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Acetylcholine content and distribution in rat cortical synaptosomes after in vivo exposure to quinolinic acid
Neuroscience Letters, 108 (1990) 219 224
219
Elsevier Scientific Publishers Ireland Ltd.
NSL 06540
Acetylcholine content and distribution in rat cortical synaptosomes after in vivo exposure to quinolinic acid Robert H. Metcalf, David L. Riddell and Roland J. Boegman Department of Pharmacology and Toxicology, Queen's University, Kingston, Ont. (Canada) (Received 13 June 1989; Accepted 22 August 1989)
Key words: Quinolinic acid; Acetylcholine; Synaptosome; Neuroexcitant; Seizure In this study, changes in the concentration and subcellular distribution of rat cortical synaptosomal acetylcholine (ACh) was investigated after a single injection of quinolinic acid (QUIN) into the nucleus basalis magnocellularis (nbM). In the P2 fraction of normal animals the ACh concentration was 235 _+18 pmol/mg protein. Of this, 64+ 10% was recovered in the particulate (P3) fraction and 24+ 1% in the soluble (SJ fraction. Cortical synaptosomes (P2) prepared 0.5 h after injecting either 600 or 1000 nmol of QUIN contained significantly higher concentrations of ACh (372 _+ 127 and 496 + 77 pmol/mg protein, respectively) when compared to the amount of ACh in control animals. The ACh concentration in the P2 fraction was still elevated 3 h after injecting 600 nmol QUIN, however, synaptosomal ACh decreased significantly 3 h after rats were treated with 1000 nmol QUIN. Determination of subcellular ACh 0.5 h after injecting QUIN revealed that neither dose of QUIN produced a change in the distribution of ACh between P3 and $3. However, 3 h after injecting QUIN, a shift in the subcellular distribution of ACh to the cytoplasmic fraction ($3) was observed with 1000 nmol QUIN. These results show that QUIN-induced depolarization of nbM neurons which project to the cortex produce both dose-dependent and time-dependent changes in synaptosomal ACh concentration and subcellular distribution.
The intermediates in the kynurenine pathway of tryptophan metabolism have been widely studied since the discovery that metabolites of this pathway exhibit both neuroexcitant and neurotoxic properties [8, 18, 23]. The convulsant characteristics of these compounds was initially described by Lapin [7] who found that in mice and frogs, the most potent kynurenines were quinolinic acid (QUIN) and kynurenic acid. Subsequently, it was found that QUIN was a potent excitant when applied to cortical neurons in the rat due to activation of N-methyl-D-aspartate (NMDA) receptors [22, 23]. When injected into the striatum or dorsal hippocampus of unanesthetized rats, QUIN produces seizures with a latency ranging from 19 to 32 min [20]. In addition, Correspondence." R.J. Boegman, Department of Pharmacology and Toxicology, Queen's University, Kingston, Ont., Canada K7L 3N6. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
