An emerging role for synaptic Zn²⁺ in substance use disorders

Abstract

Synaptic zinc (Zn²⁺) modulates dopamine and glutamate neurotransmission by binding to the dopamine transporter and glutamate receptors. Among other neurotransmitters, dopamine and glutamate critically regulate physiological processes and behaviors relevant to substance use disorders (SUDs) and addiction. In addition, Zn²⁺ interacts with inhibitory neurotransmitter systems, including GABA and glycine receptors, further influencing the excitatory-inhibitory balance within circuits relevant to addiction. Nevertheless, the specific involvement of synaptic Zn²⁺ in such processes is unknown. We propose that synaptic Zn²⁺ serves as an environmentally derived factor that can influence the vulnerability to and development of SUDs and addiction via its interaction with proteins that regulate dopamine and glutamate neurotransmission in addiction-relevant brain circuits.

1. Introduction

Zinc (Zn²⁺) is an essential trace element required by all organisms to perform diverse biological functions (Kambe et al., 2021). In fact, Zn²⁺ is the second most abundant divalent cation after calcium and is a structural component of hundreds of enzymes and proteins. As such, Zn²⁺ plays a critical role in hundreds of biological processes required for proper cellular function-- including DNA replication, transcription, protein synthesis, maintenance of cell membranes, and cellular transport across endocrine, immunological, and neuronal systems (Benarroch, 2023; Kambe et al., 2015). Loss of Zn²⁺ homeostasis is associated with immunodeficiencies, abnormal growth, as well as neurodegenerative diseases such as Alzheimer’s disease or amyotrophic lateral sclerosis (Doroszkiewicz et al., 2023; Frederickson et al., 2005; Maywald et al., 2017).

Zn²⁺ is distributed in the brain at high concentrations, and a disturbance in brain Zn²⁺ is specifically associated with neuropsychiatric or neurological manifestations that can present as altered behavior and cognition, reduced ability to learn, and depression (Anbari- Nogyni et al., 2020; Doboszewska et al., 2017; Prakash et al., 2015; Sensi et al., 2011). Approximately 10% of brain Zn²⁺ is in a histochemically-reactive chelatable pool (i.e., not bound to proteins), with highest concentrations in the cerebral cortex, hippocampus and amygdala (Frederickson et al., 2000; Shen et al., 2007). This pool of chelatable Zn²⁺ is often referred to as “vesicular” or “synaptic” Zn²⁺ since it is packaged in synaptic vesicles (Cole et al., 1999; Palmiter et al., 1996).

A growing body of evidence suggests that Zn²⁺ plays a key role in synaptic transmission and serves as an endogenous neuromodulator (Kay & Tóth, 2008). Synaptic Zn²⁺ is packaged within synaptic vesicles at glutamatergic terminal boutons by the activity of the Zn²⁺ transporter 3 (ZnT3) (Palmiter et al., 1996) and subsequently co-released into the synaptic cleft during neuronal activity following electrical or chemical stimulation (Assaf & Chung, 1984; Cole et al., 1999; Howell et al., 1984; Kalappa et al., 2015; Wu et al., 2023). In addition, studies reported Zn²⁺ release from GABAergic and glycinergic neurons (Birinyi et al., 2001; Kouvaros et al., 2020) indicating that synaptic Zn²⁺ may also be co-released from non-glutamatergic neurons. Although the exact concentration of synaptic Zn²⁺ that is released following neuronal depolarization has not been directly quantified in vivo, experimental estimates suggest that synaptic Zn²⁺ concentrations can range from ~10 nM to ~100 μM (Krall et al., 2021). Neurons that express ZnT3 are referred to as “zincergic” neurons. Once released into the synaptic cleft, Zn²⁺ can bind to a variety of ion channels or receptors (Nakashima & Dyck, 2009; Vergnano et al., 2014) or enter the postsynaptic cell where it can modulate second messenger systems. Zn²⁺ is not considered a classical neurotransmitter, the term zincergic is used here to describe neurons or projections that release synaptic Zn²⁺, which modulates different neurotransmitter systems such as glutamate, GABA, and dopamine. As such, synaptic Zn²⁺ is able to modulate neuronal function and plasticity (McAllister & Dyck, 2017).

Substance use disorder (SUD) is a chronic relapsing condition characterized by compulsive drug seeking despite negative consequences (Goldstein & Volkow, 2011; Harada et al., 2021; Lüscher et al., 2020) and is accompanied by molecular, synaptic, and structural adaptations in cortico-limbic-striatal circuitry that strengthen drug reinforcement and the transition to habitual drug taking (Nestler & Lüscher, 2019). In particular, extensive SUD-related research has shown that the dopaminergic and glutamatergic systems play a critical role in goal-directed learning and in mediating the reinforcing effects of drugs of abuse (Harada et al., 2021; Hyman et al., 2006; Vanderschuren & Kalivas, 2000). At the center of this lies the striatum, a key brain region involved in movement, motivation, drug reinforcement, learning, and decision-making (Averbeck & O’Doherty, 2022; Cox & Witten, 2019; Schultz, 2007; Smith et al., 2021; Suzuki et al., 2021; Wilhelm et al., 2023). The striatum receives dopaminergic input from the midbrain and extensive glutamatergic inputs from the cortex and limbic regions including the thalamus, hippocampus, and amygdala. The striatum is composed of dorsal (caudate putamen) and ventral (nucleus accumbens) regions (Voorn et al., 2004). Histochemical studies have shown that the distribution of synaptic Zn²⁺ within these subdivisions is heterogeneous and compartmentalized (Mengual et al., 1995). Patch-like and peripheral zones rich in vesicular Zn²⁺ have been observed in both dorsal and ventral striatum, suggesting distinct region-specific Zn²⁺ signaling pathways (Mengual et al., 1995: Sørensen et al., 1995).

Medium spiny neurons (MSNs), also known as spiny projection neurons, account for approximately 95% of all neuronal types in the striatum and are broadly segregated into two pathways (Gerfen et al., 1990), commonly referred to as the “direct” and “indirect” pathways, which express the dopamine D1 receptor (D1-MSNs, “direct”) or the dopamine D2 receptor (D2-MSNs, “indirect”) (Gerfen et al., 1990). Canonically, in the dorsal striatum, D1-MSNs facilitate locomotion, whereas D2-MSNs suppress movement (Kravitz et al., 2010). The nucleus accumbens (NAcc) contains D1-MSNs and D2-MSNs which are believed to promote rewarding and aversive behaviors, respectively (Lobo et al., 2010). However, recent studies have shown that D1- and D2-MSNs can display concurrent activation during reward-related behaviors and are therefore believed to play synergistic rather than opposing roles in movement and decision-making processes (Cui et al., 2013; Natsubori et al., 2017; Soares-Cunha et al., 2016).

Glutamatergic terminals projecting to the striatum innervate D1- and D2-MSNs, forming asymmetrical synapses onto their spines, where dopamine is also released from dopaminergic neurons innervating this region (Doig et al., 2010). Thus, glutamate can influence the activity of MSNs dependent on the extent of dopamine release (Smith et al., 1994). Studies have demonstrated that different drugs of abuse induce an increase in dopamine in the striatum (Boileau et al., 2003; Brody et al., 2009; Drevets et al., 2001; Volkow et al., 1999).

