|Home | About | Journals | Submit | Contact Us | Français|
Voltammetric analyses show that low (100–500 nM) doses of nicotine regulate striatal dopamine by inhibiting release evoked by a single stimulation to a greater extent than release evoked by high frequency stimulations. This frequency-dependent inhibition is because of nicotine desensitizing heteromeric β2 subunit-containing nicotinic acetylcholine receptor (nAChR) subtypes. Surprisingly, a high dose of nicotine (2 μM; capable of interacting with additional nAChR subtypes) produced an inhibition of dopamine evoked by high frequency stimulation, an effect that was not seen with the low dose of nicotine or the β2 antagonist, dihydro-β-erythroidine hydrobromide. This inhibition was replicated by application of α7 nAChR antagonists methyllcaconitine citrate or α-bungarotoxin in conjunction with the low dose of nicotine or dihydro-β-erythroidine hydrobromide. Blocking α7 receptor function alone produced a modest increase in dopamine evoked by single pulse stimulation while not affecting dopamine evoked by high frequency stimulation. The antagonist results were mimicked using selective α7 agonists PHA 543613 and PNU 282987. The frequency dependence of the low dose nicotine inhibition therefore requires functional α7 nAChRs, and may arise from differing levels of endogenous acetylcholine evoked by the stimulation.
Tobacco use is one of the leading causes of preventable death in the United States. Nicotine is highly addictive and accordingly tobacco use continues to pose a substantial public health risk decades after its harmful effects were first reported. However, nicotine may have beneficial aspects as well, such as reducing the incidence of Parkinson’s Disease in humans (Morens et al. 1995; Gorell et al. 1999). Increased knowledge about how the nicotinic acetylcholine receptor (nAChR) system interacts with dopamine signaling may lead to improved nicotine cessation treatments, as well as aiding in the struggle against neurodegenerative conditions like Parkinson’s Disease.
The nAChRs are expressed throughout the rodent nucleus accumbens and striatum, with expression of the relatively high nicotine affinity β2-containing heteromeric nAChRs, and the low affinity homomeric α7 nAChRs (Zoli et al. 2002; Champtiaux et al. 2003; Grady et al. 2007; Gotti et al. 2010). There is no evidence to date of α7 receptor function directly on dopamine terminals.
Active dopamine neurons fire at approximately 5 Hz under normal conditions, but exhibit sub-second duration bursts of high frequency firing at approximately 20 Hz (Clark and Chiodo 1988), which correlates with reward-based events (Schultz and Romo 1990). The application of doses of nicotine approximating the levels seen in human smokers (~100 nM) produces an inhibition of dopamine release evoked by single pulse stimulations that is mimicked by β2 receptor-selective antagonists, and is not observed in mice with the β2 subunit knocked out (Zhou et al. 2001). The nicotine-induced inhibition (through desensitization of β2-containing nAChRs) was also found to decrease in strength as the frequency of stimulation increased (Rice and Cragg 2004; Zhang and Sulzer 2004). Thus, dopamine evoked under low frequency, basal firing conditions is inhibited by nicotine to a greater extent than during high frequency burst conditions. This increases the signal to noise level of dopamine produced by the burst potentials. This frequency-dependence magnifies the apparent concentration of dopamine produced by high frequency bursts, thereby acting as a filter of dopamine signals and potentially playing a role in nicotine’s rewarding effects (Exley and Cragg 2008).
Although some evidence for α7 receptor modulation of dopamine levels exists (Kaiser and Wonnacott 2000; Livingstone et al. 2009), the impact of these receptors has not been thoroughly tested. The ability of α7 receptors to modulate evoked dopamine alone or in conjunction with nicotine was examined. We found α7 nAChR involvement in the nicotine action upon evoked dopamine.
