Alterations in neurotransmitter co-release in Parkinson’s Disease

Abstract

Parkinson’s disease is a neurological disorder characterized by degeneration of midbrain dopamine neurons, which results in numerous adaptations in basal ganglia circuits. Research over the past twenty-five years has identified that midbrain dopamine neurons of the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) co-release multiple other transmitters including glutamate and GABA, in addition to their canonical transmitter, dopamine. This review summarizes previous work characterizing neurotransmitter co-release from dopamine neurons, work examining potential changes in co-release dynamics that result in animal models of Parkinson’s disease, and future opportunities for determining how dysfunction in co-release may contribute to circuit dysfunction in Parkinson’s disease.

1. INTRODUCTION

Parkinson’s Disease (PD) is characterized by the progressive degeneration of midbrain dopamine neurons in the substantia nigra pars compacta (SNc) and the accumulation of aggregated α-synuclein in the form of Lewy bodies and neurites. SNc neurons innervate the basal ganglia and send a large projection to the dorsal striatum. Release of dopamine modulates striatal circuits and neurons, which consists of medium spiny neurons (MSNs) that project to downstream basal ganglia nuclei, as well as cholinergic (ChIs) and GABAergic interneurons (Kreitzer, 2009). Dysfunction of this circuitry is associated with many PD symptoms, including motor impairments (for review see (McGregor & Nelson, 2019)). Though dopamine depletion is thought to be the primary driver of such circuit dysfunction, the reality of disease development is much more complex. Further, common dopamine-based treatment strategies such as levodopa are unable to alleviate all symptoms and are not effective long-term solutions. Therefore, a more nuanced look at the microcircuitry and neurotransmitter profile within the striatum is required to fully understand the etiology of the disease (for review see (Sanjari Moghaddam et al., 2017)).

As PD develops, there is a characteristic prodromal decrease in the release of dopamine within the striatum. In animal models of PD, early reductions in dopamine occur prior to measurable levels of cell loss (Janezic et al., 2013; Lundblad et al., 2012), suggesting that the function and activity of dopamine neurons diminishes prior to cell death. In addition to releasing dopamine, a subset of these neurons are capable of co-releasing other transmitters including glutamate (Sulzer et al., 1998), GABA (Tritsch et al., 2012), and proteins and peptides such as Sonic hedgehog, neurotensin and CCK (German & Liang, 1993; Malave et al., 2021). Unsurprisingly, studies have suggested that changes in co-release occur alongside changes in dopamine release. Both alterations in activity and neuronal loss likely alter co-release of fast transmitters such as glutamate and GABA, which allow for a finer temporal control of synaptic activity and a more complex picture of striatal connectivity. Such changes in the pattern and extent of corelease may either be adaptive, or alternatively, underlie some of the symptoms and progression of PD. Sites of co-release therefore represent potential therapeutic targets that have yet to be fully explored.

In this review, we will briefly summarize what is known about glutamate and GABA co-release focusing on midbrain dopamine neurons, specifically the SNc neurons and their projections to the dorsal striatum. This section will include a description of the current evidence indicating whether or not co-release is independently regulated, which may have implications as to the potential for an independent time course in disease development. Then we will discuss what is known about relative vulnerability/resiliency of co-releasing dopamine neurons and evidence for adaptive changes in co-release in PD. Finally, we will pose additional avenues of exploration that remain to be investigated and that could expand our understanding of how alterations in co-release contribute to the disease.

2. NEUROTRANSMITTER CO-RELEASE

Though historical models of neuronal identity typically used the one-neuron-one-neurotransmitter framework (Eccles et al., 1954), it is now widely accepted that most neurons in the central nervous system are able to “co-release” more than one molecule (for review see (Hnasko & Edwards, 2012; Kupfermann, 1991; Svensson et al., 2018; Vaaga et al., 2014)). This function exists across a wide variety of transmitter types, including classical small molecule neurotransmitters, neuropeptides, and gaseous molecules such as nitric oxide. Varying modes of co-release allow for increased versatility and temporal control of neuronal signaling.

The capacity of a neuron to co-release more than one neurotransmitter in response to a single stimulus depends on its ability to either synthesize (or uptake) and package multiple transmitters into synaptic vesicles. Thus, synthetic enzymes and transporters are often used as markers of co-release. In addition to having the ability to co-release, the capability to co-transmit signals through more than one neurotransmitter in response to a single stimulus depends on the apposition of a neuron to multiple specialized postsynaptic sites. This process can occur through multiple mechanisms of vesicular loading (same vs. different vesicles), vesicular trafficking (same vs. different release sites), and synaptic coupling (overlapping or non-overlapping sites of receptor expression) (see (Tritsch et al., 2016) for review). Functionally speaking, different modes of co-transmission may result in similar patterns of activity, though they may be differentially regulated, and therefore, independently impacted by changes in neuronal activity and function. For simplicity in this review we will use the term co-release, even though many studies have measured the release of multiple transmitters though the activation of post-synaptic receptors.

