Introductory Review Cellscience Reviews Vol 2 No.2 ISSN 1742-8130

Synaptic actions of mesoaccumbens dopamine neurons

Nao Chuhma & Stephen Rayport

Introduction

The mesoaccumbens dopamine (DA) projection plays a crucial role in normal goal-directed behavior, as well as in pathological behaviors such as addiction, and neuropsychiatric disorders such as schizophrenia. The mesoaccumbens projection is the densest DAergic projection (Geisler and Zahm, 2005; Prensa and Parent, 2001; Swanson, 1982), and so is arguably the one that would be most affected by systemically-administered DAergic drugs. The projection is made up of DA neurons in the ventral tegmental area (VTA) that synapse on medium-spiny neurons (MSNs) in the nucleus accumbens (nAcc), the limbic striatum. MSNs comprise about 95% of nAcc neurons and give rise to all the projections from the nAcc; the remaining 5% of nAcc neurons are GABAergic and cholinergic interneurons (Pennartz et al., 1994; Steffensen et al., 1998; Wilson, 1998). MSNs receive excitatory synaptic inputs from many areas, principally the prefrontal cortex and limbic areas, including the hippocampus, basolateral amygdala, and thalamus. These convergent sets of synaptic inputs pinpoint the nAcc as a critical interface between cortical and subcortical (limbic) circuits, where motivation is translated into action (Mogenson and Yim, 1991; Wu et al., 1993). This has been refined into the current view that mesoaccumbens dopamine neurons convey incentive salience information (Robinson and Berridge, 1993) or a reward prediction error signal (Salzman et al., 2005; Tobler et al., 2005).

Recently, the dichotomy between information-laden cortical input, which is principally glutamatergic, and modulatory input from VTA DA neurons, which had principally been thought to be solely DAergic, has been blurred by increasing evidence that DA neurons exert fast excitatory actions via co-released glutamate (Figure 1). In fact, studies dating back over 50 years have hinted at the possibility (Hattori, 1993). However, it was first demonstrated unequivocally in culture just a few years ago (Joyce and Rayport, 2000; Sulzer et al., 1998). Recently, these observations have been extended to monosynaptic recordings in brain slice (Chuhma et al., 2004) and to the projections of mesocortical DA neurons in the intact brain (Lavin et al., 2005); so, glutamate cotransmission likely plays an important role in mature brain circuits. In this review, we will first summarize what is known about classical DA transmission, with an emphasis on the mesoaccumbens system, highlighting the limitations of DA as the sole DA neuron transmitter. Then, we will focus on the evidence for glutamatergic cotransmission, continue with a discussion of DA modulation of co-released glutamate, and conclude with discussion of the possible physiological role of DA neuron glutamate cotransmission.

Figure 1. Dopamine-glutamate interactions in the nucleus accumbens. In the classical view of the nAcc, information-laden glutamatergic inputs (shown in blue) impinge on medium-spiny neuron (MSN) dendritic spines (yellow) where they are subject to DA (orange) modulation. While glutamate receptors (green) are located on the postsynaptic membrane, many DA receptors (red) are located extrasynaptically, consistent with DA having a modulatory role. Recent work, however, suggests a revised view of the nAcc in which DA neurons are actually DA/glutamatergic neurons (orange/blue striped) capable of fast excitatory transmission.

DA transmission by VTA DA neurons

Separate populations of VTA DA neurons project to limbic areas and to the prefrontal cortex (PFC) (Sesack and Carr, 2002). The axons of VTA DA neurons projecting to the nAcc - the mesoaccumbens projection - course rostrally in the medial forebrain bundle, and then branch profusely after entering the nAcc, making numerous bouton-type synapses on MSNs and cholinergic interneurons, as well as varicosities without associated postsynaptic specializations (Pickel et al., 1981). DA neuron axons are thin and unmyelinated, and so have relatively slow conduction velocities. Together with the observation that many DA receptors are localized on extrasynaptic membranes (Yung et al., 1995), the implication has been that a significant component of the DA signal is mediated by what Fuxe (Zoli et al., 1999) has termed volume transmission, and is slow, being neither spatially nor temporally discrete.