Sprague–Dawley rats between 14 and 21 days old were anesthetized with halothane and killed. Coronal slices 250-μm thick were obtained and allowed to equilibrate in buffer solution for 1 h prior to placement in the recording chamber. Recordings were performed in striatal tissue in the dorsal lateral striatum from slices cut posterior 1.5–2 mm from bregma. All procedures were approved and performed in compliance with NIEHS/NIH Humane Care and Use of Animals in Research protocols.
Electrodes were fabricated by aspirating T-650 carbon fibers (courtesy of Cytec Industries, Woodland Park, NJ, USA) into glass capillaries which were then pulled in a pipette puller and cut to a length of less than 20 μm. A triangle waveform from −0.4 V to 1.0 V and then back to −0.4 V was applied at 250 V/s, and reapplied at a rate of 10 Hz. Data were recorded using the TH-1 voltammetry software (ESA Bioscience, Chelmsford, MA, USA). Local electrical stimulations were produced in the dorsal lateral striatum with a bipolar stimulating electrode placed approximately 100 μm from the recording site. Stimulations (600 μA, 1 pulse at 100 Hz) were applied every 5 min until stable dopamine release was obtained, after which alternating stimulations (1 pulse or 4 pulses at 100 Hz) were applied every 5 min for the duration of the experiment. After 30 min of pre-drug control data collection, pharmacological agents (or buffer for control) were added to the bath solution for 1 h. Statistical significance was calculated using 1 way ANOVA with Dunet’s post-testAUTHOR: Please check the word ‘Dunet’s’ here in text.. Significance was assigned with 95% confidence interval (p < 0.05).
The buffer solution used during slicing and recording consisted of 125 mM sodium chloride, 3.5 mM potassium chloride, 1.2 mM sodium phosphate, 25 mM sodium bicarbonate, 2 mM calcium chloride, 1.3 mM magnesium chlorideand 11 mM glucose. Nicotine hydrogen tartrate salt (500 nM, 2 μM), dihydro-β-erythroidine hydrobromide (DHβE; 100 nM), α-bungarotoxin (10 nM dissolved in phosphate-buffered saline; Tocris Bioscience, Ellisville, MO, USA), PHA 543613 hydrochloride (50 nM; Tocris Bioscience), PNU 282987 (100 nM in 1 eq HCl; Tocris Bioscience), 6-cyano-7-nitroquinoxaline-2,3-dione disodium (25 μM), D-(−)-2-amino-5-phosphonopentanoic acid (25 μM) and methyllcaconitine citrate (MLA; 10 nM) were used for the experiments. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in water unless otherwise noted.
We investigated whether α7-containing nAChRs, which previously were believed to play a minor role, modulate evoked dopamine. Two stimulation patterns were used; a single 600 μA electrical stimulation that previously displayed the strongest inhibition by nicotine, and a high frequency stimulation with a 4 pulse burst of 600 μA electrical pulses given at 100 Hz (Zhou et al. 2001; Rice and Cragg 2004; Zhang et al. 2009). The latter stimulation was used to model the short bursts dopamine neurons exhibit while the former models low frequency basal firing.
Evoked dopamine is a complex signal controlled by diffusion from release sites, uptake by the dopamine transporter, and dopamine release (Wu et al. 2001; Cragg and Rice 2004; Rice and Cragg 2008). The pharmacological manipulations described in this work are believed to modulate evoked dopamine signals primarily through altering dopamine release.
The ability of nicotine at both low (500 nM) and high (2 μM) doses to modulate evoked dopamine in the striatum was examined (Fig. 1). Nicotine at the low dose of 500 nM inhibited evoked dopamine to only 39 ± 5% (n = 9, p < 0.05) of pre-drug evoked dopamine. As observed previously, this effect was mimicked by the β2 receptor antagonist, DHβE, which inhibited evoked dopamine to 35 ± 6% (n = 6, p < 0.05) of pre-drug evoked dopamine (Fig. 1b). A high concentration of nicotine (2 μM), a dose which is capable of interacting with α7 nAChRs (Papke et al. 2007), produced a similar inhibition (25 ± 4%, n = 5, p < 0.05).
