (tip diameter, 10-20 Ixm) using a Picospritzer II. The duration of the pressure pulse (5 psi, 5 - 80 ms) was chosen so as to obtain a hyperpolarization of approximately 7 mV. Two to three responses were averaged immediately before, as well as 5 and 10 min after the application of NPY. NPY was applied by changing the superfusion medium by means of three-way taps. At the constant flow rate of 2 ml/min about 30 s were required until the drug reached the bath. NPY was left for 5 min in the bath when synaptic potentials were evoked and for 10 rain when nor-

473 adrenaline was pressure ejected. The peptide was washed out for at least 20 min in order to obtain a complete recovery. The drugs used were: (±)-noradrenaline hydroehloride (Sigma, Deisenhofen, FRG) and neuropeptide Y porcine (NPY; Bachem, Bubendoff, Switzerland). Means_+ SEM are given throughout. Student's paired t-test was used for comparison of means. A probability level of 0.05 or less was considered to be statistically significant.

Control

30 s 9 rain ~

~

ms NA

NPY 0.1 ymol/I 21 rain

Results 7

The present results were obtained in 11 I_C neurones with a mean membrane potential of -57.9_+1.2mV. The spontaneous firing of action potentials was prevented by passing continuous hyperpolarizing current via the recording electrode. In 6 ceils, the membrane potential did not change after a 5 min incubation with NPY (0.1 ~tmol/1). At the same time the IPSP was reduced by

~

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Washout 20 mV ms NA

15

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0 NPY 0.1 pmol/I

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NA

Fig. 2A, B. Interaction between NPY and noradren~line in LC neurones. Noradrenaline (10 retool/l) was pressure ejected. A Representative experiment. NPY (0.1 Bmol/1) was present in the superfusion medium for 10rain. The intervals between the three traces are shown. B Mean_+ SEM of 5 similar experiments as shown in A. Empty bars indicate the responses to noradrenaline both before the application of NPY, and at least 20 rain after its washout. Responses to noradrenaline after a 5 rain (hatched bars) and 10 rain (cross-hatched bars) incubation with NPY (0.1 Ixmol/1) are also indicated. * P < 0.05; significant difference from the effect of noradrenaline determined before the application of NPY (0.1 ~tmol/1). NA, noradrenaline. The duration of the pressure pulses is shown both in A and B

Washout

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0.1

10-

PSP

g

IPSP

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0

o o.1

0.1

pmol/I NPY

Fig. 1 A, B. Effect of NPY on synaptic potentials of LC neurones evoked by focal electrical stimulation. The synaptic potentials were biphasic; a fast depolarizing response (PSP) was followed by a slow hyperpolarizing response (IPSP). A Representative experiment. Four averaged synaptic potentials are shown before, during and after the application of NPY (0.1 Ixmol/1); the neuropeptide was present in the superfusion medium for 5 rain. The intervals between the three sets of traces are shown. Notice the different time-scales in the right and leftpanels within each set of traces. Stimulation artifacts were retouched in the right panels. B Mean + SEM of 6 similar experiments as shown in A. Empty bars indicate the amplitudes of synaptic potentials both before the application of NPY, and at least 20 min after its washout. Hatched bars indicate the amplitude of synaptic potentials after a 5 rain incubation with NPY (0.1 ~tmol/1). *P < 0.01; significant difference from the IPSP determined before the application of NPY (0.1 Bmol/1)

36.8_+3.4% (P0.05; Fig. l). The depression of the IPSP by NPY (0.1 Ixmol/1) was completely reversible on washout. In another 5 ceils, noradrenaline was ejected near to the site of recording. The hyperpolarizing effect of noradrenaline showed a tendency to increase after a 5 min (28.1 _+9.5%; P > 0.05), and was significantly potentiated after a 10rain (43.6+10.9%; P < 0 . 0 5 ) incubation with NPY (0.1 Bmol/l) (Fig. 2). The NPY-induced potentiation disappeared after washout.