DA transmission is modulatory

DA receptors are G-protein coupled receptors, classified in two families, D1-like and D2-like receptors. D1-like receptors comprise D1 and D5 receptors; however, in the nAcc (and striatum) there are few D5 receptors. Both D1 and D5 receptors activate Gs/olf (Missale et al., 1998), and activate adenylyl cyclase resulting in activation of protein kinase A (PKA). PKA phosphorylates protein phosphatase 1 (PP1) inhibitor DARPP 32 (Hemmings and Greengard, 1986). These PKA and DARPP-32/PP1 pathways modulate multiple cellular signaling molecules (Greengard et al., 1999), for example voltage gated Ca²⁺ channels (Surmeier et al., 1995), NMDA receptors (Snyder et al., 1998), GABAA receptors (Flores-Hernandez et al., 2000) and voltage dependent Na⁺ channels (Schiffmann et al., 1995).

D2-like receptors comprise D2, D3 and D4 receptors and activate Gi/o (Missale et al., 1998). Giα subunits inhibit adenylyl cyclase and PKA (Bonci and Hopf, 2005). Gβγ subunits of Gi/o modulate multiple intracellular targets, principally Ca²⁺ channels, K⁺ir channels and adenylyl cyclase (Albert and Robillard, 2002). This range of intracellular targets suggests that net effect of DA on the excitability of cells depends on which cascades are dominant, which will be determined by the local DA concentration, the distribution of DA receptors, the co-localization of receptors, and the regulation of post-receptor signaling cascades, which may be more or less restricted to postsynaptic microdomains. Taken together, these mechanistic insights argue further that DA’s role is principally modulatory. Presumably another signal is required if DA neurons are to convey a temporally-discrete, information-laden signal.

Tonic and phasic DA neuron activity

DA is released in target areas in two modes: tonic and phasic. Tonic release reflects the quasi-pacemaker firing of DA neurons with the additional drive provided by synaptic inputs directly on to DA neuron terminals in target areas (Floresco and Grace, 2003). The actual tonic DA level continues to be the subject of debate. Measuring DA by microdialysis is indirect and distorted in several ways by the probe used for the measurement. Taking this into account, and using the technique of no-net flux (Parsons and Justice, 1992), tonic DA levels have been estimated to be in the range of 10 nM to 100 nM (Chen, 2005a, b; Wightman and Robinson, 2002). Given both synaptic and extrasynaptic DA release, DA levels are likely to show considerable regional variation (Cragg and Rice, 2004; Sulzer and Pothos, 2000). The role of tonic DA remains unclear - one suggestion has been that it may enhance the signal-to-noise ratio to select more important, salient inputs (Nicola et al., 2004).

In contrast, phasic DA release, which is thought to convey the incentive salience signal, reflects higher-frequency DA neuron burst firing (Grace, 2000). Short bursts of DA neuron spikes (~20Hz) cause a rapid increment in DA levels to the 100 nM to 1 µM range; once released, DA action is terminated by both diffusion and reuptake (Benoit-Marand et al., 2000; Jones et al., 1995). Reuptake is mediated by the DA transporter (DAT) located in the presynaptic terminals of DA neurons (Cragg and Rice, 2004). In the nAcc, a phasic DA release event lasts several seconds (Jones et al., 1998; Jones et al., 1995), putting a limit on the temporal resolution of the information that may be conveyed.

Glutamate transmission by DA neurons

The idea that neurons might release more than one transmitter arose some time ago with observations that neurons commonly release both a small-molecule classical transmitter and a neuropeptide (Burnstock, 2004; Hökfelt et al., 1984). The idea that neurons might use two classical transmitters ran counter to the then prevailing dogma. However, the last decade has witnessed an ever increasing number of reports of colocalization of classical transmitters, including ones with opposite synaptic actions (Fung et al., 1994b; Hökfelt et al., 2000; Jo and Role, 2002; Jo and Schlichter, 1999; Johnson, 1994; Jonas et al., 1998; Nishimaru et al., 2005; Poelchen et al., 2001; Tsen et al., 2000). While two classical transmitters may seem counterintuitive or superfluous, the different input/output functions of the colocalized neurotransmitters confer on synapses a far greater dynamic range of action (Brezina and Weiss, 1997).

