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Effects of purinoceptor agonists on electrophysiological properties of rat mesencephalic trigeminal neurones in vitro
Neuroscience Letters, 92 (1988) 347 350
347
Elsevier Scientific Publishers Ireland Ltd.
NSL 05601
Effects of purinoceptor agonists on electrophysiological properties of rat mesencephalic trigeminal neurones in vitro J.T. R e g e n o l d l, H . L . H a a s 2 and P. Illes 1 ~Departrnent of Pharmacology University of Freiburg Freiburg i.Br. ( F.R.G. ) and :Department ~/" Physiology, University o["Mainz, Mainz ( F. R. G. ) (Received 19 April [988; Revised version received 15 June 1988; Accepted 15 June 1988)
Key word~': Mesencephalic trigeminal nucleus; Adenosine; Adenosine 5'-triphosphate Intracellular recordings were performed in a midpontine slice preparation of the rat brain containing the mesencephalic trigeminal nucleus (MTN). In spite of the previous demonstration of an adenosine deaminase-containing plexus terminating on this nucleus, adenosine, adenosine 5'-triphosphate (ATP), ~,[J'methylene ATP (~,p-meATP) and 2-methylthio ATP all failed to influence the membrane potential or input resistance of the MTN cells. Moreover, there was no apparent change in the shape of action potentials in the presence of these drugs, and the accomodation of the firing rate to depolarizing pulses was not affected either.
ATP was suggested to be released either as the main transmitter, or as a co-transmitter of noradrenaline and acetylcholine from terminals of peripheral nerves; its degradation product adenosine is a neuromodulator [1, 9]. In the central nervous system (CNS) adenosine inhibits both the spontaneous and evoked firing o f neurones in most brain areas. By contrast, ATP has either no effect, or produces an initial excitation followed by depression [13]. In neurones isolated from mature spinal ganglia, ATP, but not adenosine evoked an inward current carried by monovalent ions [10]. The immature neurones of spinal ganglia grown in a dissociated cell culture responded also with depolarization to ATP [11]. Adenosine had no effect on the membrane potential, but shortened the duration of calcium-dependent action potentials by reducing the voltage-sensitive calcium conductance. The M T N is described as the CNS counterpart o f the spinal ganglia. Recently, an adenosine deaminase-containing plexus was identified, projecting from the posterior
Correspondence: P. llles, Dept. of Pharmacology, University of Freiburg, Hermann-Herder-Strasse 5. D-7800 Freiburg i.Br., F.R.G. 0304-3940/88,'$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.
348 hypothalamus to the MTN. The existence of this fibre projection indicates a possible role of adenosine or its nucleotides in the synaptic regulation of neuronal activity in the MTN [12]. Thus, our aim was to study in a midpontine slice preparation of the rat brain the effects of adenosine and ATP on the electrophysiological properties of these neurones. Male Wistar rats (180-250 g) were anaesthetized with ether and decapitated. Midpontine slices of their brains containing the MTN were prepared and set up as described by Henderson et al. [7]. Briefly, slices of about 400/~m thickness were fully submerged in a continuously flowing (2 ml/min) medium of the following composition (in mmol/1): NaC1 126, KC1 5, NaH2PO4 1.2, MgCI2 1.3, CaC12 2.4, NaHCO3 25 and glucose 11. The medium was saturated with 95% 02 plus 5% CO2 and maintained at 37°C. Registration and current injection was with glass microelectrodes filled with KC1 3 mol/1 (tip resistance 60-100 MI2), and by using a high-impedance pre-amplifier and a bridge circuit. Hyperpolarizing current pulses of constant amplitude and 100 ms duration were delivered at a frequency of 0.1 Hz. Membrane input resistance was calculated from the peak potential change produced by the injected current. In some experiments pulses of different current strength and both polarities were applied in order to measure the slope conductance and to study evoked action potentials. The membrane potential was determined on withdrawal