Discussion Focal electrical stimulation in the area of the LC evokes a PSP-IPSP complex (Egan et al. 1983; Cherubini et al. 1988). When the membrane potential is recorded with KC1 filled microelectrodes, the PSP is due to the release of both an excitatory amino acid and 7-aminobutyric acid (GABA; Cherubini et al. 1988). The IPSP is most

474

probably initiated by the release of noradrenaline from the LC neurones themselves (Egan et al. 1983), although the involvement of adrenaline originating from afferent fibres of the nucleus paragigantocellularis cannot be excluded either (Williams et al. 1991). An excitatory amino acid pathway from the nucleus paragigantocellularis and a GABAergic pathway from the prepositus hypoglossi have been also described (Williams et al. 1991). NPY inhibited the IPSP, without altering the PSP. Thus a selective inhibition of noradrenergic (or adrenergic) neurotransmission occurred, without any change in the effect of excitatory or inhibitory amino acids. The action of NPY may be presynaptic, since in spite of a decrease in the IPSP amplitude, locally applied noradrenaline produced a larger hyperpolarization in the presence of the peptide than in its absence. It was shown that somatic a2-adrenoceptors and opioid p-receptors of LC neurones are coupled to an inhibitory G-protein (Aghajanian and Wang 1987) which opens potassium channels (Williams and North 1984; Williams et al. 1985); NPY-receptors appear to share the same mechanism of action (Illes and Regenold 1990). In addition, NPY potentiated only the effect of noradrenaline, but not that of opioid peptides (Regenold and Illes 1990). Hence the selective interaction between somatic NPY-receptors and a2-adrenoceptor occurs probably in the plasma membrane rather than in the common second messenger and effector systems (Illes et al. t990). At the axon terminals of LC neurones, presynaptic receptors for NPY and noradrenaline also interact with each other (Yokoo et al. 1987). Binding studies and functional investigations unequivocally confirm these findings (Agnati et al. 1989). Hence, it may be hypothesized that the amplitude of the IPSP is under a dual feedback control of the co-transmitters noradrenaline and NPY acting at the dendrites or recurrent axon collaterals of LC ceils. The firing rate of these neurones is probably regulated by mutually interacting NPY-receptors and a2adrenoceptors. Acknowledgements. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 325). We are grateful to Dr. J. Sevcik for his valuable comments on the manuscript.

References Aghajanian GK, Wang YY (1987) Common a 2- and opiate effector mechanisms in the locus coeruleus: intracellular studies in brain slices. Neuropharmacology 26:793 - 799 Agnati LF, Zoli M, Merlo-Pich E, Benfenati F, Grimaldi R, Zini I, Toffano G, Fuxe K (1989) NPY receptors and their interaction with other transmitter systems. In: Mutt V, H6kfelt T, Fuxe K, Lundberg JM (eds) Neuropeptide Y. Raven Press, New York, pp 103-114 Cherubini E, North RA, Williams JT (1988) Synaptic potentials in rat locus eoeruleus neurones. J Physiol 406:431-442 Dahlstr6m A, Fuxe K (1964) Evidence for the existence of monoaminecontaining neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brainstem neurons. Acta Physiol Scand 232:1-55 Egan TM, Henderson G, North RA, Williams JT (1983) Noradrenalinemediated synaptic inhibition in rat locus coeruleus neurones. J Physiol 345:477-488 Foote SL, Bloom FE, Aston-Jones G (1983) Nucleus locus coeruleus: new evidence of anatomical and physiological specificity. Physiol Rev 63:844-914 Holets VR, HOkfelt T, ROkaeus A, Terenius L, Goldstein M (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 24:893-906 Illes P, Regenold JT (1990) Interaction between neuropeptide Y and noradrenaline on central catecholamine neurons. Nature 344:62-63 Illes P, Weber HD, Neuburger J, Bucher B, Regenold JT, N6renberg W (1990) Receptor interactions at noradrenergic neurones. Ann NY Acad Sci 604:197-210 Regenold JT, Illes P (1990) Inhibitory adenosine Al-receptors on rat locus coeruleus neurones. An intracellular electrophysiological study. Naunyn-Schmiedeberg's Arch Pharmacol 341:225-231 Williams JT, North RA (1984) Opiate-receptor interactions on single locus coeruleus neurones. Mol Pharmacol 26:489-497 Williams JT, Henderson G, North RA (1985) Characterization of az-adrenoceptors which increase potassium conductance in rat locus coeruleus neurones. Neuroscience 14:95-101 Williams JT, Bobker DH, Harris GC (1991) Synaptic potentials in locus coeruleus neurons in brain slices. Progr Brain Res 88:167-172 Yokoo H, Schlesinger DH, Goldstein M (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 143:283-286