It is striking that the three major monoaminergic modulatory systems all show evidence for glutamate colocalization; so not only do DA neurons - the subject of the present review, but also serotonergic Raphe neurons (Fremeau et al., 2004a; Johnson, 1994) and noradrenergic locus coeruleus neurons (Fung et al., 1994a; Fung et al., 1994b). This suggests that all three crucial neuromodulatory systems transmit temporally-discrete, information-laden signals (Seamans and Yang, 2004; Sulzer and Rayport, 2000; Trudeau, 2004).

Observations in culture

When placed in microcultures, single postnatal rat VTA DA neurons make excitatory autaptic connections - synapses of a neuron back onto itself (Sulzer et al., 1998). Unlike Raphe neurons (in microcultures) where both serotonergic and glutamatergic postsynaptic signals are evident (Johnson, 1994), in DA neurons the only signal that is evident is glutamatergic. DA release is only evident indirectly through its modulation of the glutamatergic response. Most DA neurons have glutamatergic actions, so that in microcultures a glutamatergic autaptic connection provides the best means of identifying DA neurons (Jomphe et al., 2005). Consistent with this, most VTA DA neurons in culture are strongly immunoreactive for glutamate, as well as the glutamate synthetic enzyme phosphate-activated glutaminase (PAG) (Sulzer et al., 1998). When nAcc target neurons are included in micro-co-cultures, VTA DA neurons make both excitatory autaptic and synaptic connections (Joyce and Rayport, 2000), so the glutamatergic autapse again provides reliable identification of DA neurons. Interestingly, the autaptic and synaptic connections of the same DA neuron are heterogeneously modulated by DA (Joyce and Rayport, 2000). Presynaptic D2 receptors inhibit glutamate corelease (Sulzer et al., 1998) via activation of K⁺ channels (Congar et al., 2002). Moving closer to the intact circuitry, there have also been observations that substantia nigra DA neurons in explant co-cultures make fast, excitatory synaptic connections (Plenz and Kitai, 1996). However, in vitro observations of DA neuron glutamate co-transmission do not prove that DA neurons make glutamatergic synapses in the intact, mature brain, only that the neurons have the propensity to do so.

Observations in vivo

Several lines of evidence argue for DA neuron glutamate co-transmission in vivo. Ottersen and colleagues (1984) provided the first immunocytochemical evidence for high levels of glutamate in DA neurons. Although glutamate has metabolic roles and serves as a precursor to GABA, glutamatergic neurons tend to have the highest glutamate levels. Moreover, loading glutamate into non-glutamatergic neurons confers glutamatergic status (Dan et al., 1994), so observations of higher glutamate levels imply that the cells are glutamatergic. Kaneko and colleagues (1990) found that PAG is present in most DA neurons. We and others have confirmed that DA neurons are immunoreactive for glutamate, both in rat (Shiroyama et al., 1996; Sulzer et al., 1998) and monkey (Sulzer et al., 1998). In rat, when DA neuron fibers were lesioned by 6-OH DA, there was about a 20% loss in the numbers of excitatory synaptic profiles in the striatum (Ingham et al., 1998), presumably reflecting the loss of DA neuron glutamatergic varicosities; however, this could also reflect the loss of the trophic effects of DA on MSN spines, leading to a reduction in available areas for cortical glutamatergic inputs. More directly, Wilson and colleagues (1982) showed that after cortical ablation and transection of thalamic pathways, stimulation of DA neurons in the substantia nigra generated a fast monosynaptic EPSP in striatal neurons. With chronic decortication, allowing time for collateral inputs from cortical efferent fibers to degenerate, the fast EPSP disappeared, but there remained a smaller, longer-latency monosynaptic EPSP remained that the authors concluded arose from DA neurons.