Drugs of abuse also affect glutamate transmission, which plays an important role in SUDs (Márquez et al., 2017). Chronic drug use induces neuroadaptations in corticostriatal projections, leading to maladaptive behaviors and deficits in inhibitory behavioral control (Spencer & Kalivas, 2017). For example, psychostimulants such as cocaine and amphetamines enhance glutamate release and cause long-term potentiation, that is associated with drug-seeking (Scofield et al., 2016; Siemsen et al., 2020). Alcohol and other drugs of abuse upregulate glutamate receptors, including the N-methyl-D-aspartate (NMDA) receptor, and alter the function of metabotropic glutamate receptors, contributing to the neuroadaptive changes underlying addiction (Christian et al., 2012; Krystal et al., 2003; Ma et al., 2017).

In addition to increasing dopamine and glutamate neurotransmission (Bonaventura et al., 2017; Jones et al., 1995; Schmidt & Pierce, 2010; Venton et al., 2006), we recently showed that chronic cocaine also increases synaptic Zn²⁺ in the striatum (Gomez et al., 2021). However, the precise role of synaptically-released Zn²⁺ in the striatum, either in the context of cocaine exposure, or under normal conditions, is not well understood.

The role of Zn²⁺ in normal brain function and brain disorders has been reviewed elsewhere (Bitanihirwe & Cunningham, 2009; Frederickson et al., 2005; Krall et al., 2021; McAllister & Dyck, 2017; Sensi et al., 2011) but a role for Zn²⁺ with respect to the SUD neurobiology and neurocircuitry is understudied. Therefore, the goal of this review is to increase the awareness and appreciation of Zn²⁺ with respect to its potential involvement in SUDs.

2. Zn²⁺ transport and synaptic Zn²⁺ release

2.1. Zn²⁺ transport & release

Cells control their zinc homeostasis by utilizing two Zn²⁺ transporter families: the Zn²⁺ transporter (ZnT) family, and the Zn²⁺- and iron-regulated transporter-like protein (ZIP) family. The ZnT family consists of 9 transporters (Kambe et al., 2021; Yin et al., 2023), while the ZIP family consists of 14 transporters that can be broken into 4 subfamilies (Dempski, 2012; Hara et al., 2022). The ZnT and ZIP families have contrasting roles in maintaining Zn²⁺ homeostasis. ZnTs are efflux transporters that decrease cytosolic Zn²⁺ by transporting it inside organelles or shuttling it out of the cell (Huang & Tepaamorndech, 2013). In contrast, the ZIPs are influx transporters that increase cytosolic Zn²⁺ (Jeong & Eide, 2013). Together, these two families are key in maintaining cytosolic Zn²⁺ concentration and proper cellular function (Devirgiliis et al., 2007).

ZnT3 is responsible for packaging Zn2+ into synaptic vesicles of zincergic neurons (Linkous et al., 2008), which, during neurotransmission, release into the synaptic cleft, effectively rendering Zn2+ to function as a neuromodulator (McAllister & Dyck, 2017). ZnT3 is also particularly relevant to SUD neurobiology due to its localization in brain regions implicated in SUDs. ZnT3 mRNA is abundantly expressed in reward-related regions such as the prefrontal cortex (PFC), hippocampus, paraventricular thalamus (PVT), and amygdala (Kouvaros et al., 2023; Palmiter et al., 1996). As mentioned above, ZnT3 is expressed in synaptic vesicles of glutamatergic/zincergic neurons, although recent studies show that ZnT3 can also be expressed in GABAergic interneurons (Kouvaros et al., 2023; Paul et al., 2017). The exact mechanisms by which ZnT3 expression is regulated are still unknown. However, previous studies demonstrated that there are several factors which can influence ZnT3 expression, including the Zn2+ concentration itself, transcription factors, cell signaling, and environmental factors (Grabrucker et al., 2014; Salazar et al., 2004). In addition, previous research suggests that ZnT3 expression could be altered by exposure to drugs of abuse, such as cocaine (Gomez et al., 2021) which further reinforces the potential interaction between ZnT3 expression, function, and SUD neurobiology.

2.2. Synaptic Zn²⁺ clearance

Synaptic Zn²⁺ clearance is a crucial biological process that regulates postsynaptic activity and maintains proper synaptic function (Narayanan et al., 2020). The exact mechanisms that participate in the clearance of Zn²⁺ are not well understood. However, there are at least three mechanisms that could mediate this clearance: (i) Zn²⁺ uptake by Zn²⁺ transporters in presynaptic, postsynaptic or glial cells (Colvin et al., 2010; Hara et al., 2017; Hojyo & Fukada, 2016; Richard D Palmiter & Huang, 2004), (ii) Zn²⁺ entry into the postsynaptic neuron through different ion channels (Vergnano et al., 2014; Vogt et al., 2000) and (iii) interactions with protein binding sites in the postsynaptic neuron (Bancila et al., 2004; Grabrucker et al., 2011).

Reciprocal to the actions of ZnT3, ZIPs buffer the levels of synaptic Zn²⁺ in the synaptic cleft. For example, ZIP1 and ZIP3 regulate Zn²⁺ homeostasis by importing synaptic Zn²⁺ into the postsynaptic neuron (Qian et al., 2011), while ZIP3 also mediates Zn²⁺ uptake into presynaptic neurons (Bogdanovic et al., 2022). Also, a recent study showed that ZIP4 is expressed in hippocampal excitatory synapses and transports Zn²⁺ into postsynaptic neurons. Therefore, ZIP4 may also play an important role in synaptic plasticity (De Benedictis et al., 2021). In addition to ZIPs, Zn²⁺ can enter the postsynaptic cell through voltage-dependent calcium channels, and NMDA/AMPA receptors, which leads to a reduction in the levels of extracellular Zn²⁺ (Hershfinkel et al., 2009; Marin et al., 2000; Sensi et al., 2011). Furthermore, once inside the cell, Zn²⁺ acts as a second messenger that can modulate the activity of several intracellular pathways and modulate cell function (Nakashima & Dyck, 2009). However, excessive intracellular Zn²⁺ can also lead to cell death and is implicated in neurological and neurodegenerative disorders (Krall et al., 2021; Portbury & Adlard, 2017). Finally, following Zn²⁺ release from synaptic vesicles, Zn²⁺ can bind with high affinity to ionotropic NMDA and AMPA receptors (Krall et al., 2021). Zn²⁺ can also bind to matrix metalloproteinases, which are involved in synaptic plasticity and cell degradation (Hwang et al., 2011; Yong et al., 2001). In sum, Zn²⁺ concentration in the synaptic cleft depends on the balance between these clearance mechanisms.

3. Synaptic Zn²⁺ accumulates in brain circuits dysregulated in SUDs

Glutamatergic innervation to the striatum arises from brain regions associated with reward and involved in SUDs, including the PFC, hippocampus, basolateral amygdala (BLA), and the PVT (Gass & Olive, 2008; Koob & Volkow, 2010; Lüscher et al., 2020; Manza et al., 2023; Volkow et al., 2019; Zinsmaier et al., 2022) (Figure 1). Such circuits play a key role in the development and modulation of motivation and goal-directed behaviors (Cruz et al., 2023; Haber & Knutson, 2010; Zinsmaier et al., 2022). The discrete regional distribution of glutamatergic neurons and their terminals render glutamate strategically situated to interact with different neurotransmitter systems like the dopaminergic, GABAergic, glycinergic, and opioid systems, which also play critical roles in SUDs.

