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Stimulation of locomotor activity by intra-accumbens AMPA is not inhibited by neonatal 6-hydroxydopamine-induced lesions
Neuroscience Letters, 138 (1992) 265 269
265
:O t992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
NSL 08574
Stimulation of locomotor activity by intra-accumbens AMPA is not inhibited by neonatal 6-hydroxydopamine-induced lesions R.C. Boldry*, R.T. Layer, L.J. Wallace a n d N.J. U r e t s k y Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, OH 43210 (USA) (Received 19 August 1991; Revised version received 10 December 1991; Accepted 4 February 1992)
Key words: Amphetamine; Excitatory amino acid; AMPA; Dopamine; Locomotor activity; 6-Hydroxydopamine The involvement of dopamine in the hypermotility responses to amphetamine s.c. or AMPA injected into the nucleus accumbens was evaluated in adult rats depleted of dopamine as neonates with 6-hydroxydopamine. The hypermotility response to amphetamine was markedly inhibited in the lesioned animals~ whi!e that to AMPA was enhanced. In addition, the hypermotility produced by AMPA in these rats was not inhibited by sulpiride + SCH-23390; however, it was inhibited completely by ~-methyl-p-tyrosine.These results suggest that the hypermotility produced by AMPA requires endogenous dopamine, but is mediated by a different mechanism than that produced by amphetamine.
~-Amino- 3-hydroxy- 5-methylisoxazole-4-propionic acid (AMPA) is an excitatory amino acid which is thought to activate a specific subtype of glutamate receptor in the central nervous system [20]. When injected into the nucleus accumbens of rats, this compound produces an intense stimulation of coordinated locomotor activity [2, 26]. This effect is attenuated by the co-administration of antagonists of the AMPA receptor, suggesting that the hypermotility response elicited by AMPA is mediated via stimulation of these receptors. However, in addition to the activation of AMPA receptors, the hypermotility response to AMPA requires the activation of dopaminergic receptors. This conclusion is supported by the observations that the hypermotility response to AMPA is attenuated by dopaminergic receptor antagonists or by treatments that interfere with the storage and release of dopamine [3]. In addition, microdialysis experiments have shown that the local infusion of AMPA or quisqualic acid into the nucleus accumbens increases the extracellular concentration of dopamine [3, 13]. Although the involvement ofdopamine in the expression of the hypermotility response to AMPA has been demonstrated by a variety of perturbations of the dopaminergic system, it is unclear whether the *Present address: Experimental Therapeutics Branch, National Institute of" Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
Corre~spondence: N.J. Uretsky, 500 W. 12th Avenue, Columbus, OH 43210, U.S.A.
response to AMPA involves mainly a release of dopamine or an interaction between dopaminergic and AMPA receptors at sites postsynaptic to the dopaminergic synapse. Amphetamine is considered the prototype indirectly acting dopaminergic agonist, and the locomotor stimulant response to amphetamine is dependent upon increased dopaminergic neurotransmission in the nucleus accumbens [14, 15, 22]. Consistent with this conclusion is the observation that lesions of dopaminergic neurons of neonatal rats with 6-hydroxydopamine antagonize the locomotor stimulation produced by amphetamine or reverse the effect of amphetamine so that it decreases locomotor activity [8, 12, 19]. The present study compares the locomotor stimulant effect of AMPA with that of amphetamine in neonatal 6-hydroxydopamine-lesioned rats in order to determine whether the loss of dopaminergic neurons impairs the response to AMPA as it does to amphetamine. Pregnant Sprague-Dawley rats were obtained from Harlan Farms approximately 1 week antepartum. The offspring were lesioned with 6-hydroxydopamine at 3 days of age [6]. Twenty rain after treatment with desipramine (25 mg/kg, s.c.) neonates were anesthetized with ether and a midline incision was made in the scalp. Using a Hamilton syringe, 6-hydroxydopamine hydrobromide (75/~g as the fi'ee base, dissolved in 5 ¢tl of saline) or saline was injected bilaterally into the lateral ventricles. The incision was closed by applying flexible collodion to the scalp, and the animals were returned to their home cage. The rats remained housed in a temperature con-
266 7000. EZTZ SHAM TREATED ~ 6 - O H D A TREATED
6000<
5000
~ 4000 o 3000 0
2000 1000
SALINE
AMPH
SALINE
AMPH
Fig. 1. The effect of neonatal 6-hydroxydopamine (6-OHDA) lesions on the stimulation of locomotor activity by d-amphetamine. Rats injected with 6 - O H D A as neonates and sham-treated controls were injected with d-amphetamine (0.75 mg/kg, s.c.) or vehicle, and locomotor activity was measured for 1 h. Each bar represents the mean _+ S.E.M. for 4 observations. "*P < 0.01 when compared to saline-treated controls, Student's t-test.