With the cloning of the vesicular glutamate transporters (VGLUTs), Jahn and colleagues (Takamori et al., 2000) showed that the molecules are the definitive, phenotypic markers of glutamate neurons; if a GABAergic neuron was transfected with VGLUT1, it started to function as a glutamate neuron. By loading vesicles, the VGLUTs control quantal size (Wojcik et al., 2004). However, VGLUT1 was not present in DA neurons; nor was VGLUT2, at least in vivo (Fremeau et al., 2004a; Kaneko et al., 2002). VGLUT2 accounts for DA neuron cotransmission in vitro (Dal Bo et al., 2004), but it is not expressed in DA neurons in the mature brain (Fremeau et al., 2004b), possibly reflecting its developmental down regulation in postnatal life (Boulland et al., 2004; Kawano et al., 2004). Then, the cloning VGLUT3 was reported in 2002 (Fremeau et al., 2002; Gras et al., 2002; Schafer et al., 2002; Takamori et al., 2002). VGLUT3 mRNA is expressed in the DA neuron groups (Fremeau et al., 2002), and the protein is expressed both at the cell body level in the DA neuron groups and in target areas including the nAcc, with a distribution similar to that of the DA neuron marker tyrosine hydroxylase (Gras et al., 2002). However, a direct attempt to visualize VGLUT3 in DA neurons came up negative (Schafer et al., 2002). So whether VGLUT3 is present in DA neurons remains unresolved.

Glutamate co-transmission recorded in brain slice

While whole-cell patch recording in the brain slice provides the best way to do a detailed analysis of synaptic function in the relatively intact circuitry of the near-mature brain, recording the synaptic actions of DA neurons in brain slices has proved challenging because [1] the VTA is remote from the nAcc (3 mm away in mice) so that it is difficult to make slices encompassing the entire projection; [2] MSNs receive multiple excitatory inputs so it is impossible to activate just one set of inputs by local field stimulation; [3] locating the medial forebrain bundle that contains the mesoaccumbens DA neuron axons in the slice can be difficult. We overcame these difficulties by using transgenic mice with fluorescent DA neurons. These DAT-YFP mice were generated (Zhuang et al., 2005) by breeding DAT-cre mice, in which cre recombinase expression is driven by the DA transporter (DAT) promoter, with enhanced yellow fluorescent protein (YFP) reporter mice (Srinivas et al., 2001). Brain slices from DAT-YFP can be viewed under epifluorescence (Chuhma et al., 2004) to optimize parameters of slice preparation, maximize numbers of intact mesoaccumbens fibers, and guide recordings.

In the mesoaccumbens slices made from DAT-YFP mice (Figure 2), stimulation of the VTA generated monosynaptic fast excitatory postsynaptic responses in nAcc MSNs and concomitant DA release (Chuhma et al., 2004). There was also a polysynaptic late response, which was eliminated when the cortex and hippocampus were removed. Local application of glutamate to the VTA, which should activate DA neurons, but not fibers of passage, increased the frequency of synaptic events in the nAcc. Local application of the D2 receptor agonist quinpirole to the VTA reduced the response; since only DA neurons bear D2 receptors in the VTA, presumably the quinpirole selectively inhibited DA neurons (Lacey et al., 1987) and raised the threshold for their activation. These observations argue strongly that the fast excitatory response is mediated by the synapses of VTA DA neurons.

Figure 2. Excitatory responses evoked in nAcc neurons by stimulating DA neurons in brain slice from a DAT-YFP mouse. Horizontal slices encompassing the mesoaccumbens projection were made from mice with fluorescent DA neurons (DAT-YFP mice). The schematic (left panel) shows a hemi-slice. Focal electrophysiological stimulation delivered to the VTA evoked excitatory postsynaptic currents (EPSCs) in a medium-spiny neuron (MSN) in the nucleus accumbens (nAcc). Fluorescence images are shown of the living slice as seen during recordings. Yellow-fluorescent protein (YFP) labeled DA neurons are evident in the VTA (lower image). DA neurons in the young mice used are about 10 µm in diameter. A cloud of DA neuron terminals is evident in the nAcc (upper image). VTA stimulation generated two types of EPSCs in a MSN (right panel), recorded in whole-cell mode. Following the stimulus artifact (blue arrow), a short latency monosynaptic response (Early EPSC), with a short, fixed latency, was followed by a longer latency polysynaptic response (Late EPSC). Both responses were blocked by the glutamate antagonist CNQX (red). Removal of the cortex and hippocampus from the slice eliminated the late EPSC showing that it was probably mediated by collaterals of cortical pyramidal neurons, while the early EPSC remained, showing that it was mediated by direct DA neuron projections from the VTA to the nAcc.

