Excitatory glutamatergic activity in the striatum originates mainly from frontal cortex, midline and intralaminar thalamus, basal amygdala, and hippocampus (reviewed in Sesack and Grace, 2010; Stuber et al., 2012). Additionally, DA terminals release glutamate (Sulzer et al., 1998; Joyce and Rayport, 2000; Sulzer and Rayport, 2000; Chuhma et al., 2004; Dal Bo et al., 2004; Chuhma et al., 2009; Hnasko et al., 2010), and this has recently been demonstrated by way of selective optogenetic stimulation of DA terminals (Stuber et al., 2010). However, this last report demonstrates that such possibility exists only in DA terminals that reach the NAc, not the dorsal striatum. Still, some debate prevails as to this feature not being present in the adult brain (Bérubé-Carrière et al., 2009; Moss et al., 2011), or being as widespread as initially thought (Stuber et al., 2010; for a review, see Broussard, 2012).

Evidence on the potential role of glutamate as a form of local control of DA release in the striatum has long been reported (Imperato et al., 1990; Cheramy et al., 1991; Krebs et al., 1991; Desce et al., 1992) and both ionotropic and metabotropic glutamate receptors (iGluR; mGluR, respectively) have been implicated. However, most of the initial studies were performed in vivo using brain microdialysis as the measuring technique to assess DA levels as well as for local administration of glutamate receptor ligands. Such findings were of course influenced by slow temporal resolution and the effects of the ligand in a complex circuit, among other factors, making a mechanistic interpretation difficult. In vitro experimental designs, on the other hand, allowed for more direct mechanistic description while still not directly addressing whether results were equivalent to intact-tissue conditions. These distinct experimental conditions might account for what, at the time, were apparent contradictory results. Initial in vitro explorations in slices and synaptosomes accounted not only for glutamate, but for a range of neurotransmitters that could affect striatal DA release locally, including acetylcholine, GABA, glycine and opiates (reviewed in Chesselet, 1984). However, further in vivo experiments in freely moving rats were still non conclusive; i.e., activation of AMPA receptors by exogenous ligands led to a decrease in DA release, while an increase was evident only in response to the application of NMDA receptor ligands at high concentrations (Imperato et al., 1990). Blocking uptake of endogenous release, in turn, elevated DA release in a way that was sensitive to the application of either NMDA or AMPA antagonists, suggesting the involvement of both receptor types in that response (Segovia et al., 1997). Similarly, electrical stimulation of the prefrontal cortex, a putative glutamatergic input to striatum, as well as local application of kainate or NMDA increased DA release (Cheramy et al., 1991; Krebs et al., 1991). Development of electrochemical techniques, however, greatly contributed to the clarification of these mechanisms. The use of fast-scan cyclic voltammetry (FSCV) for the detection of DA in vitro allowed for better temporal resolution which was less influenced by circuit adaptive responses in the mid-term scale (minutes), which could potentially influence DA readout. Under those conditions, bath application of kainate, AMPA or NMDA elicited inhibition of DA release (Wu et al., 2000; Kulagina et al., 2001; Avshalumov et al., 2003). Moreover, electron microscopy studies were not able to demonstrate labeling of iGluRs in striatal DA terminals (Bernard et al., 1997; Bernard and Bolam, 1998). The lack of expression of iGluRs on DA terminals suggests that iGluR-mediated modulation of DA release relates to a more complex process; which may underlie interactions between multiple cellular types and/or chemical mediators. This issue, raised and investigated by Rice’s group led to the identification of H₂O₂ as a key molecule in the iGluR-mediated decrease of DA release (Avshalumov et al., 2000, 2003, 2008; Avshalumov and Rice, 2003). This model describes how glutamatergic activity on ionotropic receptors in medium spiny neurons (MSNs) triggers production and release of H₂O₂, which in turn diffuses to adjacent DA terminals and promotes opening of K_ATP channels leading to reduction of DA release (Avshalumov et al., 2008).

