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).
The α7 receptor mitigates the effect of β2 receptor desensitization with high frequency stimulation
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 α7 receptor does not modify the nicotine effect with single pulse stimulation
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 local stimulations are generating endogenous acetylcholine release
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.
The actions of the α7 antagonists are not because of non-selective binding
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