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Keywords:

  • crosstalk;
  • dopamine release;
  • muscarinic receptors;
  • nicotinic receptors;
  • nucleus accumbens;
  • synaptosomes

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Dopaminergic nerve endings in the corpus striatum possess nicotinic (nAChRs) and muscarinic cholinergic receptors (mAChRs) mediating release of dopamine (DA). Whether nAChRs and mAChRs co-exist and interact on the same nerve endings is unknown. We here investigate on these possibilities using rat nucleus accumbens synaptosomes pre-labeled with [3H]DA and exposed in superfusion to cholinergic receptor ligands. The mixed nAChR–mAChR agonists acetylcholine (ACh) and carbachol provoked [3H]DA release partially sensitive to the mAChR antagonist atropine but totally blocked by the nAChR antagonist mecamylamine. Addition of the mAChR agonist oxotremorine at the minimally effective concentration of 30 μmol/L, together with 3, 10, or 100 μmol/L (−)nicotine provoked synergistic effect on [3H]DA overflow. The [3H]DA overflow elicited by 100 μmol/L (−)nicotine plus 30 μmol/L oxotremorine was reduced by atropine down to the release produced by (−)nicotine alone and it was abolished by mecamylamine. The ryanodine receptor blockers dantrolene or 8-bromo-cADP-ribose, but not the inositol 1,4,5-trisphosphate receptor blocker xestospongin C inhibited the (−)nicotine/oxotremorine evoked [3H]DA overflow similarly to atropine. This overflow was partly sensitive to 100 nmol/L methyllycaconitine which did not prevent the synergistic effect of (−)nicotine/oxotremorine. Similarly to (−)nicotine, the selective α4β2 nAChR agonist RJR2403 exhibited synergism when added together with oxotremorine. To conclude, in rat nucleus accumbens, α4β2 nAChRs exert a permissive role on the releasing function of reportedly M5 mAChRs co-existing on the same dopaminergic nerve endings.

Abbreviations used
8-Br-cADPR

8-bromo-cADP-ribose

ACh

acetylcholine

CCh

carbachol

DA

dopamine

IP3

inositol 1,4,5-trisphosphate

KO

knockout

mAChR

muscarinic acetylcholine receptor

nAChR

nicotinic acetylcholine receptor

TTx

tetrodotoxin

VSCC

voltage-sensitive calcium channel

Acetylcholine (ACh) acts at two types of receptors: muscarinic (mAChRs) and nicotinic ACh receptors (nAChRs). Receptors of the muscarinic type are metabotropic and nAChRs are ionotropic. Both families exhibit structural and pharmacological heterogeneity: in the CNS mAChRs exist as five subtypes, termed M1 to M5, while an undefined number of nAChRs subtypes consist of pentameric ligand-gated cation channels, formed by the assembly of multiple α (α2–α7) and β (β2–β4) subunits. Subtypes M1, M3, and M5 of the mAChR are positively coupled to the phosphatidylinositol pathway while M2 and M4 are negatively coupled to adenylate cyclase. The channels of nAChRs are variously permeable to external Na+ and Ca2+ ions (see for reviews Wess 1996, 2003; Lukas et al. 1999; Eglen and Nahorski 2000; Gotti et al. 2006).

In the mammalian CNS, both mAChRs and nAChRs can display post-synaptic as well as pre-synaptic localization. The main function of pre-synaptic cholinergic receptors is to modulate neurotransmitter release from nerve endings; these receptors can modulate ACh release as pre-synaptic autoreceptors, or the release of other transmitters, as pre-synaptic heteroreceptors (see for reviews Starke et al. 1989; Langer 1997; Raiteri 2006).

Muscarinic heteroreceptors potentiating the depolarization-evoked release of dopamine (DA) were reported to exist on striatal dopaminergic terminals, based on studies with K+-depolarized striatal synaptosomes (Raiteri et al. 1982) and with K+-depolarized striatal slices (Lehmann and Langer 1982; Schoffelmeer et al. 1986; Kemel et al. 1989).

