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Inhibition by phencyclidine of excitatory amino acid-stimulated release of neurotransmitter in the nucleus accumbens
0028-3908/87 $3.00 + 0.00 Copyright 0 1987 Pergamon Journals Ltd
Neuropharmacology Vol. 26, No. 213, pp. 173479, 1987 Printed in Great Britain. All rights reserved
INHIBITION BY PHENCYCLIDINE OF EXCITATORY AMINO ACID-STIMULATED RELEASE OF NEUROTRANSMITTER IN THE NUCLEUS ACCUMBENS SUSAN M. JONES, L. D. SNELL and K. M. JOHNSON Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77550, U.S.A. (Accepted
24 March 1986)
Summary-The effects of phencyclidine (PCP) on the release of acetylcholine and dopamine, stimulated by excitatory amino acid agonists was examined in slices of nucleus accumbens of the rat. In slices incubated in [‘HIcholine or [‘Hldopamine, the amount of tritium efflux produced by 1 mM N-methyl-Daspartate (NMDA), kainic acid (KA) or quisqualic acid (QA) was compared with that produced in the presence of varying concentrations of phencyclidine. N-Methyl-D-aspartate stimulated the calcium-dependent release of both ACh and DA, which was completely inhibited by physiological concentrations of magnesium and inhibited by 2-aminophosphonovalerate (2-APV). Kainic acid- and quisqualic acid-stimulated release of ACh and DA was partially
inhibited by magnesium or by 2-APV. Phencyclidine inhibited NMDA-stimulated release of ACh and DA with IC,‘s around 100nM. Phencyclidine (0.1 PM) also significantly inhibited kainic acid and quisqualic acid-induced release of ACh in magnesium-free but not magnesium-containing buffer, suggesting that the effect of PCP on kainic acid- and possibly quisqualic acid-stimulated
release of ACh is on that part of the response which is mediated by NMDA receptors. The results suggest that the inhibition by PCP of the release of ACh and DA in the nucleus accumbens is selective for NMDA-type receptors. Key words: nucleus accumbens, acetylcholine methyl-D-aspartate, excitatory amino acids.
Phencyclidine (PCP) has been reported to have effects on several neurotransmitter systems, including acetylcholine (ACh) and the monoamines. It has previously been reported that PCP inhibited excitatory amino acid-stimulated release of ACh and dopamine (DA) from slices of striatum (Snell and Johnson, 1985, 1986). Glutamate and aspartate-stimulated release of ACh was shown to be sensitive to magnesium (Mg’+). Voltage-dependent sensitivity t.o magnesium has been demonstrated to be characteristic of responses due to activation of N-methyl-D-aspartate (NMDA) receptors (Evans, Francis and Watkins, 1978; McCulloch, Johnston, Game and Curtis, 1974; Mayer, Westbrook and Guthrie, 1984; Crunelli and Mayer, 1984). The release of ACh, stimulated by NMDA, but not by kainic acid (KA) or quisqualic acid (QA) was inhibited by PCP, demonstrating that the effect of PCP on the release of ACh was selective for the NMDA receptor subtype. However, PCP inhibited the release of DA stimulated by all three agonists, suggesting that the effects of PCP on the release of DA are mediated by non-NMDA receptors as well as by NMDA receptors (Snell and Johnson, 1986). Other PCP-like drugs inhibited the release of ACh and DA induced by NMDA in a stereoselective manner which correlated with their affinity for the PCP/sigma binding site and their effects on rotational behavior induced by PCP (Snell and Johnson, 1985; Johnson and Snell, 1985). These studies suggest that
release,
dopamine
release,
phencyclidine,
N-
the effect of PCP may be to modulate the ionic conductance mechanisms underlying activation of NMDA receptors leading to the release of ACh and DA in slices of striatum. The nucleus accumbens, like the striatum, receives a cortical input from both the allocortex and neocortex, which may be glutamatergic (Walaas and Fonnum, 1979; Fonnum and Walaas, 1980; Carter, 1980). The nucleus accumbens also receives a dopaminergic innervation, predominantly from cell bodies in the ventral tegmental area (Lindvall and Bjorklund, 1978) and contains cholinergic interneurons (Fonnum, Walaas and Iverson, 1977; Walaas and Fonnum, 1979). It has been reported that L-glutamic acid stimulates the release of [3H]DA from slices of the nucleus accumbens and striatum (Roberts and Anderson, 1979; Marien, Brien and Jhamandas, 1983; Snell and Johnson, 1985, 1986). This release was Ca2+-dependent, sensitive to Mg’+, and was decreased by the glutamate receptor antagonist, glutamic acid diethylester (Marien et al.. 1983). Recently, French, Pilapil and Quirion (1985) have reported the existence of binding sites for PCP in the nucleus accumbens. Lesions of the dopaminergic innervation to the nucleus accumbens resulted in the loss of 62% of the binding sites, suggesting that many of these sites are located on dopaminergic terminals in the nucleus accumbens. Because of the possible similarities in interactions between excitatory amino
173
SUSANM. JONESet al.
114
acids, ACh and DA in the striatum and nucleus accumbens, the effect of PCP on the ability of excitatory amino acids to stimulate the release of ACh and DA was examined, in slices of accumbens from the rat. METHODS
Male albino Sprague-Dawley rats (180-200 g) from Holtzman Co. (Madison, Wisconsin) were decapitated and the brains removed and rinsed with ice-cold 0.9% NaCl. A coronal cut was made at the rostra1 edge of the olfactory tubercle and a second cut was made 1.8-2.0 mm caudal to the first, at the rostra1 edge of the optic chiasma. This coronal slab was placed, caudal side up, on an ice-cold surface. The olfactory tubercles and all tissue subjacent to them were removed. A cut was made 0.7 mm lateral and parallel to either side of the midline of the slice. Lastly, a cut was made connecting the ventral aspect of the lateral ventricle and passing just dorsal to the anterior commissure. The resulting triangular wedges of tissue, surrounding the anterior commissure, each contained the nucleus accumbens. The tissue from two rats was pooled for each experiment. The wedges of brain tissue were sliced to a thickness of 0.4mm with a McIlwain tissue chopper, gently separated and preincubated in a modified Tyrode’s buffer (in mM: NaCl, 117; KCl, 4.7; MgCI,, 1.2; CaCl,, 1.25; NaH,PO,, 1.25; NaHCO,, 25; glucose, 11.5) and adjusted to pH 7.4 (by bubbling with a 95-5% mixture of O2 and CO*) for 10 min at 37°C. In some experiments [3H]choline hydrochloride was added (50 nM final concentration) to the buffer while, in other experiments [3H]DA was added (1OOnM final concentration) to the buffer which also contained 10 PM pargyline and 1 mM ascorbic acid. In either case the incubation continued for 30 min. At the end of the period of incubation the slices were rinsed in fresh buffer and four or five slices were placed on stainless steel mesh supports in a superfusion chamber (0.5ml volume) that was assembled in a 37’C water bath. The slices were then superfused with the buffer solution at a constant flow rate of l.Oml/min by means of a Gilson Minipuls 2 peristaltic pump. Slices incubated with [3H]choline were superfused with modified Tyrode’s buffer that also contained 10pM hemicholinium-3 to prevent the reuptake of [‘HIcholine hydrolyzed from newly released [‘H]ACh (Richardson and Szerb, 1974). Several dual-label studies were performed in which the tissue was incubated with [‘HIDA and [‘4C]choline. In these experiments, the superfusion buffer contained ascorbate, pargyline and hemicholinium-3. The results of these experiments were comparable to the single-label experiments. In agreement with others, it was found that NMDA-induced release of ACh and DA were both strongly inhibited by magnesium ions (Lehmann and Scatton, 1982; Roberts and Anderson, 1979; Snell and Johnson,
1985; 1986). Therefore, unless otherwise indicated, the superfusion buffer did not contain magnesium. The superfusate was collected in culture tubes in 3 minute fractions and the radioactivity was estimated by liquid scintillation spectrometry. Slices were superfused for 45 min to allow for baseline stabilization and the first fraction was collected between 45 and 48min. After three baseline fractions had been collected, the superfusion buffer was switched to one containing either normal buffer or to buffer containing a given concentration of the test drug. After an additional 3 fractions (9 min), the slices were superfused for 2 min with an identical buffer which con, tained an excitatory amino acid agonist (1 mM). The choice of this concentration of agonist was based on findings in the striatum where 1 mM NMDA produced maximal stimulated release of ACh and DA, while 1 mM kainic acid and quisqualic acid produced smaller, but measurable responses (Snell and Johnson, 1986). After this period, the superfusion buffer was changed back to the buffer containing the test drug only for the remainder of the collection period (30 min total). The fractional efflux of tritium was estimated as the fraction of the amount of radioactivity in the superfusate, relative to the total amount in the slices at that particular time, multiplied by 100. The radioactivity remaining in the slices at the end of the experiment was estimated by counting the radioactivity in an aliquot of the homogenate, after homogenization of the slices. The amount of release of tritium stimulated by NMDA or other amino acids was calculated by totalling the amount of fractional release above baseline in the fractions after the addition of the amino acid to the buffer. This total was divided by the baseline release (fraction 6) to account for slight variations in spontaneous efflux. This yielded a single value, representing the magnitude of stimulated release, relative to the baseline. In these experiments, under the conditions specified, the release of tritium was used as indices of the release of either ACh or DA. Several studies have demonstrated that the stimulated release of tritium in the presence of 10 PM hemicholinium-3 from slices loaded with [3H]choline is a good index of the release of ACh (Richardson and Szerb, 1974; Leventer and Johnson, 1984; Lehmann and Scatton, 1982). Similarly, tritium released from slices of striatum, incubated with a small concentration of [3H]DA in the presence of a monoamine oxidase inhibitor is thought to be indicative of the release of DA (Roberts and Anderson, 1979). Therefore, in the remainder of the text NMDA-induced release of tritium over baseline is referred to as NMDA-induced release of ACh, or NMDA-induced release of DA. Similar nomenclature is used to describe the release of tritium over baseline in response to the other amino acids. In comparisons between control and experimental conditions, statistical significance was tested by the use of independent t-tests.