trolled environment with 12 h light-dark cycles for 3-5 months before behavioral testing. Direct stereotaxic injections of drugs into the nucleus accumbens of rats anesthetized with a mixture of halothane and oxygen were performed as previously described [26]. Drugs, dissolved in 1.0 #1 of phosphate buffer, were injected bilaterally into the nucleus accumbens at a rate of 1 #l/min. d-Amphetamine sulfate was dissolved in normal saline and injected subcutaneously. The animals were then placed in cages for monitoring locomotor activity, and starting ten minutes after the injections, their locomotor activity was recorded for one hour. Locomotor activity was assessed using cages equipped with a grid of infrared beams (Opto Varimex-minor, Columbus Instrument, Columbus, OH). Ambulatory activity was determined as the number of times 2 or more consecutive beams, 3.5 cm apart, were interrupted. The data were recorded by a digital counter (Columbus Instruments). The measurement of locomotor activity was always performed between 12.00 and 16.00 h. Tissue dopamine concentrations were determined in the rats used in the amphetamine study 2 weeks after behavioral experiments. Rats were decapitated, and the brains were removed and placed on ice. The nucleus accumbens was dissected, weighed, and homogenized in 0.4 N perchloric acid. The homogenate was centrifuged, and the dopamine content of the supernatant was determined by HPLC with electrochemical detection [17]. Results of all experiments are expressed as means + S.E.M. and significance levels were determined using either analysis of variance followed by the NewmanKeuls test or the unpaired Student's t-test. The administration of d-amphetamine produced a significant stimulation of locomotor activity in rats which received sham-treatments but not in the animals which
were injected with 6-hydroxydopamine as neonates (Fig. 1). Although the animals with lesions demonstrated an increased level of spontaneous locomotor activity after direct injection with saline (compared to sham-treated controls), there was no significant change in the ambulatory activity of these animals when challenged with amphetamine. Analysis of tissue dissected from the nucleus accumbens of these animals demonstrated that treatment with 6-hydroxydopamine produced significant depletion of dopamine in comparison with sham-treated control animals. These results suggest that drugs which release dopamine may be ineffective in producing a stimulation of locomotion in rats which have been lesioned as neonates with 6-hydroxydopamine. In contrast to the effects of amphetamine, the direct injection of AMPA into the nucleus accumbens produced a significant stimulation of locomotor activity in both 6-hydroxydopamine-treated and sham-treated control animals. In fact, the stimulation of locomotion produced by AMPA was significantly greater in animals that were lesioned as neonates with 6-hydroxydopamine when compared to the sham-treated animals. To evaluate the role of endogenous dopamine in the expression of the stimulatory response to AMPA, we determined the effect of dopamine receptor antagonists and ~-methyl-p-tyrosine, drugs that produce a general impairment in dopaminergic neurotransmission throughout the brain, on the hypermotility response to AMPA in rats treated neonatally with 6-hydroxydopamine. In the first experiment, sulpiride, a dopaminergic D 2 receptor antagonist, was co-injected with AMPA into the nucleus accumbens at a dose of 10 #g/ side, while SCH-23390 (0.3 mg/kg, s.c.), a Dj receptor antagonist, was injected subcutaneously (due to its limited solubility in the injection vehicle) 20 min before the intra-accumbens injection of AMPA. The results show that these antagonists completely inhibited
TABLE I E F F E C T OF N E O N A T A L 6 - H Y D R O X Y D O P A M I N E (6-OHDA)I N D U C E D LESIONS O N T H E D O P A M I N E C O N T E N T O F T H E N U C L E U S A C C U M BENS Rats were injected into the lateral ventricles at 3 days postpartum with 75 # g of 6-OHDA/side. 14 clays after behavioral testing as adults, the rats were sacrificed and the dopamine content of the nucleus accumbens was determined. Treatment
n
[DA] nmol/g
% of control
Sham-treated 6-OHDA-treated
4 4
61.7 + 10.3 12.3 + 5.49**
19.9
**P < 0.01, Student's t-test.
267 AMPA-stimulated locomotor activity in sham-operated rats but did not significantly inhibit the stimulation of locomotion produced by A M P A in the 6-hydroxydopamine-treated animals (Fig, 2). In contrast, pretreatment of the 6-hydroxydopamine-lesioned animals with ~-methyl-p-tyrosine (250 mg/kg, i.p. 4 h and 1 h prior to the injections of A M P A - - 250 ng/side into the nucleus accumbens) virtually abolished AMPA-induced locom o t o r stimulation [AMPA alone: 11,934 (n -- 2), A M P A + ~-methyl-p-tyrosine: 956 + 469 (n -- 6)]. The present results show that the locomotor stimulation produced by A M P A was enhanced in animals treated as neonates with 6-hydroxydopamine, while the stimulatory effect of amphetamine was almost completely abolished. Thus, the effects of A M P A in animals, that were administered 6-hydroxydopamine as neonates to produce a lesion of dopaminergic neurons, differ from the effects of amphetamine, the prototype indirectly acting dopaminergic agonist. These results support the hypothesis that the locomotor stimulation produced by the administration of A M P A into the nucleus accumbens is not simply a function of dopamine release from nerve terminals at this site. However, endogenous catecholamines still appear to play an important role in the locom o t o r stimulatory effect of AMPA in animals that received a lesion as neonates, since c~-methyl-p-tyrosine, which has been shown previously to inhibit the stimulatory effects of A M P A in normal animals, also completely inhibited the effects of AMPA in the 6-hydroxydopamine treated animals. This observation is consistent with previous studies showing that the behavior of neonatal 6-hydroxydopamine-lesioned animals is markedly impaired by ~-methyl-p-tyrosine pretreatment [23, 24]. These results suggest that the mechanism of the hypermotility response to A M P A may be different than that to MK-801, an N-methyl-D-aspartate antagonist, since MK-801 [(+)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine] is reported to stimulate locomotion after both ~-methyl-p-tyrosine and reserpine [7]. One hypothesis to explain our observations can be derived from a recent proposal suggesting the presence of two distinct functional pools of dopamine, whose release is regulated by different processes [11]. One of these pools is postulated to be released in a transient or phasic manner by increasing the rate of impulse flow in dopaminergic neurons or by drugs such as amphetamine. Most of this pool appears to be lost in the rats that received a neonatal 6-hydroxydopamine-induced lesion, resulting in a marked decrease in the response to amphetamine [8, 12, 19]. The other pool of dopamine is the source of the tonic dopamine levels in the extracellular fluid and is thought to be regulated by glutamatergic re-
SHAM TREATED 6 - O H D A TREATED
14 12
_~
lo
~ a : a: o ~
o
8
z o
2
NS
SALINE
AMPA (250n9)
AMPA(250ng) SULPIR4DE SCH 23390
Fig. 2. The effect of neonatal 6-hydroxydopamine(6-OHDA)-induced lesions on the stimulation of locomotor activity by AMPA. Rats injected with 6-OHDA as neonates (hatched bars) and sham-treated controls were injected into the nucleus accumbens with vehicle, AMPA (250 ng), or a combination of AMPA and sulpiride (10,ug/side) into the nucleus accumbens (after pre-treatment with SCH-23390, 0.3 mg/kg, s.c.). Each bar represents the mean _+ S.E.M. for 4-9 observations. ANOVA F 5 , 3 4 21, P < 0.01. Newman-Keuls multiple comparison test: "P < 0.01 compared to the sham-saline and sham-AMPA-sulpirideSCH-23390 groups. **P< 0.01 compared to the 6-OHDA-saline, shamAMPA, and sham-AMPA-sulpiride-SCH-23390groups. There was no significant difference between the sham-saline and 6-OHDA-saline groups or between the 6-OHDA-AMPA and the 6-OHDA-AMPAsulpiride-SCH-23390 groups.
