- Email: [email protected]
Systemic morphine simultaneously decreases extracellular acetylcholine and increases dopamine in the nucleus accumbens of freely moving rats
Neuropharmacology Vo1.30, No.10, pp.1133-1136, 1991 Printed in Great Britain
0028-3908/91 $3.00+0.00 Pergamon Press plc
Systemic Morphine Simultaneously Decreases Extracellular Acetylcholine and Increases Dopamine in the Nucleus Accumbens of Freely Moving Rats P. Rada, G.P. Mark, E. Pothos and B.G. Hoebel
Department
of Psychology,
Princeton
University,
Princeton
NJ 08544-1010
U.S.A.
[Accepted 1 Ju&
19911
SUMMARY Microdialysis was used to measure extracellular levels of acetylcholine (ACh) and dopamine (DA) simultaneously in the nucleus accumbens (NAC) of freely moving rats. Systemic injection of morphine (20 mgfkg) significantly decreased ACh (30%, pc.01) while it increased DA (55%. p<.Ol). The effects of morphine were eliminated by naloxone. The results comfirm that morphine increases DA and in addition, demonstrate an inhibitory influence of this opiate on extracellular levels of ACh in the NAC. Key Words: Microdialysis;
Nucleus Accumbens;
Acetycholine;
Dopamine;
Morphine;
Rat
INTRODUCTION The modulation of neurotransmitter release, metabolism and. turnover by opiate receptor stimulation seems unequivocal although the exact nature of this modulation is debated. Previous research using isolated brain preparations has suggested that cholinergic activity is decreased by activation of p-receptors in the cortex (Wood & Rackman, 1981). hippocampus (Lapchak, Araujo & Collier, 1989; Wood & Rackman, 1981) and striatum (STR) (Lapchak et al., 1989). Others have achieved a decrease in ACh release by selective activation of STR 8receptors (Mulder, Wardeh, Hogenboom & Frankhuyzen, 1984). To date, however, the in vivo effects of opiates on ACh activity have not been determined. The result of opiate receptor stimulation on dopaminergic activity is well established. Early studies demonstrated an increase in STR and NAC dopamine (DA) turnover and release (as assessed by increased 3-MT) following administration of morphine which is primarily a preceptor agonist (Costa, Cheney, Racagni. 8c Zsilla. 1975; Wood, 1983). This effect was later confirmed in vivo using the microdialysis technique (DiChiara & Imperato, 1988). The NAC is particularly relevent to drug abuse due to its role in generating reward (positive reinforcement) via a dopaminergic mechanism (Koob & Bloom, 1988; Wise, 1989). The present study used microdialysis to examine the effects of systemically applied morphine on extracellular levels of ACh while monitoring DA simultaneously.
METHODS Seventeen adult maie, Sprague-Dawley rats weighing 325-375 gm were mait;tained on 12 hr reversed light-dark schedule. Subjects were anesthetized with pentobarbital mg/kg, IP) supplemented by ketamine (50 mg/kg, IP). Bilateral guide shafts aimed posterior medial NAC were stereotaxically implanted as follows: A 10.0 mm, L 1.2 mm, mm, with reference to the interaural line, midsaggital sinus, and the surface of the level respectively. Subjects were allowed to recover for a minimum of one week.
a 12(25 at the V 4.0 skull,
At least 12 hr before each experiment, microdialysis probes constructed with 200 pm o.d. by 3 mm, 6000 MW cutoff cellulose tips, were implanted in the NAC. Probes were perfused with a Ringer solution (146 mM NaCI, 4.0 mm KCI, 1.2 mM CaC12) at a flow rate of 1.8 pl/min. 1133
Preliminary Notes
1134
Neostigmine (0.5 pM) was added to the perfusion fluid to improve basal recovery of ACh. Samples were collected at 30 min intervals for 1.5 hr prior to and 2 hr after intraperitoneal injections of saline (1 cc/kg), morphine (20 mg/kg) or morphine plus naloxone (20 mg/kg, each). All injections were given between 12 noon and 3 p.m.. One half of each sample was analyzed for DA and metabolites and the remainder was assayed for ACh and choline. Dialysates were analyzed for ACh, choline, DA and metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), by reverse phase, HPLC systems with electrochemical detection. ACh and choline were separated on a 10 cm polymeric analytical column and then converted to betaine and hydrogen peroxide by an immobilized enzyme reactor (acetylcholinesterase and choline oxidase; BAS Inc., Model MF-8910). Data were converted to percent of the mean of three baseline samples and analyzed by two way analysis of variance (condition x time) followed by post-hoc t-tests when justified. Histology was performed to verify probe placement in the NAC.