220 unilateral intrastriatal injections of QUIN produce a dose-dependent increase in tonic-clonic movements of the contralateral forelimb and a loss of GABAergic and cholinergic neurons in the injection area [18]. Various investigations have shown that there are regional variations in neuronal sensitivity to QUIN [15, 19]. In the rat, the cholinergic neurons of the nucleus basalis magnocellularis (nbM) which project to the cortex are especially sensitive to the neuroexcitant and neurotoxic properties of QUIN [3, 19]. The acetylcholine (ACh) concentration in subcellular fractions of cortical synaptosomes has been reported to either increase or decrease depending on the convulsant used. Thus with lithium and pilocarpine, ACh content increases [6], while following pentylenetetrazol induced seizures, cortical ACh decreases [5]. Pentylenetetrazol treatment also produces an increase in high-affinity choline uptake (HACU) due to 'impulse flow regulation' at cholinergic nerve terminals [1, 21]. We have previously shown that injection of QUIN into the nbM of rats produces a dose- and time-dependent increase in cortical HACU [11, 16]. The present paper describes alterations in the concentration and subcellular distribution of cortical synaptosomal ACh after QUIN injection into the nbM. Under halothane anaesthesia, male Sprague Dawley rats (275 325 g) were given a single I/zl unilateral stereotaxic injection of either saline, 600 or 1000 nmol QUIN. QUIN was dissolved in 0.9% saline and adjusted to pH 7.4. Coordinates for injection were 0.8 mm posterior to bregma, 2.6 mm lateral to the midline, and 8.0 mm ventral to the surface of the skull with the incisor bar set at - 3 . 3 mm [14]. Animals were sacrificed at 0.5 and 3 h after infusion, the frontoparietal cortex dissected on ice, hand homogenized in a Potter teflon glass homogenizer with 0.32 M sucrose at a concentration of 50 mg wet wt./ml. The homogenate was centrifuged at 1000 g for l0 min and the supernatant removed and centrifuged at 17,500 g, to give the P2 synaptosoreal pellet. This pellet was resuspended in the original volume of 0.32 M sucrose. An aliquot of P2 was removed for ACh determination and the remainder used to prepare a cytoplasmic ($3) and a vesicular (P3) fraction as described previously [4, 6, 17]. Briefly, the resuspended P2 synaptosomal pellet was centrifuged at 17,500 g for 20 rain and the pellet obtained resuspended in 2 ml of double distilled water containing physostigmine (40 #M) and EGTA (0.2 mM). This was centrifuged at 100,000 g for 60 min to give a P3 and the soluble $3 fraction. P2 and P3 fractions were resuspended in 1 ml of acetone containing 15% 1 N formic acid for ACh extractions [5]. The ACh content in P> P3, and $3 was determined by gas chromatography-mass spectrometry as previously described [12], and expressed per mg protein [10]. Each value represents the mean ±S.D. of 3 separate experiments. Significant differences between treatments were determined by one-way analysis of variance. The ACh concentration in the P: fraction of normal uninjected animals was 235_+ 18 pmol/mg protein. Of this, 64_+ 10% was recovered in the P3 fraction and 24_+ 1% in the $3 fraction. Injection of 1/tl 0.9% saline resulted in a slight, non-significant decrease in ACh concentration in the P2 fraction (216_+ 56 pmol/mg protein), while the subcellular distribution remained unaltered, with 62 4-4% in P3 and 30_+ 7% in S~.
221
600
'~
500
[Z3 Normal [ZZ] Saline 12~ QUIN (0.5 h) QUIN (3.0 h)
0 400
o
xN \N
300
0 ~
=I
200
o 100
0 o
QUIN C o n c e n t r a t i o n
600
1000
(nmoles/ul)
Fig. 1. The concentration of ACh in the cortical P2 synaptosomal fraction after in vivo exposure of neurons in the nbM to QUIN. Data represents the mean + S.D. of 3 separate experiments each done in duplicate. *Significantly different from control (P ~ 0.05).