Dopamine neurons are themselves a heterogeneous population and only subsets of them exhibit co-release of glutamate and/or GABA. The mechanisms of co-release from dopamine neurons, including possible co-packaging and vesicular segregation, are not fully understood. However, dopamine neurons specifically display multiple types of terminals, raising the possibility that glutamate and GABA transmission may occur at different sites from one another and from dopamine (Hattori et al., 1991). For each transmitter, the evidence regarding co-release mechanisms are described below. These descriptions help elucidate possible mechanisms by which different types of transmission may be independently regulated.

2.1. Glutamate Co-Release

Glutamate co-release from dopamine neurons was first described using rat midbrain neurons that formed glutamatergic autapses in culture (Joyce & Rayport, 2000; Sulzer et al., 1998). Co-release was further confirmed in intact slices through activation of dopamine neurons, either by electrical stimulation within the midbrain or direct optogenetic activation of terminals. Such experiments indicated that dopamine neurons form synaptic connections with multiple striatal cell types including MSNs, ChIs, and fast spiking GABAergic interneurons (FSIs) - with varying degrees of strength (Chuhma et al., 2004, 2014, 2018, 2023; Stuber et al., 2010; Tecuapetla et al., 2010). These synaptic connections show strong regional specificity, such that glutamatergic responses are greatest in the ventral striatum and restricted to the lateral extent of the dorsal striatum (Cai & Ford, 2018; Chuhma et al., 2014).

To determine which midbrain neurons have the potential for glutamatergic co-release, studies have examined the expression of the vesicular glutamate transporter (VGLUT) which is responsible for loading glutamate into vesicles and has three isoforms (VGLUT1, VGLUT2, VGLUT3) with largely non-overlapping distributions. A proportion of midbrain neurons express VGLUT2, representing a heterogeneous population of midbrain glutamatergic neurons that can be further defined by the expression of other neuronal markers (Dal Bo et al., 2004; Kawano et al., 2006; Li et al., 2013; Root et al., 2016; Yamaguchi et al., 2007, 2015). Co-labeling for the vesicular GABA transporter (VGAT) has identified a subpopulation of glutamate-GABA neurons, which in the ventral tegmental area (VTA) display unique cellular properties to glutamate-only or GABA-only neurons (Miranda-Barrientos et al., 2021). Additionally, a subpopulation of VGLUT2⁺ cells are also putative dopamine neurons, as identified by expression of tyrosine hydroxylase (TH). While this review will focus on this latter population of cells that have the capacity to release both glutamate and dopamine, it is important to note the existence of striatal projecting glutamatergic neurons that do not also release dopamine.

Glutamatergic neurons in the midbrain exhibit a topographic patterning across the VTA and SNc (for reviews see (Morales & Margolis, 2017; Morales & Root, 2014)). The highest density of both VGLUT2⁺ only neurons and co-labeled TH/VGLUT2⁺ neurons exists in the VTA, especially in the medial nuclei of the structure (Yamaguchi et al., 2011). Conversely, the SNc has a smaller percentage of VGLUT2⁺ neurons, very few of which also express TH. Though VGLUT2⁺/TH⁻ and dual-labeled VGLUT2⁺/TH⁺ cells are much less abundant in the SNc, they are concentrated within the most lateral and dorsal extents of the SNc (substantia nigra pars lateralis and SNc dorsal tier), projecting strongly to the tail of the striatum and the dorsolateral striatum (DLS) (Poulin et al., 2018; Yamaguchi et al., 2013). These patterns hold true across mammalian brains and analysis of VGLUT2 expression in primates (including human) suggests that, in the midbrain, VGLUT2 expression is highest in the VTA region, especially within the parabrachial pigmented nucleus (PBP) (Root et al., 2016). Labelling of VGLUT2 in the primate SNc is very sparse and dual-positive TH/VGLUT2 cells represent less than 1% of the SNc neuronal population (Root et al., 2016), though a recent study found that ~10% of SNc TH⁺ neurons are VGLUT2⁺ in postmortem human tissue from male subjects (Steinkellner et al., 2022). The population of putative glutamate co-releasing dopamine neurons is sexually dimorphic, such that females have greater co-labelling of TH and VGLUT2 than males as seen in flies, rodents, and humans (Buck, Steinkellner, et al., 2021). A map of the projection targets of dopamine neurons found that the VGLUT2⁺ cells of the pars lateralis preferentially project to the caudal tail of the striatum and sparsely innervate the dorsolateral striatum (DLS) (Poulin et al., 2018). VGLUT2⁺ dopamine neurons of the VTA strongly project to the medial NAc shell and olfactory tubercle. It is important to note the potential caveat that defining populations of cells based only upon transcriptional levels may not represent the true strength of synaptic inputs. However, for glutamate co-release, the transcriptional projection patterns have been found to correspond to observed locations and strength of co-release (Chuhma et al., 2023).