Glutamate co-transmission in vivo

In rat, stimulation of the VTA in vivo generated fast excitatory synaptic responses in the prefrontal cortex (PFC, Lavin et al., 2005). This fast EPSP was unaffected by systemic DA antagonists but eliminated by systemic glutamate antagonists (Lavin et al., 2005). Infusion of NMDA into the VTA evoked a barrage of EPSCs in the PFC, which were eliminated by prior 6-OH-DA lesions of DA neurons in the VTA (Lavin et al., 2005). These observations suggest that the fast EPSP originated from VTA DA neurons, not from collaterals of cortical efferent fibers or other fibers of passage. In the nAcc, stimulation of the VTA mimicking burst firing generated fast EPSPs with a duration of ~500 ms and a short latency (Brady and O'Donnell, 2004; Goto and O'Donnell, 2001a). This EPSP was reduced but not eliminated by the combined application of D1 and D2 antagonists; what remained was presumably mediated by DA neuron glutamate co-release.

Controversies

Until recently, the possibility that DA neurons were glutamatergic was fraught with controversy for several reasons: [1] PAG does not appear to be reliably expressed in all glutamatergic neurons (Kaneko, 2000), [2] non-glutamatergic neurons express glutamate uptake transporters (Mathews and Diamond, 2003), including DA neurons (Plaitakis and Shashidharan, 2000), [3] neurons immunostained for glutamate may actually be GABAergic, or [4] simply have high levels of metabolic glutamate. Now the debate focuses on VGLUT expression in DA neurons.

In culture, DA neurons identified by tyrosine hydroxylase immunoreactivity stain for VGLUT2 (Dal Bo et al., 2004), and in early postnatal life, with diminishing intensity over the first 2 months of life (Kawano et al., 2004), suggesting that VGLUT2 may indeed confer glutamatergic status on maturing DA neurons (Kawano et al., 2004; Trudeau, 2004). VGLUT3 is apparently expressed in all monoaminergic neurons. While in serotonergic Raphe neurons, there was no doubt as the expression was robust, in DA neurons it was less clear. The initial report from el Mestikawy and colleagues (Gras et al., 2002) found VGLUT3 protein (by immunocytochemistry) in the VTA and substantia nigra (SN), but no message by in situ hybridization. The second (Takamori et al., 2002) and third (Fremeau et al., 2002) reports found VGLUT3 mRNA in the VTA and SN. The fourth report came up negative for colocalization of tyrosine hydroxylase (TH) and VGLUT3 (Schafer et al., 2002). While, the VGLUT evidence favors DA neurons being glutamatergic, it is by no means conclusive, particularly in adulthood.

Another controversy is that DA neuron glutamatergic transmission occurs only transiently during development, as the strongest data derives from immature DA neurons in culture (Sulzer et al., 1998; Trudeau, 2004), or from slice recordings done in juvenile mice (Chuhma et al., 2004). VGLUT2 is expressed at the highest levels in early postnatal life and then declines with maturation (Boulland et al., 2004). In the VTA, VGLUT2 expression was observed just after birth and expression declined during postnatal development, so that by adulthood only a fraction of VTA DA neurons showed VGLUT2 expression (Kawano et al., 2004). In postnatal VTA culture, 80% of DA neurons are immunoreactive for VGLUT2 (Dal Bo et al., 2004), which could be due to immature DA neurons maintaining high levels of VGLUT2 expression. However, the in vivo observations of fast EPSPs were obtained in adult rats (Goto and O'Donnell, 2001b; Lavin et al., 2005; Lewis and O'Donnell, 2000), and in slice, the fast EPSC was present in mice as old as 5 weeks of age (N Chuhma, unpublished observations). So, glutamatergic transmission by DA neurons is not a transient developmental phenomenon and does occur in adult brain, though it may diminish in strength with maturation.