Previous work in our laboratory has shown that the maximal increase in cortical HACU following an infusion of QUIN into the nbM occurred at 3 h after a dose of 600 nmol and 0.5 h after 1000 nmol [16]. Cortical P2 synaptosomes prepared 0.5 h after injection either dose of QUIN contained significantly higher concentrations of ACh when compared to control synaptosomes (Fig. 1). Although 1000 nmol QUIN produced a larger increase in synaptosomal ACh than did 600 nmol, the difference was not significant. At 3 h post-injection, ACh concentrations were still elevated in P2 synaptosomes prepared from rats injected with 600 nmoi QUIN. However, the cortical P2 synaptosomal ACh content decreased significantly in rats treated with 1000 nmol QUIN for 3 h (Fig. 1). Analysis of the subcellular (P3 and $3) distribution of ACh 0.5 h after injecting either dose of QUIN indicated that neither dose produced a significant change in the distribution of ACh (Fig. 2A). At 3 h after injecting 1000 nmol QUIN, a shift in the subcellular distribution of cortical ACh to the cytoplasmic fraction ($3) was observed (Fig. 2B) when compared to normal and saline injected animals. In addition, there was a significant increase in the concentration of ACh in the cytoplasmic fraction ($3) at both 600 and 1000 nmol QUIN between 0.5 h and 3 h (Fig. 2A, B). In naive animals, the distribution of synaptosomai ACh between the vesicular P3 and cytoplasmic $3 fractions was similar to that reported previously [6]. This distribution of ACh was the same in saline-treated animals, 600 nmol QUIN-injected animals at both 0.5 and 3 h, and 1000 nmol-treated animals at 0.5 h. Similar results were reported by Jope et al. [6] following convulsions induced by lithium plus pilocarpine. At 1000 nmol QUIN, a shift in ACh to the soluble fraction was observed. This may
gS2] P3 Fraction ~5~ SB Fraction
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QUIN Concentration
600
1000
(nrnoles/ul)
Fig. 2. The subcellular distribution of ACh in cortical synaptosomes after in viva exposure of neurons in the nbM to QUIN. A: ACh in P3 and $3 0.5 h after either no QUIN, saline, 600 nmot, or 1000 nmol QUIN. B: ACh in P3 and $3 3 h after exposure to QUIN. Values represent the mean _+S.D. of 3 separate experiments each done in duplicate. *Significantly different from control (P~0.05). +Significantlydifferent from similar fraction at t=0.5 h (P~0.05).
represent an inability of cholinergic nerve terminals to efficiently transport A C h into vesicles, or a shunting of A C h to a more readily releasable pool. While seizure activity resulting from cholinergic stimulation by pilocarpine alone can be antagonized by focal injections o f N M D A antagonists [13], the endogenous t r y p t o p h a n metabolite Q U I N by acting directly on N M D A receptors is also a potent convulsant and has been implicated in the etiology of seizure disorders [20]. Our results show that Q U I N stimulation o f n b M cholinergic neurons which project to the cortex, produce both dose-dependent and time-dependent changes in synaptosomal A C h concentration and subcellular distribution. The mechanism by which this occurs is, at present, u n k n o w n . However, convulsants have been reported to increase A C h content and release [24], deplete synaptosomal A C h [5], or increase
223 the activity o f m e c h a n i s m s involved in A C h synthesis, such as H A C U [1, 21]. Q U I N s t i m u l a t i o n o f n b M n e u r o n s also results in a dose- a n d t i m e - d e p e n d e n t increase in cortical H A C U suggesting a c t i v a t i o n o f A C h synthesis [11, 16]. In light o f the present findings it a p p e a r s t h a t at high doses o f Q U I N , excessive n e u r o n a l s t i m u l a t i o n m a y cause a n u n c o u p l i n g o f A C h synthesis a n d release. The present findings also s u p p o r t the h y p o t h e s i s p r o p o s e d p r e v i o u s l y t h a t A C h synthesis is r e g u l a t e d b y n e u r o n a l activity a n d n o t b y i n t r a c e l l u l a r A C h levels o r A C h release [2, 6]. In conclusion, the findings o f the p r e s e n t study suggest t h a t Q U I N s t i m u l a t i o n o f n b M cholinergic neurons p r o d u c e s changes in s y n a p t o s o m a l A C h similar to those o b s e r v e d for generalized seizures i n d u c e d by l i t h i u m - p i l o c a r p i n e t r e a t m e n t a n d that u n d e r c o n d i t i o n s o f extreme stimulation, a d i s s o c i a t i o n o f the m e c h a n i s m s involved in A C h synthesis occurs, leading to i m p r o p e r cholinergic function. This w o r k was s u p p o r t e d by a g r a n t f r o m the M e d i c a l R e s e a r c h C o u n c i l o f C a n a d a a n d a research s t u d e n t s h i p f r o m the O n t a r i o M e n t a l H e a l t h F o u n d a t i o n . 