ceptors on dopaminergic terminals [11]. This pool may be the one that is critical for the hypermotility response to AMPA. Recently, it has been shown using intracranial microdialysis that after 6-hydroxydopamine lesions in neonatal rats, the extracellular striatal dopamine levels are reduced but to a relatively small extent compared with the marked depletion of tissue dopamine [8]. Thus, the pool of dopamine that may be required for the effects of excitatory amino acids, such as AMPA, may remain relatively intact after 6-hydroxydopamine lesions, and this may account for the failure of the lesion of DA neurons to inhibit the locomotor-stimulatory effects of AMPA as it did amphetamine. However, according to this hypothesis, additional treatment with ~-methyl-p-tyrosine should deplete this pool, resulting in a complete inhibition of the locomotor stimulation produced by AMPA. The locomotor stimulatory effect of AMPA in 6-hydroxydopamine-lesioned rats may also in part be related to a functional postsynaptic supersensitivity to dopaminergic agonists that develops in response to the 6-hydroxydopamine-induced lesions [1, 4, 5, 9]. Evidence for a change in dopamine receptor sensitivity is derived from the observation that the combination of D~ and D2 dopaminergic receptor blocking agents, which antagonized the effects of AMPA in the sham-lesioned animals, did not antagonize the effects of AMPA in the neonatally lesioned animals. This effect is reminiscent of the re-
268
ported subsensitivity of neonatal lesioned rats to the effects of dopaminergic antagonists, such as haloperidol, fluphenazine, and SCH-23390 [6, 10]. At present the mechanism in which neonatal lesioned rats become refractory to the effects of dopaminergic antagonists remains unresolved. Our studies cannot exclude a role for endogenous norepinephrine in the locomotor stimulant response of neonatal-lesioned animals to AMPA. In the present study, the 6-hydroxydopamine was administered to neonatal animals after desipramine, an inhibitor of norepinephrine uptake which has been shown to protect norepinephrine neurons from the toxic effects of 6-hydroxydopamine. Therefore, the brain norepinephrine levels would remain relatively intact after 6-hydroxydopamine [19], but would be decreased by a-methyl-p-tyrosine, which might account for the ability of this latter drug to inhibit the response to AMPA. Furthermore, previous studies have shown that norepinephrine, infused into the ventricles, produces locomotor stimulation, which is enhanced in animals pretreated with 6hydroxydopamine [25, 27], and this response appears to be mediated by an action on supersensitive dopaminergic receptors [27]. However, not all the evidence supports a role for norepinephrine in the locomotor stimulant response. For example, amphetamine is a potent releaser of norepinephrine as well as dopamine [18, 21]. If the hyperactivity response were mediated through the release of norepinephrine, then the stimulatory response to amphetamine should not have been abolished in neonatal rats injected with desipramine before 6-hydroxydopamine. Regardless of the mechanism, the observation that c~methyl-p-tyrosine pretreatment completely antagonized the locomotor stimulant response to AMPA supports the concept that in neonatal 6-hydroxydopamine-lesioned animals, like in normal animals [3], the stimulation of locomotion in the nucleus accumbens produced by either AMPA or dopaminergic agonists requires the activation of both AMPA and dopaminergic receptors in the nucleus accumbens. 1 Abrous, D.N., Rivet, J.M., Le Moal, M. and Herman, J.E, Similar post-lesion receptor adjustments following the unilateral 6-hydroxydopamine lesion of the dopamine mesotelencephalic system in neonatal and adult rats, Brain Res., 526 (1990) 195-202. 2 Arnt, J., Hyperactivity following injection of a glutamate agonist and 6,7-ADTN into rat nucleus accumbens and its inhibition by THIE Life Sci., 28 (1981) 1597-1603. 3 Boldry, R.C., Willins, D.L., Wallace, L.J. and Uretsky, N.J., The role of endogenous dopamine in the hypermotility response to intraaccumbens AMPA. Brain Res. 559 (1991) 100 108.