RESULTS Systemically administered morphine resulted in a 30% decrease in extracellular ACh in the first and second samples after injection (Fig. 1; F(6,54)=4.10, pc.01). This effect was completely abolished by simultaneous injection of naloxone (F(6,66)=5.56, pc.01). Although nonsignificant, a transient increase in ACh was observed following administration of either saline or morphine plus naloxone. This effect was attributed to the stress induced by handling and injecting subjects, as has been reported by others (Nilsson, Kalen, Rosengren & Bjorklund, 1990). There was no statistically significant difference between the samples taken after saline and after morphine plus naloxone injections (F(6,48)=1.37, p=O.24). Extracellular levels of choline were unaffected by morphine injections when compared to saline (data not shown).
140 2 .i .. 'Z 3
120 -
60t a
40
0
1 30
I 80
I 90
Time
I 120
I 150
I 180
I 210
I 240
(min)
Figure 1. Extmcellulsr levels of ACh in the nucleus accumbens following 1P ivjectjons of morphine (N=7; solid line& saline (N&, dotted line) and morphineplus naloxone (N=6; dashed line). Korphme slgmficantly decreased NAC ACh in first and second samples post-injection (+=p< 0.05). This reduction was blocked by naloxonc. ACh IS expressed as a percentageof the mean of thne baseline samples.
Morphine significantly increased DA in the first, second and third samples post-injection (Fig. 2; F(6.60)= 3.84, pc.01). The maximal increase in DA occurred 1 hr after morphine whereas metabolite levels continued to increase 2 hr post-injection (data not shown). The effects of morphine on DA, DOPAC and HVA were completely reversed by coincident injection of naloxone (F(6,60)= 5.55, pc.01). Saline injections were without effect (Fig. 2). Mean, basal recoveries of ACh for groups given morphine, saline or morphine plus naloxone were 0.99 f 0.32, 0.98 f 0.37 and 1.13 f 0.39 pmoles/20 1.11.respectively. These values for DA were, 20.6 f 5.2, 12.4 f 3.4 and 11.1 f 3.4 fmoles/20 DISCUSSION In summary, these results show that morphine causes concentration in the NAC accompanied by a simultaneous changes were prevented by naloxone.
a decrease in extracellular ACh increase in extracellular DA. Both
Preliminary Notes
1135
200 -
*$:;
___& __ 8
& a
60 + Injection
6040 0
I 30
I 60
1 90
Time
I
I
I
I
,
120
150
160
210
240
(mitt)
Figure 2. Extracellular levels of dopamine in the nucleus accumbens following IP injections of morphine (N=7; solid lines), saline (N=.5;dotted lines) and morphine plus naloxone (N=5; dashed lines). Morphine significantly increased DA by a maximum of 55% 1 hr after injection (*= ~4.01, t=p
This research
was supported by USPHS grant DA-03597.
REFERENCES Cheney, D. I-., Trabucchi, M., Racagni, G., Wang, C. and Costa, E. (1974) Effects of acute and chronic morphine on regional rat brain acetylcholine turnover rate. Life Sci.,lS: 1977-1990. Costa, E., Cheney, D. L., Racagni, G. and Zsilla, G. (1975) An analysis at synaptic level of the morphine action in striatum and n. accumbens: dopamine and acetylcholine interactions. Life Sci., 17: l-8. Damsma, G., de Boer, P., Westernik, B. H. C. and Fibiger, H. C. (1990) Dopaminergic regulation of striatal cholinergic interneurons: an in vivo microdialysis study. Naunyn-Schmiedeberg’s Arch. Pharmacol., 342: 523-527. Di Chiara,
G. and Imperato, A. (1988) Opposite effects of mu and kappa opiate agonists on in the nucleus accumbens and in the dorsal caudate of freely moving rats. J. Pharmac. Exp. Therap., 244: 1067-1080. dopamine
release
Koob, G. F. and Bloom, F. E. (1988) Cellular Science, 242: 715-721.
and molecular
mechanisms
of drug dependence.