The application of 10 nM of the α7-selective antagonist MLA alone increased evoked dopamine (123 ± 7%, n = 8; p < 0.05) (Fig. 2). However, when either 500 nM nicotine or 100 nM DHβE was combined with 10 nM MLA, a significant inhibition similar to 500 nM nicotine alone was elicited (29 ± 5%, n = 7, p < 0.05; 37 ± 7%, n = 8, p < 0.05, respectively). Therefore, MLA did not appear to have an effect on either the nicotine-(low dose) or DHβE-induced decrease in evoked dopamine.
The effects of selective α7 agonists upon evoked dopamine were also tested. The agonists, 50 nM PHA 543613 and 100 nM PNU 282987 (Bodnar et al. 2005; Wishka et al. 2006; Acker et al. 2008) did not affect observed dopamine (86 ± 9%, n = 4; 81 ± 2%, n = 3, respectively) (Fig. 3). The combination of PHA 543613 with 500 nM nicotine reduced evoked dopamine to 47 ± 10% (n = 4, p < 0.05) of pre-drug evoked dopamine and PNU 282987 with nicotine reduced evoked dopamine to 28 ± 6% (n = 3, p < 0.05) of pre-drug evoked dopamine. The α7 agonists did not appear to have an effect upon the nicotine-induced decrease in evoked dopamine, which is consistent with previous findings that nicotine decreases dopamine evoked by single pulse stimulation through β2 desensitization.
In contrast to the effect on single pulse stimulations, dopamine evoked by high frequency stimulations was not significantly decreased by 500 nM nicotine (73 ± 6%, n = 9, p > 0.05) or 100 nM DHβE (75 ± 12%, n = 6, p > 0.05) (Fig. 4a and b). Unexpectedly, however, evoked dopamine was significantly reduced by the high dose (2 μM) of nicotine (38 ± 6%, n = 5, p < 0.05), which suggests that non-β2 subunit-containing nAChRs, such as the α7 nAChR, may also modulate dopamine evoked by high frequency stimulation.
The application of 10 nM MLA had no impact upon observed dopamine (106 ± 10%, n = 8) when given alone. However, when 500 nM nicotine was combined with 10 nM MLA, evoked dopamine was significantly reduced to 48 ± 6% of pre-drug evoked dopamine under high frequency stimulation (n = 7, p < 0.05) (Fig. 5a and b), which, unlike the effect of nicotine alone under these conditions, is significantly lower than control. The substitution of nicotine with 100 nM DHβE produced a similar inhibition (59 ± 9, n = 8, p < 0.05). These data suggest that when the α7 nAChR is blocked, the low (500 nM) nicotine dose will inhibit dopamine evoked by high frequency stimulation, indicating that α7 nAChR function is regulating evoked dopamine under these conditions.
Dose response data was obtained for MLA with and without 500 nM nicotine (Fig. 6). MLA alone produced a significant change in evoked dopamine for 1 pulse stimulations at 10 nM (123 ± 7%, n = 8, p < 0.05) while not producing significant changes in dopamine at 1, 5 or 50 nM (not shown). When combined with nicotine, all tested MLA doses displayed significant reductions in evoked dopamine versus control [%Pre-drug evoked dopamine (1 nM MLA: 48 ± 6%, n = 3; 5 nM MLA: 33 ± 12%, n = 3; 10 nM: 29 ± 5%, n = 7; 50 nM: 30 ± 6%, n = 3, p < 0.05)]. MLA alone did not affect dopamine evoked by 4 pulse, 100 Hz stimulations at any concentration. For high frequency stimulations, the combination of nicotine with 5, 10, and 50 nM (not shown) MLA produced significant results [%Pre-drug evoked dopamine (5 nM: 56 ± 1%, n = 3; 10 nM: 48 ± 6%, n = 7; 50 nM: 52 ± 23%, n = 3, p < 0.05)] unlike the combination of nicotine with 1 nM MLA.