Figure 1.

Schematic illustration depicting the major projections to the striatum. A) The striatum receives glutamatergic/zincergic inputs (red) from the cerebral cortex, amygdala, PVT and hippocampus, as well as dense dopaminergic inputs (blue) from the SNc/VTA. B) Glutamate and Zn²⁺ are coreleased from glutamatergic/zincergic neurons and act on AMPA-R and NMDA-R. However, Zn can bind and modulate dopaminergic transmission by binding DAT, D1R and D2R.

3.1. Striatum

Histochemical approaches using the Timm stain are used to detect the distribution of chemically reactive Zn²⁺ within synaptic vesicles (Timm, 1958). By using this technique, it was demonstrated that the striatum lacks zincergic cell bodies but receives a dense innervation of zincergic neurons from cortical and subcortical regions (Brown & Dyck, 2003; Frederickson et al., 2000; Sørensen et al., 1995). Moreover, the distribution of Zn²⁺-containing synaptic vesicles in the striatum is heterogeneous, showing a patchy striosome-matrix-like pattern (Mengual et al., 1995). In the dorsal striatum, there is higher Zn²⁺ content in the striosomes compared to the matrix. However, the Zn²⁺-rich patches do not always match with the expression of striosome markers like calbindin-D28K or acetylcholinesterase (Mengual et al., 1995). Interestingly, the NAcc receives a denser innervation of zincergic neurons than the dorsal striatum (Mengual et al., 1995). At the cellular level, these projections converge onto individual MSNs, often on the same segments of dendrites (Ding et al., 2008). While the precise effects of Zn²⁺ release onto MSNs are unknown. One previous study showed that increased dietary Zn²⁺ in mice alters cortico-striatal synaptic plasticity (Fourie et al., 2018).

3.2. Prefrontal Cortex

Previous research suggests that the PFC is associated with reward seeking and compulsive drug taking through the regulation of dopaminergic transmission in the striatum and limbic regions (Bachtell et al., 2008; Ceceli et al., 2022; Goldstein & Volkow, 2011; Harada et al., 2021; Sun & Rebec, 2006). For instance, chronic cocaine self-administration diminished basal PFC activity (Sun & Rebec, 2006). Consistent with these data, compulsive cocaine seeking in rats is associated with hypoactivity of the PFC (Chen et al., 2013) and similar findings of frontal cortical hypometabolism have been observed in chronic cocaine users (Volkow et al., 1992). Interestingly, optogenetic stimulation of the PFC decreases compulsive drug seeking behaviors (Chen et al., 2013). Altogether, these findings suggest a critical role of the PFC in cocaine as well as other SUDs (Goldstein & Volkow, 2002, 2011).

Histochemical studies showed that in the PFC, Zn²⁺-releasing glutamatergic neurons that project to the striatum are principally located between cortical layers 5 and 6, and fewer Zn²⁺-positive neurons are found in layers 2 and 3. These corticostriatal neurons are found ipsilateral and contralateral to the innervated striatum (Sørensen et al., 1995). Furthermore, the PFC neurons share reciprocal connections with different regions that contain Zn²⁺-releasing neurons, such as the BLA, the hippocampus and, the PVT (Christensen & Geneser, 1995; Sørensen et al., 1995). While these areas are involved in cognitive and emotional processing relevant to substance use disorders, the specific contribution of Zn²⁺-releasing projections to drug-related behaviors remains to be determined (Brown & Dyck, 2003). Further experimental studies are needed to establish whether synaptic Zn²⁺ modulates cortical circuits and drug seeking.

3.3. Basolateral Amygdala

Within the amygdala, the BLA is well known for its prominent role in aversion learning and fear conditioning. This region is also implicated in decision-making and in the facilitation of reward seeking and SUDs (Lee et al., 2024; Stuber et al., 2011; Wassum & Izquierdo, 2015). The BLA mediates the consolidation of both cocaine-stimulus associations and extinction learning on cue-induced cocaine-seeking behaviors (Fuchs et al., 2006). Specifically, the BLA projections to the NAcc are necessary for cue-induced reward seeking (Stuber et al., 2011). This is supported by optogenetic studies that showed that inactivation of the BLA-NAcc circuit reduced cue-induced cocaine seeking (Stefanik & Kalivas, 2013). Conversely, another study showed that optogenetic activation of the BLA-NAcc circuit reduced cue-induced alcohol seeking (Millan et al., 2017). Anatomical studies have identified glutamatergic neurons containing synaptic Zn Zn²⁺ in regions such as the BLA, PFC, hippocampus, and PVT (Christensen & Geneser, 1995; Sørensen et al., 1995) which are part of interconnected limbic circuits. The BLA sends dense glutamatergic projections to the NAcc, and the presence of synaptic Zn²⁺ in these terminals suggests that Zn²⁺ may modulate BLA-driven motivational and reward-related processes (Brown & Dyck, 2003). Future experiments need to test whether Zn²⁺ release within these pathways actively shapes behavioral responses to drugs of abuse.

3.4. Paraventricular Thalamus

The PVT is a subregion of the thalamus and is positioned in a strategic location where it integrates information it receives from different cortical and limbic regions (Zhou & Zhu, 2019). A growing body of evidence shows that PVT activity is associated with drug-seeking behavior. For instance, PVT function has been associated with different aspects of SUD phenotypes modeled using animals, such as acquisition, extinction, withdrawal, and reinstatement (Neumann et al., 2016; Pelloux et al., 2018; Zhu et al., 2016, 2018). The PVT is a potential brain region that could be sending glutamatergic projections enriched in synaptic Zn²⁺ to striatum. However, there are no histochemical studies that directly show glutamatergic/zincergic connections between these two regions. There are in situ hybridization studies reporting ZnT3 mRNA in the PVT, suggesting that a population of neurons from this region might release Zn²⁺ (Palmiter et al., 1996). Indeed, a recent study showed that the PVT has at least 5 different neuronal subtypes with different molecular identity (McGinty & Otis, 2020). Future studies are needed to confirm if the PVT sends glutamatergic/zincergic projections to the striatum and if so, whether such projections exert a modulatory role in SUD-related behaviors.

The location of glutamatergic/zincergic neurons in the PFC, BLA and the PVT and their projections to the striatum highlight the need for investigating the relationship between synaptic Zn²⁺ and its effects in these circuits to understand whether it modulates drug-seeking behaviors. Moreover, it is important to point out that not all glutamatergic neurons are zincergic neurons and therefore further studies are needed to differentiate the role of glutamate neurons that do not co-release Zn²⁺ vs. glutamate neurons that co-release Zn²⁺ and the extent to which these are involved in regulating SUD-related behaviors.