The subcellular localization of VGLUT2 in relation to the vesicular monoamine transporter (VMAT) is a subject of debate. There exist four possible modes of expression: (a) VMAT and VGLUT2 mostly segregate to separate presynaptic terminals (i.e. separate dopamine and glutamate release sites), (b) VMAT and VGLUT2 have overlapping expression (i.e. shared dopamine and glutamate release sites), (c) VMAT and VGLUT2 overlap in location but at different expression levels at different release sites, resulting in a mixed model, or (d) VMAT and VGLUT2 are located on the same synaptic vesicle. High-resolution imaging studies assessing the localization of glutamatergic and dopaminergic terminals using various markers has suggested that the two transmitters largely localize to separate release sites within the striatum, though a small proportion of sites show markers for both (Fortin et al., 2019; Onoa et al., 2010; Zhang et al., 2015). Various aspects of vesicular release including kinetics, probability, and Ca²⁺-coupling also differ for dopamine and glutamate release from dopamine neurons, further providing evidence for primarily separate vesicle populations (Silm et al., 2019), though a comparison of optogenetic activation of dopamine and glutamate co-release found similar release properties (Adrover et al., 2014). Conversely, VMAT2 and VGLUT2 co-immunoprecipitate when isolated from ventral striatal vesicles and disruption of VGLUT2 impairs dopamine levels in vesicles (Hnasko et al., 2010). As such it has been hypothesized that the expression level of VGLUT2 at potential sites of co-release may be lower than that at traditional glutamate-only release sites, possibly below the threshold level of detection (Fortin et al., 2019). Thus, likely in vivo there is some diversity in the population of dopamine vesicles with some containing glutamate and/or located spatially near other glutamatergic vesicles.

VGLUT2 expression in dopamine neurons also decreases with age (Bérubé-Carrière et al., 2009), suggesting it may be especially important in early brain development. Indeed, conditional knockout of VGLUT2 in dopamine neurons (VGLUT2-cKO) leads to impaired growth and survival of dopamine neurons and decreased striatal dopamine release (Fortin et al., 2012). However, the expression level of VGLUT2 in dopamine neurons is dynamic and lineage mapping indicates that nearly all dopamine neurons have the capacity for VGLUT2⁺ expression (Dal Bo et al., 2004; Kouwenhoven et al., 2020; Steinkellner et al., 2018). This finding has been supported in a developmental fluorescent in situ hybridization study, which similarly found that the vast majority of cells that came to express Th first expressed Vglut2 (Dumas & Wallén-Mackenzie, 2019). While dual expression of VGLUT2 and TH across the midbrain has been reported to be low in healthy adult brains, especially in primate SNc (Root et al., 2016), this low level of expression may not be representative of the levels in parkinsonian brains. Indeed, increased expression has been observed in response to neuronal injury in rodent PD models (Bérubé-Carrière et al., 2009; Buck, De Miranda, et al., 2021; Kouwenhoven et al., 2020; H. Shen et al., 2018; Steinkellner et al., 2018, 2022). Such changes in expression have led to the hypothesis that VGLUT2 expression may be neuroprotective under disease states such as PD, described further in the section on “Selective Vulnerability” found below.

In addition to proposed roles in neuronal development and survival, co-release of glutamate is thought to allow for increased temporal control of dopamine neurons in responding to stimuli, as dopamine exerts relatively slow synaptic modulation (Lapish et al., 2007). The behavioral outcomes of such a function have been assessed by examining behaviors associated with striatal dopamine release in healthy adult brains from VGLUT2-cKO mice. These mice have normal basal motor coordination (though one study suggests reduced locomotion and rotarod performance (Fortin et al., 2012)), but display altered risk-taking behavior, decreased psychostimulant-induced locomotion, and increased drug seeking (Alsiö et al., 2011; Birgner et al., 2010; Hnasko et al., 2010). An alternate approach of conditionally disrupting dopamine release from VGLUT2⁺ dopamine neurons in order to determine the function of the cells when only glutamate co-release is maintained, demonstrates that glutamate is sufficient to induce positive reinforcement of behavior (Zell et al., 2020) suggesting that disruptions in co-release could play an important role in the cognitive components of PD in addition to motor impairments. Together, these results suggest that glutamate co-release is an essential aspect of the normal functioning of the dopamine pathways in addition to its role in early development.