Using striatal synaptosomes, Raiteri et al. (1984) showed that pirenzepine, a drug originally reported to discriminate between M1 (pirenzepine sensitive) and M2 (pirenzepine insensitive) muscarinic receptor binding sites in the CNS (Hammer et al. 1980; Watson et al. 1983), was able to block the mAChRs mediating DA release. These receptors were therefore classified as M1 subtype, to distinguish them from the muscarinic autoreceptors, sited on cholinergic nerve endings (Marchi et al. 1981) which were classified as M2 (Raiteri et al. 1984). The subsequent identification of M3, M4, and M5 mAChRs and the finding that pirenzepine binding was not limited to receptors of M1 subtype led to reconsideration of the pharmacology of the mAChRs mediating DA release in the corpus striatum. Because of the limited receptor subtype selectivity of the muscarinic agonists and antagonists available (Zhang et al. 2002) approached the problem using M1-M5 mAChR knockout (KO) mice. These authors found that the oxotremorine-mediated potentiation of the K+-evoked [3H]DA release observed in striatal slices from wild-type mice was significantly reduced in M5 KO animals, suggesting that DA nerve endings in the striatum possess release-stimulating mAChRs of the M5 subtype.

Dopamine release in rodent striatum can be also modulated by pre-synaptic nAChRs present on dopaminergic terminals (Champtiaux et al. 2003; Salminen et al. 2004; Grady et al. 2007). Indeed several works carried out by monitoring [3H]DA release from striatal synaptosomes provided evidence for the heterogeneity of pre-synaptic nAChRs on DA terminals which include the α4β2* subtype (*indicates the presence of possible additional subunits) (Sharples et al. 2000; Grady et al. 2001; Jones et al. 2001; Zhou et al. 2001) and nAChRs of subunit composition α6β2* (Wonnacott et al. 2005; Gotti et al. 2006; Quik and McIntosh 2006).

Although mAChRs and nAChRs have been individually shown to be localized on DA nerve endings of the striatum, whether both receptors are co-expressed on the same terminals and, in particular, whether they are able to interact in modulating DA release is unknown.

In the present investigation, we studied the release of [3H]DA from rat nucleus accumbens synaptosomes exposed in superfusion to mAChR and nAChR agonists alone or in combination. Nucleus accumbens was chosen also considering the fundamental role that the release of DA in this brain region plays in drug seeking behavior. Our results show that nAChR activation exerts a permissive role on the activation of mAChRs co-existing on the same terminals and mediating enhancement of DA outflow.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals and brain tissue preparation

Adult male rats (Sprague–Dawley, 200–250 g) were housed at constant temperature (22 ± 1°C) and relative humidity (50%) under a regular light–dark schedule (lights on 07:00 h and off at 19:00 h). Food and water were freely available. The animals were killed by decapitation and the brain areas rapidly removed at 0–4°C. The experimental procedures were approved by the Ethical Committee of the Pharmacology and Toxicology Section, Department of Experimental Medicine, in accordance with the European legislation (European Communities Council Directive of 24 November 1986, 86/609/EEC). Crude synaptosomes of nucleus accumbens were prepared as previously described (Raiteri et al. 1984) with same minor modifications. Briefly, nucleus accumbens was homogenized in 40 volumes of 0.32 mol/L sucrose, buffered to pH 7.4 with phosphate (final concentration 0.01 M). The homogenate was centrifuged at 1000 g for 5 min, to remove nuclei and cellular debris, and crude synaptosomes were isolated from the supernatant by centrifugation at 12 000 g for 20 min. The synaptosomal pellet was then resuspended in physiological medium having the following composition (mmol/L): NaCl 125, KCl 3, MgSO4 1.2, CaCl2 1.2, NaH2PO4 1, NaHCO3 22, and glucose 10 (aeration with 95% O2 and 5% CO2), pH 7.2–7.4. In release experiments, synaptosomes were incubated 15 min at 37°C with [3H]DA (final concentration 0.05 μmol/L). Labeling with [3H]DA was performed in presence of 6-nitroquipazine and 0.1 μmol/L of the noradrenaline blocker nisoxetine.