4. Synaptic Zn²⁺ interacts with receptors dysregulated in SUDs

4.1. NMDA Receptors

NMDA receptors (NMDAR) are glutamate-gated ion channels comprised of two glycine-binding GluN1 subunits and two glutamate binding GluN2A-D subunits with the subunit transmembrane domains defining the ion channel pore (Lee et al., 2014; Stroebel & Paoletti, 2021). NMDARs play an important role in synaptic transmission as well as synaptic plasticity (Lau et al., 2009) and are expressed in brain regions important for drug-seeking behavior. In fact, NMDAR antagonists block the rewarding or reinforcing effects of drugs of abuse such as cocaine (Schenk et al., 1993). Under physiological conditions, after being co-released with glutamate, Zn²⁺ acts on postsynaptic receptors in the cortex, hippocampus and striatum (Amico- Ruvio et al., 2011; Peters et al., 1987; Westbrook & Mayer, 1987). Zn²⁺ can interact with either a low-affinity (nanomolar concentrations) voltage-dependent binding to pore-lining residues which blocks the channel, or a high-affinity (micromolar) binding to N-terminal domains of GluN2A subunits to reduce channel open probability (Karakas et al., 2009; Nozaki et al., 2011; Paoletti & Neyton, 2007; Paoletti et al., 1997; Vergnano et al., 2014). When Zn²⁺ interacts with the GluN2A subunit, it is able to modulate synaptic integration and plasticity at hippocampal mossy fibers and CA1 synapses (Vergnano et al., 2014). The influence of Zn²⁺ on neural pathways associated with NMDAR suggests that disturbances of Zn²⁺ homeostasis may have effects on SUD-related behaviors.

4.2. AMPA Receptors

AMPA receptors (AMPAR) are also ionotropic glutamate channels that mediate fast excitatory transmission in the brain and synaptic transmission (Diering & Huganir, 2018; Kessels & Malinow, 2009). Notably, this receptor is located in regions relevant for drug seeking behavior and its activity has been demonstrated to be functionally responsive to the effects of drug exposure (Bowers et al., 2010; Reimers et al., 2011). For instance, there is an increase in AMPAR function following psychostimulant exposure (Bowers et al., 2010). In line with this, AMPAR antagonist administration into the NAcc inhibits reinstated ethanol, cocaine, and heroin seeking (Cornish & Kalivas, 2000; Di Ciano & Everitt, 2001; LaLumiere & Kalivas, 2008; Sanchis- Segura et al., 2006).

Additionally, compounds that utilize AMPAR antagonism have shown in preliminary studies to decrease craving and intake of cocaine in humans (Kampman et al., 2004, 2013). Zn²⁺ is known to modulate AMPAR function and behavior (Krall et al., 2021; Kupnicka et al., 2020) and, Indeed, induces both potentiation (at low micromolar concentrations) and inhibition (at high micromolar concentrations) of AMPAR (Carrillo et al., 2020; Lin et al., 2001). One recent study showed that local injection of AMPA into the substantia nigra pars compacta, a brain region that contains dopamine cell bodies which project and release dopamine into the striatum, induced movement disturbances that were accompanied with increased Zn²⁺ levels in rats. As expected, this effect was suppressed in the presence of a Zn²⁺ chelator (Tamano et al., 2018). Future studies are needed to dissect out the exact role of Zn²⁺ and AMPAR in drug dependence.

4.3. Dopamine transporter

The dopamine transporter (DAT) is a presynaptic plasma membrane protein that regulates the extracellular dopamine concentration in the brain by the re-uptake of released dopamine. In terminal regions like the striatum, DAT is localized to presynaptic terminals, while in the ventral tegmental area (VTA) and substantia nigra, it is also expressed in dendrites and soma, where it clears somatodendritically released dopamine (Beuming et al., 2008). It is known that Zn²⁺ binds to DAT (Bonnet et al., 1994; Richfield, 1993; Srivastava et al., 2024) via four high-affinity Zn²⁺-binding sites in the DAT extracellular domain (Loland et al., 2003; Norregaard et al., 1998; Stockner et al., 2013). Cocaine and other psychostimulants promote the increase of extracellular dopamine levels in the synaptic cleft by binding to DAT and blocking its function (Beuming et al., 2008). Specifically, studies in cell culture and synaptosomes demonstrated that micromolar concentrations of Zn²⁺ bind to DAT to attenuate dopamine uptake and potentiate binding of cocaine and its analog WIN-35,428 (Norregaard et al., 1998; Richfield, 1993). Using striatal membranes from mice, we recently showed that physiological concentrations of Zn²⁺ (e.g., 10 μM) increased cocaine binding and its affinity to the DAT (Gomez et al., 2021). Moreover, we showed that Zn²⁺ increased binding of the cocaine analog WIN-35,428 to DAT in mouse tissue sections (Gomez et al., 2021) supporting the notion that Zn²⁺ binds to DAT expressed in the brain and therefore would be expected to modulate its function.

4.4. Dopamine Receptors

In addition to binding to DAT, studies showed that Zn²⁺ binds and modulates dopamine receptors (Schetz & Sibley, 1997). Specifically, micromolar concentrations of Zn²⁺ reduced the binding of [³H]SCH-23390 to D1R and [³H]methylspiperone to D2R, presumably via allosteric interactions. The binding sites responsible for the high affinity binding of Zn²⁺ to D2R are H394 and H399 in the extracellular domain (Liu et al., 2006). However, the modulation of dopamine D2-like family receptors by Zn²⁺ is subtype-specific. For instance, Zn²⁺ regulates the binding of [³H]methylspiperone to D4 receptor D4R in a noncompetitive manner, whereas the reduction in [³H]methylspiperone binding to D2R and D3 receptor via Zn²⁺ occurs via allosteric competition (Schetz et al., 1999). Nevertheless, how Zn²⁺ affects dopamine receptor signaling in the context of exposure to cocaine or other psychostimulants remains unknown.

4.5. GABA and glycine receptors

It is well known that ethanol exerts its effects by altering the function of GABA-R and glycine- receptors (Gly-R), among others (Frye et al., 1994; Söderpalm et al., 2017). For instance, the manipulation of both types of receptors leads to a reduction in ethanol self-administration (June et al., 1998; Molander et al., 2007; Solomon et al., 2019). There are a plethora of studies that show that alcohol affects Zn²⁺-regulated mechanisms in different organs, such as liver, lungs, and gut (Skalny et al., 2018). For the purposes of this review, we will focus on the effects of alcohol and Zn²⁺ in the brain. For instance, a recent study showed that mice exposed to 16 weeks of heavy alcohol consumption showed Zn²⁺ deficiency and neurotoxicity (Jones et al., 2021). Additionally, Zn²⁺ plays an important role in the modulation of inhibitory neurotransmission as micromolar concentrations of Zn²⁺ inhibit GABA receptors via an allosteric mechanism (Barberis et al., 2000). There are other studies which showed that GABA-R inhibition by endogenous Zn²⁺ occurs in the hippocampus (Ruiz et al., 2004; Xie & Smart, 1991).

Zn²⁺ binds to three different GABA domains, one is located within the ion channel and the other two are on the external amino N-terminal. Interestingly, the ablation of these three sites abolishes the Zn²⁺ inhibition (Hosie et al., 2003). Gly-Rs are modulated by Zn²⁺ in a biphasic manner. For instance, Zn²⁺ at concentrations lower than 10 μM enhances Gly-R effects, while at higher concentrations inhibits them (Bloomenthal et al., 1994; Harvey et al., 1999; Laube et al., 2000). Interestingly, low physiological concentrations of Zn²⁺ increase the ethanol-induced effects on Gly-R (McCracken et al., 2010). These results are supported by the fact that a mutation in a Zn²⁺-binding residue in the α subunit of the Gly-R blunts the effects of ethanol on Gly-R (McCracken et al., 2013). Finally, in vitro studies demonstrated that ethanol enhances the activation of Gly-function by taurine, a Gly-R agonist, and this effect is reduced by chelation of Zn²⁺ (Welsh et al., 2010) (Table 1).