of the electrode from the cell. Drug contact-time was 5-7 min; all measurements were made after 3 min incubation with the agonists. The effects of the substances were evaluated as percent changes. Drugs used were: adenosine, 2-methylthioadenosine triphosphate sodium salt (Research Biochemicals, Wayland, MA, U.S.A.); adenosine 5'-triphosphate disodium salt, c~,fl-methyleneadenosine 5'-triphosphate disodium salt (Sigma, Miinchen, F.R.G.). Results are expressed as means _ S.E.M. Student's t-test was used for comparison of the mean percentual changes with zero. Stable recordings could be obtained from 24 MTN cells for up to 3 h. The membrane potential and input resistance of these neurones were 58.5___0.9 mV and 22.6+2.1 Mr2, respectively. Accomodation of firing frequency to low-amplitude depolarizing pulses was apparent in all cells. At a larger current strength the firing continued for the duration of the whole pulse (Fig. 1). Hyperpolarizing currents of sufficient amplitude evoked a time-dependent anomalous rectification. Adenosine 100/tmol/l did not influence either the membrane potential (0.1 + 2.2%) or the input resistance ( - 7 . 0 + 8.2%, P > 0.05 both) of seven neurones (Fig. 1). The shape of action potentials (not shown), their frequency and the anomalous rectification were also unaffected in the presence of adenosine 100/tmol/1 (Fig. 1). ATP 100 gmol/l failed to alter both membrane potential ( - 0 . 3 + 1.9%) and input resistance (10.0 _ 5.2%, P > 0.05 both) of another 7 cells. ~,fl-meATP 10/tmol/1 had no effect either (5 neurones, membrane potential: 0.1_0.7%, input resistance: 8.2 ± 3.8%, P > 0.05 both). Moreover, 2-methylthio ATP 100 gmol/1 did not change the membrane potential ( - 0.4 + 1.4%) or input resistance (2.7 + 2.6%, P > 0.05 both) of 5 cells. The shape and frequency of action potentials, and the anomalous rectification were similar before and during the application of ATP 100 gmol/l, ~,fl-meATP 10 gmol/1 or 2-methylthio ATP 100/~mol/l.
349
A
1 rnin
l!l
I11111Ill llll fl ]1 l] lll If 1' ll'lllll Adenosine 100 pmol/l
B a
b
Control ~
Adenosine 100 pmol/l ~
r-
c
d
_.~
l
~
r - ] 2nA
140 mV
40 ms Fig. 1. Failure of adenosine to influence membrane potential, input resistance and firing frequency of rat mesencephalic trigeminal neurones. A: registration of membrane potential and responses to hyperpolarizing current pulses of constant amplitude. Adenosine 100/~mol/l was present in the medium for the period indicated by the horizontal bar. B: registration of the current (upper traces; a-~t) and the action potentials (lower traces; a, c) or hyperpolarizing electrotonic potentials (lower traces; b, d). a, b: before the application of adenosine (control). c, d: 3 min after the application of adenosine 100/~mol/1. Calibrations in all 4 panels of B are identical, as indicated in d. Two different brain slices were used in A and B. T h e p r e s e n t results s h o w t h a t a d e n o s i n e , A T P , ~,fl-meATP a n d 2 - m e t h y l t h i o A T P , all failed to affect the m e m b r a n e p o t e n t i a l a n d i n p u t resistance o f the M T N cells. F u r t h e r m o r e , in the presence o f these drugs, there was no a p p a r e n t c h a n g e in the shape o f a c t i o n p o t e n t i a l s a n d the a c c o m o d a t i o n o f firing to d e p o l a r i z i n g pulses. A d e n o s i n e is a P r , w h e r e a s A T P a P 2 - p u r i n o c e p t o r a g o n i s t [2]. T h e two a n a l o g u e s used, n a m e l y ~,fl-meATP a n d 2 - m e t h y l t h i o A T P are preferential for the P2x- a n d P2yreceptors, respectively. O n l y ~,fl-meATP is resistant to e n z y m a t i c d e g r a d a t i o n . A d e nosine has been s h o w n p r e v i o u s l y to h y p e r p o l a r i z e h i p p o c a m p a l p y r a m i d a l [5, 15] a n d locus coeruleus [14] n e u r o n e s o f the rat with a decrease in i n p u t resistance. In
