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Substance P increases release of acetylcholine in the dorsal striatum of freely moving rats
Brain Research, 623 (1993) 189-194 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
189
BRES 19234
Substance P increases release of acetylcholine in the dorsal striatum of freely moving rats Jeffrey J. Anderson, Thomas N. Chase and Thomas M. Engber Experimental Therapeutics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 (USA)
(Accepted 27 April 1993)
Key words: Microdialysis; Acetylcholine; Substance P; Neurokinin; Striatum; Basal ganglia
Little is known about the role that neuropeptides such as substance P play in cell-to-cell interactions in the striatum. The effect of locally perfused substance P on extracellular acetylcholine (ACh) in the dorsal striatum of awake, freely moving rats was examined using microdialysis. Neostigmine (1 /zM) was included in the perfusate to improve recovery of ACh. Basal extracellular ACh was sensitive to Na+-channel blockade with tetrodotoxin (0.3/~M) and Ca2+-channel blockade with MgC12 (10 mM) and therefore largely neuronal in origin. Local perfusion with 10 and 25/zM substance P for 20 min elevated extracellular ACh by 30% and 51%, respectively. The NK 1 receptor antagonist, CP 96,345 (10/zM), which by itself had no effect on extracellular ACh, prevented the substance P-induced increase in extracellular ACh. These results suggest that stimulation of NK 1 receptors by substance P enhances ACh release in the dorsal striatum and is consistent with anatomical evidence of a substance P-cholinergic circuit in this region.
INTRODUCTION A c e t y l c h o l i n e ( A C h ) in t h e s t r i a t u m is t h o u g h t to o r i g i n a t e p r i m a r i l y f r o m large aspiny n e u r o n s 6'36 which a r e intrinsic to this r e g i o n 9A9'24. A l t h o u g h c h o l i n e r g i c i n t e r n e u r o n s c o m p r i s e only a b o u t 2 % o f t h e t o t a l striatal cell p o p u l a t i o n in t h e rat 29, t h e y s u p p l y a d e n s e i n n e r v a t i o n 4° a n d a r e r e s p o n s i b l e for i m p a r t i n g t h e s t r i a t u m with s o m e o f t h e h i g h e s t q u a n t i t i e s o f cholinergic m a r k e r s in t h e CNS 13'17. S t r i a t a l c h o l i n e r g i c int e r n e u r o n s a p p e a r to p l a y a n i m p o r t a n t role in r e g u l a t ing s t r i a t a l f u n c t i o n since c h o l i n e r g i c a n t a g o n i s t s a m e l i o r a t e m o t o r a b n o r m a l i t i e s in P a r k i n s o n ' s d i s e a s e 8. O n e such i n t e r a c t i o n m a y b e with m e d i u m spiny striatonigral neurons containing the neurokinin substance P. T h e s e n e u r o n s c o n s t i t u t e o n e o f t h e m a j o r striatal e f f e r e n t p a t h w a y s , p r o j e c t i n g m a i n l y to t h e s u b s t a n t i a n i g r a p a r s r e t i c u l a t a . In a d d i t i o n , t h e s e n e u r o n s possess local axon c o l l a t e r a l s which a r b o r i z e within t h e s t r i a t u m 5. S u b s t a n c e P - c o n t a i n i n g n e r v e t e r m i n a l s have
b e e n shown to m a k e s y n a p t i c c o n t a c t with i d e n t i f i e d c h o l i n e r g i c n e u r o n s in t h e s t r i a t u m 7. M o r e o v e r , striatal neurons containing choline acetyltransferase mRNA s e l e c t i v e l y e x p r e s s s u b s t a n c e P ( N K l) r e c e p t o r m R N A 15. It thus a p p e a r s t h a t s u b s t a n c e P - c o n t a i n i n g axon c o l l a t e r a l s o f s t r i a t o n i g r a l n e u r o n s synapse with striatal c h o l i n e r g i c i n t e r n e u r o n s which possess subs t a n c e P ( N K l) r e c e p t o r s . It is not c l e a r w h a t a c t i o n s u b s t a n c e P m a y have on striatal c h o l i n e r g i c i n t e r n e u r o n s o r how s u b s t a n c e P may influence the release of ACh from these neurons in t h e intact brain. N e u r o k i n i n s , in g e n e r a l , a r e t h o u g h t to excite n e u r o n s 21. In s u p p o r t o f this hypothesis, septide, a s u b s t a n c e P a n a l o g selective for t h e N K 1 r e c e p tor, i n c r e a s e d t h e r e l e a s e o f A C h f r o m n e o s t r i a t a l slices ( n e u r o k i n i n A a n d n e u r o k i n i n B, n e u r o k i n i n s selective for N K 2 a n d N K 3 r e c e p t o r s , respectively, also e l i c i t e d A C h r e l e a s e ) t. A n effect o f s u b s t a n c e P on striatal A C h r e l e a s e in vivo has yet to b e d e m o n s t r a t e d . T h e p u r p o s e o f this investigation was to e x a m i n e t h e
Correspondence: J.J. Anderson, Experimental Therapeutics Branch, National Institute of Neurological Disorders and Stroke, Building 10, Room 5C215, 9000 Rockville Pike, Bethesda, MD 20892, USA. Fax: (1) (301) 496-6609.