In contrast to iGluRs, labeling of mGluRs has been reported in presynaptic profiles identified as DA axons (Paquet and Smith, 2003). Moreover, blocking glutamate uptake, or high-frequency stimulation of the cortico-striatal pathway modulates DA release, in a mGluR-dependent fashion followed by modulation of Ca⁺⁺-activated potassium channels (Zhang and Sulzer, 2003). Altogether, the existent evidence points to mGluR-mediated direct action on DA terminals, and a second MSN-mediated mechanism involving iGluR-H₂O₂ signaling.

In contrast to striatal glutamatergic activity, which originates mainly from inputs to the striatum, sources of striatal acetylcholine release are mostly from cholinergic interneurons (CINs) that account for about 2–5% of all striatal neurons (Descarries et al., 1997; Descarries and Mechawar, 2000). Additional to CINs, a recent report shows that brainstem-based cholinergic neurons send terminals to the striatum in a topographic fashion with their origin (Dautan et al., 2014). In spite of their low numbers, CINs establish prominent and intricate axonal projections that configure an extensive planar neurotransmission system (Descarries et al., 1997; Descarries and Mechawar, 2000). Electrophysiological characterization shows that CINs are tonically active neurons that fire at a relatively low rate of about 5–10 Hz (Wilson et al., 1990; Aosaki et al., 1995). This rate, however, as in the case of DA neurons, encodes behaviorally relevant reward-related events (Apicella et al., 1991, 2011; Aosaki et al., 1994, 1995; Shimo and Hikosaka, 2001; Morris et al., 2004).

Target receptors of cholinergic activity in the striatum are both of nicotinic and muscarinic types (nAChR and mAChR, respectively). While mAChRs are seven trans-membrane domain G-protein coupled receptors, nAChRs consist of five subunits arranged as homomers or heteromers that, in mammals, are formed by subfamilies II (α7) and III (α2-6, β2-4) (Le Novère et al., 2002). Particularly, striatal DA axons express a high density of α4, α5, α6, β2 and β3 subunits in an arrangement of two αβ pairs that could be α4-β2 and/or α6-β2 and/or α4-β4, plus a fifth subunit that can be α5 or β3 (Champtiaux et al., 2003; reviewed in Threlfell and Cragg, 2011). Additionally, the β2 subunit is expressed on striatal DA axons (Jones et al., 2001) and is included in all nAChRs at these terminals. This characterization is functionally relevant because some segregation exists in which predominance of different α subunits occurs between dorso-lateral striatum and the NAc. More specifically, a significant amount of work has shown that α4(non-α6)-nAChRs play a prominent role in dorsal striatum, while α4α6-nAChRs are dominant in NAc (Exley et al., 2008, 2011, 2012). Given the distinct functional role of the dorsolateral and the ventromedial striatum, it has been proposed that such differences could be taken into account as a substrate for region-specific intervention (Threlfell and Cragg, 2011).

mAChRs, in turn, are classified in two groups according to their coupling to either G_s (M₁, M₃, M₅) or G_i (M₂, M₄) subunits of G proteins, with M₂ and M₄ predominantly expressed in CINs (Yan and Surmeier, 1996). In a similar way to what has been described for nAChRs, mAChRs exhibit some dorso-ventral gradient in their ability to regulate DA release. While M₂/M₄ receptors are necessary for such regulation in the dorsal striatum, M4 is prevalent in the NAc (Threlfell et al., 2010). Additionally, expression of M₅ receptors has been reported in nigrostriatal DA neurons, although their pattern of expression on striatal DA terminals and subsequent potential role in local control of DA release remains unclear (reviewed in Threlfell and Cragg, 2011; Zhang and Sulzer, 2012).

Involvement of presynaptic cholinergic receptors on DA regulation was inferred early, mainly from experiments describing increase of DA release in response to AChR activation in slices or synaptosomes (Giorguieff et al., 1976, 1977; Wonnacott et al., 1989; Rapier et al., 1990). In a similar way to what occurred with the characterization of glutamatergic-dependent DA modulation, transition to electrochemical methods to quantify DA allowed for a better temporal resolution. Importantly, FSCV has been critical in determining a high dependence of DA release on stimulation frequency under the effect of nicotine. More specifically, in a striatal slice, the maximum peak of DA release does not change significantly through different frequencies (5, 10, 25, 50 Hz) of electrical stimulation. However, in the presence of nicotine or the nAChR antagonist mecamylamine, DA release at low frequencies is decreased, while at high frequencies release is enhanced (Rice and Cragg, 2004).