4 Breese, G.R., Napier, T.C. and Mueller, R.A., Dopamine agonistinduced locomotor activity in rats with 6-hydroxydopamine at differing ages: functional supersensitivity of D~ receptors in neonatally lesioned rats, J. Pharmacol. Exp. Ther., 234 (1985) 447-455. 5 Breese, G.R., Duncan, G.E., Napier, C., Bondy, S.C., Iorio, L.C. and Mueller, R.A., 6-Hydroxydopamine treatments enhance behavioral responses to intracerebral microinjection of D t and D 2 dopamine agonists into nucleus accumbens and striatum without changing dopamine antagonist binding, J. Pharmacol. Exp. Thcr., 240 (1987) 167-176. 6 Bruno, J.l~, Stricker, E.M. and Zigmond, M.J., Rats given dopamine-depleting brain lesions as neonates are subsensitive to dopaminergic antagonists as adults, Behav. Neurosci., 99 (1985) 771 775. 7 Carlsson, M. and Carlsson, A., The NMDA antagonist MK-801 causes marked locomQtor stimulation in monoamine-depleted mice, J. Neural Transm., 75 (1989) 221 226. 8 Casteneda, E., Whishaw, I.Q., Lermer, L. and Robinson, T.E., Dopamine depletion in neonatal rats: effects on behavior and striatal dopamine release assessed by intracerebral microdialysis during adulthood, Brain Res., 508 (1990) 30 38. 9 Dewar, K.M., Soghomonian, J.J., Bruno, J.P., Descarries, L. and Reader, T.A., Elevation of dopamine D, but not Dj receptors in adult rat neostriatum after neonatal 6-hydroxydopamine denervation, Brain Res., 536 (1990) 287 296. 10 Duncan, G.E., Criswell, H., McCown, T.J., Paul, I.A., Mueller, R.A. and Breese, G.R., Behavioral and neurochemical responses to haloperidol and SCH-23390 in rats treated neonatally or as adults with 6-hydroxydopamine, J. Pharmacol. Exp. Ther.. 243 (1987) 1027 1034. 11 Grace, A.A., Phasic versus tonic dopamine release and the modulation of dopamine system responsivity: a hypothesis for the etiology of schizophrenia, Neuroscience, 41 ( 1991 ) 1 24. 12 Heffner, T.G. and Selden, L.S., Possible involvement of serotonergic neurons in the reduction of locomotor hyperactivity caused by amphetamine in neonatal rats depleted of brain dopamine, Brain Res., 244 (1982) 81-90. 13 lmperato, A., Honore, T. and Jensen, L., Dopamine release in the nucleus caudatus and in the nucleus accumbens is under glutamatergic control through non-NMDA receptors: a study in freely moving rats, Brain Res., 530 (1990) 223 228. 14 Joyce, E.M. and Koob, G.F., Amphetamine-, scopolamine-, and caffeine-induced locomotor activity following 6-hydroxydopaminelesions of the mesolimbic dopamine system, Psychopharmacology, 73,(1981)311 313. 15 Kelly, RH., Seviour, P.W. and Iversen, S.D., Amphetamine and apomorphine responses in the rat following 6-OHDA lesions of the nucleus accumbens septi and corpus striatum, Brain Res., 94 (1975) 507 522. 16 K6nig, J.R.F. and Klippel, R.A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and the Lower Parts of the Brain Stem, Krieger, New York, 1963. 17 Kotake, C., Heffner, T., Vosmer, G. and Seiden, L., Determination ofdopamine, norepinephrine, serotonin, and their major metabolic products in rat brain by reverse-phase ion-pair high performance liquid chromatography with electrochemical detection, Pharmacol. Biochem. Behav., 22 (1985) 85 89. 18 Kuczenski, R., Biochemical actions of amphetamine and other stimulants. In 1. Creese (Ed.), Stimulants: Neurochemical, Behavioral, and Clinical Perspectives, Raven, New York, 1983, pp. 31 61. 19 Luthman, J., Fredriksson, A., Lewander, T., Jonsson, G. and
269
20
21
22
23
Archer, T., Effects of d-amphetamine and methylphenidate on hyperactivity produced by neonatal 6-hydroxydopamine treatment, Psychopharmacology, 4 (1989) 550-557. Monaghan, D.T., Bridges, R.J. and Cotman, C.W., The excitatory amino acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system, Annu. Rev. Pharmacol. Toxicol., 29 (1989) 365~,02. Moore, K.E., Amphetamines: biochemical and behavioral actions in animals. In L.L. Iversen, S.D. Iversen and S.H. Snyder (Eds.), Handbook of Psychopharmacology, Plenum, New York, 1978, pp. 41-85. Pijnenburg, A.J.J., Honig, W.M.M. and Van Rossum, J.M., Inhibition of d-amphetamine-induced locomotor activity by injection of haloperidol into the nucleus accumbens, Psychopharmacologia, 41 (1975) 87-95. Potter, B.M. and Bruno, J.P., Food intake of rats depleted of do-
24
25
26
27
pamine as neonates is impaired by inhibition of catecholamine biosynthesis, Neurosci. Lett., 107 (1990) 295-300. Rogers, D.C. and Dunnett, S.B., Hypersensitivity to c~-methyl-ptyrosine suggests that behavioral recovery of rats receiving neonatal 6-hydroxydopamine lesions is mediated by residual catecholamine neurons, Neurosci. Lett., 102 (1989) 108-113. Segal, D., McAllister, C. and Geyer, M., Ventricular infusions of norepinephrine and amphetamine: direct versus indirect action, Pharmacol. Biochem. Behav., 2 (1974) 79-86. Shreve, EE. and Uretsky, N.J., Role of quisqualic acid receptors in the hypermotility response produced by the injection of AMPA into the nucleus accumbens, Pharmacol. Biochem. Behav., 30 (1988) 379-384. Swerdlow, N.R. and Koob, G.F., Norepinephrine stimulates behavioral activation in rats following depletion of nucleus accumbens dopamine, Pharmacol. Biochem. Behav., 33 (1989) 595-599.