190 relationship between substance P and ACh release in the dorsal striatum using in vivo microdialysis in awake, freely moving rats. MATERIALS AND METHODS
Surgery and in vivo microdialysis procedure Male Sprague-Dawley rats (Taconic Farms, Germantown, NY) weighing 275-300 g were housed individually on a standard 12 h light/dark cycle with unlimited access to food and water. Prior to surgery, each animal was anesthetized with sodium pentobarbital (50 mg/kg i.p.). An intracerebral guide cannula (BAS, West Lafayette IN) directed towards the left dorsal striatum was stereotaxically implanted. The striatal coordinates relative to bregma were: 0.2 mm posterior, 2.8 mm lateral, and 4.0 mm ventral 2s. The guide cannula was secured with cranioplastic cement anchored to four screws in the skull. Rats were allowed 3-7 days to recover from the surgery. Experiments were performed between 09.00 and 16.00 h in awake, freely moving rats and each animal was utilized in only one experiment. On the day of the experiment, a microdialysis probe (3 mm length, 0.5 mm diameter, 20,000 MW cut-off; BAS) was inserted through the guide cannula which extended 3 mm beyond the tip of the guide. The probe was perfused with a buffered artificial CSF (148.3 mM NaC1, 3.0 mM KCI, 1.4 mM CaCI 2, 0.8 mM MgCI z, 1.3 mM NaH2PO 4, 0.2 mM Na2HPO4; pH 7.4) at a flow rate of 2.0 p.l/min using a CMA/100 microinjection pump (Carnegie Medicine; Stockholm, Sweden) capable of housing three different syringes. Neostigmine (1.0 /zM) was added to the artificial CSF to facilitate recovery of ACh. Following probe insertion, either a 1 h period (in the tetrodotoxin and MgC12 experiments) or 2 h period (in the substance P experiments) was allowed before baseline samples were collected to permit stabilization of ACh levels. After collection of four 20 min baseline samples, drugs were perfused through the probe for 20 or 60 min by means of a liquid switch (Carnegie Medicine, Model C M A / l l 0 ) . In the substance P experiments, a second 20 min drug perfusion period was performed 80 min after the initial drug perfusion period. Drugs that were tested included tetrodotoxin (0.3 p.M), MgCI 2 (10 mM), substance P (1, 10, and 25 /~M), and CP 96,345 (10/zM). In the MgC1z experiments, CaC12 was not included in the artificial CSF and the concentration of NaCI was adjusted to maintain iso-osmolarity. When two different concentrations of substance P were applied, the lower concentration was perfused first. All drugs were dissolved in artificial CSF containing 1 /zM neostigmine. In vitro microdialysis Immediately prior to in vivo experiments, microdialysis probes were tested for their ability to recover ACh in vitro. Probe tips were submerged in 1.5 ml polypropylene tubes containing a standard solution of ACh (50 pmol/20/zl) at room temperature and perfused with artificial CSF supplemented with neostigmine as above. ACh in both the collected dialysate and standard solution were compared to calculate the percent relative recovery. In a separate series of in vitro experiments, probes were placed in vials containing artificial CSF at room temperature and perfused with 1, 100, or 1000/xM substance P for 1 h. Later, both the solution in the vial and the perfusion fluid were assayed for substance P (see below). The percentage of substance P delivered was calculated from the ratio of substance P in the external artificial CSF relative to that in the perfusate. In this manner, an estimate of the percent of substance P which diffused out of the probe was obtained. ACh and substance P assays ACh and choline (Ch) were measured by HPLC with electrochemical detection. ACh and Ch were separated on a 10 cm polymeric analytical column (BAS) and then converted to hydrogen peroxide and betaine by an immobilized enzyme reactor (acetylcholinesterase and choline oxidase; BAS) coupled to the ana-
lytical column. The mobile phase consisted of 35 mM sodium phosphate at pH 8.5 supplemented with the antibacterial reagent Kathon CG (5 ml per 1 liter; BAS). Detection was obtained with an amperometric detector (BAS, Model LC-4C). The formed hydrogen peroxide was oxidized on a platinum electrode (BAS) held at + 0.5 V with respect to a Ag-AgCI reference electrode (BAS). The amounts of choline and ACh were calculated by comparing peak heights of these compounds in the samples with peak heights of standards. At a detector range of 20 nA full-scale, the detection limit for ACh was 1.0 pmol/20 /~1 sample. None of the drugs which were perfused through the probe in this study interfered with the assay. Substance P was measured by radioimmunoassay (RIA) using a commercially available kit (Incstar Corp., Stillwater, MN) as previously reported 12.
Histology and statistics Upon completion of the experiments, rats were killed by overdose of sodium pentobarbital and their brains were immediately removed and frozen. Brains were cut into 20 /Lm sections with a cryostat microtome in order to verify histologically the site of probe placement. Data from in vivo experiments were converted to a percent of the mean of four baseline measurements. ACh concentrations were not corrected for in vitro recovery. Data were analyzed by one factor or two-factor analysis of variance (ANOVA) with repeated measures followed by Duncan's new multiple-range test when a significant (P < 0.05) interaction F value was obtained. Comparisons to baseline were made to the fourth baseline sample. Drugs Sodium pentobarbital was purchased from Abbott Laboratories (Abbott Park, IL). Neostigmine bromide, tetrodotoxin, and substance P were purchased from Sigma (St. Louis, MO). CP 96,345 [(2S,3S)cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-]-l-azabicyclo[2,2,2]octan-3-amine] was generously supplied by Pfizer (Groton, CT).
RESULTS
In vitro recovery of ACh and delivery of substance P The in vitro recovery of ACh by microdialysis probes was 14.7 + 0.5% (mean + S.E.M.; n = 39). The percentage of substance P delivered into the surrounding medium following perfusion through the probe was 0.21% for 1 /zM substance P, 0.33% for 100 /~M substance P, and 0.25% for 1000 /zM substance P (n = 1-3 experiments).