Release experiments from synaptosomes

Identical portions of the synaptosomal suspension were then layered on microporous filters at the bottom of parallel superfusion chambers thermostated at 37°C (Superfusion System, Ugo Basile, Comerio, Varese, Italy) and superfused according to (Raiteri and Raiteri 2000). Superfusion (0.5 mL/min) was started with standard physiological solution aerated with 95% O2 and 5% CO2, at 37°C. Starting from the 36 min of superfusion four consecutive 3-min fractions were collected; in some experiments when we have monitored nicotinic/oxotremorine or RJR2403-evoked release five consecutive 1-min fractions from 36 to 41 min were collected. Agonists were present in the medium starting from the second fraction collected and antagonists were added 9 min before agonists. Samples collected and superfused synaptosomes were then counted for radioactivity. The amount of tritium released into each superfusate fraction was expressed as a percentage of the total tissue content at the start of the fraction collected. The [3H]evoked overflow was calculated by subtracting the corresponding basal release to each fraction.

When K+-evoked release was studied, synaptosomes were first superfused with standard medium for 36 min, then the following consecutive samples were collected: basal release (b1; 3 min), K+-evoked release (S; 6 min), and basal release after depolarization (b2; 3 min): Synaptosomes were exposed to the depolarizing stimulus (12 mmol/L KCl) for 90 s starting at the end of the b1. Samples collected and superfused synaptosomes were then counted for radioactivity. The amount of tritium released into each fraction was expressed as a percentage of the total tissue content at the start of the fraction collected. The K+-evoked overflow was calculated by subtracting the basal release (b1 + b2) from the K+-evoked release (S).

Statistical analysis

To analyze the significance of differences between values two-way anova followed by Newman–Keuls multiple comparison analysis was applied. Direct comparisons between two mean were performed with the two-tailed Student’s t-test. Data were considered significant for p < 0.05 at least.

Chemicals

[7,8-3H]dopamine (specific activity 42 Ci/mmol) were purchased from Amersham Radiochemical Center (Amersham, UK). (−)Nicotine bitartrate, nisoxetine, 6-nitroquipazine, oxotremorine, dihydro-β-erythroidine, mecamylamine acetylcholine, dantrolene, carbachol (CCh), methyllycaconitine citrate, xestospongin C, 8-bromo-cADP-ribose (8-Br-cADPR), and tetrodotoxin (TTx) were obtained from Sigma Chemical Co. (St Louis, MO, USA); RJR2403 from Tocris Bioscience (Bristol, UK).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Rat nucleus accumbens synaptosomes, pre-labeled with [3H]DA, were exposed in superfusion to ACh. Figure 1a and b shows the concentration–response curve of ACh (EC50 = 11.8 μmol/L) and the time course of the release caused by 100 μmol/L ACh.

image

Figure 1.  Panel (a): Concentration–response curve of ACh inducing [3H]dopamine overflow from rat accumbal synaptosomes. Panel (b): Effects of mAChR inhibitor atropine (A) and nAChR inhibitor mecamylamine (M) on the released [3H]dopamine induced by ACh. Data are mean ± SEM of at least five experiments run in triplicate. *p < 0.05; **p < 0.01; and ***p < 0.001 versus ACh two-tailed Student’s t-test. Panel (c): [3H]dopamine overflow evoked by 100 μmol/L ACh alone or in combination with 0.03 μmol/L atropine (A) and 20 μmol/L mecamylamine (M). Data are mean ± SEM of at least five experiments run in triplicate. **p < 0.01 and ***p < 0.001 versus ACh two-tailed Student’s t-test. [3H]Dopamine overflow reported in the figure was calculated for a collection time of 5 min.

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When the muscarinic receptor antagonist atropine (0.03 μmol/L) was added 9 min before ACh, the evoked release of [3H]DA was significantly inhibited (Fig. 1b and c). Figure 1 also shows that the nicotinic receptor antagonist mecamylamine (20 μmol/L) completely blocked the release evoked by 100 μmol/L ACh.