A final controversy is that both in slice and especially in vivo, stimulation of the VTA activation might activate passing fibers. The passing fibers are most likely collaterals of cortical efferents that synapse on MSNs. However, cortical efferent fibers are myelinated and so should have higher conduction velocities compared to unmyelinated DA neuron fibers, and indeed a faster EPSP was eliminated by decortication and the passage of time to allow degeneration of cortical fibers, which left a slower, long-latency monosynaptic EPSP (Wilson, 1998; Wilson et al., 1982). In more recent in vivo experiments, killing DA neurons with the DA neuron-specific toxin 6-OH-DA eliminated the VTA-evoked EPSP in the PFC in vivo (Lavin et al., 2005). Taking advantage of the fact that neuronal cell bodies but not axons bear glutamate receptors, local application of NMDA (Lewis and O'Donnell, 2000) or glutamate (Chuhma et al., 2004) to the VTA generated fast excitatory response in the PFC and in the nAcc, respectively. The converse experiment, taking further advantage of the fact that the only neurons bearing inhibitory D2 receptors in the VTA are DA neurons, we showed (Chuhma et al., 2004) that the D2 agonist quinpirole reduced the fast EPSC in slice. Taken together, these observations argue strongly against the contribution of passing fibers to the observed excitatory response, and the response being due to monosynaptic glutamatergic synaptic connections made by VTA DA neurons onto postsynaptic neurons in the nAcc and PFC.

DA modulation of the glutamatergic transmission by DA neurons

If DA and glutamate are co-released from DA neuron terminals, then the transmitters will likely regulate each other’s release. In particular, simultaneously released DA would be expected to modulate glutamate release. We will briefly summarize the reported action of DA in the nAcc, and in this context DA modulation of glutamatergic transmission.

DA modulation of the synaptic inputs in the nAcc

Application of DA or DA agonists to the nAcc have multiple electrophysiological effects (Nicola et al., 2000; Surmeier and Calabresi, 2002). DA attenuates excitatory synaptic inputs (Beurrier and Malenka, 2002; Harvey and Lacey, 1996; Nicola et al., 1996; Pennartz et al., 1992) via presynaptic D1 receptors on inputs from hippocampus or amygdala (Beurrier and Malenka, 2002; Charara and Grace, 2003; Pennartz et al., 1992) and on GABAergic inputs (Guzman et al., 2003; Nicola and Malenka, 1997; Taverna et al., 2005), and via presynaptic D2 receptors on cortical inputs (Bamford et al., 2004) and also on intrinsic GABAergic inputs (Guzman et al., 2003). Presynaptic D2 receptors on DA neuron terminals also mediate presynaptic inhibition (Benoit-Marand et al., 2001; Schmitz et al., 2002). Postsynaptically, DA application to the nAcc neurons causes slow biphasic membrane potential shifts, with D2-mediated depolarization and D1-mediated hyperpolarization (Higashi et al., 1989; Uchimura et al., 1986). D1 receptor activation couples with NMDA receptors to induce adenosine release from postsynaptic cells, which then attenuates synaptic inputs via activation of presynaptic A1 adenosine receptors (Harvey and Lacey, 1997). There is considerable evidence for D1-D2 interactions from behavioral experiments (Ikemoto et al., 1997; Phillips, 1984; Plaznik et al., 1989), from in vivo recording (Goto and O'Donnell, 2001a), and in cellular physiology (Hopf et al., 2003; O'Donnell and Grace, 1996).