Basal levels of extracellular ACh: effects of tetrodotoxin and high magnesium In this series of experiments, 1 h was allowed between probe insertion and collection of baseline sampies. Baseline concentrations of extracellular ACh were generally constant throughout the course of the experiment, although the initial two samples tended to have slightly lower levels of ACh compared to subsequent samples (Fig. 1A). Local perfusion of the dorsal striatum with the Na+-channel blocker tetrodotoxin (0.3 /xM) for 1 h reduced extracellular ACh to 21% of baseline 20 min after the start of perfusion (Fig. 1B). At 40 and 60 min after initiation of perfusion, extracellular ACh was
191 undetectable. By 100 min following discontinuation of tetrodotoxin administration, ACh levels recovered to 60% of baseline (but remained significantly decreased). Blockade of Ca 2+ channels with elevated magnesium (10 mM MgC12) also significantly reduced extracellular ACh levels (Fig. 1C). By 20 min after the start of perfusion ACh was decreased to 35% of baseline and reached a maximal reduction at 40 min (23% of
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Fig. 2. Effect of local perfusion of substance P alone and in combination with the NK l receptor antagonist CP 96,345 on levels of extracellular ACh in the dorsal striatum. Substance P (10/xM) and substance P plus CP 96,345 (10/zM each) were administered locally through the probe for 20 min. ACh levels are expressed as the percent of the mean (+S.E.M.) of the first four baseline samples collected, Baseline samples were collected beginning 2 h after insertion of the probe. The mean basal concentration of ACh was 7.2+0.7 p m o l / 2 0 / x l . * P < 0.05 compared to baseline by two-factor A N O V A with repeated measures and Duncan's multiple-range test (n = 5 experiments each).
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Time (minutes) Fig. 1. Effect of local perfusion of the Na+-channel blocker tetrodotoxin (TTX; 0.3 /zM) and effect of Ca2+-channel blockade with elevated magnesium (10 mM MgCI 2) on levels of extracellular ACh in the dorsal striatum. Ca 2+ was omitted from the perfusion buffer during perfusion with MgCI 2. Each compound was perfused through the probe for 1 h. ACh levels are expressed as the percent of the mean_+ S.E.M. (control, n = 4; TFX, n = 3; MgCI 2, n = 3) of the first four baseline samples collected. Baseline samples were collected beginning 1 h after insertion of the probe. The mean basal concentration of ACh was 8.5+0.7 p m o l / 2 0 /zl. * P < 0 . 0 5 compared baseline by two-factor A N O V A with repeated measures and Duncan's new multiple-range test.
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Effect of substance P on basal levels of extracellular ACh In these experiments a 2 h period (instead of a 1 h period) was permitted between probe insertion and collection of control samples in order to achieve more stable levels of basal ACh (Fig. 2A). At no time during
192 the control experiments did ACh levels differ significantly from baseline. Local application of substance P through the microdialysis probe produced concentration-dependent elevations in extracellular ACh compared to baseline (Fig. 3). Perfusion with 10 and 25 p,M substance P for 20 min elicited 30% and 51% increases in ACh, respectively. Perfusion with 1 /~M substance P enhanced extracellular ACh by 11%, but this effect was not statistically significant. Successive 20 rain perfusions of 10/~M substance P elicited reproducible elevations in extracellular ACh (30% increases relative to baseline) (Fig. 2B). Although ACh levels showed a tendency to remain elevated in the 20 min sample following the initial perfusion with substance P, levels returned to baseline in subsequent samples (prior to the second drug administration period). CP 96,345 (10 /zM), a substance P antagonist selective for NK 1 receptors, prevented the increase in ACh induced by 10/a,M substance P and reduced peak ACh levels by 57% (Fig. 2C). Local perfusion of 10/zM CP 96,345 alone did not alter basal extracellular ACh (Fig. 4). DISCUSSION Results from this study indicate that substance P increases extracellular ACh in the dorsal striatum of awake, freely moving rats. Baseline levels of extracellular ACh were substantially reduced by Na +- and Ca 2+-
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Substance P Fig. 3. Effect of local perfusion of substance P (1, 10 and 25/zM) on levels of extracellular A C h in the dorsal striatum. Substance P was administered locally through the probe for 20 min. A C h levels are expressed as the percent of the mean (_+S.E.M.) of the first four baseline samples collected. Baseline samples were collected starting 2 h after insertion of the probe. The m e a n basal concentration of ACh was 7.4+_ 1.0 p m o l / 2 0 /~1. * P < 0.05 compared to baseline or * P < 0.05 compared to the other two treatments by two-factor A N O V A with repeated measures and Duncan's new multiple-range test (n = 5 experiments at each concentration).
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Time (minutes) Fig. 4. Effect of the NK 1 receptor antagonist CP 96,345 on extracellular A C h in the dorsal striatum. CP 96,345 (10 ~ M ) was administered locally through the probe for 20 min. ACh levels are expressed as the percent of the mean (_+S.E.M.) of the first four baseline samples collected. Baseline samples were collected beginning 2 h after insertion of the probe. No differences were observed in basal A C h during or after perfusion of CP 96,345 by one-factor repeated measures A N O V A (n = 4).
