Muscarinic regulation of dopamine and glutamate transmission in the nucleus accumbens

Results

B6.SJL-Slc6a3^{tm1.1(cre)Bkmn}/J (DAT-cre) mice received bilateral injections of viral vector for cre-dependent expression of channelrodopsin-2 tagged with enhanced yellow fluorescent protein (ChR2-EYFP) in the ventral tegmental area (VTA) and substantia nigra pars compacta region. Expression of ChR2-EYFP was visually confirmed in the soma of midbrain DA neurons and their axonal projections in the striatum (Fig. 1A). DA transients were recorded from the NAc shell, using FSCV in an artificial cerebrospinal fluid (ACSF) solution containing blockers for the GABA, DA, and cannabinoid receptors as well as M₁ mAChRs to minimize modulation by these receptors (Methods). DA transients were evoked by local electrical or optogenetic stimulation (Fig. 1B). Electrically evoked DA (eDA) transients and optogenetically evoked DA (oDA) transients had overlapping current-voltage plots and color-plot voltammograms, as well as similar amplitudes and time courses under the same experimental conditions (Fig. S1) (20). Bath application of the nonselective mAChRs agonist oxotremorine M (oxo-m, 10 μM) depressed eDA transients by 59 ± 6% (n = 5/2; t₄ = 9.54; P < 0.001, one-sample t test; Fig. 1 C and D). However, the same concentration of oxo-m potentiated oDA by 18 ± 2% (n = 8/3; t₇ = 7.27; P < 0.001; Fig. 1 C and D). Thus, by using the same DA detection method (FSCV) within the same preparation, we were able to reveal two opposing effects of muscarinic agonists on DA transmission evoked by different stimulation techniques (electrical vs. optogenetic).

A subset of DA neurons in the VTA co-releases DA and glutamate in the NAc shell (21–23). We recently showed that the release properties and the modulation by G_i-coupled D₂ receptors are remarkably similar for DA and glutamate release from these terminals (20). We reasoned that testing the effect of oxo-m on glutamate release from DA axonal projections would provide independent evidence for its effect on neurotransmitter release from DA fibers. Indeed, oxo-m (10 μM) potentiated the amplitude of optogenetically evoked EPSCs (oEPSCs) recorded from spiny projection neurons in the NAc shell by 68 ± 9% (n = 16/7; t₁₅ = 7.970; P < 0.001; Fig. 1 C and D). However, electrically evoked EPSCs, which are caused by the recruitment of all local glutamatergic axons, were inhibited by 50 ± 10% by the same concentration of oxo-m in the same region (n = 6/2; t₅ = 5.13; P = 0.004; Fig. 1 C and D). Thus, the muscarinic agonist also potentiated glutamate transmission, similar to oDA transmission, when evoked by selective stimulation of midbrain DA axonal projections.

Similar to oxo-m, another nonselective muscarinic agonist, carbachol (10 µM), also potentiated oDA (23 ± 3%; n = 7/4; t₆ = 8.3; P < 0.001) and oEPSC (35 ± 8%; n = 7/5; t₆ = 4.4; P = 0.005) in further support for a role of mAChRs in these potentiating effects. We tested whether endogenously released ACh can induce a similar potentiation of oDA transients and oEPSC. Because CINs are tonically active in vitro (24, 25), bath application of acetylcholinesterase inhibitors will increase synaptic ACh levels. Two different inhibitors of acetylcholinesterase potentiated oDA transients (29 ± 7% and 20 ± 3% for 10 µM physostigmine and 1 µM ambenonium, respectively; n = 6–9/3–3; t > 4.1; P < 0.003), which were reversed by scopolamine (1 µM) (Fig. 2A for physostigmine only). Similarly, physostigmine potentiated oEPSCs by 37 ± 7%, which was also reversed by the nonselective mAChRs antagonist scopolamine (n = 8/4; t₇ = 5.4; P = 0.001; Fig. 2B). Thus, endogenously released ACh also modulates DA and glutamate release from DA axonal projections. Taken together, these results provide strong evidence that mAChRs within the NAc trigger potentiation of neurotransmitter release from midbrain DA neuron projection.

In addition to potentiating DA transient amplitude, the mAChR agonists increased the decay time constant of oDA transients. A series of experiments indicated that the increase in this decay time constant is a result of action on the DA reuptake mechanism but cannot account for the potentiation of the peak amplitude of the transient (Fig. S2).