265
:O t992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
NSL 08574
Stimulation of locomotor activity by intra-accumbens AMPA is not inhibited by neonatal 6-hydroxydopamine-induced lesions R.C. Boldry*, R.T. Layer, L.J. Wallace a n d N.J. U r e t s k y Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, OH 43210 (USA) (Received 19 August 1991; Revised version received 10 December 1991; Accepted 4 February 1992)
Key words: Amphetamine; Excitatory amino acid; AMPA; Dopamine; Locomotor activity; 6-Hydroxydopamine The involvement of dopamine in the hypermotility responses to amphetamine s.c. or AMPA injected into the nucleus accumbens was evaluated in adult rats depleted of dopamine as neonates with 6-hydroxydopamine. The hypermotility response to amphetamine was markedly inhibited in the lesioned animals~ whi!e that to AMPA was enhanced. In addition, the hypermotility produced by AMPA in these rats was not inhibited by sulpiride + SCH-23390; however, it was inhibited completely by ~-methyl-p-tyrosine.These results suggest that the hypermotility produced by AMPA requires endogenous dopamine, but is mediated by a different mechanism than that produced by amphetamine.
~-Amino- 3-hydroxy- 5-methylisoxazole-4-propionic acid (AMPA) is an excitatory amino acid which is thought to activate a specific subtype of glutamate receptor in the central nervous system [20]. When injected into the nucleus accumbens of rats, this compound produces an intense stimulation of coordinated locomotor activity [2, 26]. This effect is attenuated by the co-administration of antagonists of the AMPA receptor, suggesting that the hypermotility response elicited by AMPA is mediated via stimulation of these receptors. However, in addition to the activation of AMPA receptors, the hypermotility response to AMPA requires the activation of dopaminergic receptors. This conclusion is supported by the observations that the hypermotility response to AMPA is attenuated by dopaminergic receptor antagonists or by treatments that interfere with the storage and release of dopamine [3]. In addition, microdialysis experiments have shown that the local infusion of AMPA or quisqualic acid into the nucleus accumbens increases the extracellular concentration of dopamine [3, 13]. Although the involvement ofdopamine in the expression of the hypermotility response to AMPA has been demonstrated by a variety of perturbations of the dopaminergic system, it is unclear whether the *Present address: Experimental Therapeutics Branch, National Institute of" Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
Corre~spondence: N.J. Uretsky, 500 W. 12th Avenue, Columbus, OH 43210, U.S.A.
response to AMPA involves mainly a release of dopamine or an interaction between dopaminergic and AMPA receptors at sites postsynaptic to the dopaminergic synapse. Amphetamine is considered the prototype indirectly acting dopaminergic agonist, and the locomotor stimulant response to amphetamine is dependent upon increased dopaminergic neurotransmission in the nucleus accumbens [14, 15, 22]. Consistent with this conclusion is the observation that lesions of dopaminergic neurons of neonatal rats with 6-hydroxydopamine antagonize the locomotor stimulation produced by amphetamine or reverse the effect of amphetamine so that it decreases locomotor activity [8, 12, 19]. The present study compares the locomotor stimulant effect of AMPA with that of amphetamine in neonatal 6-hydroxydopamine-lesioned rats in order to determine whether the loss of dopaminergic neurons impairs the response to AMPA as it does to amphetamine. Pregnant Sprague-Dawley rats were obtained from Harlan Farms approximately 1 week antepartum. The offspring were lesioned with 6-hydroxydopamine at 3 days of age [6]. Twenty rain after treatment with desipramine (25 mg/kg, s.c.) neonates were anesthetized with ether and a midline incision was made in the scalp. Using a Hamilton syringe, 6-hydroxydopamine hydrobromide (75/~g as the fi'ee base, dissolved in 5 ¢tl of saline) or saline was injected bilaterally into the lateral ventricles. The incision was closed by applying flexible collodion to the scalp, and the animals were returned to their home cage. The rats remained housed in a temperature con-
266 7000. EZTZ SHAM TREATED ~ 6 - O H D A TREATED
6000<
5000
~ 4000 o 3000 0
2000 1000
SALINE
AMPH
SALINE
AMPH
Fig. 1. The effect of neonatal 6-hydroxydopamine (6-OHDA) lesions on the stimulation of locomotor activity by d-amphetamine. Rats injected with 6 - O H D A as neonates and sham-treated controls were injected with d-amphetamine (0.75 mg/kg, s.c.) or vehicle, and locomotor activity was measured for 1 h. Each bar represents the mean _+ S.E.M. for 4 observations. "*P < 0.01 when compared to saline-treated controls, Student's t-test.