channel blockade, indicating that basal concentrations of ACh recovered by microdialysis were predominantly of neuronal origin in agreement with previous reports ~1'23. Local perfusion of the dorsal striatum with substance P induced concentration-dependent elevations in extracellular ACh. The substance P antagonist CP 96,345, which selectively blocks NK 1 receptors 25'35, attenuated the substance P-induced increase in ACh. CP 96,345 alone had no effect on basal levels of extracellular ACh suggesting that NK~ receptors on cholinergic neurons are not tonically stimulated. Taken together these data suggest that substance P, through activation of NK 1 receptors, stimulates the release of ACh in the dorsal striatum. The present data also show that, under ideal in vitro conditions, only a small fraction (0.21%-0.33%) of substance P perfused through the probe actually passes through the dialysis membrane into the external environment. Under in vivo conditions, delivery of substance P is probably even less due to high tortuosity and low volume fraction - factors which limit diffusion in brain tissue 3. The relatively large size of substance P (MW 1347), charge interactions, and molecular weight cut-off of the dialysis membrane (20,000 Da) are also factors which limit local application of this peptide. As a result of the low fractional delivery of substance P through the microdialysis probe, administration of micromolar concentrations of substance P was required in order to elicit effects on ACh release. It has recently been reported that CP 96,345 in addition to being a potent NK~ receptor antagonist may also act as an antagonist of L-type Ca 2+ channels 34. Although these channels are thought to be located on cell bodies and proximal dendrites 39 some evidence indicates their involvement in neurotransmitter re-
193 lease 3°. We have shown, however, that CP 96,345 alone does not alter basal release of ACh at a concentration which attenuated substance P-induced ACh release. Hence, it appears that CP 96,345 at the concentration employed here blocks NK t receptors with little, if any, effect by itself on ACh release. Findings from this investigation are in agreement with studies in neostriatal slices in which septide, a NK 1 agonist, elicited release of ACh a. Substance P, like septide, displays highest affinity to the NK a receptor (compared to NK 2 and NK 3 receptors) although substance P is slightly more potent than septide aa. Arenas and coworkers I observed maximal release of ACh at greater than 1 /xM concentrations of septide with an ECs0 of approximately 12 nM; very much in line with effective concentrations employed in the present study and with estimates of actual concentrations of substance P delivered locally into the striatum. The ability of substance P to stimulate striatal ACh release is consistent with anatomical evidence showing synaptic contact between substance P-containing terminals and cholinergic neurons in the striatum 7 and presence of substance P (NK l) receptors on striatal cholinergic neurons tS. The source of substance P-containing nerve terminals in the striatum is not entirely certain. However, substance P is present in large amounts in medium spiny striatonigral neurons. Moreover, since a large number of these neurons reside in the striatum and they characteristically show widespread axonal arborization, it seems likely that striatal substance P originates from local collaterals of striatonigral neurons. Receptor binding studies show a moderate density of NK 1 receptor binding sites in the dorsal striatum and a relatively sparse number of NK 1 binding sites in the substantia nigra pars reticulata 22'32. Because substantia nigra pars reticulata contains the highest density of substance P in brain 26 yet is almost devoid of NK t receptors indicates a mismatch between substance P and its receptor in this region. However, the present findings point to a physiological role for substance P and NK 1 receptors in the dorsal striatum in the regulation of ACh release. The functional significance of substance P-induced ACh release in the striatum can only be postulated. Muscarinic receptor antagonists have long been known as moderately effective therapeutic agents in Parkinson's disease 8. Administration of muscarinic antagonists can potentiate stereotypy caused by dopamine receptor agonists 2'1s'33. Hence, ACh and dopamine appear to have opposing actions in the striatum. Recent microdialysis studies suggest that a tonic inhibitory influence of dopamine on ACh release is mediated by dopamine D 2 receptors since D 2 antagonists increase
ACh release a't°. Our findings suggest that substance P does not tonically alter ACh release, since the NK t antagonist alone did not affect extracellular ACh levels. Conceivably, substance P modulates or regulates the tonic effect of dopamine on striatal ACh release. A second possibility is that ACh release induced by substance P from striatonigral collaterals may serve as a functional link between striatonigral and striatopallidal output pathways 16. Dopamine is thought to directly modulate striatonigral neurons primarily via D~ receptors 14'31 and striatopallidal neurons predominantly via D 2 receptors 14'2°. Stimulation of striatonigral neurons may stimulate local release of substance P in the striatum which binds to NK 1 receptors on cholinergic neurons and enhances ACh release. ACh, in turn, may stimulate muscarinic receptors on striatopallidal neurons 38 to alter activity of striatopallidal output. This hypothetical mechanism may help to explain synergy between D~ and D 2 receptor stimulation as observed in behavioral, biochemical, and electrophysiological studies 27'37 even though these receptors may be segregated on different striatal output pathways. In conclusion, these results suggest that substance P, through activation of striatal NK 1 receptors, stimulates release of ACh in the dorsal striatum of awake, freely moving rats and supports anatomical evidence of a substance P-cholinergic circuit in this region. Acknowledgements. The authors wish to thank Shirley Kuo for performing the substance P radioimmunoassay and Pfizer Inc. for their generous supply of CP 96,345.