Considerable heterogeneity among midbrain DA neurons has been reported with regard to the expression profiles (26, 27). A small subset of tyrosine-hydroxylase-positive (TH) neurons also express vGluT₂ mRNA (TH/vGluT₂ neurons) and co-release glutamate. This area also contains neurons lacking TH that express vGluT₂ mRNA (vGluT₂-only neurons) and send projections to the NAc (23, 26). We tested whether the mAChR-dependent potentiation of DA transmission occurs in all midbrain axonal projections to the NAc or is, rather, restricted to a subset of them. Cre-dependent ChR2-EYFP was expressed in the VTA/substantia nigra pars compacta of Slc17a6^tm2(cre)Lowl/J (vGluT₂-cre) mice to selectively stimulate axonal projections from TH/vGluT₂ and vGluT₂-only midbrain neurons. The mean amplitude of oDA transients was smaller in these mice (129 ± 10 nM from n = 5/2) compared with in DAT-cre mice (unpaired t test, t₁₁ = 11.19; P < 0.001). However, the degree of oxo-m potentiation was similar to that in DAT-cre mice (21 ± 2% from n = 5/2; t₁₁ = 1.139; P = 0.279; Fig. 3A). These results suggest the mAChR-dependent potentiation occurs in axonal projections from both TH/vGluT₂ and TH-only midbrain neurons. The mAChRs antagonist scopolamine reversed the oxo-m potentiation and brought levels below the baseline in these experiments, suggesting that under some circumstances, mAChRs are constitutively activated by tonic release of ACh in the slice.

In contrast, the mean amplitude of oEPSCs was larger in vGluT₂-cre mice (154 ± 21 pA; n = 9/3) compared with in DAT-cre mice (t₂₄ = 3.2; P = 0.004), whereas the degree of oxo-m potentiation was smaller in vGluT₂-cre mice (25 ± 9%; n= 9/3; Fig. 3B) compared with in DAT-cre mice (68 ± 9%; t₂₃ = 3.3; P = 0.003). These results suggest vGluT₂-only inputs from the midbrain to the NAc are not potentiated by the muscarinic agonist.

We next sought to investigate the mechanisms responsible for the differential effects of muscarinic agonists on DA transmission between electrical and optogenetic stimulation. We used amperometry for DA detection, as this method provides a higher temporal resolution (5 kHz vs. 10 Hz for FSCV), which allowed us to observe synaptic events more precisely (Fig. 4). Amperometry recordings showed that oDA transients had a single component with an onset delay of 2.5 ± 0.1 ms (n = 15/6; Fig. 4C). To the contrary, eDA transients were biphasic and showed a first component with an onset delay of 3.1 ± 0.1 ms (n = 6/4) and a second component with an onset delay of 8.0 ± 0.2 ms (n = 13/6; Fig. 4D; see ref. 28). The nAChRs antagonist dihydro-β-erythroidine hydrobromide (DHβE) (1 µM) selectively abolished the second component of the eDA transient and had no effect on the first component, causing a reduction of 46 ± 4% in the total peak of eDA transient (n = 5/2; t₄ = 11.25; P < 0.001; Fig. 4D). In contrast, DHβE produced a very small decrease in the oDA transients (7 ± 2%; n = 5/2; t₄ = 4.359; P = 0.012; Fig. 4C). These results demonstrate a significant contribution of nAChRs to DA transients evoked by electrical stimulation, but not by optogenetic stimulation. Thus, optogenetic stimulation selectively activates DA axonal projections to trigger DA transients with a short delay by the monosynaptic direct mechanism that is independent of nAChRs activation (Fig. 4A). In contrast, the biphasic nature of the eDA transients can be explained by a combination of a monosynaptic DA release triggered by direct stimulation of DA axonal projections (monosynaptic direct mechanism) and a disynaptic DA release triggered by stimulation of CINs, release of Ach, and the subsequent activation of nAChRs on DA terminals triggering DA release (disynaptic nAChR-dependent mechanism) (Fig. 4B).

Oxo-m depressed eDA transients by 46 ± 3% (n = 5/2; t₄ = 14.64; P < 0.001; Fig. 4 F and H), comparable to the depression seen with FSCV (t₈ = 0.1283; P = 0.901). However, similar to DHβE, oxo-m selectively abolished the second component of the eDA transients, suggesting the muscarinic agonist targets the disynaptic nAChR-dependent mechanism of DA release. In contrast, oDA transients were potentiated by oxo-m (30 ± 6%; n = 7/2; t₆ = 5.169; P = 0.0021; Fig. 4 E and G), as seen with oDA transients measured with FSCV, suggesting the muscarinic agonist also targets the monosynaptic mechanism of DA release. Scopolamine (1 µM) reversed both the oxo-m-mediated depression of eDA and the potentiation of oDA transients, indicating that each actions of oxo-m are mediated by potentially different mAChRs located on different axonal fibers (Fig. 4 E–H).