The selective α7 agonists, PHA 543613 and PNU 282987, both displayed similar results to 10 nM MLA alone, and when paired with 500 nM nicotine (Fig. 7). Both 50 nM PHA and 100 nM PNU doses failed to modulate evoked dopamine (96 ± 10%, n = 4; 107 ± 2%, n = 3, respectively). When combined with 500 nM nicotine, PHA 543613 reduced evoked dopamine to 52 ± 10% (n = 4, p < 0.05) of pre-drug evoked dopamine. Nicotine combined with PNU 282987 decreased evoked dopamine to 64 ± 3% (n = 5, p < 0.05) of pre-drug evoked dopamine. These results indicate that the agonists are desensitizing α7 nAChRs as they are producing a reduction in dopamine evoked by high frequency stimulation when combined with nicotine, similar to the effect of direct antagonism of α7 nAChRs with MLA.
The selective α7 nAChR antagonist, α-bungarotoxin (10 nM) had no impact upon dopamine evoked by single pulse (103 ± 7%, n = 4) or high frequency (95 ± 4%, n = 4) stimulation (Fig. 8). When combined with the low 500 nM dose of nicotine, α-bungarotoxin significantly inhibited dopamine evoked by single pulse (51 ± 4%, n = 4, p < 0.05) and high frequency (53 ± 7%, n = 4, p < 0.05), an effect similar to that seen with MLA. These data provide further evidence that the inhibition of dopamine evoked by high frequency stimulation involves the α7 nAChR.
Slices were pre-treated with 25 μM 6-cyano-7-nitroquinoxaline-2,3-dione disodium and 25 μM D-(−)-2-amino-5-phosphonopentanoic acid for 1 h prior to addition of 500 nM nicotine. Dopamine evoked by 1 pulse stimulations was significantly decreased to 60 ± 9% (n = 4, p < 0.05) of pre-nicotine level. With 4 pulse, 100 Hz stimulations, evoked dopamine was not significantly altered from pre-nicotine evoked dopamine (79 ± 13%, n = 4). The block of α-amino-3-hydroxy-5-methylisoxazole-4-propionate and NMDA signaling was unable to modulate the effect of 500 nM nicotine administration upon evoked dopamine release.
The previously reported findings that nicotine produces a frequency-dependent inhibition of evoked dopamine are critical to understanding how nAChRs modulate dopamine neurotransmission in the striatum. Nanomolar doses of nicotine strongly inhibit dopamine evoked by single pulse stimulation, and the inhibition diminishes as the number of stimulus pulses and stimulus frequency are increased. Though much of the current debate has focused upon characterizing the mechanisms behind the inhibition, no investigation of the mechanisms behind the frequency-dependence has been reported. Here, we have shown that the α7 nAChR is required for the frequency-dependence of the inhibition (i.e. the lack of effect seen under high frequency stimulations).
Unlike dopamine evoked by single pulse stimulation, dopamine evoked by high frequency stimulation is not significantly inhibited by β2 nAChR desensitization or antagonism. However, when applied at a high dose (2 μM), nicotine significantly inhibits dopamine release under both single and 4 pulse, 100 Hz stimulus paradigms. This high dose of nicotine is capable of interacting with and desensitizing β2 and α7 nAChRs, and the inhibition it produced suggests that the frequency-dependent inhibition of evoked dopamine by nicotine is more complex than simply β2 nAChR desensitization.
Antagonism of α7 nAChRs alone did not impact evoked dopamine, however the combination of α7 antagonists with 500 nM nicotine or DHβE produced an inhibition of dopamine evoked by high frequency stimulation. Agonism of the α7 nAChR with PHA 543613 or PNU 282987 mimicked the results observed with the antagonists, MLA and α-bungarotoxin, which suggests that the agonist effects upon evoked dopamine are because of desensitization of the α7 receptors. From this observation, we conclude that the loss of β2 nAChR function through either antagonism or desensitization inhibits evoked dopamine release regardless of stimulus paradigm. Instead, the α7 nAChR blocks or compensates for the inhibition of dopamine during high frequency stimulation, and is responsible for the frequency-dependent aspect of the inhibition of evoked dopamine by nanomolar nicotine doses.