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substance P (NK t) receptor antagonist, Science, 251 (1991) 437439. 26 Nicoll, R.A., Schenker, C. and Leeman, S.E., Substance P as a neurotransmitter candidate, Annu. Rev. Neurosci., 3 (1980) 227268. 27 Paul, M.L., Graybiel, A.M., David, J.-C. and Robertson, H.A., Dl-like and D2-1ike dopamine receptors synergistically activate rotation and c-los expression in the dopamine-depleted striatum in a rat model of Parkinson's disease, J. Neurosci., 12 (1992) 3729-3742. 28 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, Sydney, 1986. 29 Phelps, P.E., Houser, C.R. and Vaughn, J.E., lmmunocytochemical localization of choline acetyltransferase within the rat neostriatum: a correlated light and electron microscopic study of cholinergic neurons and synapses, J. Comp. Neurol., 238 (1985) 286-307. 30 Pin, J.-P. and Bockaert, J., Omega-conotoxin GVIA and dihydropyridines discriminate two types of Ca 2+ channels involved in GABA release from striatal neurons in culture, Eur. J. Pharmacol., 188 (1990) 81-84. 31 Robertson, G.S., Vincent, S.R. and Fibiger, H.C., Striato-nigral projection neurons contain D1 dopamine receptor-activated c-los, Brain Res., 523 (1990) 288-290. 32 Saffroy, M., Beaujouan, J.-C., Torrens, Y., Besseyre, J., Bergstrom, L. and Glowinski, J., Localization of tachykinin binding sites (NKI, NK2, NK 3 ligands) in the rat brain, Peptides, 9 (1988) 227-241. 33 Scheel-Kruger, J., Central effects of anticholinergic drugs measured by the apomorphine gnawing test in mice, Acta Pharmacol. Toxicol., 28 (1970) 1-16. 34 Schmidt, A.W., McLean, S. and Heym, J., The substance P receptor antagonist CP-96,345 interacts with Ca 2÷ channels, Eur. J. Pharmacol., 219 (1992) 491-492. 35 Snider, R.M., Constantine, J.W., Lowe, III J.A., Longo, K.P., Lebel, W.S., Woody, H.A., Drozda, S.E., Desai, M.C., Vinick, F.J., Spencer, R.W. and Hess, H.-J., A potent nonpeptide antagonist of the substance P (NK 1) receptor, Science, 251 (1991) 435-437. 36 Wainer, B.H., Bolam, J.P., Freund, T.F., Henderson, Z., Totterdell, S. and Smith, A.D., Cholinergic synapses in the rat brain: a correlated light and electron microscopic immunohistochemical study employing a monoclonal antibody against choline acetyltransferase, Brain Res., 308 (1984) 69-76. 37 Waiters, J.R., Bergstrom, D.A., Carlson, J.H., Chase, T.N. and Braun, A.R., D 1 dopamine receptor activation required for postsynaptic expression of D 2 agonist effects, Science, 236 (1987) 719-722. 38 Weiner, D.M., Levey, A.I. and Brann, M.R., Expression of muscarinic acetylcholine and dopamine receptor mRNAs in rat basal ganglia, Proc. NatL Acad. Sci. USA, 87 (1990) 7050-7054. 39 Westenbroek, R.E., Ahlijanian, M.K. and Catterall, W.A., Clustering of L-type Ca 2+ channels at the base of major dendrites in hippocampal pyramidal neurons, Nature, 347 (1990) 281-284. 40 Wilson, C.J., Chang, H.T. and Kitai, S.T., Firing patterns and synaptic potentials of identified giant aspiny interneurons in the rat neostriatum, J. Neurosci., 10 (1990) 508-519. 41 Wormser, U., Laufer, R., Hart, Y., Chorev, M., Gilon, C., and Selinger, Z., Highly selective agonists for substance P receptor subtypes, EMBO J., 5 (1986) 2805-2808.
189
BRES 19234
Substance P increases release of acetylcholine in the dorsal striatum of freely moving rats Jeffrey J. Anderson, Thomas N. Chase and Thomas M. Engber Experimental Therapeutics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 (USA)
(Accepted 27 April 1993)
Key words: Microdialysis; Acetylcholine; Substance P; Neurokinin; Striatum; Basal ganglia
Little is known about the role that neuropeptides such as substance P play in cell-to-cell interactions in the striatum. The effect of locally perfused substance P on extracellular acetylcholine (ACh) in the dorsal striatum of awake, freely moving rats was examined using microdialysis. Neostigmine (1 /zM) was included in the perfusate to improve recovery of ACh. Basal extracellular ACh was sensitive to Na+-channel blockade with tetrodotoxin (0.3/~M) and Ca2+-channel blockade with MgC12 (10 mM) and therefore largely neuronal in origin. Local perfusion with 10 and 25/zM substance P for 20 min elevated extracellular ACh by 30% and 51%, respectively. The NK 1 receptor antagonist, CP 96,345 (10/zM), which by itself had no effect on extracellular ACh, prevented the substance P-induced increase in extracellular ACh. These results suggest that stimulation of NK 1 receptors by substance P enhances ACh release in the dorsal striatum and is consistent with anatomical evidence of a substance P-cholinergic circuit in this region.
INTRODUCTION A c e t y l c h o l i n e ( A C h ) in t h e s t r i a t u m is t h o u g h t to o r i g i n a t e p r i m a r i l y f r o m large aspiny n e u r o n s 6'36 which a r e intrinsic to this r e g i o n 9A9'24. A l t h o u g h c h o l i n e r g i c i n t e r n e u r o n s c o m p r i s e only a b o u t 2 % o f t h e t o t a l striatal cell p o p u l a t i o n in t h e rat 29, t h e y s u p p l y a d e n s e i n n e r v a t i o n 4° a n d a r e r e s p o n s i b l e for i m p a r t i n g t h e s t r i a t u m with s o m e o f t h e h i g h e s t q u a n t i t i e s o f cholinergic m a r k e r s in t h e CNS 13'17. S t r i a t a l c h o l i n e r g i c int e r n e u r o n s a p p e a r to p l a y a n i m p o r t a n t role in r e g u l a t ing s t r i a t a l f u n c t i o n since c h o l i n e r g i c a n t a g o n i s t s a m e l i o r a t e m o t o r a b n o r m a l i t i e s in P a r k i n s o n ' s d i s e a s e 8. O n e such i n t e r a c t i o n m a y b e with m e d i u m spiny striatonigral neurons containing the neurokinin substance P. T h e s e n e u r o n s c o n s t i t u t e o n e o f t h e m a j o r striatal e f f e r e n t p a t h w a y s , p r o j e c t i n g m a i n l y to t h e s u b s t a n t i a n i g r a p a r s r e t i c u l a t a . In a d d i t i o n , t h e s e n e u r o n s possess local axon c o l l a t e r a l s which a r b o r i z e within t h e s t r i a t u m 5. S u b s t a n c e P - c o n t a i n i n g n e r v e t e r m i n a l s have
b e e n shown to m a k e s y n a p t i c c o n t a c t with i d e n t i f i e d c h o l i n e r g i c n e u r o n s in t h e s t r i a t u m 7. M o r e o v e r , striatal neurons containing choline acetyltransferase mRNA s e l e c t i v e l y e x p r e s s s u b s t a n c e P ( N K l) r e c e p t o r m R N A 15. It thus a p p e a r s t h a t s u b s t a n c e P - c o n t a i n i n g axon c o l l a t e r a l s o f s t r i a t o n i g r a l n e u r o n s synapse with striatal c h o l i n e r g i c i n t e r n e u r o n s which possess subs t a n c e P ( N K l) r e c e p t o r s . It is not c l e a r w h a t a c t i o n s u b s t a n c e P m a y have on striatal c h o l i n e r g i c i n t e r n e u r o n s o r how s u b s t a n c e P may influence the release of ACh from these neurons in t h e intact brain. N e u r o k i n i n s , in g e n e r a l , a r e t h o u g h t to excite n e u r o n s 21. In s u p p o r t o f this hypothesis, septide, a s u b s t a n c e P a n a l o g selective for t h e N K 1 r e c e p tor, i n c r e a s e d t h e r e l e a s e o f A C h f r o m n e o s t r i a t a l slices ( n e u r o k i n i n A a n d n e u r o k i n i n B, n e u r o k i n i n s selective for N K 2 a n d N K 3 r e c e p t o r s , respectively, also e l i c i t e d A C h r e l e a s e ) t. A n effect o f s u b s t a n c e P on striatal A C h r e l e a s e in vivo has yet to b e d e m o n s t r a t e d . T h e p u r p o s e o f this investigation was to e x a m i n e t h e
Correspondence: J.J. Anderson, Experimental Therapeutics Branch, National Institute of Neurological Disorders and Stroke, Building 10, Room 5C215, 9000 Rockville Pike, Bethesda, MD 20892, USA. Fax: (1) (301) 496-6609.
