Naunyn-Schmiedeberg's Arch Pharmacol (1992) 346:472- 474
Pharmacology © Springer-Verlag 1992
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
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.
30 s 9 rain ~
NPY 0.1 ymol/I 21 rain
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
~' ms NA
Washout 20 mV ms NA
._g 10 Control
0 NPY 0.1 pmol/I
~umo[/I NPY 5-80
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
~ 5 0
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% (P<0.01), while the PSP was not altered (4.5_+11.0%; P>0.05; 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
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, with...