Figure 2 illustrates the effects of atropine and mecamylamine on the [3H]DA release and overflow elicited by the mixed mAChR/nAChR agonist CCh, added at 30 μmol/L to the superfusion solution. The concentration–response curve of CCh (EC50 = 8.87 μmol/L) (Fig. 2a) was very similar to that of ACh (Fig. 2a) and the effect of atropine and mecamylamine (Fig. 2b and c) closely resembles those reported in Fig. 1.

image

Figure 2.  Panel (a): Concentration–response curve of CCh inducing [3H]dopamine overflow from rat accumbal synaptosomes. Panel (b): Effects of mAChR inhibitor atropine (A) and nAChR inhibitor mecamylamine (M) on the released [3H]dopamine induced by CCh from rat accumbal synaptosomes. Data are mean ± SEM of at least five experiments run in triplicate. *p < 0.05; **p < 0.01; and ***p < 0.001 versus CCh two-tailed Student’s t-test. Panel (c): [3H]dopamine overflow evoked by CCh alone or in combination with 0.03 μmol/L atropine (A) and 20 μmol/L mecamylamine (M). Data are mean ± SEM of at least five experiments run in triplicate. **p < 0.01 and ***p < 0.001 versus CCh two-tailed Student’s t-test. [3H]Dopamine overflow reported in the figure was calculated for a collection time of 5 min.

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The mAChR agonist oxotremorine (30 μmol/L) caused only a modest overflow of [3H]DA from nucleus accumbens synaptosomes, however, when it was added to the superfusion solution together with 3, 10, or 100 μmol/L (−)nicotine the evoked overflow of the [3H]catecholamine was significantly higher than the sum of the overflows produced by the two agonists alone (Fig. 3a and b).

image

Figure 3.  Panel (a): Effects of (−)nicotine (N) at different concentrations, alone or in combination with mAChR agonist oxotremorine (O) on [3H]dopamine overflow from rat accumbal synaptosomes.. Data are mean ± SEM of three to six experiments run in triplicate. °p < 0.05 versus (−)nicotine (3 μmol/L); #p < 0.05 versus (−)nicotine (10 μmol/L); and *p < 0.05 versus (−)nicotine (100 μmol/L) two tailed Student’s t-test Panel (b): Time course of (−)nicotine (N) alone or in combination with oxotremorine (O). Data are mean ± SEM of three to six experiments run in triplicate. *p < 0.05 versus (−)nicotine (100 μmol/L) two tailed Student’s t-test. [3H]Dopamine overflow reported in the figure was calculated for a collection time of 5 min.

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The dose–response effect of oxotremorine in the presence and in the absence of nicotine is reported in Fig. 4a. The figure shows that when the concentration of oxotremorine is relatively low (10–30 μmol/L) nicotine produced an overadditive effect but with high concentration of oxotremorine (300 μmol/L) the nicotinic facilitation is no longer present.

image

Figure 4.  Panel (a): Dose–response effect of oxotremorine (O) (1–300 μmol/L (O1-O300) in presence and in absence of (−)nicotine (N) (100 μmol/L). Data are mean ± SEM of at least three experiments run in triplicate. *p < 0.05 versus (−)nicotine (100 μmol/L) two tailed Student’s t-test. Panel (b): Effects of nAChR inhibitor mecamylamine (M) (20 μmol/L) and mAChR inhibitor atropine (A) (0.03 μmol/L) on [3H]dopamine overflow evoked by (−)nicotine (N) (100 μmol/L) alone or in combination with oxotremorine (O) (30 or 300 μmol/L). Data are mean ± SEM of three to six experiments run in triplicate. *p < 0.05 and ***p < 0.001 versus (−)nicotine (100 μmol/L); ‡‡‡p < 0.001 versus (−)nicotine (100 μmol/L) plus oxotremorine (30 μmol/L); °°°p < 0.001 versus (−)nicotine (100 μmol/L) plus oxotremorine (300 μmol/L); and ¥¥versus oxotremorine (300 μmol/L). Two-way anova followed by Newman–Keuls. [3H]Dopamine overflow reported in the figure was calculated for a collection time of 5 min.