190 relationship between substance P and ACh release in the dorsal striatum using in vivo microdialysis in awake, freely moving rats. MATERIALS AND METHODS
Surgery and in vivo microdialysis procedure Male Sprague-Dawley rats (Taconic Farms, Germantown, NY) weighing 275-300 g were housed individually on a standard 12 h light/dark cycle with unlimited access to food and water. Prior to surgery, each animal was anesthetized with sodium pentobarbital (50 mg/kg i.p.). An intracerebral guide cannula (BAS, West Lafayette IN) directed towards the left dorsal striatum was stereotaxically implanted. The striatal coordinates relative to bregma were: 0.2 mm posterior, 2.8 mm lateral, and 4.0 mm ventral 2s. The guide cannula was secured with cranioplastic cement anchored to four screws in the skull. Rats were allowed 3-7 days to recover from the surgery. Experiments were performed between 09.00 and 16.00 h in awake, freely moving rats and each animal was utilized in only one experiment. On the day of the experiment, a microdialysis probe (3 mm length, 0.5 mm diameter, 20,000 MW cut-off; BAS) was inserted through the guide cannula which extended 3 mm beyond the tip of the guide. The probe was perfused with a buffered artificial CSF (148.3 mM NaC1, 3.0 mM KCI, 1.4 mM CaCI 2, 0.8 mM MgCI z, 1.3 mM NaH2PO 4, 0.2 mM Na2HPO4; pH 7.4) at a flow rate of 2.0 p.l/min using a CMA/100 microinjection pump (Carnegie Medicine; Stockholm, Sweden) capable of housing three different syringes. Neostigmine (1.0 /zM) was added to the artificial CSF to facilitate recovery of ACh. Following probe insertion, either a 1 h period (in the tetrodotoxin and MgC12 experiments) or 2 h period (in the substance P experiments) was allowed before baseline samples were collected to permit stabilization of ACh levels. After collection of four 20 min baseline samples, drugs were perfused through the probe for 20 or 60 min by means of a liquid switch (Carnegie Medicine, Model C M A / l l 0 ) . In the substance P experiments, a second 20 min drug perfusion period was performed 80 min after the initial drug perfusion period. Drugs that were tested included tetrodotoxin (0.3 p.M), MgCI 2 (10 mM), substance P (1, 10, and 25 /~M), and CP 96,345 (10/zM). In the MgC1z experiments, CaC12 was not included in the artificial CSF and the concentration of NaCI was adjusted to maintain iso-osmolarity. When two different concentrations of substance P were applied, the lower concentration was perfused first. All drugs were dissolved in artificial CSF containing 1 /zM neostigmine. In vitro microdialysis Immediately prior to in vivo experiments, microdialysis probes were tested for their ability to recover ACh in vitro. Probe tips were submerged in 1.5 ml polypropylene tubes containing a standard solution of ACh (50 pmol/20/zl) at room temperature and perfused with artificial CSF supplemented with neostigmine as above. ACh in both the collected dialysate and standard solution were compared to calculate the percent relative recovery. In a separate series of in vitro experiments, probes were placed in vials containing artificial CSF at room temperature and perfused with 1, 100, or 1000/xM substance P for 1 h. Later, both the solution in the vial and the perfusion fluid were assayed for substance P (see below). The percentage of substance P delivered was calculated from the ratio of substance P in the external artificial CSF relative to that in the perfusate. In this manner, an estimate of the percent of substance P which diffused out of the probe was obtained. ACh and substance P assays ACh and choline (Ch) were measured by HPLC with electrochemical detection. ACh and Ch were separated on a 10 cm polymeric analytical column (BAS) and then converted to hydrogen peroxide and betaine by an immobilized enzyme reactor (acetylcholinesterase and choline oxidase; BAS) coupled to the ana-
lytical column. The mobile phase consisted of 35 mM sodium phosphate at pH 8.5 supplemented with the antibacterial reagent Kathon CG (5 ml per 1 liter; BAS). Detection was obtained with an amperometric detector (BAS, Model LC-4C). The formed hydrogen peroxide was oxidized on a platinum electrode (BAS) held at + 0.5 V with respect to a Ag-AgCI reference electrode (BAS). The amounts of choline and ACh were calculated by comparing peak heights of these compounds in the samples with peak heights of standards. At a detector range of 20 nA full-scale, the detection limit for ACh was 1.0 pmol/20 /~1 sample. None of the drugs which were perfused through the probe in this study interfered with the assay. Substance P was measured by radioimmunoassay (RIA) using a commercially available kit (Incstar Corp., Stillwater, MN) as previously reported 12.