2.2. GABA Co-Release

GABA co-release is a common occurrence in the CNS and has been observed in many different neuronal types, including midbrain dopamine neurons (for review see (Tritsch et al., 2016)). GABA co-release has been characterized following optogenetic stimulation of striatal DA terminals in slices. Photoactivated dopamine terminals inhibit the firing of MSNs through the activation of GABA_A receptors (Tritsch et al., 2012). Unlike glutamate co-release, GABA co-release does not show mediolateral heterogeneity and the associated GABA_A-mediated IPSCs can be measured across all subregions of the striatum. Though the proportion of DA neurons that are able to co-release GABA is unknown, it is thought to be a majority as ~90% of mouse midbrain neurons express the membrane GABA transporter (mGAT), allowing them to uptake GABA for release (Tritsch et al., 2014). The connectivity of GABAergic synapses of dopamine neurons is cell-type specific, such that large GABA_A-mediated IPSCs can be measured from MSNs and ChIs while fast-spiking interneurons exhibit much weaker responses (Chuhma et al., 2023).

Dopamine neurons express a non-canonical mechanism for the synthesis and expression of GABA. The GABA synthetic enzyme glutamate decarboxylase (GAD) is not highly expressed in dopamine neurons, suggesting that they rely on a different synthetic pathway and/or extracellular uptake. Evidence for both alternatives exist, as GABA co-release is dependent on both uptake through mGAT and synthesis through aldehyde dehydrogenase (ALDH) (Kim et al., 2015; Tritsch et al., 2014); though a subsequent study has suggested that co-released GABA relies solely on uptake and not de novo synthesis through the ALDH pathway (Melani & Tritsch, 2022).

In addition to atypical GABA synthesis, dopamine neurons also display differential vesicular loading of GABA. While typical GABAergic neurons rely on the vesicular GABA transport (VGAT) to package GABA into vesicles, GABA release from dopamine neurons is maintained in VGAT KO mice (Tritsch et al., 2012). Intriguingly, it was shown that vesicular loading of GABA within dopamine neuron terminals instead depends on the vesicular monoamine transporter (VMAT2) (Tritsch et al., 2012, 2014). Such studies suggest that GABA and dopamine may be loaded into the same vesicles, as they use the same transporter, though the evidence for such a model has only been indirectly measured through electrophysiology and no studies have as of yet demonstrated the chemically identified of the released transmitter to be GABA. Imaging studies have provided further evidence that GABA and VMAT2 colocalize within the same terminals in TH+ neurons (Stensrud et al., 2014).

Despite both GABA and dopamine likely being in the same vesicle, a recent study suggested that dopamine-GABA co-transmission from SNc neurons is independently modulated (Zych & Ford, 2022). By simultaneously measuring D2- and GABA_A-mediated postsynaptic events in D2-MSNs in the striatum following optogenetic activation of dopamine terminals it was found that the release of the two transmitters display different sensitivities to calcium and presynaptic modulation through D2-autoreceptors or kappa opioid receptors. GABA and dopamine transmission were also found to differ in their probability of release and dependence on RIM proteins, which are an integral part of release machinery at dopamine release sites (Liu et al., 2018). These differences may result from differential vesicular packaging, however, given that there is evidence of GABA and dopamine being packaged into the same vesicles, there is likely also a role for separate postsynaptic targets that transmit the GABA and/or dopamine signals. These postsynaptic specialties may also differentially interact with presynaptic release sites to impact vesicular dynamics such as the release machinery and active zone vesicular sorting. The possibility that the release of GABA and dopamine may be independently regulated raises the possibility that differences in the dynamics of co-transmission may be differentially altered in PD.

3. CO-RELEASE IN PARKINSON’S DISEASE

Pathologically, PD is characterized by a degeneration of dopamine neurons that is accompanied by the formation of Lewy bodies, which are aggregates whose main molecular component is the synaptic protein alpha-synuclein (α-synuclein) (Sharma & Burré, 2023; Sulzer & Edwards, 2019). In the presymptomatic prodromal phase of the disease, dopamine release is thought to diminish before gross degeneration is observed. In rodent models, this decrease in transmitter release can also be induced by α-synuclein overexpression (Janezic et al., 2013; Lundblad et al., 2012). Importantly, the effects of PD development are not uniform across the midbrain and specific dopamine subpopulations are more or less vulnerable to parkinsonian insult (Buck, De Miranda, et al., 2021; Gaertner et al., 2022; Kashani et al., 2007; H. Shen et al., 2018; Steinkellner et al., 2018). The following sections will discuss how the disease-induced changes in dopamine neuron health and survival may impact the co-release of glutamate and GABA, as well as how altered co-release relates to PD etiology.