1 Atweh, S,, Simon, J.R. and Kuhar, M.J., Utilization of sodium-dependent high affinity choline uptake in vitro as a measure of the activity of cholinergic neurons in vivo, Life Sci., 17 (1975) 1535-1544. 2 Collier, B., Kwok, Y.N. and Welner, S.A., Increased acetylcholine synthesis and release following presynaptic activity in a sympathetic ganglion, J. Neurochem., 40 (1983) 91 98. 3 EI-Defrawy, S.R., Coloma, F., Jhamandas, K,, Boegman, R.J., Beninger, R.J. and Wirsching, B.A., Functional and neurochemical cortical cholinergic impairment following neurotoxic lesions of the nucleus basalis magnocellularis in the Rat, Neurobiol. Aging 6 (1985) 325-330. 4 Jope, R.S., Acetylcholine turnover and compartmentation in rat brain synaptosomes, J. Neurochem., 36(1981) 1712 1721. 5 Jope, R.S., Effects of phosphatidylcholine administration to rats on choline in blood and choline and acetylcholine in brain, J. Pharmacol. Exp. Ther., 220 (1982) 322-328. 6 Jope, R.S., Simonata, M. and Lally, K., Acetylcholine content in rat brain is elevated by status epilepticus induced by lithium and pilocarpine, J. Neurochem, 49 (1987) 944-951. 7 Lapin, I.P., Stimulant and convulsant effects of kynurenines injected into brain ventricles in mice, J. Neurol. Transm., 42 (1978) 3743. 8 Lapin, I.P., Kynurenines and seizures, Epilepsia, 22 (1981) 257 265. 9 Lehmann, J., Nagy, J.I., Atmadja, S. and Fibiger, H.C., The nucleus basalis magnocellularis: the origin ofa cholinergic projection to the neocortex of the rat, Neuroscience, 5 (1980) 1161-1174. 10 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 11 Metcalf, R.H., Boegman, R.J., Quirion, R., Riopelle, R.J. and Ludwin, S.K., Effect of quinolinic acid in the nucleus basalis magnocellularis on cortical high affinity choline uptake, J. Neurochem., 49 (1987) 639~44. 12 Metcalf, R.H. and Boegman, R.J., Release of acetylcholine from tissue slices of the rat nucleus basalis magnocellularis, J. Neurochem., 52 (1989) 1143-1148. 13 Patal, S., DeSarro, G.B. and Meldrum, B.S., Regulation of seizure threshold by excitatory amino acids in the striatum and entopeduncular nucleus of rats, Neuroscience, 27 (3) (1988) 837 850. 14 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coorindates, Academic Press, Toronto, 1986. 15 Perkins, M.N. and Stone, T.W., Quinolinic acid: regional variations in neuronal sensitivity, Brain Res., 259 (1983) 172-176. 16 Riddell, D.L. and Boegman, R.J., Excitotoxin-induced changes in cortical high affinity choline uptake, J. Neurochem., submitted.
224 17 Salehmoghaddam, S.H. and Collier, B., The relationship between acetylcholine release from brain slices and the acetylcholine content of subcellular fractions prepared from brain, J. Neurochem., 27 (1976) 71 76. 18 Schwarcz, R., Whetsell, W.O., Jr. and Mangano, R.M., Quinolinic acid: an endogenous metabolite that produces axon-sparing lesions in the rat brain, Science, 219 (1983) 316 318. 19 Schwarcz, R. and Kohler, C., Differential vulnerability of central neurons of the rat to quinolinic acid, Neurosci. Lett., 38 (1983) 85 90. 20 Schwarcz, R., Brush, G.S., Foster, A.C. and French, A.D., Seizure activity and lesions after intrahippocampal quinolinic acid injection, Exp. Neurot., 84 (1984) 1 17. 21 Simon, J.R. and Kuhar, M.J., Impulse-flow regulation of high affinity choline uptake in brain cholinergic nerve terminals, Nature (Lond.), 255 (1975) 162 163. 22 Stone, T.W., The relative potencies of(-)-2-amino-5-phosphonovalerate and (---)-2-amino-7-phosphonoheptanoate as antagonists of N-methylaspartate and quinolinic acids and repetitive spikes in rat hippocampal slices, Brain Res., 381 (1986) 195 198. 23 Stone, T.W. and Perkins, M.N., Quinolinic acid: a potent endogenous excitant at amino acid receptors in CNS, Europ. J. Pharmacol., 72 (1981) 411412. 24 Yaksh, T.L. and Yamamura, H.I., The release in vivo of [3H]-acetylcholine from cat caudate nucleus and cerebral cortex by atropine, pentylenetetrazole, K +-depolarization and electrical stimulation, J. Neurochem., 25 (1975) 123 130.