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The presence of atropine (0.03 μmol/L) inhibited the overflow provoked by 100 μmol/L (−)nicotine plus oxotremorine down to the value observed when 100 μmol/L (−)nicotine was added with atropine (Fig. 4b). Figure 4b also shows that the 100 μmol/L (−)nicotine/30 μmol/L oxotremorine-evoked overflow of [3H]DA was completely abolished by 20 μmol/L mecamylamine. The concentration of atropine used (0.03 μmol/L) did not modify the stimulatory effect of 100 μmol/L (−)nicotine and completely counteracted the 300 μmol/L oxotremorine evoked [3H]DA overflow (Fig. 4b). At higher concentration (0.1 μmol/L), atropine slightly inhibited (−15%) the stimulatory effect of 100 μmol/L (−)nicotine (not shown). The [3H]DA overflow evoked by 100 μmol/L (−)nicotine plus 300 μmol/L oxotremorine was inhibited down to the effect of 300 μmol/L oxotremorine alone by 20 μmol/L mecamylamine (Fig. 4b).

Figure 5a shows that the overflow of [3H]DA evoked by 100 μmol/L (−)nicotine was slightly, although significantly inhibited by the nAChR antagonist methyllycaconitine added at 100 nmol/L. The presence of this antagonist did not seem to affect the synergism observed during exposure of accumbal synaptosomes to (−)nicotine plus oxotremorine.

image

Figure 5.  Panel (a): Effect of methyllycaconitine (MLA) (100 nmol/L) on [3H]dopamine overflow induced by (−)nicotine (N) (100 μmol/L), oxotremorine (O) (30 μmol/L) or by (−)nicotine (N) (100 μmol/L)/oxotremorine (O) (30 μmol/L) combination from isolated rat nerve endings of nucleus accumbens. Data are mean ± SEM of at least five experiments run in triplicate. *p < 0.05 versus (−)nicotine two-tailed Student’s t-test. Panel (b): Effect of nAChR agonist RJR2403 at different concentrations on [3H]dopamine overflow; counteracting effects of nAChR inhibitors dihydro-β-erythroidine (DHβE) and mecamylamine (M) on [3H]dopamine overflow evoked by RJR2403 (100 μmol/L). ##p < 0.01 versus RJR2403 (100 μmol/L) two-tailed Student’s t-test. Panel (c): Effect of RJR2403 (R) (100 μmol/L) alone or in combination with oxotremorine (O) (30 μmol/L) on [3H]dopamine overflow from isolated rat nerve endings of nucleus accumbens. Data are mean ± SEM of at least five experiments run in triplicate. °p < 0.05 versus RJR2403 (100 μmol/L); ###p < 0.001 versus RJR2403 plus oxotremorine two-tailed Student’s t-test. [3H]Dopamine overflow reported in the figure was calculated for a collection time of five min.

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RJR2403 is a relatively selective agonist at the nAChRs of the α4β2 subtype (Bencherif et al. 1996; Papke et al. 2000) although its effect on α6β2* nAChR subtypes has not yet been demonstrated. As shown in Fig. 5b, the compound evoked a concentration-dependent overflow of tritium from accumbal synaptosomes pre-labeled with [3H]DA. The stimulatory effect of 100 μmol/L RJR2403 was inhibited by 1 μmol/L dihydro-β-erythroidine and 20 μmol/L mecamylamine. The releasing effect of 100 μmol/L RJR2403 was doubled when 30 μmol/L oxotremorine was added and this combination was completely blocked by 20 μmol/L mecamylamine (Fig. 5c).