PCP effects on ACh and DA release in nucleus
accumbens
175
from New England Nuclear (Boston, Massachusetts). All other drugs and chemicals were obtained from commerical sources. RESULTS
Excitatory
_L
NMUA +
TTX
0
Ca
+
A+O++
+
Mg APV
TTX
Ca
Mg
APV
Fig. 1. Effects of different experimental conditions on the release of [3H]ACh stimulated by 1 mM N-methyl-oaspartate (NMDA) or 1 mM kainate (KA). After preincubation, the slices were superfused with magnesium-free buffer (open bars) or buffer containing 0.5 PM tetrodotoxin (TTX), buffer with CaCl, excluded plus the addition of 1 mM ethylenebis(oxyethylenenitrile)-tetra acetic acid (EGTA) (0 Ca), buffer containing 1.2 mM MgCl, (+ Mg), or 300 PM 2-aminophosphonovalerate (APV). Fractional release is the increase in the efflux of [3H]ACh above baseline stimulated by NMDA or kainic acid as described in the text. Values are the mean ( f SE) of independent experiments.
The N values for kainic acid and kainic acid + Mg were 11 and 13, respectively, while all others ranged between 4 and 7. (*Significantly different from control (NMDA alone), P < 0.05; **significantly different from control (NMDA or kainic acid), P -c0.01; Student’s r-test). Phencyclidine was obtained from Dr Robert Willette of the National Institute on Drug Abuse (Bethesda, Maryland). The drugs 3,4-[7-[3H]dihydroxyphenylethylamine (27 Ci/mmol) and [methyl‘HI-choline chloride (80 Ci/mmol) were purchased
amino acid-stimulated
release of [3H]ACh
An increase in the release of ACh above baseline was stimulated by 1 mM NMDA, quisqualic acid and kainic acid with NMDA producing the largest increase in fractional release in Mg-free buffer. The EC& for NMDA-stimulated released of ACh was 75pM (95% CL. of 51-110 PM). The release of ACh, stimulated by 1 mM NMDA, was completely inhibited when 0.5 PM tetrodotoxin (TTX; P < 0.01) or magnesium (1.2 mM MgCl,; P < 0.01) was added to the superfusion buffer (Fig. 1). The release of ACh, stimulated by 1 mM NMDA, was also inhibited 48% by 300 PM 2-aminophosphonovalerate (ZAPV; P -z 0.05), an antagonist of NMDA receptors. Three mM 2-APV inhibited NMDA-stimulated release of ACh by 94% (data not shown). Pencyclidine inhibited NMDA-stimulated release of ACh in a concentration-dependent manner with an IC,, of 0.17pM (95 CL. of 0.094.30pM; Fig. 2). The release stimulated by NMDA was greatly reduced when CaCl, was omitted and 1 mM EGTA was added to the magnesium-free buffer (Fig. 1). However, under these conditions the spontaneous release of ACh increased almost two-fold, possibly due to the lack of both calcium and magnesium. The release of ACh stimulated by 1 mM kainic acid was also inhibited by PCP, with 0.1 p M PCP (the IC,, concentration for NMDA-stimulated release of ACh) producing 47% inhibition (P < 0.01; Table 1). This concentration also appeared to be maximal, because
* t+x q
NMDA
o KA
6
z
zl
l
1.5P--/k-_
z! 1.0
2 .g
l
l
*
l
*
0.5
t
**
F
lJ_
*
0
0
-7
-6
Log
CPCPI
-6
-5
(M )
Fig. 2. Effect of PCP on the release of [‘H]ACh stimulated by 1 mM NMDA or 1 mM kainic acid (KA). After preincubation with [3H] choline, the slices were superfused in magnesium-free buffer. Then, PCP was added at the beginning of the fourth fraction and release stimulated by the addition of NMDA or kainic acid to the buffer at fraction seven. Fractional release is the amount of stimulated release above baseline as described in the text. Each point is the mean ( &- SE) of 54 (drug conditions), 7 (NMDA control) or 11 (kainic acid control) independent experiments. (**significantly different from control condition, P < 0.01; Student’s t-test).
176
SUSAN M. JONESet al.
Table 1. The effect of PCP on the release of [‘H]ACh stimulated NMDA, kainic acid (KA) and quisqualic acid (QA) Stimulus
Control
1 mM NMDA 1mMKA 1mMQA
Fractional N
2.67 f 0.36 1.37f0.08 0.75+0.11
7 11 5
release (x 100) 0.1 uMPCP 1.3650.14 0.72 +0.16 0.39 +0.06
by
N 5 5 5
* ** * l
After preincubation with [‘HIcholine, the slices were superfused in magnesium-free buffer. Phencyclidine (PCP) was added beginning at the fourth fraction collected, and excitatory amino acids were added to stimulate release, beginning at the seventh fraction. Fractional release is the stimulated release above baseline in the absence or presence of PCP. All values are the mean + SE of the indicated number of independent experiments. Significant differences from corresponding controls: *P < 0.05, **P < 0.01 (Student’s r-test).