Table 1.

Zinc’s Role in Receptor Function and Drug Interaction

Receptor	Type of Receptor	Role	Interaction with Zn2+	References
NMDA	Ionotropic glutamate receptor (channel)	Synaptic transmission and plasticity	- Low affinity binding blocks channel. - High affinity binding to GluN2A reduces channel open probability.	Amico-Ruvio et al., 2011; Karakas et al., 2009; Lau et al., 2009; Lee et al., 2014; Nozaki et al., 2011; Paoletti & Neyton, 2007; Paoletti et al., 1997; Peters et al., 1987; Schenk et al., 1993; Stroebel & Paoletti, 2021; Vergnano et al., 2014; Westbrook & Mayer, 1987
AMPA	Ionotropic glutamate receptor (channel)	Mediates synaptic transmission and fast excitatory transmission.	- Induces potentiation at low Zn2+ levels and inhibition at high levels. - Increased Zn2+ levels in SNc after AMPA injection caused movement disturbances.	Bowers et al., 2010; Carrillo et al., 2020; Cornish & Kalivas, 2000; Di Ciano & Everitt, 2001; Diering & Huganir, 2018; Kampman et al., 2004; Kampman et al., 2013; Kessels & Malinow, 2009; Kupnicka et al., 2020; LaLumiere & Kalivas, 2008; Lin et al., 2001; Reimers et al., 2011; Sanchis-Segura et al., 2006; Tamano et al., 2018
DAT	Presynaptic plasma membrane protein	Regulates extracellular dopamine by re-uptake into presynaptic terminals.	- Zn2+ binds to DAT, attenuating dopamine uptake. - Potentiates cocaine binding and increases its affinity for DAT. - Zn2+ increases binding of cocaine analogs to DAT.	Beuming et al., 2008; Bonnet et al., 1994; Gomez et al., 2021; Loland et al., 2003; Norregaard et al., 1998; Richfield, 1993; Srivastava et al., 2024; Stockner et al., 2013
Dopamine Receptors	Metabotropic receptor	Modulation of dopamine signaling.	- Zn2+ reduces binding to D1R and D2R. - High affinity binding to D2R occurs extracellularly. - Modulates D2-like family receptors and binds noncompetitively to D4R.	Liu et al., 2006; Schetz & Sibley, 1997; Schetz et al., 1999
GABA and Glycine receptors	Ionotropic receptors (channel)	Modulation of GABA or glycine signaling	- Zn2+ inhibits GABA-R via allosteric mechanism; binds to multiple domains. - Zn2+ modulates GlyRs biphasically and interacts with ethanol-induced effects.	Barberis et al., 2000; Frye et al., 1994; Hosie et al., 2003; Jones et al., 2021; June et al., 1998; Laube et al., 2000; McCracken et al., 2010; 2013; Molander et al., 2007; Ruiz et al., 2004; Skalny et al., 2018; Söderpalm et al., 2017; Solomon et al., 2019; Welsh et al., 2010; Xie & Smart, 1991

5. Zn²⁺ and intracellular signaling pathways in the context of SUDs

As discussed before, Zn²⁺ acts as a potent neuromodulator by targeting multiple postsynaptic receptors and ion channels. Zn²⁺ can inhibit or modulate the activity of NMDA, AMPA, and GABA receptors, depending on receptor subunit composition and local concentration (Paoletti et al., 2009; Vergnano et al., 2014). These interactions alter postsynaptic ion fluxes that initiate different intracellular signaling cascades that regulate neuronal plasticity.

Zn²⁺ modulates several intracellular pathways that are essential for synaptic function and structural remodeling. It promotes the activation of the MAPK/ERK pathway, leading to ERK1/2 phosphorylation and subsequent regulation of nuclear targets involved in plasticity and gene expression (Bossy-Wetzel et al., 2004; Hwang et al., 2005). It also influences the PI3K/Akt pathway, which contributes to neuronal survival, synaptogenesis, and dendritic architecture (Nakashima & Dyck, 2009). In addition, by altering calcium influx through NMDA receptors, Zn²⁺ can modulate CaMKII and CREB phosphorylation which are key mechanisms in long-term memory consolidation and drug-induced plasticity (Gao et al., 2011; Yu et al., 2013).

Experimental evidence supports the role of Zn²⁺ in modulating ERK signaling in vivo. For example, acute administration of Zn²⁺ induces a rapid but transient activation of ERK in the prefrontal cortex, and this activation is necessary for its short-lasting antidepressant-like effects in rodents (Pochwat et al., 2017). In contrast, long-acting NMDA receptor antagonists such as Ro 25–6981 induce a more sustained ERK activation, which is required for their longer-lasting behavioral effects (Pochwat et al., 2017). These findings underscore the functional relevance of ERK modulation by Zn²⁺ and suggest a dynamic role for Zn²⁺ in regulating experience-dependent plasticity.

Interestingly, these same signaling pathways, MAPK/ERK, PI3K/Akt, and CaMKII/CREB, are disrupted in the brains of individuals with substance use disorders and have been implicated in the long-lasting neuroadaptations that underlie craving, relapse, and compulsive drug-seeking behavior. Preclinical studies have shown altered ERK and CREB activity in the striatum, prefrontal cortex, and hippocampus following exposure to cocaine, opioids, and alcohol (Carlezon et al., 2005; Koob & Volkow, 2010; Lu et al., 2006; Nestler & Lüscher, 2019; Russo et al., 2009).

Through its interaction with synaptic receptors and modulation of these intracellular pathways, Zn²⁺ may influence the same plasticity-related signaling mechanisms that are dysregulated in addiction. While this area remains underexplored, the available evidence suggests that zinc could modulate or even counteract some of the molecular alterations induced by chronic drug exposure. A better understanding of these downstream mechanisms is essential for elucidating Zn²⁺’s role in addiction-related neuroplasticity and may uncover novel targets for therapeutic intervention.

6. Zn²⁺ deficits in humans and animals in the context of drug and alcohol exposure

Accumulating evidence points to a key role for Zn²⁺ in the pathophysiology of diseases of the central nervous system (Frederickson et al., 2005; Krall et al., 2021; Kumar et al., 2021; Zhang et al., 2022). Alterations in the levels of Zn²⁺ and ZnT3 polymorphisms are associated with different neurological and neuropsychiatric disorders (Jelen et al., 2022; Torres-Vega et al., 2012). Experimental studies in humans and animals (Gomez et al., 2021; Kupnicka et al., 2020; Ordak et al., 2018; Ruiz Martínez et al., 1990) have documented dysregulation of Zn²⁺ homeostasis following the consumption of drugs of abuse (Table 2) and we review these in detail below.

Table 2.

