Actu Plysiol Simd
1990,138, 581-582
Morphine withdrawal responses of rat locus coeruleus neurons are blocked by an excitatory amino-acid antagonist C.-S. TUNG", J. G R E N H O F F and T. H. S V E N S S O N Department of Pharmacology, Karolinska Institute, Stockholm, Sweden Several lines of evidence implicate activation of the brain stem noradrenergic nucleus locus coeruleus (LC) in the behavioural and physiological reactions of the opiate withdrawal syndrome (see Redmond & Krystal 1984). The ability of the opioid antagonist naloxone to elicit a withdrawal reaction is considered a sign of physical dependence on opiates. In electrophysiological single-cell recording experiments carried out in morphine-dependent rats, LC neurons have been found to be markedly activated by naloxone (Aghajanian 1978). In contrast, when L C neurons from morphine-dependent rats are recorded in an inziirro brain slice preparation, naloxone does not affect neuronal firing (Andrade et al. 1983),indicating that the L C activation produced by systemic naloxone in ziz*o is critically dependent on an afferent neuronal input to the L C cells. Recent neuroanatomical and electrophysiological findings indicate that an excitatory amino acid (EAA) pathway from the medullary nucleus paragigantocellularis is a major excitatory input to the L C (Ennis & Aston-Jones 1988),and that an EAA input tonically controls the firing pattern of I,C neurons in vivo (Svensson et al. 1989).Therefore, we have investigated if such an EAA input could also be responsible for the excitation of L C neurons observed in the morphine withdrawal reaction. Singlecell recording in vivo was used to analyse the effect of the selective EAA antagonist kynurenate on naloxoneprecipitated activation of L C neurons in the morphinedependent rat. Eight male albino Sprague-Dawley rats weighing 240-260 g (Alab, Sollentuna, Sweden) were injected with morphine HCI (Apoteksbolaget, Stockholm, Sweden) twice daily for 5 days in gradually increasing doses (5, 10,20, 40 and 50 mg kg-' i.p,), a schedule Received 23 January 1990, accepted 23 January
'990. Key roords :excitatory amino acids, locus coeruleus, morphine, noradrenaline. Correspondence : Torgny H. Svensson MD, Department of Pharmacology, Karolinska Institute, PO Box 60400, S-10401 Stockholm, Sweden. * Present address : Department of Pharmacology, National Defense Medical Center, Taipei, The Republic of China on Taiwan.
which has previously been shown to produce physical dependence in rats (Thollander et ul. 1989). The experiments were made on the first day following the morphine treatment period. Single-cell recording was performed as previously described (Svensson et nl. 1989). Briefly, rats anaesthetized with chloral hydrate throughout the experiment were inserted in a stereotaxic instrument. Following surgery, a glass microelectrode with an impedance of 2 M R at 135 H z was lowered into the brain until typical L C cell activity was encountered. Single-cell activity was analysed from interspike time interval histograms created by an Apple I1 +computer, permitting quantitative analysis of the firing rate, burst firing and regularity of firing of the recorded cell. Burst firing was measured as the ratio between spikes in bursts and total number of spikes. Burst onset was signalled by an interspike interval < 80 ms, burst termination by an interval > 160ms. Regularity of firing was assessed from the variation coefficient, i.e. the ratio betwccn standard deviation and mean interval value of a histogram of 300 consecutive spikes. Kynurenate (kynurenic acid, Sigma, St Louis, MO, USA) in 0.15 M NaC1, set to pH 7.2 with NaOH, was infused through a steel cannula into the lateral ventricle. Naloxone HCI (Endo, Garden City, NY, USA), in saline, was injected into the jugular vein. Comparison of firing rates and variation coefficients between cells recorded before and after drug administration was madc with one-way ANOVA followed by Student's t-test. A twotailed P < 0.05 was considered significant. T h e spontaneous activity of L C cells in morphinetreated rats appeared suppressed when compared with cells in untreated rats. Thus, the firing rate was lower, the activity was more regular (lower variation coefficient) and no burst firing was seen in cells from morphine-treated rats (Table I). Naloxone (0.01 mg kg-' i.v.) markedly changed cell firing by producing increased firing rate, deregularization and burst firing (Fig. I).This naloxone-induced activation was absent when kynurenate was given before naloxone (Fig. I ) . Kynurenate alone produced a regularization of firing (variation coefficient before 29.I ",), after 22.6y0,P < 0.05, Student's t-test). Kynurenate was also able to reverse the naloaone-induced activation (data not shown).