1.Op M PCP did not produce greater inhibition (Fig. 2). Kainic acid-stimulated release of ACh was inhibited 48% by the presence of 0.5 PM tetrodotoxin (P < 0.05; Fig. 1). Kainic acid-stimulated release of ACh was also inhibited 31% by 300 PM 2-APV (P < O.Ol), but was not significantly inhibited by the presence of Mg2+ (Fig. 1). Kainic acid stimulated release of ACh was also significantly reduced by the exclusion of calcium from the magnesium-free buffer (Fig. 1). The release of ACh stimulated by 1 mM quisqualic acid was inhibited 48% by 0.1 PM PCP (P < 0.05; Table l), and was reduced 79% and 49% by tetrodotoxin (P < 0.01) and Mg2+ (P < 0.05) respectively (data not shown). Because sensitivity to magnesium and 2-APV are indicative of an action on NMDA receptors, the effects of PCP on kainic acidstimulated release of ACh were examined in the presence of magnesium, when presumably responses to NMDA would be blocked. Under these conditions, the fractional release of [3H]ACh, stimulated
+
0
TTX
Co
+
KA
+
+ TTX
Mg APV
0
+
Co Mg
+ APV
Fig. 4. The release of [‘HIDA stimulated by 1 mM NMDA or 1 mM kainic acid and the effects of different experiment conditions. After preincubation, the slices were superfused with magnesium-free buffer (open bars) or buffer containing 0.5pM tetrodotoxin (TTX), buffer with CaCl, excluded plus the addition of 1 mM EGTA (0 Ca), buffer containing 1.2 mM MgCl, (+ Mg), or 300 p M 2-aminophosphonovalerate (APV). Fractional release is the increase in the efflux of [‘HIDA above baseline stimulated by NMDA or kainic acid as described in the text. The Nvalues for NMDA and kainic acid were 16 and 19, respectively, while all others ranged between 4 and 8. (*significantly different from control (kainic acid alone), P < 0.05; **significantly different from respective control, P < 0.01; Student’s r-test).
by 1 mM kainic acid (1.27 f 0.09, n = 4), was not significantly inhibited by 0.1 PM PCP (1.19 f 0.16, n = 8). The finding that PCP did not inhibit kainic acid-stimulated release of ACh in the presence of magnesium suggests that in the absence of magnesium, the inhibition of kainic acid-stimulated release by PCP may have been of that part of the response due to activation of NMDA receptors.
q
NMDA
0 KA
I=
/ -10 ’
I
-9 Log
I
I
-8 CPCPI
-7
I
1
-6
-5
(Ml
Fig. 3. Effect of PCP on the release of [3H]DA induced by 1mM NMDA or 1 mM kainic acid (KA). After preincubation with [rH]DA, the slices were superfused in magnesium-free buffer; PCP was added at the beginning of the fourth fraction and release stimulated by the addition of NMDA or kainic acid to the buffer at fraction seven. Fractional release is the amount of stimulated release above baseline as described in the text. Each point is the mean ( f SE) of 5-l 1 (drug conditions), 16 (NMDA control) or 19 (kainic acid control) independent experiments. (*significantly different from control condition, P < 0.05; **significantly different from control, P < 0.01; Student’s r-test).
PCP
effects on ACh and DA release in nucleus accumbens
Table 2. Effect of PCP on the release of [‘HIDA stimulated by NMDA, kainic acid (KA) and quisqualic acid (QA) Stimulus 1 mM NMDA ImMKA ImMQA
Control
Fractional release (x 100) N 0.1 PMPCP A’
5.09 f 0.39 0.91 20.09 0.98 * 0.17
16 19 5
3.04 kO.51 0.92 & 0.07 0.63 jI 0.12
5 11 5
After preincubation with [sH]DA, the slices were superfused in magnesium-free buffer. Phencyclidine (PCP) was added beginning at the fourth fraction, and the excitatory amino acids were added to stimmate release, beginning in the seventh fraction. Fractional release is the release above baseline in the absence or presence of PCP. All values are the mean f SE of the indicated number of independent experiments. Significant difference from corresponding control: *P < 0.05 (Student’s t-test).
Excitatory amino acid-stimulated release of [‘HIDA
The release of DA was also stimulated by I mM NMDA, kainic acid or quisqualic acid. The EC,, of NMDA-stimulated release of DA was 120~M (95% C.L. of 9&170~M). The release of DA stimulated by 1 mM NMDA was inhibited by PCP with an I&, of 0.11 PM (95% CL. of 0.07-0.18 FM; Fig. 3). This release was completely inhibited by Mg2+ (P < O.Ol), and the presence of tetrodotoxin reduced the release by 74% (P < 0.01; Fig. 4). 2-Aminophosphonovalerate (300 PM) inhibited the NMDA-stimulated release of DA 84% (P < 0.01; Fig. 4) and 3 mM 2-APV produced 100% inhibition (data not shown). The release of DA stimulated by kainic acid was not inhibited by PCP (Fig. 3). However, 1OnM PCP produced a slight enhancement (44%; P < 0.01) of kainic acid-stimulated release of DA. There was no effect with larger concentrations of PCP (Fig. 3). The release of DA stimulated by kainic acid was inhibited 49% by tetrodotoxin (P < O.OS), 40% by Mg2+ ions (P ~0.05) and 41% by 300,~M 2-APV (P <0.05; Fig. 4). The release of DA stimulated by quisqualic acid was not affected by 0.1 p M PCP (Table 2). The release of DA stimulated by both NMDA and kainic acid was inhibited in magnesium and calcium-free buffer, although the response to NMDA was considerably more sensitive to Mg2+ than was the response to kainic acid (Fig. 4). DISCUSSION
The excitatory amino acids NMDA, kainic acid and quisqualic acid stimulate the release of neurotransmitters in several regions of the brain. In the nucleus accumbens, the release of ACh, stimulated by NMDA and kainic acid, was greatly reduced by the addition of tetrodotoxin to the buffer. Although excitatory amino acids open Na+ channels which are not sensitive to tetrodotoxin (Luini, Goldberg and Teichberg, 1981), and therefore could affect the membrane potential and release ACh (or DA) in the presence of tetrodotoxin, the sensitivity to tetrodotoxin of the present responses indicated that the release of ACh, stimulated by these excitatory amino acids, involved the activation of voltage-dependent sodium channels and the propagation of an action
177
potential, possibly through activation of receptors located on the cell body or dendrites of the cholinergic interneurons. The release of DA was also very sensitive to tetrodotoxin. However, since the dopaminergic innervation of the nucleus accumbens arises from cell bodies in the ventral tegmental area, the interpretation of the sensitivity to tetrodotoxin in this case is more difficult. Two possibilities are that either part of the release of DA, stimulated by NMDA and kainic acid, involves an inte~e~on or that the NMDA receptors are located on axons or terminals in such a way that their effect still requires the ultimate activation of voltage-dependent sodium channels. The release of [3H]DA stimulated by L-glutamate, reported by Marien et al. (1983), was insensitive to tetrodotoxin. The reason for the discrepancy between these two studies is unknown, since L-glutamate would be expected to be acting at one or more of these receptor subtypes. In the striatum, the inhibitory effect of PCP on the release of ACh induced by excitatory amino acids was specific for NMDA-induced release {Snell and Johnson, 1986). However, in the nucleus accumbens, PCP was effective at inhibiting release of ACh induced by NMDA, kainic acid or quisqualic acid. Although complete concentration-responses were not measured for PCP against all three agonists, the approximate IC,, concentration of PCP against NMDA-stimuIated release also inhibited the release of kainic acid and quisqualic acid by about 50% However, the effect of PCP on the response to kainic acid did not appear to be concentration-dependent. Also, kainic acid-induced release of ACh was significantly less sensitive to the effects of tetrodotoxin, removal of calcium, and magnesium ions, thus suggesting an action mediated by different mechanisms. Two characteristics reported to distinguish NMDA receptors from kainic acid or quisqualic acid type receptors are the voltage-dependent blockade of NMDA receptors by small concentrations of magnesium (Nowak, Bregestovski and Ascher, 1984; Mayer ef al., 1984) and the selective blockade of NMDA receptors by the antagonist 2-APV (Watkins and Evans, 1981). The inhibition of kainic acid- and quisqualic acid-stimulated release of ACh by magnesium ions and 2-APV suggests an action of these agonists at NMDA-type receptors. Consistent with this hypothesis was the finding that PCP was no longer effective at inhibiting kainic acid-stimulated release of ACh in the presence of magnesium, when responses to NMDA were totally blocked. Alternatively, part of the kainic acid- or quisqualic acidstimulated release could be due to the release of endogenous glutamate or aspartate which then activated NMDA receptors (McBean and Roberts, 1981; Ferkany and Coyle, 1983). Nevertheless, it appears that the effects of PCP on excitatory amino acidinduced release of ACh in the nucleus accumbens, as well as in the striatum, is coupled mechanistically to activation of the NMDA subtype of receptor.