350 a d d i t i o n it e n h a n c e d a c c o m m o d a t i o n in the p y r a m i d a l cells [6] a n d i n h i b i t e d calcium spikes [5]. A d e n o s i n e also depressed the a m p l i t u d e o f e x c i t a t o r y s y n a p t i c p o t e n t i a l s (EPSPs) elicited b y s t i m u l a t i o n o f the s t r a t u m r a d i a t u m [15]. I n the M T N focal stim u l a t i o n e v o k e d o n l y a local d e p o l a r i z a t i o n , b u t n o E P S P [7]. Thus, the effect o f adenosine c o u l d n o t be investigated o n s y n a p t i c potentials. In spite o f m o r p h o l o g i c a l findings, which d e m o n s t r a t e d a p u r i n e r g i c i n n e r v a t i o n o f the M T N [12], the p r e s e n t e l e c t r o p h y s i o l o g i c a l investigation fails to s u p p o r t the f u n c t i o n a l significance o f this p a t h w a y . O n l y p e r i p h e r a l (see I n t r o d u c t i o n ) , b u t n o t central p r i m a r y afferent n e u r o n e s seem to be sensitive to the a p p l i c a t i o n o f p u r i n o c e p t o r agonists. H o w e v e r , v a r i o u s o t h e r p u t a t i v e t r a n s m i t t e r s , such as glycine, acetylcholine, n o r a d r e n a l i n e , d o p a m i n e , 7 - a m i n o b u t y r a t e [3], g l u t a m a t e [7] a n d substance P [4] were also ineffective on the firing frequency o f the M T N . Thus, it is n o t yet clear which n e u r o t r a n s m i t t e r is utilized by the t e r m i n a l s f o r m i n g synapses on the n e u r o n e s o f this nucleus [8]. This s t u d y was s u p p o r t e d by the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t ( S F B 325). 1
2 3 4 5 6 7
8 9 10 11 12
13 14 15
Burnstock, G., The co-transmitter hypothesis, with special reference to the storage and release of ATP with noradrenaline and acetylcholine. In A.C. Cuello (Ed.), Co-transmission, Macmillan, London, 1982, pp. 151 163. Burnstock, G. and Kennedy, C., Is there a basis for distinguishing two types of P2-purinoceptor?, Gen. Pharmacol., 16 (1985) 433440. De Montigny, C. and Lund, J.P., A microiontophoretic study of the action of kainic acid and putative neurotransmitters in the rat mesencephalic trigeminal nucleus, Neuroscience, 5 (1980) 1621-1628. Guyenet, P.G. and Aghajanian, G.K., Excitation of neurones in the locus coeruleus by substance P and related peptides, Brain Res., 136 (1977) 178 184. Greene, R.W. and Haas, H.L., Adenosine actions on CA1 pyramidal neurones in rat hippocampal slices, J. Physiol. (Lond.), 366 (1985) 119 127. Haas, H.L. and Greene, R.W., Adenosine enhances afterhyperpolarization and accomodation in hippocampal pyramidal cells, Pfl/igers Arch., 402 (1984) 244-247. Henderson, G., Pepper, C,M. and Shefner, S.A., Electrophysiological properties of neurones contained in the locus coeruleus and mesencephalic nucleus of the trigeminal nerve in vitro, Exp. Brain Res., 45 (1982) 29-37. Hinrichsen, C.F. and Larramendi, L.M.H., Synapses and cluster formation of the mouse mesencephalic fifth nucleus, Brain Res., 7 (1968) 296-299. llles, P., Jackisch, R. and Regenold, J.T., Presynaptic Pl-purinoceptors in jejunal branches of the rabbit mesenteric artery and their possible function, J. Physiol. (Lond.), 397 (1988) 13-29. Krishtal, O.A., Marchenko, S.M. and Pidoplichko, V.I., Receptor for ATP in the membrane of mammalian sensory neurones, Neurosci. Lett., 35 (1983) 41-45. Macdonald, R.L., Skerrit, J.H. and Werz, M.A., Adenosine agonists reduce voltage-dependent calcium conductance of mouse sensory neurones in cell culture, J. Physiol. (Lond.), 370 (1986) 75-90. Nagy, J.l., Buss, M. and Daddona, P.E., On the innervation of trigeminal mesencephalic primary afferent neurons by adenosine deaminase-containing projections from the hypothalamus in the rat, Neuroscience, 17 (1986) 141-156. Phillis, J.W. and Wu, P.H., The role of adenosine and its nucleotides in central synaptic transmission, Prog. Neurobiol., 16 (1981) 187-239. Shefner, S.A. and Chiu, T.H., Adenosine inhibits locus coeruleus neurons: an intracellular study in a rat brain slice preparation, Brain Res., 366 (1986) 364-368. Siggins, G.R. and Schubert, P., Adenosine depression of hippocampal neurons in vitro: an intracellular study of dose-dependent actions on synaptic and membrane potentials, Neurosci. Lett., 23 (1981) 55 60.