219
Elsevier Scientific Publishers Ireland Ltd.
NSL 06540
Acetylcholine content and distribution in rat cortical synaptosomes after in vivo exposure to quinolinic acid Robert H. Metcalf, David L. Riddell and Roland J. Boegman Department of Pharmacology and Toxicology, Queen's University, Kingston, Ont. (Canada) (Received 13 June 1989; Accepted 22 August 1989)
Key words: Quinolinic acid; Acetylcholine; Synaptosome; Neuroexcitant; Seizure In this study, changes in the concentration and subcellular distribution of rat cortical synaptosomal acetylcholine (ACh) was investigated after a single injection of quinolinic acid (QUIN) into the nucleus basalis magnocellularis (nbM). In the P2 fraction of normal animals the ACh concentration was 235 _+18 pmol/mg protein. Of this, 64+ 10% was recovered in the particulate (P3) fraction and 24+ 1% in the soluble (SJ fraction. Cortical synaptosomes (P2) prepared 0.5 h after injecting either 600 or 1000 nmol of QUIN contained significantly higher concentrations of ACh (372 _+ 127 and 496 + 77 pmol/mg protein, respectively) when compared to the amount of ACh in control animals. The ACh concentration in the P2 fraction was still elevated 3 h after injecting 600 nmol QUIN, however, synaptosomal ACh decreased significantly 3 h after rats were treated with 1000 nmol QUIN. Determination of subcellular ACh 0.5 h after injecting QUIN revealed that neither dose of QUIN produced a change in the distribution of ACh between P3 and $3. However, 3 h after injecting QUIN, a shift in the subcellular distribution of ACh to the cytoplasmic fraction ($3) was observed with 1000 nmol QUIN. These results show that QUIN-induced depolarization of nbM neurons which project to the cortex produce both dose-dependent and time-dependent changes in synaptosomal ACh concentration and subcellular distribution.
The intermediates in the kynurenine pathway of tryptophan metabolism have been widely studied since the discovery that metabolites of this pathway exhibit both neuroexcitant and neurotoxic properties [8, 18, 23]. The convulsant characteristics of these compounds was initially described by Lapin [7] who found that in mice and frogs, the most potent kynurenines were quinolinic acid (QUIN) and kynurenic acid. Subsequently, it was found that QUIN was a potent excitant when applied to cortical neurons in the rat due to activation of N-methyl-D-aspartate (NMDA) receptors [22, 23]. When injected into the striatum or dorsal hippocampus of unanesthetized rats, QUIN produces seizures with a latency ranging from 19 to 32 min [20]. In addition, Correspondence." R.J. Boegman, Department of Pharmacology and Toxicology, Queen's University, Kingston, Ont., Canada K7L 3N6. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
220 unilateral intrastriatal injections of QUIN produce a dose-dependent increase in tonic-clonic movements of the contralateral forelimb and a loss of GABAergic and cholinergic neurons in the injection area [18]. Various investigations have shown that there are regional variations in neuronal sensitivity to QUIN [15, 19]. In the rat, the cholinergic neurons of the nucleus basalis magnocellularis (nbM) which project to the cortex are especially sensitive to the neuroexcitant and neurotoxic properties of QUIN [3, 19]. The acetylcholine (ACh) concentration in subcellular fractions of cortical synaptosomes has been reported to either increase or decrease depending on the convulsant used. Thus with lithium and pilocarpine, ACh content increases [6], while following pentylenetetrazol induced seizures, cortical ACh decreases [5]. Pentylenetetrazol treatment also produces an increase in high-affinity choline uptake (HACU) due to 'impulse flow regulation' at cholinergic nerve terminals [1, 21]. We have previously shown that injection of QUIN into the nbM of rats produces a dose- and time-dependent increase in cortical HACU [11, 16]. The present paper describes alterations in the concentration and subcellular distribution of cortical synaptosomal ACh after QUIN injection into the nbM. Under halothane anaesthesia, male Sprague Dawley rats (275 325 g) were given a single I/zl unilateral stereotaxic injection of either saline, 600 or 1000 nmol QUIN. QUIN was dissolved in 0.9% saline and adjusted to pH 7.4. Coordinates for injection were 0.8 mm posterior to bregma, 2.6 mm lateral to the midline, and 8.0 mm ventral to the surface of the skull with the incisor bar set at - 3 . 3 mm [14]. Animals were sacrificed at 0.5 and 3 h after infusion, the frontoparietal cortex dissected on ice, hand homogenized in a Potter teflon glass homogenizer with 0.32 M sucrose at a concentration of 50 mg wet wt./ml. The homogenate was centrifuged at 1000 g for l0 min and the supernatant removed and centrifuged at 17,500 g, to give the P2 synaptosoreal pellet. This pellet was resuspended in the original volume of 0.32 M sucrose. An aliquot of P2 was removed for ACh determination and the remainder used to prepare a cytoplasmic ($3) and a vesicular (P3) fraction as described previously [4, 6, 17]. Briefly, the resuspended P2 synaptosomal pellet was centrifuged at 17,500 g for 20 rain and the pellet obtained resuspended in 2 ml of double distilled water containing physostigmine (40 #M) and EGTA (0.2 mM). This was centrifuged at 100,000 g for 60 min to give a P3 and the soluble $3 fraction. P2 and P3 fractions were resuspended in 1 ml of acetone containing 15% 1 N formic acid for ACh extractions [5]. The ACh content in P> P3, and $3 was determined by gas chromatography-mass spectrometry as previously described [12], and expressed per mg protein [10]. Each value represents the mean ±S.D. of 3 separate experiments. Significant differences between treatments were determined by one-way analysis of variance. The ACh concentration in the P: fraction of normal uninjected animals was 235_+ 18 pmol/mg protein. Of this, 64_+ 10% was recovered in the P3 fraction and 24_+ 1% in the $3 fraction. Injection of 1/tl 0.9% saline resulted in a slight, non-significant decrease in ACh concentration in the P2 fraction (216_+ 56 pmol/mg protein), while the subcellular distribution remained unaltered, with 62 4-4% in P3 and 30_+ 7% in S~.
221
600
'~
500
[Z3 Normal [ZZ] Saline 12~ QUIN (0.5 h) QUIN (3.0 h)
0 400
o
xN \N
300
0 ~
=I
200
o 100
0 o
QUIN C o n c e n t r a t i o n
600
1000
(nmoles/ul)
Fig. 1. The concentration of ACh in the cortical P2 synaptosomal fraction after in vivo exposure of neurons in the nbM to QUIN. Data represents the mean + S.D. of 3 separate experiments each done in duplicate. *Significantly different from control (P ~ 0.05).