Effects of Zn²⁺ on Neurobiological Processes Relevant to Substance Use and Addiction

Substance	Role of Zn2+	Key Findings
Psychostimulants	Zn2+ influences dopamine transmission and cocaine effects.	- Zn2+ levels inversely correlate with cocaine metabolites. - Chronic cocaine increases Zn2+ in brain regions (e.g., NAcc, hippocampus, PFC). - Zn2+ deficiency reduces cocaine sensitization and preference. - Prenatal Zn2+ affects dopamine turnover.	Gomez et al., 2021; Okada et al., 2013; Schoen et al., 2019
Opioids	Zn2+ modulates opioid receptor binding and affects behavior.	- Inhibits enkephalin and mu-opioid receptor binding. - Zn2+ deficiency increases receptor binding and opioid effects. - Zn2+ enhances or inhibits morphine effects. - Zn2+ supplementation may reduce opioid use.	Akbari et al., 2015; Chavez & Rigg, 2020; Ciubotariu et al., 2015; Ciubotariu et al., 2017; Elnimr et al., 1996; Essatara et al., 1984; Fowler et al., 2004; Hanissian & Tejwani, 1988; Larson et al., 2000; Mahboub et al., 2021; Mesbahzadeh et al., 2019; Ogawa et al., 1985; Ruiz Martínez et al., 1990; Sadlik et al., 2000; Stengaard-Pedersen, 1982; Stengaard-Pedersen et al., 1981; Tantillo et al., 2021; Tejwani & Hanissian, 1990
Alcohol	Zn2+ interacts with alcohol metabolism and dopamine release.	- Mutations in Zn2+related pathways reduce alcohol consumption. - Zn2+ chelators decrease dopamine release post-alcohol intake. - Chronic alcohol use lowers Zn2+ in brain and blood. - Zn2+ aids alcohol metabolism via alcohol dehydrogenase.	Baj et al., 2020; Bode et al., 1988; Bogden & Troiano, 1978; Dinsmore et al., 1985; Gandigawad & Hiremath, 2017; Hartoma et al., 1977; Kasarskis et al., 1985; Lieber, 2000; McClain et al., 1986; McCracken et al., 2013; Menzano & Carlen, 1994; Morud et al., 2015; Skalny et al., 2018; Spanagel, 2009; Valberg et al., 1985; Welsh et al., 2010

6.1. Psychostimulants

We recently examined Zn²⁺ levels in human postmortem striatal tissue from 20 male subjects whose cause of death was attributed to cocaine use. The cocaine users had lower striatal Zn²⁺ compared to matched controls. Moreover, we demonstrated that striatal Zn²⁺ levels inversely correlated to plasma levels of benzoylecgonine (a cocaine metabolite) in these subjects, indicating that striatal Zn²⁺ levels correlated with the extent of recent cocaine use (Gomez et al., 2021).

In the same study we found that chronic cocaine exposure increased Zn²⁺ levels in the caudate putamen (i.e., dorsal striatum) and the NAcc compared to vehicle-treated mice. In addition, we studied ⁶⁵Zn uptake using positron emission tomography, and found that chronic cocaine exposure increased ⁶⁵Zn uptake in the NAcc, hippocampus, amygdala, and PFC (Gomez et al., 2021). Moreover, using fast scan cyclic voltammetry, we demonstrated that repeated and escalating doses of cocaine induced lower electrically-evoked extracellular dopamine release in ZnT3 KO compared to wild type mice. These findings suggested that synaptic Zn²⁺ potentiates cocaine’s effects in vivo by binding to DAT to modulate dopaminergic transmission (Gomez et al., 2021).

Locomotor sensitization to cocaine is a well-known behavior that is associated with changes in dopaminergic transmission (Gomez et al., 2021). We demonstrated that ZnT3 KO mice develop locomotor sensitization to cocaine. However, the locomotor response to cocaine was lower than that observed in WT mice. Interestingly, when we studied the expression of cocaine locomotor sensitization, we found that ZnT3 deletion reduced the locomotor response to cocaine. Furthermore, using conditioned place preference (CPP), we studied the effect of ZnT3 deletion on cocaine reward. We found that ZnT3 KO mice did not show cocaine CPP across varying doses. In addition, ZnT3 KO mice exhibited reduced intravenous self-administration to cocaine compared to their WT counterparts (Gomez et al., 2021). Besides targeting the ZnT3 gene, another experimental approach to reduce Zn²⁺ levels in the brain consists of providing a Zn²⁺-deficient diet to the animals (Schoen et al., 2019). In line with the effect of ZnT3 deletion, dietary Zn²⁺ deficiency reduced the expression of cocaine locomotor sensitization compared to animals with a normal diet. Similarly, Zn²⁺-deficient mice showed lower cocaine CPP. In line with these findings, a previous study showed that prenatal exposure to zinc oxide nanoparticles altered dopamine transmission by increasing dopamine turnover in the PFC, CPu, NAcc and amygdala in mouse offspring (Okada et al., 2013). Taken together, these results suggest that synaptic Zn²⁺ is necessary for the expression of cocaine-induced behaviors and cocaine-induced behavioral plasticity.

6.2. Opioids

Opioid receptors are widely distributed throughout the brain, where they regulate brain functions by acting on different regions involved in reward, motivation, and analgesia (Le Merrer et al., 2009; Waldhoer et al., 2004). Clinical and preclinical evidence shows that opioid receptors have an important role in the neurobiological substrates underlying drug seeking (Trigo et al., 2010).

Early experiments showed that physiological concentrations of Zn²⁺ inhibit enkephalin binding to opioid receptors in the rat basal ganglia, hippocampus, and the cerebral cortex (Stengaard- Pedersen, 1981; 1982) by reducing both the affinity and number of binding sites (Ogawa et al., 1985). In addition, binding studies performed in brain membranes from rats demonstrated that Zn²⁺ inhibits the binding of [³H]DAMGO to mu-opioid receptors, but not the binding of [³H]DSTLE and [³H]EKC to delta- and kappa-opioid receptors, respectively (Fowler et al., 2004; Tejwani & Hanissian, 1990). This effect was due to the interaction of Zn²⁺ with sulfhydryl groups on mu-opioid receptors (Tejwani & Hanissian, 1990). Zn²⁺ was also able to inhibit the binding of [³H]naloxone to opioid receptors in rat membranes in a dose-dependent manner. This effect was due to a decrease in receptor affinity, without changing the number of binding sites (Hanissian & Tejwani, 1988). Further studies using brain membranes from Zn²⁺-deficient rats reported that [³H]naloxone binding to opioid receptors was significantly increased (Essatara et al., 1984).

In vivo pharmacological studies in animals have also shed light on the role of Zn²⁺ in the opioid system. Previous research has shown that even though opioids provide effective analgesia, there are important concerns about opioid tolerance and dependence (Fujita et al., 2019). One study showed that intrathecal administration of Zn²⁺ in mice inhibited the development of acute morphine tolerance and morphine-induced analgesia (Larson et al., 2000). Studies performed in rats reported that the intracerebroventricular or intraperitoneal administration of Zn²⁺ enhanced morphine-induced CPP during the expression phase. Interestingly, this effect was reversed by the pretreatment with D1R and D2R antagonists as well as with a serotonin 5HT-1A receptor antagonist. Together, these findings suggest that the Zn²⁺ effects on morphine CPP might be mediated partially by dopaminergic and serotonergic systems (Mesbahzadeh et al., 2019). However, further studies are needed to directly test this hypothesis. On the other hand, a recent study showed that zinc sulphate oral administration decreased naloxone-induced conditioned place aversion in a dose-dependent manner (Ciubotariu et al., 2017). The aforementioned studies suggest an important role of Zn²⁺ in the modulation of opioid-induced behaviors (Ciubotariu et al., 2015). However, further work studying the role of Zn²⁺ in other behavioral procedures, such as opioid self-administration, are needed.