581
C. S. Tung et nl.
j8z Table
I.
Firing properties of locus coeruleus neurons in untreated and morphine-treated rats Cntreated
-
~
~~.
~~
Firing rate (I-iz) \-ariation coefficient ("(,) Burst firing ('I(,) No. o f cells
..
\lorphine (see test) .. . - .-
~~
3.0s-0.;
43.2 -t 2.j 8 '5
I .z
~
~~~
.
~~~~~~
0. I**
30.6:t I .6'** 0**
I8
\lean SE11,Student's /-test (firing rate and variation coefficient). %lean. 1Vilcoxon two-sample test (burst firing). **P < 0.01. ***P < 0.001 4.
ct ( I / . 1989) and the pharmacological elTect of systemically administered nicotine (Tung et a/. 1989). Furthermore, it adds new perspectives to the treatment of opiate withdrawal, in which L C activation has been postulated to pla! a significant role.
:.;
This work was supported by the Swedish Medical Research Council (project no. 4747). the Council for Tobacco Research-USA, Inc. (grant no. 2192), Torsten och Ragnar Soderbergs Stiftelser and the Karolinska Institute. Dr Tung was supported by an IBRO/L;NESCO fellowship. We thank D r M. Thollander for his help with the morphine treatment schedule.
REFERENCES . ~ G H A J A N I A NG.K. ,
v
r
_______
.~
3asal
Na
Fig. I . Changes in firing rate (a) and variation coefficient (b) values in locus coeruleus neurons in morphine-dependent rats produced by nalosone (0.01mg kg ' i . \ , ) in control cells ( 0 ,n = 1 1 ) and cells recorded after kynurenate administration (A, n = 7 ) . .in increased \-ariation coefficient implies deregularization of cell firing. The nalovone response, which includes burst firing, is abolished afi-er klnurenate treatment. \lean SElI. **' P < 0.001.
The present results clearly indicate that nalosorieprecipitated morphine \vithdra\val responses in LC neurons are not due to actions on opiate receptors on these neurons, but depend on an E.i.1 input to the I L neurons. This finding underlines the significance of the F,.\.\ innervation in the regulation of LC neuroiial actit it!. as previousl! sho\\n for sensor! stimulation (Ennis & Aston-Jones 1988). burst firing (Svensson
1978. Tolerance of locus coeruleus neurones to morphine and suppression of withdrawal responses by clonidine. N a t u r e 276,186-188. .\NDRADE, R., VANDERMAELEN, C.P. & AGHAJANIAN, G.K. 1983. Morphine tolerance and dependence in the locus coeruleus: single cell studies in brain slices. Eur 3 Pharmacol 91, 161-169. ENSIS, 11. & ASTON-JONES, G. 1988. Activation of locus coeruleus neurons from nucleus paragigantocellularis: a new excitatory amino acid pathway in brain. 3 Keieurosci 8, 364413657, RELNOSD JR, D.E. & KRYSTAL, J.H. 1984. Multiple mechanisms of withdrawal from opioid drugs. /Inn Rei. .Vrrimsci 7, 43-478. SVESSSON,T.H., ENGBERG,G., TLNG,C.-S. & GRENHOFF, J. 1989. Pacemaker-like firing of noradrenergic locus coeruleus neurons in z.iz.o induced by the excitatory amino acid antagonist kynurenate in the rat. Acts Physiol Scand 135, 421-422. THOLL.%SDER, &HELLSTROM, I., P.M. & SVENSSON, T.H. 1989. Suppression of small intestinal motility and morphine withdrawal diarrhoea by clonidine : peripheral site of action. .4cta Physiol Scand 137, 385-392. TLSG,C.-S., UGEDO, L., GRENHOFF, J., ENGBERG, G. 8r SVENSSON, T . H . 1989. Peripheral induction of burst firing in locus coeruleus neurons by nicotine mediated via excitatory amino acids. Sjjnapse 4, 3 I 3-3 I 8.