SUSAN M. JONES et al.
178
In an earlier study, in the striatum, it was found that PCP inhibited the release of DA induced by NMDA, quisqualic acid and kainic acid (Snell and Johnson, 1986). Since NMDA-induced release of DA in the striatum was completely inhibited by MgZf, and PCP also inhibited the release of DA induced by quisqualic acid and kainic acid in the presence of magnesium, it was necessary to conclude that PCP could inhibit release induced by excitatory amino acids acting at receptor subtypes other than the NMDA type. However, in the present study, in the nucleus accumbens it was found that PCP had no inhibitory effect on kainic acid-stimulated release of DA. In fact, it was found that 10 nM PCP consistently enhanced kainic acid-induced release of DA, a phenomenon for which the authors have no explanation. Nonetheless, the inhibitory effects of PCP in the nucleus accumbens appear to be related only to release activated by an action on NMDA receptors. Thus, even though a small portion of kainic acidinduced release of DA was sensitive to magnesium and 2-APV (suggesting partial activation through NMDA receptors), PCP had no inhibitory effect. It is likely that the failure to observe an effect of PCP on the portion of the kainic acid-induced response that may be mediated by NMDA receptors is due to the fact that this signal is very small. That is, fractional release of DA induced by 1 mM NMDA and kainic acid is about 5.1 and 0.9, respectively. The magnesium-sensitive components are about 5.0 and 0.3, respectively. Thus, the NMDA component of the response to kainic acid is only about 6% of the analogous portion of the NMDA response. To summarize, it has been observed that PCP inhibited the release of DA and ACh, induced by excitatory amino acids in the striatum (Snell and Johnson, 1985, 1986) and in the nucleus accumbens (this study). Inhibition of the release of transmitter in the striatum by various PCP-like drugs was stereoselective and was highly correlated with their affinity for the PCP/sigma receptor site (Snell and Johnson, 1985, 1986). Further, inhibition of excitatory amino acid-induced release of ACh in the striatum and the release of DA and ACh in the nucleus accumbens appears to be associated with the activation of NMDA subtypes of receptor. However, evidence obtained in a study of the release of DA in the striatum suggests that PCP/sigma receptors may not be exclusively associated with NMDA receptors. Nevertheless, this study confirms and extends to the nucleus accumbens the observation initiallv made in the spinal cord, that PCP and similar drugs selectively antagonize the excitatory effects of glutamate-like amino acids by activating a site associated with the NMDA receptor (Anis, Berry, Burton and Lodge, 1983; Lodge and Berry, 1984). REFERENCES
Anis N. A., Berry S. C., Burton N. R. and Lodge D. (1983) The dissociative
anaesthetics,
ketamine
and phencyclidine
selectively reduce excitation of central mammalian neurones by N-methyl-aspartate. Br. J. Pharmac. 19: 565-575. Carter C. J. (1980) Glutamatergic pathways from the medial prefrontal cortex to the anterior striatum, nucleus accumbens and substantia nigra. Proc. B.P.S., pp. 50-51. Crunelli V. and Mayer M. L. (1984) Mg’+ dependence of membrane resistance increases evoked by NMDA in hippocampal neurones. Brain Res. 311: 392-396. Evans R. H., Francis A. A. and Watkins J. C. (1978) Mg*+-like selective antagonism of excitatory amino acid-induced responses by a,c-diaminopimelic acid, D-a-aminoadipate and HA-966 in isolated spinal cord of frog and immature rat. Brain Res. 148: 536542. Ferkany J. W. and Coyle J. T. (1983) Kainic acid selectively stimulates the release of endogenous excitatory amino acids. J. Pharmac. exp. Ther. 225: 3999406.
Fonnum F. and Walaas I. (1980) Localization of neurotransmitters in nucleus accumbens. In: The Neurobiology of the Nucleus Accumbens (Chronister R. B. and DeFrance D. F., Eds), pp. 259-272. Haer Inst., Brunswick. Fonnum F., Walaas I. and Iverson E. (1977) Localization of GABAergic, cholinergic and aminergic structures in the mesolimbic system. J. Neurochem. 29: 221-230. French E. D., Pilapil C. and Quirion R. (1985) Phencyclidine binding sites in the nucleus accumbens and phencyclidine-induced hyperactivity are decreased following lesions of the mesolimbic dopamine system. Eur. J. Pharmac. 116: I-9. Johnson K. M. and Snell L. D. (1985) Effects of phencyclidine (PCP)-like drugs on turning behavior, ‘H-dopamine uptake and ‘H-PCP binding. Pharmac. Biochem. Behav. 22: 731-735. Lehmann J. and Scatton B. (1982) Characterization of the excitatory amino acid receptor-mediated release of 3[H]acetylcholine from rat striatal slices. Brain Res. 252: 77-89. Leventer S. M. and Johnson K. M. (1984) Phencyclidineinduced inhibition of striatal acetylcholine release: comparison with mu, kappa and sigma opiate agonists. Life Sci. 34, 793-801. Lindvall 0. and Bjorklund A. (1978) Anatomy of the dopaminergic neuron systems in the rat brain. In: Advances in Biochemical Psychopharmacology, Dopamine, Vol. 19 (Roberts P. J., Woodruff G. N., Iverson L. L., Eds), pp. l-23. Raven Press, New York. Lodge D. and Berry S. C. (1984) Psychotomimetic effects of sigma opiates may be mediated by block of central excitatory synapses utilizing receptors for aspartate-like amino acids. In: Modulation of Sensorimotor Activity During Alterations in Behavioral States, pp. 503-518. Liss, New York. Luini A., Goldberg 0. and Teichberg V. I. (1981) Distinct pharmacological properties of excitatory amino acid receptors in the rat striatum: study bv Na+ et&x assay. . Proc. natn. Aead. Sci. U.S.A. 7fkV3250-3254.
Marien M.. Brien J. and Jhamandas K. (1983) Regional release of [‘Hldopamine from rat brain in vitro: effects of opioids on release induced by potassium, nicotine and L-glutamic acid. Can. J. Physiol. Pharmac. 61: 43-60. Mayer M. L., Westbrook G. L. and Guthrie P. B. (1984) Voltage-dependent block by Mg*+ of NMDA responses in spinal cord neurones. Nature 309: 261-263. McBean G. J. and Roberts P. J. (1981) Glutamate preferring receptors regulate the release of D-‘H-aspartate from rat hippocampal slices. Nature 291: 593-594. McCulloch R. M., Johnston G. A. R., Game C. J. A. and Curtis D. R. (1974) The differential sensitivity of spinal interneurons and Renshaw cells to kainate and Nmethyl-D-aspartate. Exp. Brain Res. 21: 515-518. Nowak L., Bregestovski P. and Ascher P. (1984) Mag-
PCP effects on ACh and DA release in nucleus accumbens nesium gates glutamate-activated channels in mouse central neurones. Nature 307: 462-465. Richardson W. and Szerb J. C. (1974) The release of labelled acetylcholine and choline from cerebral cortical slices stimulated electrically. Er. J. Pharmac. 52: 449-507. Roberts P. J. and Anderson S. D. (1979) Stimulatory effect of L-glutamate and related amino acids on 3H-dopamine release from rat striatum: an in t;itro model for glutamate actions. J. ~e~r~chern. 32: 1539-1545. Sneh L. D. and Johnson K. M. (1985) Antagonism of NMDA-induced transmitter release in the rat striatum by
119
phencyclidine-like drugs and its relationship to turning behavior. J. Pharmac. exp. Ther. 235: 50-57. Snell L. D. and Johnson K. M. (1986) Characterization of the inhibition of excitatory amino acid-induced neurotransmitter release in the rat striatum by phencyclidinelike drugs. J. Pharmac. exp. Ther. 238: 938-946. Walaas 1. and Fonnum F. (1979) The effects of surgical and chemical lesions on neurotransm~tter candidates in the nucleus accumbens of the rat. ~~ro~c~e~ce 4: 209-216. Watkins J. C. and Evans R. H. (1981) Excitatory amino acid neurotmnsmitters. Ann. Ret’. Pharmac. Tax. 21: 162-204.