Previous work in our laboratory has shown that the maximal increase in cortical HACU following an infusion of QUIN into the nbM occurred at 3 h after a dose of 600 nmol and 0.5 h after 1000 nmol [16]. Cortical P2 synaptosomes prepared 0.5 h after injection either dose of QUIN contained significantly higher concentrations of ACh when compared to control synaptosomes (Fig. 1). Although 1000 nmol QUIN produced a larger increase in synaptosomal ACh than did 600 nmol, the difference was not significant. At 3 h post-injection, ACh concentrations were still elevated in P2 synaptosomes prepared from rats injected with 600 nmoi QUIN. However, the cortical P2 synaptosomal ACh content decreased significantly in rats treated with 1000 nmol QUIN for 3 h (Fig. 1). Analysis of the subcellular (P3 and $3) distribution of ACh 0.5 h after injecting either dose of QUIN indicated that neither dose produced a significant change in the distribution of ACh (Fig. 2A). At 3 h after injecting 1000 nmol QUIN, a shift in the subcellular distribution of cortical ACh to the cytoplasmic fraction ($3) was observed (Fig. 2B) when compared to normal and saline injected animals. In addition, there was a significant increase in the concentration of ACh in the cytoplasmic fraction ($3) at both 600 and 1000 nmol QUIN between 0.5 h and 3 h (Fig. 2A, B). In naive animals, the distribution of synaptosomai ACh between the vesicular P3 and cytoplasmic $3 fractions was similar to that reported previously [6]. This distribution of ACh was the same in saline-treated animals, 600 nmol QUIN-injected animals at both 0.5 and 3 h, and 1000 nmol-treated animals at 0.5 h. Similar results were reported by Jope et al. [6] following convulsions induced by lithium plus pilocarpine. At 1000 nmol QUIN, a shift in ACh to the soluble fraction was observed. This may
gS2] P3 Fraction ~5~ SB Fraction
~0o ]
A
sod
T
i
604 4O4
o ,m £j ,-4
[
o
-',-~
o
2O4
/;
d Oi
;::L 100 s
]~
o © S
@ 6od
404
204
o] Normal
Saline
QUIN Concentration
600
1000
(nrnoles/ul)
Fig. 2. The subcellular distribution of ACh in cortical synaptosomes after in viva exposure of neurons in the nbM to QUIN. A: ACh in P3 and $3 0.5 h after either no QUIN, saline, 600 nmot, or 1000 nmol QUIN. B: ACh in P3 and $3 3 h after exposure to QUIN. Values represent the mean _+S.D. of 3 separate experiments each done in duplicate. *Significantly different from control (P~0.05). +Significantlydifferent from similar fraction at t=0.5 h (P~0.05).
represent an inability of cholinergic nerve terminals to efficiently transport A C h into vesicles, or a shunting of A C h to a more readily releasable pool. While seizure activity resulting from cholinergic stimulation by pilocarpine alone can be antagonized by focal injections o f N M D A antagonists [13], the endogenous t r y p t o p h a n metabolite Q U I N by acting directly on N M D A receptors is also a potent convulsant and has been implicated in the etiology of seizure disorders [20]. Our results show that Q U I N stimulation o f n b M cholinergic neurons which project to the cortex, produce both dose-dependent and time-dependent changes in synaptosomal A C h concentration and subcellular distribution. The mechanism by which this occurs is, at present, u n k n o w n . However, convulsants have been reported to increase A C h content and release [24], deplete synaptosomal A C h [5], or increase