Histology and statistics Upon completion of the experiments, rats were killed by overdose of sodium pentobarbital and their brains were immediately removed and frozen. Brains were cut into 20 /Lm sections with a cryostat microtome in order to verify histologically the site of probe placement. Data from in vivo experiments were converted to a percent of the mean of four baseline measurements. ACh concentrations were not corrected for in vitro recovery. Data were analyzed by one factor or two-factor analysis of variance (ANOVA) with repeated measures followed by Duncan's new multiple-range test when a significant (P < 0.05) interaction F value was obtained. Comparisons to baseline were made to the fourth baseline sample. Drugs Sodium pentobarbital was purchased from Abbott Laboratories (Abbott Park, IL). Neostigmine bromide, tetrodotoxin, and substance P were purchased from Sigma (St. Louis, MO). CP 96,345 [(2S,3S)cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-]-l-azabicyclo[2,2,2]octan-3-amine] was generously supplied by Pfizer (Groton, CT).
RESULTS
In vitro recovery of ACh and delivery of substance P The in vitro recovery of ACh by microdialysis probes was 14.7 + 0.5% (mean + S.E.M.; n = 39). The percentage of substance P delivered into the surrounding medium following perfusion through the probe was 0.21% for 1 /zM substance P, 0.33% for 100 /~M substance P, and 0.25% for 1000 /zM substance P (n = 1-3 experiments).
Basal levels of extracellular ACh: effects of tetrodotoxin and high magnesium In this series of experiments, 1 h was allowed between probe insertion and collection of baseline sampies. Baseline concentrations of extracellular ACh were generally constant throughout the course of the experiment, although the initial two samples tended to have slightly lower levels of ACh compared to subsequent samples (Fig. 1A). Local perfusion of the dorsal striatum with the Na+-channel blocker tetrodotoxin (0.3 /xM) for 1 h reduced extracellular ACh to 21% of baseline 20 min after the start of perfusion (Fig. 1B). At 40 and 60 min after initiation of perfusion, extracellular ACh was
191 undetectable. By 100 min following discontinuation of tetrodotoxin administration, ACh levels recovered to 60% of baseline (but remained significantly decreased). Blockade of Ca 2+ channels with elevated magnesium (10 mM MgC12) also significantly reduced extracellular ACh levels (Fig. 1C). By 20 min after the start of perfusion ACh was decreased to 35% of baseline and reached a maximal reduction at 40 min (23% of
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Fig. 2. Effect of local perfusion of substance P alone and in combination with the NK l receptor antagonist CP 96,345 on levels of extracellular ACh in the dorsal striatum. Substance P (10/xM) and substance P plus CP 96,345 (10/zM each) were administered locally through the probe for 20 min. ACh levels are expressed as the percent of the mean (+S.E.M.) of the first four baseline samples collected, Baseline samples were collected beginning 2 h after insertion of the probe. The mean basal concentration of ACh was 7.2+0.7 p m o l / 2 0 / x l . * P < 0.05 compared to baseline by two-factor A N O V A with repeated measures and Duncan's multiple-range test (n = 5 experiments each).
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Time (minutes) Fig. 1. Effect of local perfusion of the Na+-channel blocker tetrodotoxin (TTX; 0.3 /zM) and effect of Ca2+-channel blockade with elevated magnesium (10 mM MgCI 2) on levels of extracellular ACh in the dorsal striatum. Ca 2+ was omitted from the perfusion buffer during perfusion with MgCI 2. Each compound was perfused through the probe for 1 h. ACh levels are expressed as the percent of the mean_+ S.E.M. (control, n = 4; TFX, n = 3; MgCI 2, n = 3) of the first four baseline samples collected. Baseline samples were collected beginning 1 h after insertion of the probe. The mean basal concentration of ACh was 8.5+0.7 p m o l / 2 0 /zl. * P < 0 . 0 5 compared baseline by two-factor A N O V A with repeated measures and Duncan's new multiple-range test.
baseline). By 100 min after MgCI 2 perfusion was discontinued, extracellular ACh had essentially returned to baseline levels.
Effect of substance P on basal levels of extracellular ACh In these experiments a 2 h period (instead of a 1 h period) was permitted between probe insertion and collection of control samples in order to achieve more stable levels of basal ACh (Fig. 2A). At no time during
192 the control experiments did ACh levels differ significantly from baseline. Local application of substance P through the microdialysis probe produced concentration-dependent elevations in extracellular ACh compared to baseline (Fig. 3). Perfusion with 10 and 25 p,M substance P for 20 min elicited 30% and 51% increases in ACh, respectively. Perfusion with 1 /~M substance P enhanced extracellular ACh by 11%, but this effect was not statistically significant. Successive 20 rain perfusions of 10/~M substance P elicited reproducible elevations in extracellular ACh (30% increases relative to baseline) (Fig. 2B). Although ACh levels showed a tendency to remain elevated in the 20 min sample following the initial perfusion with substance P, levels returned to baseline in subsequent samples (prior to the second drug administration period). CP 96,345 (10 /zM), a substance P antagonist selective for NK 1 receptors, prevented the increase in ACh induced by 10/a,M substance P and reduced peak ACh levels by 57% (Fig. 2C). Local perfusion of 10/zM CP 96,345 alone did not alter basal extracellular ACh (Fig. 4). DISCUSSION Results from this study indicate that substance P increases extracellular ACh in the dorsal striatum of awake, freely moving rats. Baseline levels of extracellular ACh were substantially reduced by Na +- and Ca 2+-
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Time (minutes) Fig. 4. Effect of the NK 1 receptor antagonist CP 96,345 on extracellular A C h in the dorsal striatum. CP 96,345 (10 ~ M ) was administered locally through the probe for 20 min. ACh levels are expressed as the percent of the mean (_+S.E.M.) of the first four baseline samples collected. Baseline samples were collected beginning 2 h after insertion of the probe. No differences were observed in basal A C h during or after perfusion of CP 96,345 by one-factor repeated measures A N O V A (n = 4).