Lapchak, P. A., Araujo, D. M. and Collier, B. (1989) Regulation of endogenous release from mammalian brain slices by opiate receptors: hippocampus, striatum cortex of guinea-pig and rat. Neuroscience, 3 1: 3 13-325.
acetylcholine and cerebral
1136
Preliminary Notes
Matthews, R. T. and German, D. C. (1984) Electrophysiological evidence for excitation ventral tegmental area dopamine neurons by morphine. Neurosci.,ll: 617-625.
of rat
Mulder, A. H., Wardeh, G., Hogenboom, F. and Frankhuyzen, A. L. (1984) Kappa and deltaopioid receptor agonists differentially inhibit striatal dopamine and acetylcholine release. Nature, 308: 278-280. Nilsson, O., Kalen, P., Rosengren, E. and Bjorklund, A. (1990) Acetylcholine release in the rat hippocampus as studied by microdialysis is dependent on axonal impulse flow and increases during behavioral activation. Neurosci., 36: 325-338. Pollard, H., Llorens-Cortes, C., Bonnet, J., Costentin, J. and Schwartz, J. C. (1978) receptors on mesolimbic dopaminergic neurons. Neurosci. Lett., 7: 295299.
Opiate
Russell, V. A., Allin, R., Lamm, M. C. L. and Taljaard, 3. J. F. (1989) Increased dopamine D2 receptor-mediated inhibition of [14C] acetylcholine release in the dorsomedial part of the nucleus accumbens. Neurochem. Res., 14: 877-881. Wise, R.A. (1989) Opiate reward: 133.
Sites and substrates.
Neurosci.
Biobehav.
Review,
13: 129-
Wood, P. L. and Rackman, A. (1981) Actions of kappa, sigma and partial mu narcotic receptor agonists on rat brain acetylcholine turnover. Neurosci. Lett., 23: 75-80. Wood, P.L. (1983) Opiod regulation of CNS dopaminergic pathways: A review of methodology, receptor types, regional variations in species differences. Peptides, 4: 595-601.
0028-3908/91 $3.00+0.00 Pergamon Press plc
Systemic Morphine Simultaneously Decreases Extracellular Acetylcholine and Increases Dopamine in the Nucleus Accumbens of Freely Moving Rats P. Rada, G.P. Mark, E. Pothos and B.G. Hoebel
Department
of Psychology,
Princeton
University,
Princeton
NJ 08544-1010
U.S.A.
[Accepted 1 Ju&
19911
SUMMARY Microdialysis was used to measure extracellular levels of acetylcholine (ACh) and dopamine (DA) simultaneously in the nucleus accumbens (NAC) of freely moving rats. Systemic injection of morphine (20 mgfkg) significantly decreased ACh (30%, pc.01) while it increased DA (55%. p<.Ol). The effects of morphine were eliminated by naloxone. The results comfirm that morphine increases DA and in addition, demonstrate an inhibitory influence of this opiate on extracellular levels of ACh in the NAC. Key Words: Microdialysis;
Nucleus Accumbens;
Acetycholine;
Dopamine;
Morphine;
Rat
INTRODUCTION The modulation of neurotransmitter release, metabolism and. turnover by opiate receptor stimulation seems unequivocal although the exact nature of this modulation is debated. Previous research using isolated brain preparations has suggested that cholinergic activity is decreased by activation of p-receptors in the cortex (Wood & Rackman, 1981). hippocampus (Lapchak, Araujo & Collier, 1989; Wood & Rackman, 1981) and striatum (STR) (Lapchak et al., 1989). Others have achieved a decrease in ACh release by selective activation of STR 8receptors (Mulder, Wardeh, Hogenboom & Frankhuyzen, 1984). To date, however, the in vivo effects of opiates on ACh activity have not been determined. The result of opiate receptor stimulation on dopaminergic activity is well established. Early studies demonstrated an increase in STR and NAC dopamine (DA) turnover and release (as assessed by increased 3-MT) following administration of morphine which is primarily a preceptor agonist (Costa, Cheney, Racagni. 8c Zsilla. 1975; Wood, 1983). This effect was later confirmed in vivo using the microdialysis technique (DiChiara & Imperato, 1988). The NAC is particularly relevent to drug abuse due to its role in generating reward (positive reinforcement) via a dopaminergic mechanism (Koob & Bloom, 1988; Wise, 1989). The present study used microdialysis to examine the effects of systemically applied morphine on extracellular levels of ACh while monitoring DA simultaneously.