347
Elsevier Scientific Publishers Ireland Ltd.
NSL 05601
Effects of purinoceptor agonists on electrophysiological properties of rat mesencephalic trigeminal neurones in vitro J.T. R e g e n o l d l, H . L . H a a s 2 and P. Illes 1 ~Departrnent of Pharmacology University of Freiburg Freiburg i.Br. ( F.R.G. ) and :Department ~/" Physiology, University o["Mainz, Mainz ( F. R. G. ) (Received 19 April [988; Revised version received 15 June 1988; Accepted 15 June 1988)
Key word~': Mesencephalic trigeminal nucleus; Adenosine; Adenosine 5'-triphosphate Intracellular recordings were performed in a midpontine slice preparation of the rat brain containing the mesencephalic trigeminal nucleus (MTN). In spite of the previous demonstration of an adenosine deaminase-containing plexus terminating on this nucleus, adenosine, adenosine 5'-triphosphate (ATP), ~,[J'methylene ATP (~,p-meATP) and 2-methylthio ATP all failed to influence the membrane potential or input resistance of the MTN cells. Moreover, there was no apparent change in the shape of action potentials in the presence of these drugs, and the accomodation of the firing rate to depolarizing pulses was not affected either.
ATP was suggested to be released either as the main transmitter, or as a co-transmitter of noradrenaline and acetylcholine from terminals of peripheral nerves; its degradation product adenosine is a neuromodulator [1, 9]. In the central nervous system (CNS) adenosine inhibits both the spontaneous and evoked firing o f neurones in most brain areas. By contrast, ATP has either no effect, or produces an initial excitation followed by depression [13]. In neurones isolated from mature spinal ganglia, ATP, but not adenosine evoked an inward current carried by monovalent ions [10]. The immature neurones of spinal ganglia grown in a dissociated cell culture responded also with depolarization to ATP [11]. Adenosine had no effect on the membrane potential, but shortened the duration of calcium-dependent action potentials by reducing the voltage-sensitive calcium conductance. The M T N is described as the CNS counterpart o f the spinal ganglia. Recently, an adenosine deaminase-containing plexus was identified, projecting from the posterior
Correspondence: P. llles, Dept. of Pharmacology, University of Freiburg, Hermann-Herder-Strasse 5. D-7800 Freiburg i.Br., F.R.G. 0304-3940/88,'$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.
348 hypothalamus to the MTN. The existence of this fibre projection indicates a possible role of adenosine or its nucleotides in the synaptic regulation of neuronal activity in the MTN [12]. Thus, our aim was to study in a midpontine slice preparation of the rat brain the effects of adenosine and ATP on the electrophysiological properties of these neurones. Male Wistar rats (180-250 g) were anaesthetized with ether and decapitated. Midpontine slices of their brains containing the MTN were prepared and set up as described by Henderson et al. [7]. Briefly, slices of about 400/~m thickness were fully submerged in a continuously flowing (2 ml/min) medium of the following composition (in mmol/1): NaC1 126, KC1 5, NaH2PO4 1.2, MgCI2 1.3, CaC12 2.4, NaHCO3 25 and glucose 11. The medium was saturated with 95% 02 plus 5% CO2 and maintained at 37°C. Registration and current injection was with glass microelectrodes filled with KC1 3 mol/1 (tip resistance 60-100 MI2), and by using a high-impedance pre-amplifier and a bridge circuit. Hyperpolarizing current pulses of constant amplitude and 100 ms duration were delivered at a frequency of 0.1 Hz. Membrane input resistance was calculated from the peak potential change produced by the injected current. In some experiments pulses of different current strength and both polarities were applied in order to measure the slope conductance and to study evoked action potentials. The membrane potential was determined on withdrawal of the electrode from the cell. Drug contact-time was 5-7 min; all measurements were made after 3 min incubation with the agonists. The