Neuropharmacology Vol. 26, No. 213, pp. 173479, 1987 Printed in Great Britain. All rights reserved
INHIBITION BY PHENCYCLIDINE OF EXCITATORY AMINO ACID-STIMULATED RELEASE OF NEUROTRANSMITTER IN THE NUCLEUS ACCUMBENS SUSAN M. JONES, L. D. SNELL and K. M. JOHNSON Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77550, U.S.A. (Accepted
24 March 1986)
Summary-The effects of phencyclidine (PCP) on the release of acetylcholine and dopamine, stimulated by excitatory amino acid agonists was examined in slices of nucleus accumbens of the rat. In slices incubated in [‘HIcholine or [‘Hldopamine, the amount of tritium efflux produced by 1 mM N-methyl-Daspartate (NMDA), kainic acid (KA) or quisqualic acid (QA) was compared with that produced in the presence of varying concentrations of phencyclidine. N-Methyl-D-aspartate stimulated the calcium-dependent release of both ACh and DA, which was completely inhibited by physiological concentrations of magnesium and inhibited by 2-aminophosphonovalerate (2-APV). Kainic acid- and quisqualic acid-stimulated release of ACh and DA was partially
inhibited by magnesium or by 2-APV. Phencyclidine inhibited NMDA-stimulated release of ACh and DA with IC,‘s around 100nM. Phencyclidine (0.1 PM) also significantly inhibited kainic acid and quisqualic acid-induced release of ACh in magnesium-free but not magnesium-containing buffer, suggesting that the effect of PCP on kainic acid- and possibly quisqualic acid-stimulated
release of ACh is on that part of the response which is mediated by NMDA receptors. The results suggest that the inhibition by PCP of the release of ACh and DA in the nucleus accumbens is selective for NMDA-type receptors. Key words: nucleus accumbens, acetylcholine methyl-D-aspartate, excitatory amino acids.
Phencyclidine (PCP) has been reported to have effects on several neurotransmitter systems, including acetylcholine (ACh) and the monoamines. It has previously been reported that PCP inhibited excitatory amino acid-stimulated release of ACh and dopamine (DA) from slices of striatum (Snell and Johnson, 1985, 1986). Glutamate and aspartate-stimulated release of ACh was shown to be sensitive to magnesium (Mg’+). Voltage-dependent sensitivity t.o magnesium has been demonstrated to be characteristic of responses due to activation of N-methyl-D-aspartate (NMDA) receptors (Evans, Francis and Watkins, 1978; McCulloch, Johnston, Game and Curtis, 1974; Mayer, Westbrook and Guthrie, 1984; Crunelli and Mayer, 1984). The release of ACh, stimulated by NMDA, but not by kainic acid (KA) or quisqualic acid (QA) was inhibited by PCP, demonstrating that the effect of PCP on the release of ACh was selective for the NMDA receptor subtype. However, PCP inhibited the release of DA stimulated by all three agonists, suggesting that the effects of PCP on the release of DA are mediated by non-NMDA receptors as well as by NMDA receptors (Snell and Johnson, 1986). Other PCP-like drugs inhibited the release of ACh and DA induced by NMDA in a stereoselective manner which correlated with their affinity for the PCP/sigma binding site and their effects on rotational behavior induced by PCP (Snell and Johnson, 1985; Johnson and Snell, 1985). These studies suggest that
release,
dopamine
release,
phencyclidine,
N-
the effect of PCP may be to modulate the ionic conductance mechanisms underlying activation of NMDA receptors leading to the release of ACh and DA in slices of striatum. The nucleus accumbens, like the striatum, receives a cortical input from both the allocortex and neocortex, which may be glutamatergic (Walaas and Fonnum, 1979; Fonnum and Walaas, 1980; Carter, 1980). The nucleus accumbens also receives a dopaminergic innervation, predominantly from cell bodies in the ventral tegmental area (Lindvall and Bjorklund, 1978) and contains cholinergic interneurons (Fonnum, Walaas and Iverson, 1977; Walaas and Fonnum, 1979). It has been reported that L-glutamic acid stimulates the release of [3H]DA from slices of the nucleus accumbens and striatum (Roberts and Anderson, 1979; Marien, Brien and Jhamandas, 1983; Snell and Johnson, 1985, 1986). This release was Ca2+-dependent, sensitive to Mg’+, and was decreased by the glutamate receptor antagonist, glutamic acid diethylester (Marien et al.. 1983). Recently, French, Pilapil and Quirion (1985) have reported the existence of binding sites for PCP in the nucleus accumbens. Lesions of the dopaminergic innervation to the nucleus accumbens resulted in the loss of 62% of the binding sites, suggesting that many of these sites are located on dopaminergic terminals in the nucleus accumbens. Because of the possible similarities in interactions between excitatory amino
173
SUSANM. JONESet al.
114
acids, ACh and DA in the striatum and nucleus accumbens, the effect of PCP on the ability of excitatory amino acids to stimulate the release of ACh and DA was examined, in slices of accumbens from the rat. METHODS
Male albino Sprague-Dawley rats (180-200 g) from Holtzman Co. (Madison, Wisconsin) were decapitated and the brains removed and rinsed with ice-cold 0.9% NaCl. A coronal cut was made at the rostra1 edge of the olfactory tubercle and a second cut was made 1.8-2.0 mm caudal to the first, at the rostra1 edge of the optic chiasma. This coronal slab was placed, caudal side up, on an ice-cold surface. The olfactory tubercles and all tissue subjacent to them were removed. A cut was made 0.7 mm lateral and parallel to either side of the midline of the slice. Lastly, a cut was made connecting the ventral aspect of the lateral ventricle and passing just dorsal to the anterior commissure. The resulting triangular wedges of tissue, surrounding the anterior commissure, each contained the nucleus accumbens. The tissue from two rats was pooled for each experiment. The wedges of brain tissue were sliced to a thickness of 0.4mm with a McIlwain tissue chopper, gently separated and preincubated in a modified Tyrode’s buffer (in mM: NaCl, 117; KCl, 4.7; MgCI,, 1.2; CaCl,, 1.25; NaH,PO,, 1.25; NaHCO,, 25; glucose, 11.5) and adjusted to pH 7.4 (by bubbling with a 95-5% mixture of O2 and CO*) for 10 min at 37°C. In some experiments [3H]choline hydrochloride was added (50 nM final concentration) to the buffer while, in other experiments [3H]DA was added (1OOnM final concentration) to the buffer which also contained 10 PM pargyline and 1 mM ascorbic acid. In either case the incubation continued for 30 min. At the end of the period of incubation the slices were rinsed in fresh buffer and four or five slices were placed on stainless steel mesh supports in a superfusion chamber (0.5ml volume) that was assembled in a 37’C water bath. The slices were then superfused with the buffer solution at a constant flow rate of l.Oml/min by means of a Gilson Minipuls 2 peristaltic pump. Slices incubated with [3H]choline were superfused with modified Tyrode’s buffer that also contained 10pM hemicholinium-3 to prevent the reuptake of [‘HIcholine hydrolyzed from newly released [‘H]ACh (Richardson and Szerb, 1974). Several dual-label studies were performed in which the tissue was incubated with [‘HIDA and [‘4C]choline. In these experiments, the superfusion buffer contained ascorbate, pargyline and hemicholinium-3. The results of these experiments were comparable to the single-label experiments. In agreement with others, it was found that NMDA-induced release of ACh and DA were both strongly inhibited by magnesium ions (Lehmann and Scatton, 1982; Roberts and Anderson, 1979; Snell and Johnson,
1985; 1986). Therefore, unless otherwise indicated, the superfusion buffer did not contain magnesium. The superfusate was collected in culture tubes in 3 minute fractions and the radioactivity was estimated by liquid scintillation spectrometry. Slices were superfused for 45 min to allow for baseline stabilization and the first fraction was collected between 45 and 48min. After three baseline fractions had been collected, the superfusion buffer was switched to one containing either normal buffer or to buffer containing a given concentration of the test drug. After an additional 3 fractions (9 min), the slices were superfused for 2 min with an identical buffer which con, tained an excitatory amino acid agonist (1 mM). The choice of this concentration of agonist was based on findings in the striatum where 1 mM NMDA produced maximal stimulated release of ACh and DA, while 1 mM kainic acid and quisqualic acid produced smaller, but measurable responses (Snell and Johnson, 1986). After this period, the superfusion buffer was changed back to the buffer containing the test drug only for the remainder of the collection period (30 min total). The fractional efflux of tritium was estimated as the fraction of the amount of radioactivity in the superfusate, relative to the total amount in the slices at that particular time, multiplied by 100. The radioactivity remaining in the slices at the end of the experiment was estimated by counting the radioactivity in an aliquot of the homogenate, after homogenization of the slices. The amount of release of tritium stimulated by NMDA or other amino acids was calculated by totalling the amount of fractional release above baseline in the fractions after the addition of the amino acid to the buffer. This total was divided by the baseline release (fraction 6) to account for slight variations in spontaneous efflux. This yielded a single value, representing the magnitude of stimulated release, relative to the baseline. In these experiments, under the conditions specified, the release of tritium was used as indices of the release of either ACh or DA. Several studies have demonstrated that the stimulated release of tritium in the presence of 10 PM hemicholinium-3 from slices loaded with [3H]choline is a good index of the release of ACh (Richardson and Szerb, 1974; Leventer and Johnson, 1984; Lehmann and Scatton, 1982). Similarly, tritium released from slices of striatum, incubated with a small concentration of [3H]DA in the presence of a monoamine oxidase inhibitor is thought to be indicative of the release of DA (Roberts and Anderson, 1979). Therefore, in the remainder of the text NMDA-induced release of tritium over baseline is referred to as NMDA-induced release of ACh, or NMDA-induced release of DA. Similar nomenclature is used to describe the release of tritium over baseline in response to the other amino acids. In comparisons between control and experimental conditions, statistical significance was tested by the use of independent t-tests.