While electrical stimulation combined with pharmacological and genetic manipulations have produced a wealth of information on cholinergic control of DA release (Giorguieff et al., 1977; Cheramy et al., 1991; Krebs et al., 1991; Desce et al., 1992; Tremblay et al., 1992; Chéramy et al., 1996; Schmitz et al., 2003; Zhang and Sulzer, 2003, 2012; Exley and Cragg, 2008; Exley et al., 2011; reviewed by Cragg, 2006; Rice et al., 2011; Threlfell and Cragg, 2011; Zhang and Sulzer, 2012), the advent of optogenetics offered the previously unseen possibility of selective control of CINs. This would allow inducing AChR activation by means of endogenous release of ACh, obtained by selective stimulation of striatal CINs. Taking advantage of in vitro slices, electrochemistry, and optogenetics, both the laboratory of Stephanie Cragg (Threlfell et al., 2012) and ours (Cachope et al., 2012) were interested in characterizing changes in striatal DA levels in response to endogenous AChR activation. Interestingly, both research groups were advancing on characterizing similar phenomena in functionally different striatal areas, showing how endogenous release of ACh directly triggers DA release in the dorsal striatum and the NAc, respectively. Additionally to demonstrating how selective activation of CINs is enough to trigger DA release in striatum and NAc, respectively, both reports confirmed the role of nAChRs and mAChRs in modulating such output. Moreover, Cragg’s report very nicely unveiled circuital mechanisms by which thalamic input synchronizes CIN firing, subsequently promoting DA release (Threlfell et al., 2012). Our experiments focused instead on the possibility of CINs-triggered DA release in vivo (Cachope et al., 2012). Consistently with the in vitro data, optogenetic stimulation of CINs in the anesthetized mouse was sufficient to trigger elevation of DA concentrations in the NAc. Also, following Sabatini’s group’s report regarding the ability of CINs to evoke glutamatergic responses (Higley et al., 2011), we showed that ACh-evoked DA release is sensitive to AMPA blockers (Cachope et al., 2012).

In the case of mAChRs, they can also locally modulate DA release in the striatum. In vitro experiments with FSCV show how a wide range mAChR antagonist (oxotremorine) decreases DA release evoked by single pulse electrical stimulation, but enhances DA levels in response to train stimulation (Threlfell et al., 2010). A similar effect was observed using selective optogenetic stimulation, in which single pulse optical stimulation did not affect DA release, but instead 5 and 10 Hz stimulation enhanced DA release under application of the mAChR antagonist scopolamine (Cachope et al., 2012; Threlfell et al., 2012).

A complex interaction between diverse neurotransmission and neuromodulatory systems takes place in the control of striatal DA release. Although we have focused on the effect of glutamatergic and cholinergic systems, a handful other receptors have been identified as able to alter striatal DA levels; including GABA, cannabinoid, purinergic and opioid. Interestingly (and, up to some point expected), the possibilities for diversity on this local control are dependent on the type of receptor, not just the type of transmitter/modulator being released. Both in the case of glutamate and acetylcholine, different receptors lead to distinct and even opposite effects. As illustrated in Figure 1, mGluR activation on DA terminals and iGluR activation on MSNs result both in modulation of K conductances decreasing DA release. In contrast, activation of nAChRs on DA terminals leads to increased DA release, while activation of mAChR autoreceptors expectedly result in decreased DA release. More importantly, all these results demonstrate that firing rate at the VTA and SNc does not entirely determine striatal DA output, leaving enough room for control mechanisms driven by input from other areas (glutamatergic), as well as by interneurons (cholinergic), which might exert considerable impact on it.