DA modulation is significantly influenced by postsynaptic MSN membrane potential. MSNs fluctuate between down and up states; the down state refers to the very hyperpolarized resting potential of the neurons of about -80 mV, while the upstate refers to a quasi-stable more-depolarized potential of about -60 mV. Transitions to the up state are driven by hippocampal or cortical glutamatergic inputs (Calabresi et al., 1990; O'Donnell and Grace, 1995; Wilson and Kawaguchi, 1996). DA has different modulatory effects when MSNs are in the down or upstate (Surmeier and Calabresi, 2002; Nicola et al., 2004). For example, when MSNs are in the downstate, D1 receptor activation reduces the excitability of MSNs through K⁺ir channel activation (Pacheco-Cano et al., 1996), while in the upstate, D1 receptors increase excitability through activation of L type Ca²⁺ channels (Surmeier et al., 1995). This should selectively enhance excitatory synaptic inputs when MSNs are in the up state.

All of the effects, and many others initiated by DA receptor activation are mediated via two second messenger pathways, one involves DARPP-32, which when knocked out eliminates about half of DA’s effects, and PP-1 (Fienberg et al., 1998). These pathways are differentially regulated by glutamate providing a further substrate for the interaction of the effects of the two neurotransmitters (Greengard, 2001), but one that is beyond the scope of this review.

DA modulation of the glutamatergic transmission by DA neurons

VTA stimulation in patterns mimicking the burst firing of DA neurons drives nAcc neurons into the up state in vivo (Brady and O'Donnell, 2004; Goto and O'Donnell, 2001a) and in the in vitro slice (N Chuhma, unpublished observations). This depolarization is reduced by application of D1 and D2 antagonists together, but never completely blocked (Goto and O'Donnell, 2001a), suggesting that the glutamatergic component of the DA neuron signal - which would remain after the DA receptor blockade - was enhanced by concomitant DA release. As might be predicted, the DA enhancement was much robust when DA neurons fired in burst-like patterns. This was borne out directly in the mesoaccumbens slice, as the depolarization evoked in MSNs neurons by VTA stimulation was reduced by co-application of D1 and D2 antagonists and enhanced by DAT blockade (Chuhma and Rayport, 2005). When postsynaptic K⁺ channels were blocked with a Cs⁺-based intracellular solution, DA agonists reduced the DA neuron-evoked excitation, and this reduction was reversed by D2 antagonists (Chuhma and Rayport, 2005). So, DA apparently modulates the coreleased glutamate transmission via presynaptic inhibition and postsynaptic facilitation. Presynaptic D2 receptors inhibit DA releasing terminals (Benoit-Marand et al., 2001; Schmitz et al., 2003) and so they apparently also inhibit DA neuron glutamate-releasing terminals. The postsynaptic enhancement is more prominent with burst firing (Chuhma and Rayport, 2005), suggesting that only stronger responses which are not blocked by the presynaptic inhibition would be enhanced by postsynaptic DA action. This contrast enhancement mechanism would favor transmission of stronger DA neuron signals presumably mediating incentive salience.

Physiological role of DA neuron glutamatergic transmission

DA neurons are thought to signal a reward prediction error signal involved in goal-directed learning (Bonci et al., 2003; Montague et al., 2004; Salzman et al., 2005; Schultz, 2002; Tobler et al., 2005). This acts as teaching signal in postsynaptic areas to update goal representations based on past experience. In particular, the DA teaching signal apparently binds the evaluation of rewards (the goal) to the sequence of actions required to obtain the reward (goal-directed behavior) (Berridge and Robinson, 1998; McHaffie et al., 2005). This DA signal is presumably mediated by phasic DA neuron firing, which occurs with short latency (50 -110 msec) from the timing of the expected reward, and is short lasting (<200 ms) (Redgrave et al., 1999). Redgrave et al. argue that the short-latency, short-lasting DA signal serves to reallocate cognitive or behavioral processing towards unexpected but important events by disinhibition of the “winning” processes and sustained inhibition of “losing” processes.