trolled environment with 12 h light-dark cycles for 3-5 months before behavioral testing. Direct stereotaxic injections of drugs into the nucleus accumbens of rats anesthetized with a mixture of halothane and oxygen were performed as previously described [26]. Drugs, dissolved in 1.0 #1 of phosphate buffer, were injected bilaterally into the nucleus accumbens at a rate of 1 #l/min. d-Amphetamine sulfate was dissolved in normal saline and injected subcutaneously. The animals were then placed in cages for monitoring locomotor activity, and starting ten minutes after the injections, their locomotor activity was recorded for one hour. Locomotor activity was assessed using cages equipped with a grid of infrared beams (Opto Varimex-minor, Columbus Instrument, Columbus, OH). Ambulatory activity was determined as the number of times 2 or more consecutive beams, 3.5 cm apart, were interrupted. The data were recorded by a digital counter (Columbus Instruments). The measurement of locomotor activity was always performed between 12.00 and 16.00 h. Tissue dopamine concentrations were determined in the rats used in the amphetamine study 2 weeks after behavioral experiments. Rats were decapitated, and the brains were removed and placed on ice. The nucleus accumbens was dissected, weighed, and homogenized in 0.4 N perchloric acid. The homogenate was centrifuged, and the dopamine content of the supernatant was determined by HPLC with electrochemical detection [17]. Results of all experiments are expressed as means + S.E.M. and significance levels were determined using either analysis of variance followed by the NewmanKeuls test or the unpaired Student's t-test. The administration of d-amphetamine produced a significant stimulation of locomotor activity in rats which received sham-treatments but not in the animals which
were injected with 6-hydroxydopamine as neonates (Fig. 1). Although the animals with lesions demonstrated an increased level of spontaneous locomotor activity after direct injection with saline (compared to sham-treated controls), there was no significant change in the ambulatory activity of these animals when challenged with amphetamine. Analysis of tissue dissected from the nucleus accumbens of these animals demonstrated that treatment with 6-hydroxydopamine produced significant depletion of dopamine in comparison with sham-treated control animals. These results suggest that drugs which release dopamine may be ineffective in producing a stimulation of locomotion in rats which have been lesioned as neonates with 6-hydroxydopamine. In contrast to the effects of amphetamine, the direct injection of AMPA into the nucleus accumbens produced a significant stimulation of locomotor activity in both 6-hydroxydopamine-treated and sham-treated control animals. In fact, the stimulation of locomotion produced by AMPA was significantly greater in animals that were lesioned as neonates with 6-hydroxydopamine when compared to the sham-treated animals. To evaluate the role of endogenous dopamine in the expression of the stimulatory response to AMPA, we determined the effect of dopamine receptor antagonists and ~-methyl-p-tyrosine, drugs that produce a general impairment in dopaminergic neurotransmission throughout the brain, on the hypermotility response to AMPA in rats treated neonatally with 6-hydroxydopamine. In the first experiment, sulpiride, a dopaminergic D 2 receptor antagonist, was co-injected with AMPA into the nucleus accumbens at a dose of 10 #g/ side, while SCH-23390 (0.3 mg/kg, s.c.), a Dj receptor antagonist, was injected subcutaneously (due to its limited solubility in the injection vehicle) 20 min before the intra-accumbens injection of AMPA. The results show that these antagonists completely inhibited
TABLE I E F F E C T OF N E O N A T A L 6 - H Y D R O X Y D O P A M I N E (6-OHDA)I N D U C E D LESIONS O N T H E D O P A M I N E C O N T E N T O F T H E N U C L E U S A C C U M BENS Rats were injected into the lateral ventricles at 3 days postpartum with 75 # g of 6-OHDA/side. 14 clays after behavioral testing as adults, the rats were sacrificed and the dopamine content of the nucleus accumbens was determined. Treatment
n
[DA] nmol/g
% of control
Sham-treated 6-OHDA-treated
4 4
61.7 + 10.3 12.3 + 5.49**
19.9
**P < 0.01, Student's t-test.
267 AMPA-stimulated locomotor activity in sham-operated rats but did not significantly inhibit the stimulation of locomotion produced by A M P A in the 6-hydroxydopamine-treated animals (Fig, 2). In contrast, pretreatment of the 6-hydroxydopamine-lesioned animals with ~-methyl-p-tyrosine (250 mg/kg, i.p. 4 h and 1 h prior to the injections of A M P A - - 250 ng/side into the nucleus accumbens) virtually abolished AMPA-induced locom o t o r stimulation [AMPA alone: 11,934 (n -- 2), A M P A + ~-methyl-p-tyrosine: 956 + 469 (n -- 6)]. The present results show that the locomotor stimulation produced by A M P A was enhanced in animals treated as neonates with 6-hydroxydopamine, while the stimulatory effect of amphetamine was almost completely abolished. Thus, the effects of A M P A in animals, that were administered 6-hydroxydopamine as neonates to produce a lesion of dopaminergic neurons, differ from the effects of amphetamine, the prototype indirectly acting dopaminergic agonist. These results support the hypothesis that the locomotor stimulation produced by the administration of A M P A into the nucleus accumbens is not simply a function of dopamine release from nerve terminals at this site. However, endogenous catecholamines still appear to play an important role in the locom o t o r stimulatory effect of AMPA in animals that received a lesion as neonates, since c~-methyl-p-tyrosine, which has been shown previously to inhibit the stimulatory effects of A M P A in normal animals, also completely inhibited the effects of AMPA in the 6-hydroxydopamine treated animals. This observation is consistent with previous studies showing that the behavior of neonatal 6-hydroxydopamine-lesioned animals is markedly impaired by ~-methyl-p-tyrosine pretreatment [23, 24]. These results suggest that the mechanism of the hypermotility response to A M P A may be different than that to MK-801, an N-methyl-D-aspartate antagonist, since MK-801 [(+)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine] is reported to stimulate locomotion after both ~-methyl-p-tyrosine and reserpine [7]. One hypothesis to explain our observations can be derived from a recent proposal suggesting the presence of two distinct functional pools of dopamine, whose release is regulated by different processes [11]. One of these pools is postulated to be released in a transient or phasic manner by increasing the rate of impulse flow in dopaminergic neurons or by drugs such as amphetamine. Most of this pool appears to be lost in the rats that received a neonatal 6-hydroxydopamine-induced lesion, resulting in a marked decrease in the response to amphetamine [8, 12, 19]. The other pool of dopamine is the source of the tonic dopamine levels in the extracellular fluid and is thought to be regulated by glutamatergic re-
SHAM TREATED 6 - O H D A TREATED
14 12
_~
lo
~ a : a: o ~
o
8
z o
2
NS
SALINE
AMPA (250n9)
AMPA(250ng) SULPIR4DE SCH 23390
Fig. 2. The effect of neonatal 6-hydroxydopamine(6-OHDA)-induced lesions on the stimulation of locomotor activity by AMPA. Rats injected with 6-OHDA as neonates (hatched bars) and sham-treated controls were injected into the nucleus accumbens with vehicle, AMPA (250 ng), or a combination of AMPA and sulpiride (10,ug/side) into the nucleus accumbens (after pre-treatment with SCH-23390, 0.3 mg/kg, s.c.). Each bar represents the mean _+ S.E.M. for 4-9 observations. ANOVA F 5 , 3 4 21, P < 0.01. Newman-Keuls multiple comparison test: "P < 0.01 compared to the sham-saline and sham-AMPA-sulpirideSCH-23390 groups. **P< 0.01 compared to the 6-OHDA-saline, shamAMPA, and sham-AMPA-sulpiride-SCH-23390groups. There was no significant difference between the sham-saline and 6-OHDA-saline groups or between the 6-OHDA-AMPA and the 6-OHDA-AMPAsulpiride-SCH-23390 groups.