effects of the substances were evaluated as percent changes. Drugs used were: adenosine, 2-methylthioadenosine triphosphate sodium salt (Research Biochemicals, Wayland, MA, U.S.A.); adenosine 5'-triphosphate disodium salt, c~,fl-methyleneadenosine 5'-triphosphate disodium salt (Sigma, Miinchen, F.R.G.). Results are expressed as means _ S.E.M. Student's t-test was used for comparison of the mean percentual changes with zero. Stable recordings could be obtained from 24 MTN cells for up to 3 h. The membrane potential and input resistance of these neurones were 58.5___0.9 mV and 22.6+2.1 Mr2, respectively. Accomodation of firing frequency to low-amplitude depolarizing pulses was apparent in all cells. At a larger current strength the firing continued for the duration of the whole pulse (Fig. 1). Hyperpolarizing currents of sufficient amplitude evoked a time-dependent anomalous rectification. Adenosine 100/tmol/l did not influence either the membrane potential (0.1 + 2.2%) or the input resistance ( - 7 . 0 + 8.2%, P > 0.05 both) of seven neurones (Fig. 1). The shape of action potentials (not shown), their frequency and the anomalous rectification were also unaffected in the presence of adenosine 100/tmol/1 (Fig. 1). ATP 100 gmol/l failed to alter both membrane potential ( - 0 . 3 + 1.9%) and input resistance (10.0 _ 5.2%, P > 0.05 both) of another 7 cells. ~,fl-meATP 10/tmol/1 had no effect either (5 neurones, membrane potential: 0.1_0.7%, input resistance: 8.2 ± 3.8%, P > 0.05 both). Moreover, 2-methylthio ATP 100 gmol/1 did not change the membrane potential ( - 0.4 + 1.4%) or input resistance (2.7 + 2.6%, P > 0.05 both) of 5 cells. The shape and frequency of action potentials, and the anomalous rectification were similar before and during the application of ATP 100 gmol/l, ~,fl-meATP 10 gmol/1 or 2-methylthio ATP 100/~mol/l.
349
A
1 rnin
l!l
I11111Ill llll fl ]1 l] lll If 1' ll'lllll Adenosine 100 pmol/l
B a
b
Control ~
Adenosine 100 pmol/l ~
r-
c
d
_.~
l
~
r - ] 2nA
140 mV
40 ms Fig. 1. Failure of adenosine to influence membrane potential, input resistance and firing frequency of rat mesencephalic trigeminal neurones. A: registration of membrane potential and responses to hyperpolarizing current pulses of constant amplitude. Adenosine 100/~mol/l was present in the medium for the period indicated by the horizontal bar. B: registration of the current (upper traces; a-~t) and the action potentials (lower traces; a, c) or hyperpolarizing electrotonic potentials (lower traces; b, d). a, b: before the application of adenosine (control). c, d: 3 min after the application of adenosine 100/~mol/1. Calibrations in all 4 panels of B are identical, as indicated in d. Two different brain slices were used in A and B. T h e p r e s e n t results s h o w t h a t a d e n o s i n e , A T P , ~,fl-meATP a n d 2 - m e t h y l t h i o A T P , all failed to affect the m e m b r a n e p o t e n t i a l a n d i n p u t resistance o f the M T N cells. F u r t h e r m o r e , in the presence o f these drugs, there was no a p p a r e n t c h a n g e in the shape o f a c t i o n p o t e n t i a l s a n d the a c c o m o d a t i o n o f firing to d e p o l a r i z i n g pulses. A d e n o s i n e is a P r , w h e r e a s A T P a P 2 - p u r i n o c e p t o r a g o n i s t [2]. T h e two a n a l o g u e s used, n a m e l y ~,fl-meATP a n d 2 - m e t h y l t h i o A T P are preferential for the P2x- a n d P2yreceptors, respectively. O n l y ~,fl-meATP is resistant to e n z y m a t i c d e g r a d a t i o n . A d e nosine has been s h o w n p r e v i o u s l y to h y p e r p o l a r i z e h i p p o c a m p a l p y r a m i d a l [5, 15] a n d locus coeruleus [14] n e u r o n e s o f the rat with a decrease in i n p u t resistance. In