Table 1 shows that [3H]DA overflow elicited by 100 μmol/L (−)nicotine was completely insensitive to 1 μmol/L TTx. The overflow of [3H]DA provoked by 100 μmol/L (−)nicotine plus 30 μmol/L oxotremorine was inhibited down to effect of (−)nicotine alone by 10 μmol/L dantrolene, an antagonist at the ryanodine receptors. Table 1 also shows that the [3H]DA overflow was inhibited by 10 μmol/L 8-Br-cADPR, an antagonist of cADPR, the likely endogenous agonist at ryanodine receptors (Walseth and Lee 1993), while it was insensitive to 0.5 μmol/L xestospongin C, an antagonist at inositol 1,4,5-trisphosphate (IP3) receptors (Gafni et al. 1997). Dantrolene, 8-Br-cADPR, and xestospongin C were inactive at this concentration on basal tritium release and did not modify the overflow of [3H]DA elicited by 100 μmol/L (−)nicotine or 12 mmol/L KCl (not shown). Table 1 also reports that 300 μmol/L oxotremorine-evoked overflow was almost totally prevented by dantrolene or by 8-Br-cADPR, but it was not modified significantly by xestospongin C.

Table 1.   Effects of ryanodine and IP3 receptor blockers on [3H]dopamine overflow evoked by different agonists
Drugs[3H]dopamine overflow (%)
  1. Data are mean ± SEM of six experiments run in triplicate. For experimental details see Materials and methods. °p < 0.05 versus nicotine 100 μmol/L; §p < 0.05 and §§p < 0.01 versus nicotine 100 μmol/L + oxotremorine 30 μmol/L; and *p < 0.05 and ***p < 0.001 versus oxotremorine 300 μmol/L. Two-way anova followed by Newman–Keuls. IP3, inositol 1,4,5-trisphosphate; TTx, tetrodotoxin; 8-Br-cADPR, 8-bromo-cADP-ribose.

Nicotine (100 μmol/L)0.56 ± 0.05
Nicotine (100 μmol/L) + TTx (1 μmol/L) 0.53 ± 0.04
Oxotremorine (30 μmol/L)0.09 ± 0.01
Nicotine (100 μmol/L) + Oxotremorine (30 μmol/L) 0.89 ± 0.10°
Nicotine (100 μmol/L) + Oxotremorine (30 μmol/L) + Dantrolene (10 μmol/L)0.54 ± 0.05§§
Nicotine (100 μmol/L) + Oxotremorine (30 μmol/L) + 8-Br-cADPR (10 μmol/L)0.61 ± 0.09§
Nicotine (100 μmol/L) + Oxotremorine (30 μmol/L) + Xestospongin C (0.5 μmol/L)0.79 ± 0.17
Oxotremorine (300 μmol/L)0.49 ± 0.03
Oxotremorine (300 μmol/L) + Dantrolene (10 μmol/L) 0.06 ± 0.02***
Oxotremorine (300 μmol/L) + 8-Br-cADPR (10 μmol/L) 0.08 ± 0.05*
Oxotremorine (300 μmol/L) + Xestospongin C (0.5 μmol/L) 0.41 ± 0.07

The [3H]DA overflow elicited by 12 mmol/L KCl was significantly increased when 30 μmol/L oxotremorine was present in the superfusion solution; this potentiating effect was significantly inhibited by dantrolene or by 8-Br-cADPR (Table 2).

Table 2.   Effects of ryanodine and IP3 receptor blockers on KCl evoked [3H]dopamine overflow
Drugs[3H]dopamine overflow (%)
  1. Data are mean ± SEM of four experiments run in triplicate. For experimental details see Materials and methods. °°p < 0.01 versus KCl (12 mmol/L); and §p < 0.05 and §§p < 0.01 versus KCl (12 mmol/L) + Oxotremorine (30 μmol/L). Two-way anova followed by Newman–Keuls. IP3, inositol 1,4,5-trisphosphate; 8-Br-cADPR, 8-bromo-cADP-ribose.