To further test the hypothesis that oxo-m inhibits the disynaptic nAChR-dependent mechanism of DA release, we first tested whether the oxo-m-induced depression could be prevented by blocking the nAChR-dependent DA release with DHβE (Fig. 5A), using FSCV. As expected, application of DHβE resulted in a large depression in the eDA transients (75 ± 4%; n = 8/3; t₇ = 17.19; P < 0.001) (29). Subsequent application of oxo-m failed to depress eDA transients but, interestingly, potentiated the remaining eDA transients to a similar extent as oDA transients (18 ± 2% of the baseline in DHβE; n = 7/3; t₆ = 8.285; P < 0.001; Fig. 5A). Second, we tested whether the G_i-coupled M₂ and M₄ mAChRs, which are expressed in CINs (14, 15), are required for the oxo-m depression of eDA transients. Strikingly, in M₂/M₄ double-knockout mice, oxo-m failed to depress eDA transients, indicating that these mAChR subtypes are required for the oxo-m-mediated depression of electrically evoked DA release. Furthermore, oxo-m potentiated eDA transients in these mice (17 ± 4%; n = 10/3; t₉ = 3.77; P = 0.004; Fig. 5B). This effect was reversed by scopolamine (102 ± 4% of the baseline; t₉ = 0.469; P = 0.65). Subsequent application of DHβE depressed eDA transients by 60 ± 4% in M₂/M₄ double-knockout mice (t₉ = 17.35; P < 0.001), as it did in control animals, confirming that the nAChR-dependent mechanism of DA release is still intact in the M₂/M₄ double-knockout mice, even in the absence of oxo-m-mediated depression. Taken together, these results indicate that oxo-m depresses eDA transients by inhibiting the disynaptic nAChR-dependent mechanism of DA release through activation of M₂ and M₄ mAChRs, possibly via autoreceptor-mediated feedback inhibition of ACh release from CINs (15).

Interestingly, in the absence of oxo-m-mediated depression of the nAChR-dependent mechanism of DA release (e.g., in the presence of nAChRs antagonist and in M₂/M₄ double-knockout mice), oxo-m potentiated eDA transients, as seen in oDA transients. These observations suggest that the oxo-m-mediated depression of the disynaptic nAChR-dependent mechanism masks the oxo-m-mediated potentiation of the monosynaptic direct mechanism of DA release, and that other, non-M₂/M₄ mAChR subtypes are responsible for the oxo-m potentiation of DA release. M₅ mAChR transcripts have been detected in midbrain DA neurons (12, 17), and M₅ knockout (M₅KO) mice show impaired muscarinic agonist-induced potentiation of [³H]-DA release (8). However, most recently, M₅ mAChRs have also been implicated in depressing DA transmission in the striatum (11). To clarify the role of M₅ mAChRs in modulating DA transmission in the striatum, M₅KO mice were crossed with DAT-cre mice to selectively activate DA axonal projections with optogenetic stimulation. The amplitudes of oDA transients and oEPSCs recorded in the M₅KO/DAT-cre mice were comparable to those measured in DAT-cre mice under the same conditions [oDA: 438 ± 48 vs. 394 ± 17 nM in M₅KO and DAT-cre, respectively (t₁₃ = 0.91; P = 0.38); oEPSC: 78 ± 9 vs. 83 ± 12 pA in M₅KO and DAT-cre mice, respectively (t₂₂ = 0.26; P = 0.8)]. However, in contrast to in DAT-cre mice, oxo-m now failed to potentiate oDA transients and oEPSCs in M₅KO/DAT-cre mice. Indeed, oxo-m produced a small depression of oDA transients and oEPSCs in M₅KO mice [oDA: 7 ± 2% (n = 8/3; t₇ = 2.826; P = 0.026); oEPSC: 12 ± 5% (n = 7/3; t₆ = 2.321; P = 0.059)], which was reversed by scopolamine (Fig. 6 A and B). At the same time, the oxo-m-mediated depression of eDA transients was intact in the M₅ mAChR-deficient mice (Fig. S3A). In the presence of DHβE, which blocks the nAChR-dependent component of the eDA transient, oxo-m failed to potentiate eDA transients in M₅KO mice but, rather, caused a small depression, as seen with oDA transients (Fig. S3B). Thus, M₅ mAChRs are required for muscarinic agonist-induced potentiation of both oDA and oEPSC from DA axonal projections.