Although no studies have reported Zn²⁺ levels in postmortem human brain samples of individuals with opioid use disorders (OUD), several studies have examined the levels of Zn²⁺ in body fluids (Ciubotariu et al., 2015; Elnimr et al., 1996; Potkin et al., 1982; Ruiz Martínez et al., 1990). Previous studies examining whole blood Zn²⁺ levels showed a decrease of Zn²⁺ in heroin-dependent individuals compared to non-users. Interestingly, the Zn²⁺ levels were inversely correlated with the period of heroin exposure (Elnimr et al., 1996). Other studies found low Zn²⁺ levels in plasma and serum in heroin addicts (Ruiz Martínez et al., 1990; Sadlik et al., 2000) and one study found no differences in serum Zn²⁺ levels between heroin users and non-users subjects (Akbari et al., 2015). One report showed lower Zn²⁺ in the cerebrospinal fluid in ex-heroin users compared to non-users (Potkin et al., 1982). In regard to OUD, once the opioids begin to take a more prominent role in the drug user’s life, a notable concern is whether this affects the intake of necessary nutritional elements (Chavez & Rigg, 2020). As an essential element, Zn²⁺ must be acquired via diet and malnutrition is a common problem in SUDs (Mahboub et al., 2021). Thus, it is possible that the altered Zn²⁺ levels in SUD might be, in part, a consequence of nutritional deficiency (Ciubotariu et al., 2015). Indeed, a retrospective study conducted in post-operative total hip arthroplasty patients showed that Zn²⁺ deficiency may potentiate opioid consumption and this study suggested that Zn²⁺ supplementation could help reduce opioid addiction and dependence (Tantillo et al., 2021).

6.3. Alcohol

Prior work studied the effect of a mutation at the Zn²⁺ binding site of the Gly-R on alcohol consumption and preference in mice. By using a two-bottle choice drinking paradigm, the authors found that mutant mice displayed both lower consumption of and preference to alcohol. Interestingly, this effect was observed in female but not male mice (McCracken et al., 2013). These results were not likely due to alterations in the motor performance of the animals because WT and mutant mice showed similar motor incoordination induced by alcohol (McCracken et al., 2013). Further studies showed that zinc sulfate (18 mg/kg; orally) did not affect alcohol consumption in rats (Gandigawad & Hiremath, 2017). Another study showed that Zn²⁺ dietary deficiency does not affect voluntary alcohol consumption, but it does affect the excitatory transmission in the NAcc (Morud et al., 2015). It is well known that ethanol increases extracellular dopamine levels in the striatum (Spanagel, 2009). Microdialysis experiments showed that local injection of Zn²⁺ chelators into the NAcc reduced basal and alcohol-induced extracellular dopamine levels (Morud et al., 2015). These results suggest that the increased extracellular dopamine levels following ethanol consumption require the interaction of Zn²⁺ and alcohol, possibly at Gly-R and GABA-R (Morud et al., 2015; Welsh et al., 2010).

Studies using postmortem brain tissue reported that chronic alcohol intake induces a decrease in the levels of Zn²⁺ in the globus pallidus, substantia nigra, putamen, amygdala, and hippocampus (Kasarskis et al., 1985). It has been suggested that the altered levels of Zn²⁺ could cause neuronal damage and brain dysfunction (Baj et al., 2020; Menzano & Carlen, 1994). Numerous studies have demonstrated that chronic alcohol consumption alters Zn²⁺ levels in serum, plasma, and hepatic tissue (McClain et al., 1986; Skalny et al., 2018). Early studies showed low serum Zn²⁺ levels in alcoholics (Hartoma et al., 1977) and in patients with delirium tremens (Banach & Morasiewicz, 1994). Meanwhile, patients with alcoholic cirrhosis and patients with alcohol withdrawal syndrome presented lower plasma Zn²⁺ levels than controls (Bode et al., 1988; Bogden & Troiano, 1978). There are different reasons that could explain the imbalance in Zn²⁺ homeostasis in alcohol users. For instance, studies in humans using ⁶⁵Zn as a radiotracer to examine Zn²⁺ uptake demonstrated that alcohol consumption decreased Zn²⁺ absorption (Dinsmore et al., 1985) and increased Zn²⁺ excretion in urine (Valberg et al., 1985). Interestingly, Zn²⁺ has a catalytic role in the metabolism of alcohol by interacting with the alcohol dehydrogenase enzyme (Lieber, 2000). Further studies are necessary to clearly establish the role of synaptic Zn²⁺ in the context of alcohol exposure and alcohol use disorders.

7. Synaptic Zn²⁺, sex differences, and SUD vulnerability

Human and animal studies have reported sexually dimorphic behavioral responses to drugs of abuse (Becker & Hu, 2008; Hernandez- Avila et al., 2004; Van Etten & Anthony, 2001). Previous studies have shown that ZnT3 is regulated by exogenous and endogenous factors (Ackland et al., 2007; Iguchi et al., 2004; Saito et al., 2000; Salazar et al., 2004; Suphioglu et al., 2010). Studies indicate that ovariectomized mice showed an increase in ZnT3 and synaptic Zn²⁺ levels in the brain, while estrogen replacement reduced these levels (Lee et al., 2004). These changes were associated with transcriptional changes of the adaptor complex AP-3, which is known to regulate the levels of ZnT3 (Lee et al., 2004). Together these results show that estrogen modulates ZnT3 as well as synaptic Zn²⁺. Interestingly, a previous study reported that there are sex-related differences on the risk of developing schizophrenia in female patients carrying a rare allele for single nucleotide polymorphisms located in the SLC30A3 (ZnT3) gene (Perez-Becerril et al., 2016). In support to the potential modulatory role of estradiol on ZnT3 and Zn²⁺, previous research found sex-specific effects of deleting ZnT3 on sensorimotor behaviors (Thackray et al., 2017). Furthermore, male but not female ZnT3 KO mice displayed impaired social interaction (Yoo et al., 2016). A similar sex-specific behavioral phenotype was observed in prenatal Zn²⁺-deficient mice (Grabrucker et al., 2016). The exact physiological mechanisms that underlie these differences remain unclear; however, a possible mechanism could be related to the interaction of Zn²⁺ with matrix metalloproteinases and/or Shank proteins (Grabrucker et al., 2016; Yoo et al., 2016).