PCP effects on ACh and DA release in nucleus
accumbens
175
from New England Nuclear (Boston, Massachusetts). All other drugs and chemicals were obtained from commerical sources. RESULTS
Excitatory
_L
NMUA +
TTX
0
Ca
+
A+O++
+
Mg APV
TTX
Ca
Mg
APV
Fig. 1. Effects of different experimental conditions on the release of [3H]ACh stimulated by 1 mM N-methyl-oaspartate (NMDA) or 1 mM kainate (KA). After preincubation, the slices were superfused with magnesium-free buffer (open bars) or buffer containing 0.5 PM tetrodotoxin (TTX), buffer with CaCl, excluded plus the addition of 1 mM ethylenebis(oxyethylenenitrile)-tetra acetic acid (EGTA) (0 Ca), buffer containing 1.2 mM MgCl, (+ Mg), or 300 PM 2-aminophosphonovalerate (APV). Fractional release is the increase in the efflux of [3H]ACh above baseline stimulated by NMDA or kainic acid as described in the text. Values are the mean ( f SE) of independent experiments.
The N values for kainic acid and kainic acid + Mg were 11 and 13, respectively, while all others ranged between 4 and 7. (*Significantly different from control (NMDA alone), P < 0.05; **significantly different from control (NMDA or kainic acid), P -c0.01; Student’s r-test). Phencyclidine was obtained from Dr Robert Willette of the National Institute on Drug Abuse (Bethesda, Maryland). The drugs 3,4-[7-[3H]dihydroxyphenylethylamine (27 Ci/mmol) and [methyl‘HI-choline chloride (80 Ci/mmol) were purchased
amino acid-stimulated
release of [3H]ACh
An increase in the release of ACh above baseline was stimulated by 1 mM NMDA, quisqualic acid and kainic acid with NMDA producing the largest increase in fractional release in Mg-free buffer. The EC& for NMDA-stimulated released of ACh was 75pM (95% CL. of 51-110 PM). The release of ACh, stimulated by 1 mM NMDA, was completely inhibited when 0.5 PM tetrodotoxin (TTX; P < 0.01) or magnesium (1.2 mM MgCl,; P < 0.01) was added to the superfusion buffer (Fig. 1). The release of ACh, stimulated by 1 mM NMDA, was also inhibited 48% by 300 PM 2-aminophosphonovalerate (ZAPV; P -z 0.05), an antagonist of NMDA receptors. Three mM 2-APV inhibited NMDA-stimulated release of ACh by 94% (data not shown). Pencyclidine inhibited NMDA-stimulated release of ACh in a concentration-dependent manner with an IC,, of 0.17pM (95 CL. of 0.094.30pM; Fig. 2). The release stimulated by NMDA was greatly reduced when CaCl, was omitted and 1 mM EGTA was added to the magnesium-free buffer (Fig. 1). However, under these conditions the spontaneous release of ACh increased almost two-fold, possibly due to the lack of both calcium and magnesium. The release of ACh stimulated by 1 mM kainic acid was also inhibited by PCP, with 0.1 p M PCP (the IC,, concentration for NMDA-stimulated release of ACh) producing 47% inhibition (P < 0.01; Table 1). This concentration also appeared to be maximal, because
* t+x q
NMDA
o KA
6
z
zl
l
1.5P--/k-_
z! 1.0
2 .g
l
l
*
l
*
0.5
t
**
F
lJ_
*
0
0
-7
-6
Log
CPCPI
-6
-5
(M )
Fig. 2. Effect of PCP on the release of [‘H]ACh stimulated by 1 mM NMDA or 1 mM kainic acid (KA). After preincubation with [3H] choline, the slices were superfused in magnesium-free buffer. Then, PCP was added at the beginning of the fourth fraction and release stimulated by the addition of NMDA or kainic acid to the buffer at fraction seven. Fractional release is the amount of stimulated release above baseline as described in the text. Each point is the mean ( &- SE) of 54 (drug conditions), 7 (NMDA control) or 11 (kainic acid control) independent experiments. (**significantly different from control condition, P < 0.01; Student’s t-test).
176
SUSAN M. JONESet al.
Table 1. The effect of PCP on the release of [‘H]ACh stimulated NMDA, kainic acid (KA) and quisqualic acid (QA) Stimulus
Control
1 mM NMDA 1mMKA 1mMQA
Fractional N
2.67 f 0.36 1.37f0.08 0.75+0.11
7 11 5
release (x 100) 0.1 uMPCP 1.3650.14 0.72 +0.16 0.39 +0.06
by
N 5 5 5
* ** * l
After preincubation with [‘HIcholine, the slices were superfused in magnesium-free buffer. Phencyclidine (PCP) was added beginning at the fourth fraction collected, and excitatory amino acids were added to stimulate release, beginning at the seventh fraction. Fractional release is the stimulated release above baseline in the absence or presence of PCP. All values are the mean + SE of the indicated number of independent experiments. Significant differences from corresponding controls: *P < 0.05, **P < 0.01 (Student’s r-test).