KCl (12 mmol/L)2.84 ± 0.37
Oxotremorine (30 μmol/L)0.07 ± 0.02
KCl (12 mmol/L) + Oxotremorine (30 μmol/L)4.16 ± 0.32°°
KCl (12 mmol/L) + Oxotremorine (30 μmol/L) + Dantrolene (10 μmol/L)2.89 ± 0.17§§
KCl (12 mmol/L) + Oxotremorine (30 μmol/L) + 8-Br-cADPR (10 μmol/L)3.21 ± 0.22§

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Nucleus accumbens synaptosomes exposed in superfusion to ACh or to CCh under basal conditions released DA; this event was in part inhibited by atropine but completely prevented by mecamylamine, indicating that it originates from the activation of both mAChRs and nAChRs. More important, the results with the antagonists seem compatible with the co-existence of release-enhancing mAChRs and nAChRs on the same dopaminergic nerve endings where the two cholinergic receptor types would interact in modulating DA release. The characteristics of the technique used to monitor release (a monolayer of synaptosomes up-down perfused in conditions preventing indirect effects; see Raiteri and Raiteri 2000) favor this interpretation of the results obtained; in fact, indirect mechanisms involving neuronal loops that should have been considered if such results had been obtained with more complex brain tissue preparations, including brain slices, are much less likely in our experimental set up. That mAChRs and nAChRs co-exist on the same dopaminergic nerve endings is suggested by the finding that the releasing effects of both ACh and CCh were in part sensitive to the muscarinic receptor antagonist atropine but totally abrogated by the nicotinic receptor antagonist mecamylamine. This result also suggests the possibility that activation of mAChRs is dependent on the activation of nAChRs.

To verify this hypothesis, varying concentrations of (−)nicotine (3, 10, and 100 μmol/L) were added to the superfusion solution together with the selective mAChR agonist oxotremorine at a concentration (30 μmol/L) able to cause only a weak release of DA. Moreover, a dose–response effect of oxotremorine was also studied in the presence and in the absence of nicotine. The concomitant presence of (−)nicotine and oxotremorine provoked more than additive overflow of DA. These results are compatible with the idea that activation of nAChRs triggers activation of mAChRs co-existing on the same DA-releasing terminals.

Accordingly, the mAChR antagonist atropine inhibited in part the overflow provoked by (−)nicotine/oxotremorine, whereas the nAChRs antagonists mecamylamine practically abolished it. Thus it can be concluded that, in the rat nucleus accumbens, dopaminergic terminals co-express mAChRs and nAChRs; synaptic ACh released onto these terminals, or reaching them during non-synaptic transmission (Vizi et al. 2004), is expected to activate nAChRs and, subsequently, mAChRs leading to DA overflow significantly higher than that elicited through activation of nAChRs only.

What is the mechanism by which activation of nAChRs permits activation of co-existing mAChRs? It has to be recalled that mAChR agonists were originally found to potentiate the release of DA only during depolarization of striatal synaptosomes (Raiteri et al. 1982, 1984) or striatal slices (Lehmann and Langer 1982; Schoffelmeer et al. 1986; Kemel et al. 1989). Also recently oxotremorine was reported to evoke release of DA from K+ depolarized slices prepared from mAChRs KO (Zhang et al. 2002). It has been established that the nAChRs situated on DA terminal are Na+-permeant non-α7 receptors whose activation causes plasma membrane depolarization, opening of voltage-sensitive calcium channels (VSCCs) and DA exocytosis (Wonnacott 1997; Zhou et al. 2001; Salminen et al. 2004; Quik and McIntosh 2006). It can therefore be assumed that (−)nicotine, acting at nAChRs, depolarizes accumbal DA terminals enough to permit activation of mAChRs.

The finding that the mAChR mediated component of the overflow induced by (−)nicotine/oxotremorine was inhibited by dantrolene is compatible with the involvement of ryanodine receptors. Accordingly, the mAChR-mediated component of the release also was inhibited by 8-Br-cADPR, an antagonist of cADPR. The apparent involvement of the cADPR–ryanodine receptor system in the mAChR-evoked DA release from nucleus accumbens nerve endings is in a way unexpected. It is generally accepted that muscarinic M1, M3, and M5 receptors couple preferentially via the Gq/11 protein to phospholipase C (Caulfield 1993); their activation leads to the formation of IP3 which mobilizes Ca2+ from internal stores (Berridge 1997). Although evidence has been provided that M5 mAChRs can couple to this signally pathway, it has also been shown that mAChR subtypes are promiscuous in their coupling to G proteins (see, for a review Eglen and Nahorski 2000).