channel blockade, indicating that basal concentrations of ACh recovered by microdialysis were predominantly of neuronal origin in agreement with previous reports ~1'23. Local perfusion of the dorsal striatum with substance P induced concentration-dependent elevations in extracellular ACh. The substance P antagonist CP 96,345, which selectively blocks NK 1 receptors 25'35, attenuated the substance P-induced increase in ACh. CP 96,345 alone had no effect on basal levels of extracellular ACh suggesting that NK~ receptors on cholinergic neurons are not tonically stimulated. Taken together these data suggest that substance P, through activation of NK 1 receptors, stimulates the release of ACh in the dorsal striatum. The present data also show that, under ideal in vitro conditions, only a small fraction (0.21%-0.33%) of substance P perfused through the probe actually passes through the dialysis membrane into the external environment. Under in vivo conditions, delivery of substance P is probably even less due to high tortuosity and low volume fraction - factors which limit diffusion in brain tissue 3. The relatively large size of substance P (MW 1347), charge interactions, and molecular weight cut-off of the dialysis membrane (20,000 Da) are also factors which limit local application of this peptide. As a result of the low fractional delivery of substance P through the microdialysis probe, administration of micromolar concentrations of substance P was required in order to elicit effects on ACh release. It has recently been reported that CP 96,345 in addition to being a potent NK~ receptor antagonist may also act as an antagonist of L-type Ca 2+ channels 34. Although these channels are thought to be located on cell bodies and proximal dendrites 39 some evidence indicates their involvement in neurotransmitter re-
193 lease 3°. We have shown, however, that CP 96,345 alone does not alter basal release of ACh at a concentration which attenuated substance P-induced ACh release. Hence, it appears that CP 96,345 at the concentration employed here blocks NK t receptors with little, if any, effect by itself on ACh release. Findings from this investigation are in agreement with studies in neostriatal slices in which septide, a NK 1 agonist, elicited release of ACh a. Substance P, like septide, displays highest affinity to the NK a receptor (compared to NK 2 and NK 3 receptors) although substance P is slightly more potent than septide aa. Arenas and coworkers I observed maximal release of ACh at greater than 1 /xM concentrations of septide with an ECs0 of approximately 12 nM; very much in line with effective concentrations employed in the present study and with estimates of actual concentrations of substance P delivered locally into the striatum. The ability of substance P to stimulate striatal ACh release is consistent with anatomical evidence showing synaptic contact between substance P-containing terminals and cholinergic neurons in the striatum 7 and presence of substance P (NK l) receptors on striatal cholinergic neurons tS. The source of substance P-containing nerve terminals in the striatum is not entirely certain. However, substance P is present in large amounts in medium spiny striatonigral neurons. Moreover, since a large number of these neurons reside in the striatum and they characteristically show widespread axonal arborization, it seems likely that striatal substance P originates from local collaterals of striatonigral neurons. Receptor binding studies show a moderate density of NK 1 receptor binding sites in the dorsal striatum and a relatively sparse number of NK 1 binding sites in the substantia nigra pars reticulata 22'32. Because substantia nigra pars reticulata contains the highest density of substance P in brain 26 yet is almost devoid of NK t receptors indicates a mismatch between substance P and its receptor in this region. However, the present findings point to a physiological role for substance P and NK 1 receptors in the dorsal striatum in the regulation of ACh release. The functional significance of substance P-induced ACh release in the striatum can only be postulated. Muscarinic receptor antagonists have long been known as moderately effective therapeutic agents in Parkinson's disease 8. Administration of muscarinic antagonists can potentiate stereotypy caused by dopamine receptor agonists 2'1s'33. Hence, ACh and dopamine appear to have opposing actions in the striatum. Recent microdialysis studies suggest that a tonic inhibitory influence of dopamine on ACh release is mediated by dopamine D 2 receptors since D 2 antagonists increase
ACh release a't°. Our findings suggest that substance P does not tonically alter ACh release, since the NK t antagonist alone did not affect extracellular ACh levels. Conceivably, substance P modulates or regulates the tonic effect of dopamine on striatal ACh release. A second possibility is that ACh release induced by substance P from striatonigral collaterals may serve as a functional link between striatonigral and striatopallidal output pathways 16. Dopamine is thought to directly modulate striatonigral neurons primarily via D~ receptors 14'31 and striatopallidal neurons predominantly via D 2 receptors 14'2°. Stimulation of striatonigral neurons may stimulate local release of substance P in the striatum which binds to NK 1 receptors on cholinergic neurons and enhances ACh release. ACh, in turn, may stimulate muscarinic receptors on striatopallidal neurons 38 to alter activity of striatopallidal output. This hypothetical mechanism may help to explain synergy between D~ and D 2 receptor stimulation as observed in behavioral, biochemical, and electrophysiological studies 27'37 even though these receptors may be segregated on different striatal output pathways. In conclusion, these results suggest that substance P, through activation of striatal NK 1 receptors, stimulates release of ACh in the dorsal striatum of awake, freely moving rats and supports anatomical evidence of a substance P-cholinergic circuit in this region. Acknowledgements. The authors wish to thank Shirley Kuo for performing the substance P radioimmunoassay and Pfizer Inc. for their generous supply of CP 96,345.
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