1.Op M PCP did not produce greater inhibition (Fig. 2). Kainic acid-stimulated release of ACh was inhibited 48% by the presence of 0.5 PM tetrodotoxin (P < 0.05; Fig. 1). Kainic acid-stimulated release of ACh was also inhibited 31% by 300 PM 2-APV (P < O.Ol), but was not significantly inhibited by the presence of Mg2+ (Fig. 1). Kainic acid stimulated release of ACh was also significantly reduced by the exclusion of calcium from the magnesium-free buffer (Fig. 1). The release of ACh stimulated by 1 mM quisqualic acid was inhibited 48% by 0.1 PM PCP (P < 0.05; Table l), and was reduced 79% and 49% by tetrodotoxin (P < 0.01) and Mg2+ (P < 0.05) respectively (data not shown). Because sensitivity to magnesium and 2-APV are indicative of an action on NMDA receptors, the effects of PCP on kainic acidstimulated release of ACh were examined in the presence of magnesium, when presumably responses to NMDA would be blocked. Under these conditions, the fractional release of [3H]ACh, stimulated
+
0
TTX
Co
+
KA
+
+ TTX
Mg APV
0
+
Co Mg
+ APV
Fig. 4. The release of [‘HIDA stimulated by 1 mM NMDA or 1 mM kainic acid and the effects of different experiment conditions. After preincubation, the slices were superfused with magnesium-free buffer (open bars) or buffer containing 0.5pM tetrodotoxin (TTX), buffer with CaCl, excluded plus the addition of 1 mM EGTA (0 Ca), buffer containing 1.2 mM MgCl, (+ Mg), or 300 p M 2-aminophosphonovalerate (APV). Fractional release is the increase in the efflux of [‘HIDA above baseline stimulated by NMDA or kainic acid as described in the text. The Nvalues for NMDA and kainic acid were 16 and 19, respectively, while all others ranged between 4 and 8. (*significantly different from control (kainic acid alone), P < 0.05; **significantly different from respective control, P < 0.01; Student’s r-test).
by 1 mM kainic acid (1.27 f 0.09, n = 4), was not significantly inhibited by 0.1 PM PCP (1.19 f 0.16, n = 8). The finding that PCP did not inhibit kainic acid-stimulated release of ACh in the presence of magnesium suggests that in the absence of magnesium, the inhibition of kainic acid-stimulated release by PCP may have been of that part of the response due to activation of NMDA receptors.
q
NMDA
0 KA
I=
/ -10 ’
I
-9 Log
I
I
-8 CPCPI
-7
I
1
-6
-5
(Ml
Fig. 3. Effect of PCP on the release of [3H]DA induced by 1mM NMDA or 1 mM kainic acid (KA). After preincubation with [rH]DA, the slices were superfused in magnesium-free buffer; PCP was added at the beginning of the fourth fraction and release stimulated by the addition of NMDA or kainic acid to the buffer at fraction seven. Fractional release is the amount of stimulated release above baseline as described in the text. Each point is the mean ( f SE) of 5-l 1 (drug conditions), 16 (NMDA control) or 19 (kainic acid control) independent experiments. (*significantly different from control condition, P < 0.05; **significantly different from control, P < 0.01; Student’s r-test).
PCP
effects on ACh and DA release in nucleus accumbens
Table 2. Effect of PCP on the release of [‘HIDA stimulated by NMDA, kainic acid (KA) and quisqualic acid (QA) Stimulus 1 mM NMDA ImMKA ImMQA
Control
Fractional release (x 100) N 0.1 PMPCP A’
5.09 f 0.39 0.91 20.09 0.98 * 0.17
16 19 5
3.04 kO.51 0.92 & 0.07 0.63 jI 0.12
5 11 5
After preincubation with [sH]DA, the slices were superfused in magnesium-free buffer. Phencyclidine (PCP) was added beginning at the fourth fraction, and the excitatory amino acids were added to stimmate release, beginning in the seventh fraction. Fractional release is the release above baseline in the absence or presence of PCP. All values are the mean f SE of the indicated number of independent experiments. Significant difference from corresponding control: *P < 0.05 (Student’s t-test).
Excitatory amino acid-stimulated release of [‘HIDA
The release of DA was also stimulated by I mM NMDA, kainic acid or quisqualic acid. The EC,, of NMDA-stimulated release of DA was 120~M (95% C.L. of 9&170~M). The release of DA stimulated by 1 mM NMDA was inhibited by PCP with an I&, of 0.11 PM (95% CL. of 0.07-0.18 FM; Fig. 3). This release was completely inhibited by Mg2+ (P < O.Ol), and the presence of tetrodotoxin reduced the release by 74% (P < 0.01; Fig. 4). 2-Aminophosphonovalerate (300 PM) inhibited the NMDA-stimulated release of DA 84% (P < 0.01; Fig. 4) and 3 mM 2-APV produced 100% inhibition (data not shown). The release of DA stimulated by kainic acid was not inhibited by PCP (Fig. 3). However, 1OnM PCP produced a slight enhancement (44%; P < 0.01) of kainic acid-stimulated release of DA. There was no effect with larger concentrations of PCP (Fig. 3). The release of DA stimulated by kainic acid was inhibited 49% by tetrodotoxin (P < O.OS), 40% by Mg2+ ions (P ~0.05) and 41% by 300,~M 2-APV (P <0.05; Fig. 4). The release of DA stimulated by quisqualic acid was not affected by 0.1 p M PCP (Table 2). The release of DA stimulated by both NMDA and kainic acid was inhibited in magnesium and calcium-free buffer, although the response to NMDA was considerably more sensitive to Mg2+ than was the response to kainic acid (Fig. 4). DISCUSSION
The excitatory amino acids NMDA, kainic acid and quisqualic acid stimulate the release of neurotransmitters in several regions of the brain. In the nucleus accumbens, the release of ACh, stimulated by NMDA and kainic acid, was greatly reduced by the addition of tetrodotoxin to the buffer. Although excitatory amino acids open Na+ channels which are not sensitive to tetrodotoxin (Luini, Goldberg and Teichberg, 1981), and therefore could affect the membrane potential and release ACh (or DA) in the presence of tetrodotoxin, the sensitivity to tetrodotoxin of the present responses indicated that the release of ACh, stimulated by these excitatory amino acids, involved the activation of voltage-dependent sodium channels and the propagation of an action
177
potential, possibly through activation of receptors located on the cell body or dendrites of the cholinergic interneurons. The release of DA was also very sensitive to tetrodotoxin. However, since the dopaminergic innervation of the nucleus accumbens arises from cell bodies in the ventral tegmental area, the interpretation of the sensitivity to tetrodotoxin in this case is more difficult. Two possibilities are that either part of the release of DA, stimulated by NMDA and kainic acid, involves an inte~e~on or that the NMDA receptors are located on axons or terminals in such a way that their effect still requires the ultimate activation of voltage-dependent sodium channels. The release of [3H]DA stimulated by L-glutamate, reported by Marien et al. (1983), was insensitive to tetrodotoxin. The reason for the discrepancy between these two studies is unknown, since L-glutamate would be expected to be acting at one or more of these receptor subtypes. In the striatum, the inhibitory effect of PCP on the release of ACh induced by excitatory amino acids was specific for NMDA-induced release {Snell and Johnson, 1986). However, in the nucleus accumbens, PCP was effective at inhibiting release of ACh induced by NMDA, kainic acid or quisqualic acid. Although complete concentration-responses were not measured for PCP against all three agonists, the approximate IC,, concentration of PCP against NMDA-stimuIated release also inhibited the release of kainic acid and quisqualic acid by about 50% However, the effect of PCP on the response to kainic acid did not appear to be concentration-dependent. Also, kainic acid-induced release of ACh was significantly less sensitive to the effects of tetrodotoxin, removal of calcium, and magnesium ions, thus suggesting an action mediated by different mechanisms. Two characteristics reported to distinguish NMDA receptors from kainic acid or quisqualic acid type receptors are the voltage-dependent blockade of NMDA receptors by small concentrations of magnesium (Nowak, Bregestovski and Ascher, 1984; Mayer ef al., 1984) and the selective blockade of NMDA receptors by the antagonist 2-APV (Watkins and Evans, 1981). The inhibition of kainic acid- and quisqualic acid-stimulated release of ACh by magnesium ions and 2-APV suggests an action of these agonists at NMDA-type receptors. Consistent with this hypothesis was the finding that PCP was no longer effective at inhibiting kainic acid-stimulated release of ACh in the presence of magnesium, when responses to NMDA were totally blocked. Alternatively, part of the kainic acid- or quisqualic acidstimulated release could be due to the release of endogenous glutamate or aspartate which then activated NMDA receptors (McBean and Roberts, 1981; Ferkany and Coyle, 1983). Nevertheless, it appears that the effects of PCP on excitatory amino acidinduced release of ACh in the nucleus accumbens, as well as in the striatum, is coupled mechanistically to activation of the NMDA subtype of receptor.
SUSAN M. JONES et al.