223 the activity o f m e c h a n i s m s involved in A C h synthesis, such as H A C U [1, 21]. Q U I N s t i m u l a t i o n o f n b M n e u r o n s also results in a dose- a n d t i m e - d e p e n d e n t increase in cortical H A C U suggesting a c t i v a t i o n o f A C h synthesis [11, 16]. In light o f the present findings it a p p e a r s t h a t at high doses o f Q U I N , excessive n e u r o n a l s t i m u l a t i o n m a y cause a n u n c o u p l i n g o f A C h synthesis a n d release. The present findings also s u p p o r t the h y p o t h e s i s p r o p o s e d p r e v i o u s l y t h a t A C h synthesis is r e g u l a t e d b y n e u r o n a l activity a n d n o t b y i n t r a c e l l u l a r A C h levels o r A C h release [2, 6]. In conclusion, the findings o f the p r e s e n t study suggest t h a t Q U I N s t i m u l a t i o n o f n b M cholinergic neurons p r o d u c e s changes in s y n a p t o s o m a l A C h similar to those o b s e r v e d for generalized seizures i n d u c e d by l i t h i u m - p i l o c a r p i n e t r e a t m e n t a n d that u n d e r c o n d i t i o n s o f extreme stimulation, a d i s s o c i a t i o n o f the m e c h a n i s m s involved in A C h synthesis occurs, leading to i m p r o p e r cholinergic function. This w o r k was s u p p o r t e d by a g r a n t f r o m the M e d i c a l R e s e a r c h C o u n c i l o f C a n a d a a n d a research s t u d e n t s h i p f r o m the O n t a r i o M e n t a l H e a l t h F o u n d a t i o n . 1 Atweh, S,, Simon, J.R. and Kuhar, M.J., Utilization of sodium-dependent high affinity choline uptake in vitro as a measure of the activity of cholinergic neurons in vivo, Life Sci., 17 (1975) 1535-1544. 2 Collier, B., Kwok, Y.N. and Welner, S.A., Increased acetylcholine synthesis and release following presynaptic activity in a sympathetic ganglion, J. Neurochem., 40 (1983) 91 98. 3 EI-Defrawy, S.R., Coloma, F., Jhamandas, K,, Boegman, R.J., Beninger, R.J. and Wirsching, B.A., Functional and neurochemical cortical cholinergic impairment following neurotoxic lesions of the nucleus basalis magnocellularis in the Rat, Neurobiol. Aging 6 (1985) 325-330. 4 Jope, R.S., Acetylcholine turnover and compartmentation in rat brain synaptosomes, J. Neurochem., 36(1981) 1712 1721. 5 Jope, R.S., Effects of phosphatidylcholine administration to rats on choline in blood and choline and acetylcholine in brain, J. Pharmacol. Exp. Ther., 220 (1982) 322-328. 6 Jope, R.S., Simonata, M. and Lally, K., Acetylcholine content in rat brain is elevated by status epilepticus induced by lithium and pilocarpine, J. Neurochem, 49 (1987) 944-951. 7 Lapin, I.P., Stimulant and convulsant effects of kynurenines injected into brain ventricles in mice, J. Neurol. Transm., 42 (1978) 3743. 8 Lapin, I.P., Kynurenines and seizures, Epilepsia, 22 (1981) 257 265. 9 Lehmann, J., Nagy, J.I., Atmadja, S. and Fibiger, H.C., The nucleus basalis magnocellularis: the origin ofa cholinergic projection to the neocortex of the rat, Neuroscience, 5 (1980) 1161-1174. 10 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 11 Metcalf, R.H., Boegman, R.J., Quirion, R., Riopelle, R.J. and Ludwin, S.K., Effect of quinolinic acid in the nucleus basalis magnocellularis on cortical high affinity choline uptake, J. Neurochem., 49 (1987) 639~44. 12 Metcalf, R.H. and Boegman, R.J., Release of acetylcholine from tissue slices of the rat nucleus basalis magnocellularis, J. Neurochem., 52 (1989) 1143-1148. 13 Patal, S., DeSarro, G.B. and Meldrum, B.S., Regulation of seizure threshold by excitatory amino acids in the striatum and entopeduncular nucleus of rats, Neuroscience, 27 (3) (1988) 837 850. 14 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coorindates, Academic Press, Toronto, 1986. 15 Perkins, M.N. and Stone, T.W., Quinolinic acid: regional variations in neuronal sensitivity, Brain Res., 259 (1983) 172-176. 16 Riddell, D.L. and Boegman, R.J., Excitotoxin-induced changes in cortical high affinity choline uptake, J. Neurochem., submitted.
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