3.1. Selective Resiliency of VGLUT2+ dopamine Neurons

While a significant fraction of dopamine neurons are lost in PD, a subpopulation is resilient to parkinsonian degeneration (Gaertner et al., 2022). Because dopamine neurons of the midbrain represent a heterogeneous population (Poulin et al., 2014), many studies have investigated whether there are cellular markers that confer a level of resiliency to neurons in PD. VGLUT2 is one hypothesized marker, as studies have suggested that VGLUT2 expression may be neuroprotective (Buck, Steinkellner, et al., 2021; H. Shen et al., 2018). Consistent with this role, VGLUT2 expression is high in the VTA, which is largely spared in PD, and PD is associated with a proportional increase in the number of SNc TH⁺ neurons that are VGLUT2⁺. This increase is observed in post-mortem human brain tissue (Kashani et al., 2007; Steinkellner et al., 2022) and across multiple mouse models of PD, including 6-OHDA lesion (Dal Bo et al., 2008; Steinkellner et al., 2022), rotenone induced degeneration (Buck, Steinkellner, et al., 2021), and injection of pre-formed fibrils (Steinkellner et al., 2022).

The mechanism underlying the increase in VGLUT2⁺ dopamine neurons can be attributed to either a decrease in vulnerability of these cells to parkinsonian degeneration or to a dynamic increase in the expression of VGLUT2 (Kouwenhoven et al., 2020; Steinkellner et al., 2022). While the majority of adult SNc dopamine neurons lack VGLUT2 expression (Bérubé-Carrière et al., 2009; Dal Bo et al., 2004), cell-fate mapping and in situ hybridization suggests that most embryonic dopamine neurons express VGLUT2 (Dumas & Wallén-Mackenzie, 2019; Fougère et al., 2021; Kouwenhoven et al., 2020; Steinkellner et al., 2018). This expression profile suggests that many adult dopamine neurons have the capacity to express VGLUT2, which may be induced under certain conditions such as parkinsonian insult. Interestingly, cultured midbrain dopamine neurons display varied levels of VMAT and VGLUT2 depending on the identity of cells they are co-cultured with (Fortin et al., 2019) and it has been hypothesized that the microenvironment surrounding dopamine terminals impacts the expression of vesicular transporters. For example, VGLUT2 and VMAT expression are suppressed for dopamine neurons terminating in the dorsal and ventral striatum, respectively (Fortin et al., 2019). The idea that VGLUT2 expression dynamically shifts in PD was supported in a recent rodent model using A53T mutant human α-synuclein. In these animals, the fractional expression of VGLUT2⁺ TH neurons increased without a concomitant decrease in total cell numbers (Steinkellner et al., 2022), suggesting cells were reprogramming before the onset of gross degeneration. Thus, putative glutamate co-releasing neurons are conferred a level of resiliency and neuroprotection associated with their expression of VGLUT2.

While it is clear that VGLUT2 expression is correlated with cellular survival, the mechanism of neuroprotection and the behavioral consequences of increased expression are not understood. VGLUT2 expression is associated with neuronal development and has been shown to increase axonal outgrowth and promote reinnervation after injury (Bérubé-Carrière et al., 2009; Kouwenhoven et al., 2020). It also is hypothesized to maintain dopamine synapses. Supporting this hypothesis, VGLUT2-cKO mice have both reduced dopamine release and altered expression of dopamine receptors (Alsiö et al., 2011; Fortin et al., 2012). Interestingly, VGLUT2-cKO mice have normal basal motor function, but display augmented motor dysfunction after MPTP exposure (H. Shen et al., 2018). These results suggest that the role of VGLUT2 in maintaining motor behavior is induced after parkinsonian insult, but not required in healthy adult brains. In addition to maintaining dopamine synapses, VGLUT2 may directly mediate dopamine release by influencing vesicular loading of dopamine. VGLUT2 promotes acidification of synaptic vesicles, which increases the packaging of dopamine through a process known as “vesicular synergy” (Aguilar et al., 2017; El Mestikawy et al., 2011; Hnasko & Edwards, 2012). Therefore, the increased expression of VGLUT2 in PD may promote motor function by maintaining dopamine synapses, promoting regrowth, and enhancing dopamine release by increasing its vesicular loading. Whether changes in the expression of VGLUT2 also result in altered glutamatergic co-release (e.g. increased proportion of neurons able to co-release, increased synaptic co-release sites, etc.) and whether these changes are neuroprotective is not known, though evidence for altered synaptic activity of glutamate is described below.