ceptors on dopaminergic terminals [11]. This pool may be the one that is critical for the hypermotility response to AMPA. Recently, it has been shown using intracranial microdialysis that after 6-hydroxydopamine lesions in neonatal rats, the extracellular striatal dopamine levels are reduced but to a relatively small extent compared with the marked depletion of tissue dopamine [8]. Thus, the pool of dopamine that may be required for the effects of excitatory amino acids, such as AMPA, may remain relatively intact after 6-hydroxydopamine lesions, and this may account for the failure of the lesion of DA neurons to inhibit the locomotor-stimulatory effects of AMPA as it did amphetamine. However, according to this hypothesis, additional treatment with ~-methyl-p-tyrosine should deplete this pool, resulting in a complete inhibition of the locomotor stimulation produced by AMPA. The locomotor stimulatory effect of AMPA in 6-hydroxydopamine-lesioned rats may also in part be related to a functional postsynaptic supersensitivity to dopaminergic agonists that develops in response to the 6-hydroxydopamine-induced lesions [1, 4, 5, 9]. Evidence for a change in dopamine receptor sensitivity is derived from the observation that the combination of D~ and D2 dopaminergic receptor blocking agents, which antagonized the effects of AMPA in the sham-lesioned animals, did not antagonize the effects of AMPA in the neonatally lesioned animals. This effect is reminiscent of the re-
268
ported subsensitivity of neonatal lesioned rats to the effects of dopaminergic antagonists, such as haloperidol, fluphenazine, and SCH-23390 [6, 10]. At present the mechanism in which neonatal lesioned rats become refractory to the effects of dopaminergic antagonists remains unresolved. Our studies cannot exclude a role for endogenous norepinephrine in the locomotor stimulant response of neonatal-lesioned animals to AMPA. In the present study, the 6-hydroxydopamine was administered to neonatal animals after desipramine, an inhibitor of norepinephrine uptake which has been shown to protect norepinephrine neurons from the toxic effects of 6-hydroxydopamine. Therefore, the brain norepinephrine levels would remain relatively intact after 6-hydroxydopamine [19], but would be decreased by a-methyl-p-tyrosine, which might account for the ability of this latter drug to inhibit the response to AMPA. Furthermore, previous studies have shown that norepinephrine, infused into the ventricles, produces locomotor stimulation, which is enhanced in animals pretreated with 6hydroxydopamine [25, 27], and this response appears to be mediated by an action on supersensitive dopaminergic receptors [27]. However, not all the evidence supports a role for norepinephrine in the locomotor stimulant response. For example, amphetamine is a potent releaser of norepinephrine as well as dopamine [18, 21]. If the hyperactivity response were mediated through the release of norepinephrine, then the stimulatory response to amphetamine should not have been abolished in neonatal rats injected with desipramine before 6-hydroxydopamine. Regardless of the mechanism, the observation that c~methyl-p-tyrosine pretreatment completely antagonized the locomotor stimulant response to AMPA supports the concept that in neonatal 6-hydroxydopamine-lesioned animals, like in normal animals [3], the stimulation of locomotion in the nucleus accumbens produced by either AMPA or dopaminergic agonists requires the activation of both AMPA and dopaminergic receptors in the nucleus accumbens. 1 Abrous, D.N., Rivet, J.M., Le Moal, M. and Herman, J.E, Similar post-lesion receptor adjustments following the unilateral 6-hydroxydopamine lesion of the dopamine mesotelencephalic system in neonatal and adult rats, Brain Res., 526 (1990) 195-202. 2 Arnt, J., Hyperactivity following injection of a glutamate agonist and 6,7-ADTN into rat nucleus accumbens and its inhibition by THIE Life Sci., 28 (1981) 1597-1603. 3 Boldry, R.C., Willins, D.L., Wallace, L.J. and Uretsky, N.J., The role of endogenous dopamine in the hypermotility response to intraaccumbens AMPA. Brain Res. 559 (1991) 100 108.