Discussion

This study tackles a long-standing controversy regarding the effect of muscarinic agonists on DA transmission in the striatum. We first replicated the disparate findings of depression and potentiation of DA transmission by mAChR agonists in the same in vitro slice preparation by using two different modes of stimulation: electrical or optogenetic. We then demonstrated that G_i-coupled M₂/M₄ autoreceptors mediate the depression of DA transmission and that G_q-coupled M₅ receptors, most likely expressed on DA neuron terminals (12), mediate the potentiation of DA transmission in the striatum (Fig. 7). Thus, our results demonstrate that different mAChR subtypes mediate depression or potentiation of two different mechanisms of DA release in this brain region.

Importantly, our findings also support the idea that electrically evoked DA transients in the striatum are the result of a combination of two mechanisms of DA release: a direct monosynaptic mechanism evoked by direct stimulation of DA axonal projections with short onset delay, and a disynaptic nAChR-dependent mechanism evoked by stimulation of CIN fibers within the striatum with long onset delay. The disynaptic mechanism of DA release is triggered by activation of nAChRs on DA terminals independent of action potential in DA neurons. In contrast, optogenetic stimulation of DA neuron projections in the NAc triggers the direct monosynaptic mechanism, which is independent of ACh and nAChRs activation. Clearly, these findings have implications beyond the striatal actions of muscarinic agonists and need to be considered when interpreting data obtained using electrical stimulation to evoke DA transients. In light of our findings, it should be important to revisit, and potentially reinterpret, previous data on the modulation of DA transmission.

The potentiation of the peak DA transient by muscarinic agonists was accompanied by an increase in the decay time constant, which is indicative of a reduced clearance mechanism (diffusion and/or reuptake). Garcia-Olivares et al. (30) showed that G protein βγ subunits inhibit DAT activity, which might explain the increase in the decay time constant we observed after mAChRs activation. A reduced clearance mechanism can itself cause an increase in peak DA transients. Thus, we performed several experiments to determine the contribution of the increase in the decay time constant to the potentiation of DA transients. Several lines of evidence clearly indicated that the effect on the decay time constants cannot account for the potentiation of DA transmission by muscarinic agonists.

DA and glutamate are co-released from VTA DA neurons (21–23) and share several release properties (20). We observed that the M₅ mAChR-mediated potentiation affects oEPSCs recorded from spiny projection neurons in a similar fashion as oDA transients evoked by direct optogenetic stimulation of DA axonal projections (Fig. S4). This finding indicates that the axonal projections from midbrain DA neurons that release DA and glutamate are under the same modulation by muscarinic agonists and supports the concept that both neurotransmitters are released from the same terminals. Yamaguchi et al. (26) showed the existence of three different types of projections from VTA to NAc: DA (TH-only), DA/glutamate (TH/vGluT₂), and glutamate (vGluT₂-only) projection neurons. We found that the degree of potentiation of oDA transients in vGluT₂-cre mice (DA/glutamate fibers) was indistinguishable from the corresponding response observed with DAT-cre mice (DA and DA/glutamate fibers). However, the degree of potentiation of oEPSCs by muscarinic agonists was significantly reduced in vGluT₂-cre compared with DAT-cre mice, suggesting M₅ mAChRs activity preferentially regulates DA releasing neurons. Recently, Zhang et al. (23) reported that dopaminergic and glutamatergic vesicular systems coexist, but segregate to different compartments of the same axonal projections. This observation raises the possibility that the number and/or coupling efficiency of M₅ mAChRs can be differentially expressed at these two compartments within the same axonal projection, favoring glutamate over DA transmission (Fig. 1 and Fig. 6).

Cholinergic transmission in the striatum is known to play an important role in modulating synaptic plasticity and the behaviors controlled by the basal ganglia (31, 32). Our study presents an insight into how cholinergic transmission affects plasticity by potentiating neurotransmitter release from midbrain DA neurons. Our findings strongly suggest that the mAChR-dependent potentiation of DA/glutamate transmission from midbrain neurons, together with nAChR-mediated enhancement of DA release, leads to a profound increase in striatal DA transmission on ACh release. Clearly, these data provide a rational basis for the development of novel, mAChR subtype-selective drugs aimed at modulating dopaminergic signaling for therapeutic purposes.

Methods

Supplementary Material