Together, studies in consideration of neuroprotective properties suggest that VGLUT2 is a promising marker of resilience. This resilience may be related to maintaining dopamine synapses and promoting vesicular loading of dopamine, though other mechanisms are possible. However, the increase in VGLUT2 expression within dopamine neurons as PD progresses is expected to maintain and possibly increase co-release as the disease progresses. This may result in changes in glutamatergic co-release, which are explored further below. Conversely, the vulnerability of GABA co-releasing dopamine neurons is unknown.

3.2. Selective accumulation of α-synuclein at release sites

Another major hallmark of PD is the formation of Lewy bodies, which consist of prion-like aggregates of the synaptic protein α-synuclein in dopamine neurons. These aggregates are hypothesized to be a causative agent of PD, as α-synuclein mutation or increased copy number are associated with familial PD (Nalls et al., 2019; Singleton et al., 2003) and, in parallel, some rodent models of PD are achieved using these human genetic mutations, protein overexpression, or injection of preformed fibrils (PFFs) (Simons & Fleming, 2023). Clusters of α-synuclein can be observed in dopamine cell bodies as well as in terminals in the striatum. The latter locus of accumulation may induce damage, as the presence of α-synuclein at presynaptic sites is associated with impaired neurotransmitter release and altered expression of vesicular release machinery (Nemani et al., 2010; Volpicelli-Daley et al., 2011). However, recent studies have suggested that α-synuclein aggregates form more readily at specific synapse types. In particular, VGLUT1⁺ terminals in the basolateral amygdala displayed an increase in aggregates as compared to VGLUT2⁺ terminals in the thalamus (Chen et al., 2022). Importantly, this selective aggregation was correlated with selective effects on glutamatergic transmission. This study suggests that VGLUT2⁺ terminals may be protected from accumulation of α-synuclein, though such a hypothesis has not been tested across brain areas and, in particular, is not known for VGLUT2⁺ sites of co-release from dopamine neurons. Because α-synuclein regulates synaptic transmission, a decrease in susceptibility to α-synuclein accumulation may contribute to the ability of VGLUT2⁺ to promote the maintenance of synapses, providing another level of neuroprotection. Thus, it is important to investigate the pattern of α-synuclein accumulation at different types of synaptic specializations (including sites of GABA co-release in addition to VGLUT2⁺ terminals), as this aspect of PD development may allow for differential effects on neurotransmitter release.

3.3. PD-induced changes in synaptic function

In the prodromal phase of PD, the function of dopamine neurons begins to diminish before significant degeneration or overt motor symptoms are observed. This stage of disease development is marked by changes in intrinsic activity and synaptic function (Shen et al., 2022), most notably the reduction in dopamine release. Though some of the major motor symptoms are explained by this loss of dopamine, as evidenced by the efficacy of dopamine replacement therapies such as L-DOPA, there is evidence that the full disease etiology is impacted by other transmitter systems. In order to develop a more complete understanding of how these synaptic changes manifest PD symptoms and progression, it is essential to investigate the direct impact on glutamate and GABA co-release as well as the overall balance in signaling through different neurotransmitter systems within the striatum.

3.3.1. Striatal glutamate.

Glutamate is the primary excitatory neurotransmitter in the nervous system and the striatum receives glutamate input from multiple sources. The primary glutamate afferents to the striatum originate in the cortex, thalamus, and subthalamic nucleus. Along with co-released glutamate, these inputs form an architecture of glutamatergic synapses throughout the striatum. PD progression is linked to glutamatergic synaptic changes in general, and the potential for these to include changes in co-released glutamate specifically remain to be determined (for review see (Bonsi et al., 2005)). For example, dopaminergic denervation as a result of 6-OHDA lesion in rodents is associated with increased striatal glutamate release, increased spontaneous AMPAR-mediated currents in striatal neurons, changes in glutamatergic synapse density, altered subunit expression of NMDARs, increased expression of mGluR2/3 within the striatum, and dampened glutamatergic long term potentiation (Calabresi et al., 1993; Dunah et al., 2000; Gardoni et al., 2006; Ingham et al., 1998; Lindefors & Ungerstedt, 1990; Meshul et al., 1999; Picconi et al., 2002, 2003). A recent study also found a change in spontaneous EPSCs recorded from MSNs after treatment with α-synuclein pre-formed fibrils, which was rescued by treatment with L-DOPA (Tozzi et al., 2021). Importantly, some of these changes in glutamatergic synaptic function can be attributed to alterations in glutamatergic afferents as studies have demonstrated that parkinsonian models induce specific changes in corticostriatal and thalamostriatal circuitry; cortical inputs onto iMSNs are pruned and the strength of thalamic inputs onto iMSNs is enhanced (Fieblinger et al., 2014; Tanimura et al., 2019). However, future studies are required to determine what (if any) changes occur at co-release sites, especially with respect to presynaptic release properties and postsynaptic receptor distribution and composition. This potential for changes in co-released glutamate in PD is purely speculative as few studies have addressed such a role.