4 Breese, G.R., Napier, T.C. and Mueller, R.A., Dopamine agonistinduced locomotor activity in rats with 6-hydroxydopamine at differing ages: functional supersensitivity of D~ receptors in neonatally lesioned rats, J. Pharmacol. Exp. Ther., 234 (1985) 447-455. 5 Breese, G.R., Duncan, G.E., Napier, C., Bondy, S.C., Iorio, L.C. and Mueller, R.A., 6-Hydroxydopamine treatments enhance behavioral responses to intracerebral microinjection of D t and D 2 dopamine agonists into nucleus accumbens and striatum without changing dopamine antagonist binding, J. Pharmacol. Exp. Thcr., 240 (1987) 167-176. 6 Bruno, J.l~, Stricker, E.M. and Zigmond, M.J., Rats given dopamine-depleting brain lesions as neonates are subsensitive to dopaminergic antagonists as adults, Behav. Neurosci., 99 (1985) 771 775. 7 Carlsson, M. and Carlsson, A., The NMDA antagonist MK-801 causes marked locomQtor stimulation in monoamine-depleted mice, J. Neural Transm., 75 (1989) 221 226. 8 Casteneda, E., Whishaw, I.Q., Lermer, L. and Robinson, T.E., Dopamine depletion in neonatal rats: effects on behavior and striatal dopamine release assessed by intracerebral microdialysis during adulthood, Brain Res., 508 (1990) 30 38. 9 Dewar, K.M., Soghomonian, J.J., Bruno, J.P., Descarries, L. and Reader, T.A., Elevation of dopamine D, but not Dj receptors in adult rat neostriatum after neonatal 6-hydroxydopamine denervation, Brain Res., 536 (1990) 287 296. 10 Duncan, G.E., Criswell, H., McCown, T.J., Paul, I.A., Mueller, R.A. and Breese, G.R., Behavioral and neurochemical responses to haloperidol and SCH-23390 in rats treated neonatally or as adults with 6-hydroxydopamine, J. Pharmacol. Exp. Ther.. 243 (1987) 1027 1034. 11 Grace, A.A., Phasic versus tonic dopamine release and the modulation of dopamine system responsivity: a hypothesis for the etiology of schizophrenia, Neuroscience, 41 ( 1991 ) 1 24. 12 Heffner, T.G. and Selden, L.S., Possible involvement of serotonergic neurons in the reduction of locomotor hyperactivity caused by amphetamine in neonatal rats depleted of brain dopamine, Brain Res., 244 (1982) 81-90. 13 lmperato, A., Honore, T. and Jensen, L., Dopamine release in the nucleus caudatus and in the nucleus accumbens is under glutamatergic control through non-NMDA receptors: a study in freely moving rats, Brain Res., 530 (1990) 223 228. 14 Joyce, E.M. and Koob, G.F., Amphetamine-, scopolamine-, and caffeine-induced locomotor activity following 6-hydroxydopaminelesions of the mesolimbic dopamine system, Psychopharmacology, 73,(1981)311 313. 15 Kelly, RH., Seviour, P.W. and Iversen, S.D., Amphetamine and apomorphine responses in the rat following 6-OHDA lesions of the nucleus accumbens septi and corpus striatum, Brain Res., 94 (1975) 507 522. 16 K6nig, J.R.F. and Klippel, R.A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and the Lower Parts of the Brain Stem, Krieger, New York, 1963. 17 Kotake, C., Heffner, T., Vosmer, G. and Seiden, L., Determination ofdopamine, norepinephrine, serotonin, and their major metabolic products in rat brain by reverse-phase ion-pair high performance liquid chromatography with electrochemical detection, Pharmacol. Biochem. Behav., 22 (1985) 85 89. 18 Kuczenski, R., Biochemical actions of amphetamine and other stimulants. In 1. Creese (Ed.), Stimulants: Neurochemical, Behavioral, and Clinical Perspectives, Raven, New York, 1983, pp. 31 61. 19 Luthman, J., Fredriksson, A., Lewander, T., Jonsson, G. and
269
20
21
22
23
Archer, T., Effects of d-amphetamine and methylphenidate on hyperactivity produced by neonatal 6-hydroxydopamine treatment, Psychopharmacology, 4 (1989) 550-557. Monaghan, D.T., Bridges, R.J. and Cotman, C.W., The excitatory amino acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system, Annu. Rev. Pharmacol. Toxicol., 29 (1989) 365~,02. Moore, K.E., Amphetamines: biochemical and behavioral actions in animals. In L.L. Iversen, S.D. Iversen and S.H. Snyder (Eds.), Handbook of Psychopharmacology, Plenum, New York, 1978, pp. 41-85. Pijnenburg, A.J.J., Honig, W.M.M. and Van Rossum, J.M., Inhibition of d-amphetamine-induced locomotor activity by injection of haloperidol into the nucleus accumbens, Psychopharmacologia, 41 (1975) 87-95. Potter, B.M. and Bruno, J.P., Food intake of rats depleted of do-
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25
26
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pamine as neonates is impaired by inhibition of catecholamine biosynthesis, Neurosci. Lett., 107 (1990) 295-300. Rogers, D.C. and Dunnett, S.B., Hypersensitivity to c~-methyl-ptyrosine suggests that behavioral recovery of rats receiving neonatal 6-hydroxydopamine lesions is mediated by residual catecholamine neurons, Neurosci. Lett., 102 (1989) 108-113. Segal, D., McAllister, C. and Geyer, M., Ventricular infusions of norepinephrine and amphetamine: direct versus indirect action, Pharmacol. Biochem. Behav., 2 (1974) 79-86. Shreve, EE. and Uretsky, N.J., Role of quisqualic acid receptors in the hypermotility response produced by the injection of AMPA into the nucleus accumbens, Pharmacol. Biochem. Behav., 30 (1988) 379-384. Swerdlow, N.R. and Koob, G.F., Norepinephrine stimulates behavioral activation in rats following depletion of nucleus accumbens dopamine, Pharmacol. Biochem. Behav., 33 (1989) 595-599.