350 a d d i t i o n it e n h a n c e d a c c o m m o d a t i o n in the p y r a m i d a l cells [6] a n d i n h i b i t e d calcium spikes [5]. A d e n o s i n e also depressed the a m p l i t u d e o f e x c i t a t o r y s y n a p t i c p o t e n t i a l s (EPSPs) elicited b y s t i m u l a t i o n o f the s t r a t u m r a d i a t u m [15]. I n the M T N focal stim u l a t i o n e v o k e d o n l y a local d e p o l a r i z a t i o n , b u t n o E P S P [7]. Thus, the effect o f adenosine c o u l d n o t be investigated o n s y n a p t i c potentials. In spite o f m o r p h o l o g i c a l findings, which d e m o n s t r a t e d a p u r i n e r g i c i n n e r v a t i o n o f the M T N [12], the p r e s e n t e l e c t r o p h y s i o l o g i c a l investigation fails to s u p p o r t the f u n c t i o n a l significance o f this p a t h w a y . O n l y p e r i p h e r a l (see I n t r o d u c t i o n ) , b u t n o t central p r i m a r y afferent n e u r o n e s seem to be sensitive to the a p p l i c a t i o n o f p u r i n o c e p t o r agonists. H o w e v e r , v a r i o u s o t h e r p u t a t i v e t r a n s m i t t e r s , such as glycine, acetylcholine, n o r a d r e n a l i n e , d o p a m i n e , 7 - a m i n o b u t y r a t e [3], g l u t a m a t e [7] a n d substance P [4] were also ineffective on the firing frequency o f the M T N . Thus, it is n o t yet clear which n e u r o t r a n s m i t t e r is utilized by the t e r m i n a l s f o r m i n g synapses on the n e u r o n e s o f this nucleus [8]. This s t u d y was s u p p o r t e d by the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t ( S F B 325). 1
2 3 4 5 6 7
8 9 10 11 12
13 14 15
Burnstock, G., The co-transmitter hypothesis, with special reference to the storage and release of ATP with noradrenaline and acetylcholine. In A.C. Cuello (Ed.), Co-transmission, Macmillan, London, 1982, pp. 151 163. Burnstock, G. and Kennedy, C., Is there a basis for distinguishing two types of P2-purinoceptor?, Gen. Pharmacol., 16 (1985) 433440. De Montigny, C. and Lund, J.P., A microiontophoretic study of the action of kainic acid and putative neurotransmitters in the rat mesencephalic trigeminal nucleus, Neuroscience, 5 (1980) 1621-1628. Guyenet, P.G. and Aghajanian, G.K., Excitation of neurones in the locus coeruleus by substance P and related peptides, Brain Res., 136 (1977) 178 184. Greene, R.W. and Haas, H.L., Adenosine actions on CA1 pyramidal neurones in rat hippocampal slices, J. Physiol. (Lond.), 366 (1985) 119 127. Haas, H.L. and Greene, R.W., Adenosine enhances afterhyperpolarization and accomodation in hippocampal pyramidal cells, Pfl/igers Arch., 402 (1984) 244-247. Henderson, G., Pepper, C,M. and Shefner, S.A., Electrophysiological properties of neurones contained in the locus coeruleus and mesencephalic nucleus of the trigeminal nerve in vitro, Exp. Brain Res., 45 (1982) 29-37. Hinrichsen, C.F. and Larramendi, L.M.H., Synapses and cluster formation of the mouse mesencephalic fifth nucleus, Brain Res., 7 (1968) 296-299. llles, P., Jackisch, R. and Regenold, J.T., Presynaptic Pl-purinoceptors in jejunal branches of the rabbit mesenteric artery and their possible function, J. Physiol. (Lond.), 397 (1988) 13-29. Krishtal, O.A., Marchenko, S.M. and Pidoplichko, V.I., Receptor for ATP in the membrane of mammalian sensory neurones, Neurosci. Lett., 35 (1983) 41-45. Macdonald, R.L., Skerrit, J.H. and Werz, M.A., Adenosine agonists reduce voltage-dependent calcium conductance of mouse sensory neurones in cell culture, J. Physiol. (Lond.), 370 (1986) 75-90. Nagy, J.l., Buss, M. and Daddona, P.E., On the innervation of trigeminal mesencephalic primary afferent neurons by adenosine deaminase-containing projections from the hypothalamus in the rat, Neuroscience, 17 (1986) 141-156. Phillis, J.W. and Wu, P.H., The role of adenosine and its nucleotides in central synaptic transmission, Prog. Neurobiol., 16 (1981) 187-239. Shefner, S.A. and Chiu, T.H., Adenosine inhibits locus coeruleus neurons: an intracellular study in a rat brain slice preparation, Brain Res., 366 (1986) 364-368. Siggins, G.R. and Schubert, P., Adenosine depression of hippocampal neurons in vitro: an intracellular study of dose-dependent actions on synaptic and membrane potentials, Neurosci. Lett., 23 (1981) 55 60.