178
In an earlier study, in the striatum, it was found that PCP inhibited the release of DA induced by NMDA, quisqualic acid and kainic acid (Snell and Johnson, 1986). Since NMDA-induced release of DA in the striatum was completely inhibited by MgZf, and PCP also inhibited the release of DA induced by quisqualic acid and kainic acid in the presence of magnesium, it was necessary to conclude that PCP could inhibit release induced by excitatory amino acids acting at receptor subtypes other than the NMDA type. However, in the present study, in the nucleus accumbens it was found that PCP had no inhibitory effect on kainic acid-stimulated release of DA. In fact, it was found that 10 nM PCP consistently enhanced kainic acid-induced release of DA, a phenomenon for which the authors have no explanation. Nonetheless, the inhibitory effects of PCP in the nucleus accumbens appear to be related only to release activated by an action on NMDA receptors. Thus, even though a small portion of kainic acidinduced release of DA was sensitive to magnesium and 2-APV (suggesting partial activation through NMDA receptors), PCP had no inhibitory effect. It is likely that the failure to observe an effect of PCP on the portion of the kainic acid-induced response that may be mediated by NMDA receptors is due to the fact that this signal is very small. That is, fractional release of DA induced by 1 mM NMDA and kainic acid is about 5.1 and 0.9, respectively. The magnesium-sensitive components are about 5.0 and 0.3, respectively. Thus, the NMDA component of the response to kainic acid is only about 6% of the analogous portion of the NMDA response. To summarize, it has been observed that PCP inhibited the release of DA and ACh, induced by excitatory amino acids in the striatum (Snell and Johnson, 1985, 1986) and in the nucleus accumbens (this study). Inhibition of the release of transmitter in the striatum by various PCP-like drugs was stereoselective and was highly correlated with their affinity for the PCP/sigma receptor site (Snell and Johnson, 1985, 1986). Further, inhibition of excitatory amino acid-induced release of ACh in the striatum and the release of DA and ACh in the nucleus accumbens appears to be associated with the activation of NMDA subtypes of receptor. However, evidence obtained in a study of the release of DA in the striatum suggests that PCP/sigma receptors may not be exclusively associated with NMDA receptors. Nevertheless, this study confirms and extends to the nucleus accumbens the observation initiallv made in the spinal cord, that PCP and similar drugs selectively antagonize the excitatory effects of glutamate-like amino acids by activating a site associated with the NMDA receptor (Anis, Berry, Burton and Lodge, 1983; Lodge and Berry, 1984). REFERENCES
Anis N. A., Berry S. C., Burton N. R. and Lodge D. (1983) The dissociative
anaesthetics,
ketamine
and phencyclidine
selectively reduce excitation of central mammalian neurones by N-methyl-aspartate. Br. J. Pharmac. 19: 565-575. Carter C. J. (1980) Glutamatergic pathways from the medial prefrontal cortex to the anterior striatum, nucleus accumbens and substantia nigra. Proc. B.P.S., pp. 50-51. Crunelli V. and Mayer M. L. (1984) Mg’+ dependence of membrane resistance increases evoked by NMDA in hippocampal neurones. Brain Res. 311: 392-396. Evans R. H., Francis A. A. and Watkins J. C. (1978) Mg*+-like selective antagonism of excitatory amino acid-induced responses by a,c-diaminopimelic acid, D-a-aminoadipate and HA-966 in isolated spinal cord of frog and immature rat. Brain Res. 148: 536542. Ferkany J. W. and Coyle J. T. (1983) Kainic acid selectively stimulates the release of endogenous excitatory amino acids. J. Pharmac. exp. Ther. 225: 3999406.
Fonnum F. and Walaas I. (1980) Localization of neurotransmitters in nucleus accumbens. In: The Neurobiology of the Nucleus Accumbens (Chronister R. B. and DeFrance D. F., Eds), pp. 259-272. Haer Inst., Brunswick. Fonnum F., Walaas I. and Iverson E. (1977) Localization of GABAergic, cholinergic and aminergic structures in the mesolimbic system. J. Neurochem. 29: 221-230. French E. D., Pilapil C. and Quirion R. (1985) Phencyclidine binding sites in the nucleus accumbens and phencyclidine-induced hyperactivity are decreased following lesions of the mesolimbic dopamine system. Eur. J. Pharmac. 116: I-9. Johnson K. M. and Snell L. D. (1985) Effects of phencyclidine (PCP)-like drugs on turning behavior, ‘H-dopamine uptake and ‘H-PCP binding. Pharmac. Biochem. Behav. 22: 731-735. Lehmann J. and Scatton B. (1982) Characterization of the excitatory amino acid receptor-mediated release of 3[H]acetylcholine from rat striatal slices. Brain Res. 252: 77-89. Leventer S. M. and Johnson K. M. (1984) Phencyclidineinduced inhibition of striatal acetylcholine release: comparison with mu, kappa and sigma opiate agonists. Life Sci. 34, 793-801. Lindvall 0. and Bjorklund A. (1978) Anatomy of the dopaminergic neuron systems in the rat brain. In: Advances in Biochemical Psychopharmacology, Dopamine, Vol. 19 (Roberts P. J., Woodruff G. N., Iverson L. L., Eds), pp. l-23. Raven Press, New York. Lodge D. and Berry S. C. (1984) Psychotomimetic effects of sigma opiates may be mediated by block of central excitatory synapses utilizing receptors for aspartate-like amino acids. In: Modulation of Sensorimotor Activity During Alterations in Behavioral States, pp. 503-518. Liss, New York. Luini A., Goldberg 0. and Teichberg V. I. (1981) Distinct pharmacological properties of excitatory amino acid receptors in the rat striatum: study bv Na+ et&x assay. . Proc. natn. Aead. Sci. U.S.A. 7fkV3250-3254.
Marien M.. Brien J. and Jhamandas K. (1983) Regional release of [‘Hldopamine from rat brain in vitro: effects of opioids on release induced by potassium, nicotine and L-glutamic acid. Can. J. Physiol. Pharmac. 61: 43-60. Mayer M. L., Westbrook G. L. and Guthrie P. B. (1984) Voltage-dependent block by Mg*+ of NMDA responses in spinal cord neurones. Nature 309: 261-263. McBean G. J. and Roberts P. J. (1981) Glutamate preferring receptors regulate the release of D-‘H-aspartate from rat hippocampal slices. Nature 291: 593-594. McCulloch R. M., Johnston G. A. R., Game C. J. A. and Curtis D. R. (1974) The differential sensitivity of spinal interneurons and Renshaw cells to kainate and Nmethyl-D-aspartate. Exp. Brain Res. 21: 515-518. Nowak L., Bregestovski P. and Ascher P. (1984) Mag-
PCP effects on ACh and DA release in nucleus accumbens nesium gates glutamate-activated channels in mouse central neurones. Nature 307: 462-465. Richardson W. and Szerb J. C. (1974) The release of labelled acetylcholine and choline from cerebral cortical slices stimulated electrically. Er. J. Pharmac. 52: 449-507. Roberts P. J. and Anderson S. D. (1979) Stimulatory effect of L-glutamate and related amino acids on 3H-dopamine release from rat striatum: an in t;itro model for glutamate actions. J. ~e~r~chern. 32: 1539-1545. Sneh L. D. and Johnson K. M. (1985) Antagonism of NMDA-induced transmitter release in the rat striatum by
119
phencyclidine-like drugs and its relationship to turning behavior. J. Pharmac. exp. Ther. 235: 50-57. Snell L. D. and Johnson K. M. (1986) Characterization of the inhibition of excitatory amino acid-induced neurotransmitter release in the rat striatum by phencyclidinelike drugs. J. Pharmac. exp. Ther. 238: 938-946. Walaas 1. and Fonnum F. (1979) The effects of surgical and chemical lesions on neurotransm~tter candidates in the nucleus accumbens of the rat. ~~ro~c~e~ce 4: 209-216. Watkins J. C. and Evans R. H. (1981) Excitatory amino acid neurotmnsmitters. Ann. Ret’. Pharmac. Tax. 21: 162-204.