1990,138, 581-582
Morphine withdrawal responses of rat locus coeruleus neurons are blocked by an excitatory amino-acid antagonist C.-S. TUNG", J. G R E N H O F F and T. H. S V E N S S O N Department of Pharmacology, Karolinska Institute, Stockholm, Sweden Several lines of evidence implicate activation of the brain stem noradrenergic nucleus locus coeruleus (LC) in the behavioural and physiological reactions of the opiate withdrawal syndrome (see Redmond & Krystal 1984). The ability of the opioid antagonist naloxone to elicit a withdrawal reaction is considered a sign of physical dependence on opiates. In electrophysiological single-cell recording experiments carried out in morphine-dependent rats, LC neurons have been found to be markedly activated by naloxone (Aghajanian 1978). In contrast, when L C neurons from morphine-dependent rats are recorded in an inziirro brain slice preparation, naloxone does not affect neuronal firing (Andrade et al. 1983),indicating that the L C activation produced by systemic naloxone in ziz*o is critically dependent on an afferent neuronal input to the L C cells. Recent neuroanatomical and electrophysiological findings indicate that an excitatory amino acid (EAA) pathway from the medullary nucleus paragigantocellularis is a major excitatory input to the L C (Ennis & Aston-Jones 1988),and that an EAA input tonically controls the firing pattern of I,C neurons in vivo (Svensson et al. 1989).Therefore, we have investigated if such an EAA input could also be responsible for the excitation of L C neurons observed in the morphine withdrawal reaction. Singlecell recording in vivo was used to analyse the effect of the selective EAA antagonist kynurenate on naloxoneprecipitated activation of L C neurons in the morphinedependent rat. Eight male albino Sprague-Dawley rats weighing 240-260 g (Alab, Sollentuna, Sweden) were injected with morphine HCI (Apoteksbolaget, Stockholm, Sweden) twice daily for 5 days in gradually increasing doses (5, 10,20, 40 and 50 mg kg-' i.p,), a schedule Received 23 January 1990, accepted 23 January
'990. Key roords :excitatory amino acids, locus coeruleus, morphine, noradrenaline. Correspondence : Torgny H. Svensson MD, Department of Pharmacology, Karolinska Institute, PO Box 60400, S-10401 Stockholm, Sweden. * Present address : Department of Pharmacology, National Defense Medical Center, Taipei, The Republic of China on Taiwan.
which has previously been shown to produce physical dependence in rats (Thollander et ul. 1989). The experiments were made on the first day following the morphine treatment period. Single-cell recording was performed as previously described (Svensson et nl. 1989). Briefly, rats anaesthetized with chloral hydrate throughout the experiment were inserted in a stereotaxic instrument. Following surgery, a glass microelectrode with an impedance of 2 M R at 135 H z was lowered into the brain until typical L C cell activity was encountered. Single-cell activity was analysed from interspike time interval histograms created by an Apple I1 +computer, permitting quantitative analysis of the firing rate, burst firing and regularity of firing of the recorded cell. Burst firing was measured as the ratio between spikes in bursts and total number of spikes. Burst onset was signalled by an interspike interval < 80 ms, burst termination by an interval > 160ms. Regularity of firing was assessed from the variation coefficient, i.e. the ratio betwccn standard deviation and mean interval value of a histogram of 300 consecutive spikes. Kynurenate (kynurenic acid, Sigma, St Louis, MO, USA) in 0.15 M NaC1, set to pH 7.2 with NaOH, was infused through a steel cannula into the lateral ventricle. Naloxone HCI (Endo, Garden City, NY, USA), in saline, was injected into the jugular vein. Comparison of firing rates and variation coefficients between cells recorded before and after drug administration was madc with one-way ANOVA followed by Student's t-test. A twotailed P < 0.05 was considered significant. T h e spontaneous activity of L C cells in morphinetreated rats appeared suppressed when compared with cells in untreated rats. Thus, the firing rate was lower, the activity was more regular (lower variation coefficient) and no burst firing was seen in cells from morphine-treated rats (Table I). Naloxone (0.01 mg kg-' i.v.) markedly changed cell firing by producing increased firing rate, deregularization and burst firing (Fig. I).This naloxone-induced activation was absent when kynurenate was given before naloxone (Fig. I ) . Kynurenate alone produced a regularization of firing (variation coefficient before 29.I ",), after 22.6y0,P < 0.05, Student's t-test). Kynurenate was also able to reverse the naloaone-induced activation (data not shown).