3.3.2. Striatal GABA.

In opposition to glutamate, GABA is the primary inhibitory neurotransmitter in the nervous system. The local interneuron population and MSN collaterals are major sources of GABA within the striatum (Boccalaro et al., 2019; Wilson, 2007), in addition to GABA co-released from dopamine neurons (Tritsch et al., 2012, 2014). There is also some evidence for the regulation of GABAergic signaling by ChIs, both through GABA co-release from ChIs themselves (Lozovaya et al., 2018) and through disynaptic activation of GABA release from interneurons (English et al., 2011) and dopamine terminals (Nelson et al., 2014). Dysfunction of GABAergic neurotransmission is associated with the development of PD (Emir et al., 2012; Kish et al., 1986; O’Gorman Tuura et al., 2018), including in non-motor symptoms (Murueta-Goyena et al., 2019) and rodent models of PD display altered GABAergic transmission (Gittis & Kreitzer, 2012). For example, 6-OHDA lesion induces remodeling of striatal circuitry, increasing the connectivity between fast-spiking interneurons and indirect-pathway MSNs (iMSNs) (Gittis et al., 2011) and decreasing the connectivity of MSN collaterals (Taverna et al., 2008). In consideration of such altered connectivity resulting from dopamine depletion, it is unsurprising that such treatment also impacts both the frequency and kinetics of GABA receptor-mediated miniature IPSCs recorded from MSNs (Boccalaro et al., 2020; Dehorter et al., 2009). Though these effects have been attributed to changes in the striatal interneurons and MSN collaterals, GABA co-release from dopamine neurons has not been considered. Interestingly a mouse model of α-synuclein overexpression (OVX mice), displays augmented GABAergic transmission in the striatum (Roberts et al., 2020) which may be due to a downregulation of mGAT leading to enhanced GABAergic tone and thus enhanced tonic inhibition of SNc terminals via presynaptic GABAergic receptors (Kramer et al., 2020; Lopes et al., 2019). However, because co-released GABA from dopamine neurons is specifically reduced in OVX mice, this effect likely results from other sources of GABA and it remains unknown if the reduction in GABA co-release contributes to feedback inhibition of dopamine release (see review from (Roberts et al., 2021)). As described for changes in glutamate function, the above changes in GABAergic function in PD have not been attributed to co-release and therefore that form of transmission specifically would benefit from further exploration.

3.3.3. Excitatory/Inhibitory (E/I) Balance.

Throughout the nervous system, the balance between excitatory and inhibitory synaptic strength is essential for normal neuronal function. Imbalances in glutamate- and GABA-mediated neuronal modulation are associated with several neurological disorders (for review see (Sohal & Rubenstein, 2019)). Because PD is marked by changes in glutamate and GABA signaling (as described above), such an imbalance is likely to occur. Additionally, impairments of glutamatergic plasticity have been observed within the striatum in PD models (Durante et al., 2019; Kurz et al., 2010; Paillé et al., 2010; Tozzi et al., 2012, 2016), further impacting the ability of the striatum to dynamically respond to changes in excitatory or inhibitory drive. Thus, future studies should consider how various inputs are integrated onto different striatal cell types in order to regulate their excitability and downstream signaling. As the striatum receives both excitatory and inhibitory inputs in response to the activation of dopamine neurons, it is important to consider the strength, density, and connectivity of those synapses in the context of driving striatal pathways.

3.3.4. Dopamine modulation and feedback.

Dopamine transmits signals through GPCRs, resulting in a modulatory influence over neuronal activity (Gerfen & Surmeier, 2011; Kreitzer & Malenka, 2008). This function allows it to regulate cellular excitability and impact the net effect of fast excitatory and inhibitory transmission. MSNs within the striatum can be defined by their expression of dopamine receptor isoforms, with direct pathway MSNs (dMSNs) expressing D1-like G_s/olf-coupled receptors (D₁Rs) and indirect pathway MSNs (iMSNs) expressing D2-like G_i/o-coupled receptors (D₂Rs). As these two pathways are thought to bidirectionally facilitate or suppress movement, the loss of dopamine in PD likely drives imbalances between these two pathways that result in a net suppression of movement. D₁Rs and D₂Rs also influence glutamatergic synaptic plasticity in opposing ways, allowing them to have a greater overall effect on MSN activity (Lovinger, 2010). MSNs express LTP and LTD through D₁R- and D₂Rdependent mechanisms, respectively. Therefore, as dopamine release diminishes, the capacity for plasticity mechanisms to occur is also diminished. Indeed, rodent models of PD are deficient for LTP. It is unknown how such changes in glutamatergic modulation may impact specifically sites of co-release.