The loss of β2 receptor function via either desensitization induced by a low nicotine dose, or use of the β2 antagonist DHβE, significantly reduces dopamine evoked by single pulse stimulation, an effect similar to that observed with a high nicotine dose. Loss of α7 receptor function alone using the specific antagonist MLA produces a modest yet significant increase in dopamine evoked by single pulse stimulation. The application of α-bungarotoxin alone did not affect evoked dopamine, nor did either of the selective α7 agonists, PHA 543613 or PNU 282987. Therefore, the α7 receptor has little to no discernable effect upon dopamine evoked by single pulse stimulation, alone or in the presence of nicotine.
The inhibition of evoked dopamine by nAChR antagonists (β2 and β2/α7 combination) for high frequency but not single pulse stimulations suggests that the stimulations are evoking the release of significant levels of endogenous acetylcholine (ACh). The striatum contains the terminals of cholinergic interneurons that will be affected along with dopamine terminals by the local electrical stimulation. The two stimulation paradigms are potentially producing two distinctly different neurochemical environments with unique levels of α7 nAChR activity. For single pulse stimulations, we suggest that the α7 receptors are not saturated and nanomolar concentrations of nicotine strongly inhibit release, while the α7 receptors may be saturated during high frequency stimulations and nanomolar concentrations of nicotine appear to have no effect. By removing the influence of α7 receptors with selective antagonists or desensitization, nicotine produced similar inhibitions for both stimulus paradigms. The frequency dependence of the nicotine inhibition may therefore occur because the high frequency stimulation produces an ACh concentration capable of activating α7 receptors, while the single pulse stimulation evokes an ACh level insufficient to saturate the α7 receptors.
At higher concentrations than those used here, MLA has been shown to lose selectivity for α7 nAChRs and also bind to β2 receptors with the highest affinity for the α6β2 receptor (Klink et al. 2001). Though still a matter of debate, the α6β2 receptor has been specifically implicated as the primary β2 nAChR responsible for inhibition of dopamine evoked by low frequency stimulation by nanomolar nicotine concentrations and DHβE (Exley et al. 2008). Non-specific antagonism of the α6β2 nAChR is an unlikely explanation for the observed inhibitions of dopamine evoked by high frequency stimulation for multiple reasons. First, the α6β2 receptor is likely desensitized or antagonized by both 500 nM nicotine and 100 nM DHβE administration (respectively), conditions under which dopamine evoked by the high frequency stimulation is not significantly affected. Second, MLA did not inhibit dopamine evoked by single pulse stimulation as would be expected if β2 nAChRs were also blocked. Lastly, the ability of the α7 nAChR to modulate evoked dopamine was confirmed by substitution of α-bungarotoxin and two selective agonists for the α7 nAChR. Therefore, we conclude that the inhibition of dopamine evoked by high frequency stimulation that we have observed is because of antagonism of α7 (and not α6β2) nAChRs.
Direct block or desensitization of striatal α7 receptors would remove the frequency-dependence of the nicotine inhibition and greatly diminish dopamine evoked by both burst and basal firing patterns, potentially reducing the rewarding effect of nicotine. Though the receptors are expressed in the midbrain in a subset of dopamine neurons (Klink et al. 2001; Zoli et al. 2002), there is currently a lack of evidence for functional α7 nAChR expression directly on dopamine terminals. It is thought that pre-synaptic α7 nAChRs in the striatum are primarily located on cholinergic and glutamatergic terminals, and may modulate dopamine indirectly (Kaiser and Wonnacott 2000; Quik et al. 2005; Livingstone et al. 2009).
Research was supported by the Intramural Research Program of the NIEHS/NIH. We are grateful to Drs. Christian Erxleben and Steve Simons for valuable suggestions in the writing of this manuscript.