Our results suggest that the mAChRs involved (probably M5 subtype) mediate DA release not through IP3 receptor activation, but by ryanodine receptor-dependent mobilization of intraterminal Ca2+. One possible scenario is the following: (−)nicotine acting at depolarizing nAChRs on DA terminals causes entry of Ca2+ through VSCCs, in a TTx insensitive manner, and stimulates DA exocytosis; mAChR agonists cause elevation of cADPR and mobilization of cytosolic Ca2+ insufficient to evoke DA release, but sufficient to reinforce the external Ca2+-dependent DA exocytosis provoked by (−)nicotine alone or KCl depolarization. Another possibility is that Ca2+ ions entering through VSCCs enable mAChR agonists to evoke internal Ca2+-dependent DA release. In fact, Ca2+ influx through VSCCs may trigger a calcium-induced calcium release by cADPR-primed ryanodine receptor activation (Berridge 1998). An alternative explanation for the interaction between nicotinic and muscarinic receptors might be because of changes in the voltage sensitivity of receptor affinity for agonists (Ben-Chaim et al. 2006). Thus, both nicotinic-induced and KCl depolarization might increase affinity for oxotremorine leading to potentiation of [3H] DA release.

As to the pharmacology of the pre-synaptic receptors involved in the observed synergism between nAChRs and mAChRs, the results available from the literature regarding striatal preparations and the present data with accumbal synaptosomes are compatible with co-existence and interaction between nAChRs of the α4β2* subtype and mAChRs of the M5 subtype. Indeed, immunoprecipitation and in situ mRNA hybridization studies have demonstrated that the M5 receptor represents the only mAChR subtype expressed by the DA-containing neurons of the ventral tegmental area, the region which provides the major dopaminergic innervation of nucleus accumbens (Vilaro et al. 1990; Weiner et al. 1990). Moreover, in vitro DA release studies demonstrate a strong reduction of DA release from striatal slices of M5 KO mice (Yamada et al. 2003). Therefore, the mAChRs up-regulated by nAChRs, originally classified as M1 subtype (Raiteri et al. 1984), when only M1 and M2 receptors had been identified, probably belong to the M5 subtype (see for a review Eglen and Nahorski 2000; Zhang et al. 2002).

The small but significant inhibition by 100 nmol/L methyllycaconitine of the (−)nicotine-evoked release from DA terminals previously reported to lack α7 nAChRs (Kulak et al. 1997) is consistent with the presence on accumbal DA terminals also of nAChRs with subunit composition α6α4β2β3 (Mogg et al. 2002). However, the nAChRs/mAChRs synergism remained almost unmodified in the presence of 100 nmol/L methyllycaconitine (Fig. 5), suggesting that α4β2* receptors play a major role in up-regulating mAChR function. This view is strengthened by the finding that the relatively selective α4β2* receptor agonist RJR2403 behaved like (−)nicotine (in presence of methyllycaconitine) when added in combination with oxotremorine (Figs 5 and 3).

There are indications that M5 mAChR can mediate potentiation of rewarding effects of drugs of abuse (Fink-Jensen et al. 2003; Yamada et al. 2003; Thomsen et al. 2005; Mark et al. 2006) and may be considered as useful targets for candidate treatments. The present results suggest that in vivo administration of nicotine may over-stimulate release of DA from accumbal terminals through both direct and indirect mechanisms. Nicotine can directly evoke DA release by acting at nAChRs sited on DA terminals. In addition, nicotine can enhance release of ACh through nACh autoreceptors present on cholinergic terminals. Finally, activation of nAChRs on DA nerve endings permits or up-regulates the function of mAChRs co-existing on the same terminals, contributing to the elevated efflux of dopamine that probably occurs in drug addition.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by Italian MIUR. The authors wish to thank Mrs Maura Agate for editorial assistance.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References