METHODS Seventeen adult maie, Sprague-Dawley rats weighing 325-375 gm were mait;tained on 12 hr reversed light-dark schedule. Subjects were anesthetized with pentobarbital mg/kg, IP) supplemented by ketamine (50 mg/kg, IP). Bilateral guide shafts aimed posterior medial NAC were stereotaxically implanted as follows: A 10.0 mm, L 1.2 mm, mm, with reference to the interaural line, midsaggital sinus, and the surface of the level respectively. Subjects were allowed to recover for a minimum of one week.
a 12(25 at the V 4.0 skull,
At least 12 hr before each experiment, microdialysis probes constructed with 200 pm o.d. by 3 mm, 6000 MW cutoff cellulose tips, were implanted in the NAC. Probes were perfused with a Ringer solution (146 mM NaCI, 4.0 mm KCI, 1.2 mM CaC12) at a flow rate of 1.8 pl/min. 1133
Preliminary Notes
1134
Neostigmine (0.5 pM) was added to the perfusion fluid to improve basal recovery of ACh. Samples were collected at 30 min intervals for 1.5 hr prior to and 2 hr after intraperitoneal injections of saline (1 cc/kg), morphine (20 mg/kg) or morphine plus naloxone (20 mg/kg, each). All injections were given between 12 noon and 3 p.m.. One half of each sample was analyzed for DA and metabolites and the remainder was assayed for ACh and choline. Dialysates were analyzed for ACh, choline, DA and metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), by reverse phase, HPLC systems with electrochemical detection. ACh and choline were separated on a 10 cm polymeric analytical column and then converted to betaine and hydrogen peroxide by an immobilized enzyme reactor (acetylcholinesterase and choline oxidase; BAS Inc., Model MF-8910). Data were converted to percent of the mean of three baseline samples and analyzed by two way analysis of variance (condition x time) followed by post-hoc t-tests when justified. Histology was performed to verify probe placement in the NAC.
RESULTS Systemically administered morphine resulted in a 30% decrease in extracellular ACh in the first and second samples after injection (Fig. 1; F(6,54)=4.10, pc.01). This effect was completely abolished by simultaneous injection of naloxone (F(6,66)=5.56, pc.01). Although nonsignificant, a transient increase in ACh was observed following administration of either saline or morphine plus naloxone. This effect was attributed to the stress induced by handling and injecting subjects, as has been reported by others (Nilsson, Kalen, Rosengren & Bjorklund, 1990). There was no statistically significant difference between the samples taken after saline and after morphine plus naloxone injections (F(6,48)=1.37, p=O.24). Extracellular levels of choline were unaffected by morphine injections when compared to saline (data not shown).
140 2 .i .. 'Z 3
120 -
60t a
40
0
1 30
I 80
I 90
Time
I 120
I 150
I 180
I 210
I 240
(min)
Figure 1. Extmcellulsr levels of ACh in the nucleus accumbens following 1P ivjectjons of morphine (N=7; solid line& saline (N&, dotted line) and morphineplus naloxone (N=6; dashed line). Korphme slgmficantly decreased NAC ACh in first and second samples post-injection (+=p< 0.05). This reduction was blocked by naloxonc. ACh IS expressed as a percentageof the mean of thne baseline samples.