Assuming then that phasic (short-lasting) DA neuron firing is the teaching signal, then how does DA neuron activity actually convey this information to target neurons? DA action, as discussed above, is predominantly modulatory, without the necessary temporal or spatial resolution. Even if the argument can be made that the intracellular cascade from DA binding to activation of postreceptor target molecules occurs in less than 50 msec, as has been documented in the presynaptic inhibitory actions of released DA on DA neuron terminals in the nAcc and striatum (Phillips et al., 2002; Schmitz et al., 2002), DA actions take seconds to wane (Benoit-Marand et al., 2001; Schmitz et al., 2002), and probably persist for much longer times (Lavin et al., 2005). So while a burst of DA neuron firing could transmit the onset of an event relatively quickly, it could not transmit events involving any significant frequency. However true these limitations may be for the mesoaccumbens projection, they are more of a limitation for DA’s role in the mesocortical projection where the less dense DA projection and weaker DA uptake system will translates into a much slower onset and offset of DA release events. Moreover, postsynaptic DA actions are likely to be much slower than presynaptic actions. So, if patterns of DA neuron activity are to convey a meaningful signal, a faster transmitter is required, and as we have argued, the best candidate for this is glutamate.

What then would the merit be of having DA and glutamate released simultaneously? Presumably, the information contained in short-lasting DA neuron bursts is conveyed to target cells via glutamatergic cotransmission. Another view of DA neuron glutamate cotransmission is that the glutamatergic signals provide a timed substrate for DA modulation (Figure 3). In this view, the DA neuron glutamatergic signal drives MSNs into the upstate thereby allowing other glutamatergic afferents to drive MSNs and simultaneously providing DA to shape those inputs. While the onset of the DA signal may be sufficiently rapid, one may argue that the DA signal needs also to be terminated discretely, and this might be handled by the return of MSNs to their down state. So the glutamate cotransmission would serve to provide a timed substrate for DA modulation MSN activity.

Figure 3. Hypothesized role of DA neuron glutamate cotransmission. The DA neuron glutamate signal may create a timed window for DA modulation of cortical inputs. (A) We would argue that DA action is principally modulatory, so that its action is most evident in the enhancement of other glutamatergic inputs. (B) In the absence of other synaptic inputs, DA produces only a subtle modulation of the postsynaptic cell membrane potential, which may be either depolarizing (as shown here) or hyperpolarizing, depending on the distribution of postsynaptic DA receptors and postreceptor signaling cascades. (C) If DA neurons only release DA (left), then the postsynaptic cell will show just a slow change in membrane potential with a brief application of DA. However, if DA neuron co-release glutamate, DA neuron excitability is converted to timed depolarization of the target neuron (right), putting it into the up state. In the upstate other excitatory inputs (not shown) would be capable of driving the postsynaptic cell till the cell dropped back down into the down (resting) state.

Several lines of evidence suggest that long-term potentiation of glutamatergic inputs plays a crucial role in the transition to the drug dependent state, and by analogy to pathological sensitization in progressive disorders such as schizophrenia (Thomas and Malenka, 2003). So far, such observations have focused on inputs to the DA neurons; however, an intriguing possibility is that DA neuron glutamatergic synapses might also show pathological potentiation. By virtue of the corelease of the two key transmitters involved in long-term potentiation, DA neuron synapses might be especially vulnerable. These are highly speculative views that raise several intriguing hypotheses that will probably best be addressed in transgenic mice with a DA neuron specific knock out of glutamatergic transmission.

Summary

DA neuron glutamate cotransmission has been debated for many years. Now, several lines of evidence make this likely. In particular, the observations that activation of VTA DA neurons produces a fast glutamatergic response in the mesoaccumbens slice and in the PFC in vivo, and the cloning of VGLUT3, which may be expressed in DA neurons, all argue for DA neuron glutamate cotransmission. Many critical issues remain, in particular whether VGLUT3 is responsible for the glutamate cotransmission in the intact brain, the relative importance of the cotransmission during development and in adulthood, and most importantly the role of the DA neuron glutamate co-transmission. At this point, we would argue that the glutamatergic co-transmission in the mesoaccumbens projection provides a timed short-lasting excitation to MSNs communicating the salience signal necessary to process associated information.

Acknowledgements

We gratefully acknowledge the support of NARSAD (NC, SR), NIDA (SR) and NIMH (SR).

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