3.3.5. Dopamine/Acetylcholine (ACh) balance.

One of the major cell types within the striatum is the ACh-releasing cholinergic interneuron (ChI). Though ChIs represent only ~1–2% of the striatal neuron population, they exert a large influence over striatal activity through their widespread and dense arborizations (Gonzales & Smith, 2015). The motor dysfunction observed in PD has been attributed to changes in MSN activity that result not solely from the loss of dopaminergic input but also possibly from a change in the balance between dopamine and ACh (Ztaou & Amalric, 2019). ChIs and dopamine neurons have reciprocal connectivity such that synchronous firing of ChIs activates presynaptic nicotinic ACh receptors to induce dopamine release. In turn, dopamine pauses ChI firing through the activation of postsynaptic D2 receptors (Cai & Ford, 2018; Chuhma et al., 2014; Threlfell et al., 2012). However, dopamine neurons that co-release glutamate proffer an additional level of regulation through activation of postsynaptic glutamate receptors expressed on ChIs. There is a regional heterogeneity in this regulation that correlates with the regional heterogeneity in co-release sites. In the ventral striatum where co-release is the highest, activation of dopamine terminals initiates a burst in firing in ChIs that is mediated through AMPA and NMDA receptors (Chuhma et al., 2014). In the dorsal striatum, where co-release of glutamate is limited to the dorsal subregion, activation of dopamine terminals likewise initiates a burst in firing of ChIs, though it is instead mediated by mGluRs (Cai & Ford, 2018; Chuhma et al., 2014; Straub et al., 2014). DMS ChIs do not exhibit a glutamate-mediated response to this stimulation. Because glutamate co-release is able to drive ChI activity, changes in co-release in PD may therefore contribute to the changes in dopamine/ACh balance.

Interestingly, a recent study found that the glutamate-dependent burst in ChI firing activity within the DLS is altered in a mouse model of PD (Cai et al., 2021). Specifically, after a 6-OHDA lesion, DLS ChIs no longer exhibited mGluR-mediated burst firing in response to optogenetic activation of dopamine terminals. This loss was attributed to changes in mGluR1 expression within ChIs, rather than changes in co-release, and both burst firing and normal motor function could be rescued by increased exogenous expression of mGluR1. Notably, the alterations of glutamatergic synaptic activity were specific to co-released dopamine and no changes in ChIs were observed at ionotropic glutamatergic synapses originating from cortex or thalamus (Aceves Buendia et al., 2019; Cai et al., 2021). Together, these results suggest that the induction of adaptive changes in the expression of mGluRs targeted by co-transmitted glutamate occur in response to neuronal injury and that these changes may underlie some of the motor dysfunction that is characteristic of PD.

4. CONCLUSION

PD is a debilitating neurological disorder affecting many individuals every year. It primarily impacts dopamine neurons of the midbrain that project to the striatum, which represent a heterogeneous population of cells that variably release dopamine, glutamate, and GABA. In order to fully understand the impacts of SNc neuron degeneration, it is important to consider all of these modes of transmission.

At all levels of PD development, there is evidence suggesting that co-release of glutamate and/or GABA are affected (Figure 1). As dopamine neurons become unhealthy, it is widely accepted that dopamine release decreases (even before motor symptoms emerge). Because there is evidence that co-release of glutamate and GABA are independently regulated, it is unclear if the signaling of dopamine neurons through those pathways is similarly impacted, and, if so, if changes occur along the same timeframe. Additionally, alterations in postsynaptic receptor composition at GABA and glutamate synapses have been observed in PD models, suggesting a dynamic shift in the signaling through those pathways.

Fig. 1.

Potential alterations in co-release in Parkinson’s disease.

In addition to possible changes in the release probability, synaptic strength, and density at co-release sites, there is evidence for resilience of glutamate co-releasing synapses and neurons in the face of dopamine degeneration. Specifically, VGLUT2 is upregulated in surviving neurons in parkinsonian brains and there is evidence that α-synuclein accumulation may be less prevalent at VGLUT2⁺ terminals. Overall, these results may indicate that glutamate co-release is protective and/or that glutamatergic drive is enhanced in PD.

Future studies are required to determine how these various factors come together to alter the balance of neurotransmitter systems in the striatum. Understanding the impacts of changes in co-release, along with changes in dopamine signaling, may help elucidate the synaptic and circuit alterations that drive the motor and cognitive deficits in PD. Addressing outstanding questions such as how co-release may be differentially regulated compared to canonical dopamine release, if deficits in co-release contribute to PD symptomology and, if co-release can be leveraged to slow or alleviate disease progression will likely lead to new understanding of the complexity of the disease and avenues for improved therapeutics.