581
C. S. Tung et nl.
j8z Table
I.
Firing properties of locus coeruleus neurons in untreated and morphine-treated rats Cntreated
-
~
~~.
~~
Firing rate (I-iz) \-ariation coefficient ("(,) Burst firing ('I(,) No. o f cells
..
\lorphine (see test) .. . - .-
~~
3.0s-0.;
43.2 -t 2.j 8 '5
I .z
~
~~~
.
~~~~~~
0. I**
30.6:t I .6'** 0**
I8
\lean SE11,Student's /-test (firing rate and variation coefficient). %lean. 1Vilcoxon two-sample test (burst firing). **P < 0.01. ***P < 0.001 4.
ct ( I / . 1989) and the pharmacological elTect of systemically administered nicotine (Tung et a/. 1989). Furthermore, it adds new perspectives to the treatment of opiate withdrawal, in which L C activation has been postulated to pla! a significant role.
:.;
This work was supported by the Swedish Medical Research Council (project no. 4747). the Council for Tobacco Research-USA, Inc. (grant no. 2192), Torsten och Ragnar Soderbergs Stiftelser and the Karolinska Institute. Dr Tung was supported by an IBRO/L;NESCO fellowship. We thank D r M. Thollander for his help with the morphine treatment schedule.
REFERENCES . ~ G H A J A N I A NG.K. ,
v
r
_______
.~
3asal
Na
Fig. I . Changes in firing rate (a) and variation coefficient (b) values in locus coeruleus neurons in morphine-dependent rats produced by nalosone (0.01mg kg ' i . \ , ) in control cells ( 0 ,n = 1 1 ) and cells recorded after kynurenate administration (A, n = 7 ) . .in increased \-ariation coefficient implies deregularization of cell firing. The nalovone response, which includes burst firing, is abolished afi-er klnurenate treatment. \lean SElI. **' P < 0.001.
The present results clearly indicate that nalosorieprecipitated morphine \vithdra\val responses in LC neurons are not due to actions on opiate receptors on these neurons, but depend on an E.i.1 input to the I L neurons. This finding underlines the significance of the F,.\.\ innervation in the regulation of LC neuroiial actit it!. as previousl! sho\\n for sensor! stimulation (Ennis & Aston-Jones 1988). burst firing (Svensson
1978. Tolerance of locus coeruleus neurones to morphine and suppression of withdrawal responses by clonidine. N a t u r e 276,186-188. .\NDRADE, R., VANDERMAELEN, C.P. & AGHAJANIAN, G.K. 1983. Morphine tolerance and dependence in the locus coeruleus: single cell studies in brain slices. Eur 3 Pharmacol 91, 161-169. ENSIS, 11. & ASTON-JONES, G. 1988. Activation of locus coeruleus neurons from nucleus paragigantocellularis: a new excitatory amino acid pathway in brain. 3 Keieurosci 8, 364413657, RELNOSD JR, D.E. & KRYSTAL, J.H. 1984. Multiple mechanisms of withdrawal from opioid drugs. /Inn Rei. .Vrrimsci 7, 43-478. SVESSSON,T.H., ENGBERG,G., TLNG,C.-S. & GRENHOFF, J. 1989. Pacemaker-like firing of noradrenergic locus coeruleus neurons in z.iz.o induced by the excitatory amino acid antagonist kynurenate in the rat. Acts Physiol Scand 135, 421-422. THOLL.%SDER, &HELLSTROM, I., P.M. & SVENSSON, T.H. 1989. Suppression of small intestinal motility and morphine withdrawal diarrhoea by clonidine : peripheral site of action. .4cta Physiol Scand 137, 385-392. TLSG,C.-S., UGEDO, L., GRENHOFF, J., ENGBERG, G. 8r SVENSSON, T . H . 1989. Peripheral induction of burst firing in locus coeruleus neurons by nicotine mediated via excitatory amino acids. Sjjnapse 4, 3 I 3-3 I 8.