Morphine significantly increased DA in the first, second and third samples post-injection (Fig. 2; F(6.60)= 3.84, pc.01). The maximal increase in DA occurred 1 hr after morphine whereas metabolite levels continued to increase 2 hr post-injection (data not shown). The effects of morphine on DA, DOPAC and HVA were completely reversed by coincident injection of naloxone (F(6,60)= 5.55, pc.01). Saline injections were without effect (Fig. 2). Mean, basal recoveries of ACh for groups given morphine, saline or morphine plus naloxone were 0.99 f 0.32, 0.98 f 0.37 and 1.13 f 0.39 pmoles/20 1.11.respectively. These values for DA were, 20.6 f 5.2, 12.4 f 3.4 and 11.1 f 3.4 fmoles/20 DISCUSSION In summary, these results show that morphine causes concentration in the NAC accompanied by a simultaneous changes were prevented by naloxone.
a decrease in extracellular ACh increase in extracellular DA. Both
Preliminary Notes
1135
200 -
*$:;
___& __ 8
& a
60 + Injection
6040 0
I 30
I 60
1 90
Time
I
I
I
I
,
120
150
160
210
240
(mitt)
Figure 2. Extracellular levels of dopamine in the nucleus accumbens following IP injections of morphine (N=7; solid lines), saline (N=.5;dotted lines) and morphine plus naloxone (N=5; dashed lines). Morphine significantly increased DA by a maximum of 55% 1 hr after injection (*= ~4.01, t=p
This research
was supported by USPHS grant DA-03597.
REFERENCES Cheney, D. I-., Trabucchi, M., Racagni, G., Wang, C. and Costa, E. (1974) Effects of acute and chronic morphine on regional rat brain acetylcholine turnover rate. Life Sci.,lS: 1977-1990. Costa, E., Cheney, D. L., Racagni, G. and Zsilla, G. (1975) An analysis at synaptic level of the morphine action in striatum and n. accumbens: dopamine and acetylcholine interactions. Life Sci., 17: l-8. Damsma, G., de Boer, P., Westernik, B. H. C. and Fibiger, H. C. (1990) Dopaminergic regulation of striatal cholinergic interneurons: an in vivo microdialysis study. Naunyn-Schmiedeberg’s Arch. Pharmacol., 342: 523-527. Di Chiara,
G. and Imperato, A. (1988) Opposite effects of mu and kappa opiate agonists on in the nucleus accumbens and in the dorsal caudate of freely moving rats. J. Pharmac. Exp. Therap., 244: 1067-1080. dopamine
release
Koob, G. F. and Bloom, F. E. (1988) Cellular Science, 242: 715-721.
and molecular
mechanisms
of drug dependence.
Lapchak, P. A., Araujo, D. M. and Collier, B. (1989) Regulation of endogenous release from mammalian brain slices by opiate receptors: hippocampus, striatum cortex of guinea-pig and rat. Neuroscience, 3 1: 3 13-325.
acetylcholine and cerebral
1136
Preliminary Notes
Matthews, R. T. and German, D. C. (1984) Electrophysiological evidence for excitation ventral tegmental area dopamine neurons by morphine. Neurosci.,ll: 617-625.
of rat
Mulder, A. H., Wardeh, G., Hogenboom, F. and Frankhuyzen, A. L. (1984) Kappa and deltaopioid receptor agonists differentially inhibit striatal dopamine and acetylcholine release. Nature, 308: 278-280. Nilsson, O., Kalen, P., Rosengren, E. and Bjorklund, A. (1990) Acetylcholine release in the rat hippocampus as studied by microdialysis is dependent on axonal impulse flow and increases during behavioral activation. Neurosci., 36: 325-338. Pollard, H., Llorens-Cortes, C., Bonnet, J., Costentin, J. and Schwartz, J. C. (1978) receptors on mesolimbic dopaminergic neurons. Neurosci. Lett., 7: 295299.
Opiate
Russell, V. A., Allin, R., Lamm, M. C. L. and Taljaard, 3. J. F. (1989) Increased dopamine D2 receptor-mediated inhibition of [14C] acetylcholine release in the dorsomedial part of the nucleus accumbens. Neurochem. Res., 14: 877-881. Wise, R.A. (1989) Opiate reward: 133.
Sites and substrates.
Neurosci.
Biobehav.
Review,
13: 129-
Wood, P. L. and Rackman, A. (1981) Actions of kappa, sigma and partial mu narcotic receptor agonists on rat brain acetylcholine turnover. Neurosci. Lett., 23: 75-80. Wood, P.L. (1983) Opiod regulation of CNS dopaminergic pathways: A review of methodology, receptor types, regional variations in species differences. Peptides, 4: 595-601.