Preclinical studies showed that females exhibit more robust psychostimulant-induced sensitization (Sircar & Kim, 1999; van Haaren & Meyer, 1991) and are more prone to cocaine self-administration than males (Jackson et al., 2006; Lynch et al., 2001). Consistent with these results, estradiol increases cocaine self-administration in ovariectomized animals (Lynch et al., 2001). Estradiol acts on estrogen receptors, which are expressed in limbic-related areas and midbrain areas such as the VTA (Küppers & Beyer, 1999; Osterlund & Hurd, 2001; Gillies et al., 2014). Estrogens have been demonstrated to modulate dopamine neurotransmission through activation of ERs that are expressed in striatal and midbrain neurons (Almey et al., 2012, 2016, 2022; Milner et al., 2010; Shughrue, 2004). Although there is no research to date that has studied the interaction of sex and synaptic Zn²⁺ in SUDs, available evidence shows that the aforementioned data, imply that sex hormones regulate ZnT3 expression and synaptic Zn²⁺ levels. Given the complex and multifactorial nature of sex differences in addiction, which involve hormonal, genetic, neuroimmune, and environmental factors, it is unlikely that Zn²⁺ is a primary driver. Instead, Zn²⁺ signaling may act as a modulatory component within broader systems. The relationships between sex, Zn²⁺ metabolism, and SUD-related behaviors remain associative. Causal mechanisms will need to be addressed in future studies using sex-specific and circuit-targeted manipulations of synaptic Zn²⁺ signaling.

8. Therapeutic implications and future directions

Evidence suggests that synaptic Zn²⁺ and ZnT3 can modulate neurotransmission in brain regions involved in reward and motivation, such as the striatum and PFC (Gomez et. al. 2021). These findings show a potential modulatory role in behaviors relevant to substance use disorders (SUDs), but additional research is required to demonstrate whether targeting Zn²⁺ signaling can causally influence drug-related behaviors or for its treatment. As such, a deeper understanding of ZnT3 function and synaptic Zn²⁺ neurobiology and neurocircuitry and its interaction with the neurotransmitter systems known to be affected in SUDs may provide an important framework towards the development of SUD therapeutics. Although Zn²⁺ is present in brain regions involved in reinforcement, such as the striatum, amygdala, and PFC, the specific circuits through which it modulates drug-seeking behavior are still unclear. It is not known whether Zn²⁺ release alters synaptic plasticity within these pathways or how its dynamics change with drug exposure. Dissecting these mechanisms is critical to understand how Zn²⁺ contributes to the development and persistence of addictive behaviors.

Previous studies have associated ZnT3 and synaptic Zn²⁺ function with brain diseases comorbid with SUDs such as schizophrenia (Jelen et al., 2022; Perez- Becerril et al., 2016), attention-deficit/hyperactivity disorder (ADHD) or diseases which target dopamine neurons such as Parkinson’s (Olesen et al., 2016; Rafalo-Ulinska et al., 2016). In SUD, the specific regulation of synaptic Zn²⁺ by ZnT3 could have beneficial effects by affecting the responses to substance of abuse. However, progress in this area is currently limited by the lack of pharmacological agents that target ZnT3. Accordingly, the identification or development of selective chemical agents that can modulate ZnT3 function may accentuate our understanding of SUD neurobiology and potentially facilitate the development of novel SUD therapeutics. This strategy offers therapeutic promise. However, it is important to consider potential off-target effect, as Zn²⁺ is involved in several physiological processes beyond reward and motivation, (Sindreu et al., 2011; Krall et al., 2021).

It is well known that alcoholism affects Zn²⁺ content in the body, which can be attributed to the reduced intake or absorption of Zn²⁺. These altered levels of Zn²⁺ could lead to different metabolic disturbances and also may play a role in alcohol-seeking behaviors. In the case of alcohol-use disorder, it may be beneficial to supplement Zn²⁺ in the patient’s diet, although it cannot be excluded to also be beneficial for other SUDs (Ciubotariu et al., 2015). Zn²⁺ levels are influenced by diet, environment, and health status, which can confound interpretations of its role in addiction (King, 2011; Prasad, 2013). These factors should be considered in both clinical and preclinical studies to avoid misleading conclusions and to improve the translational value of zinc-based interventions.

While experimental data support the idea that synaptic Zn²⁺ modulates dopamine and glutamate transmission, its role in driving drug-related behaviors remains largely unexplored. Human studies are especially lacking. It is still unknown whether individual differences in dietary zinc intake are linked to psychological traits associated with addiction risk. Addressing these gaps could help identify vulnerable populations and guide more targeted prevention strategies. Studies examining the clinical utility of zinc supplementation are scarce. To date, only one study has examined the efficacy of zinc supplementation as an aid to SUD treatment. Among patients receiving methadone maintenance treatment, oral zinc supplementation significantly lowered the probability of opioid relapse and improved symptoms of depression and anxiety (Amini & HeidariFarsani, 2023). While this initial finding is promising, research in human populations remains limited. Most of the current knowledge on synaptic Zn²⁺ and its relevance to SUD comes from animal models or molecular studies. There is a need for more human-based research to determine whether these preclinical findings translate to clinical populations with SUD (Amini & HeidariFarsani, 2023; Gomez et al., 2021).

Psychiatric disorders frequently co-occur among individuals with substance use disorder, and anxiety and depressive symptomatology have been linked to higher relapse rates (Clark et al., 2015; Sliedrecht, et al., 2019.) Therefore, effective treatment of underlying mental health issues may aid addiction treatment. The antidepressant and anxiolitic effects of zinc monotherapy in rodents on the forced swim test, tail suspension test, and elevated plus maze have been demonstrated by several laboratories (Kroczka et al., 2000; Nowak et al., 2003; Joshi, et al., 2012; Samardžić et al., 2013). Furthermore, zinc appears to potentiate the effects of other antidepressant treatments, including imipramine, citalopram, and fluoxetine (Szewczyk et al., 2002; Cunha et al., 2008; Joshi et al., 2012).

Along these lines, zinc hydro-aspartate supplements have been shown to increase the efficacy of the antidepressant imipramine among patients with treatment-resistant depression (Siwek et al., 2009). Additionally, oral zinc in conjunction with SSRIs reduces symptom severity in patients with major depressive disorder (Ranjbar et al., 2013; Ranjbar et al., 2014). Zinc supplementation may therefore serve as an indirect treatment for SUDs through its antidepressant and anxiolytic effects, as treatment of co-occurring psychiatric disorders may lower the likelihood of substance relapse (Amini & HeidariFarsani, 2023; De Filippis et al., 2025).

Dietary zinc supplementation during pregnancy may also be beneficial for individuals with SUDs (Summers et al., 2009). Prenatal substance use presents a significant public health concern due to the harmful effects it may have on the developing fetus. Though abstinence from drugs and alcohol during pregnancy is highly preferable, treatments to mitigate physical and behavioral teratogenicity may be useful among individuals with SUDs that are unable to maintain abstinence (Summers et al., 2009).

Offspring born to mice exposed to ethanol during pregnancy show higher rates of physical abnormalities and postnatal mortality (Summers et al., 2009). However, when fed a zinc-supplemented diet, the offspring of pregnant mice exposed to ethanol show health outcomes similar to their non-exposed counterparts. Additionally, prenatal zinc supplementation rescued object recognition and spatial memory in ethanol-exposed animals (Summers et al., 2008).

Another important factor in using zinc supplementation to treat SUD is aging. Previous studies using the stable isotopes, ⁶⁷Zn and ⁷⁰Zn, showed that zinc absorption is significantly lower in elderly men compared to younger men, with absorption rates averaging 17% in older adults versus 31% in younger ones (Turnlund et al., 1986). However, the net difference between zinc intake and excretion does not differ between age groups, indicating that older adults may adapt to lower zinc intake more effectively than younger individuals (Turnlund et al., 1986). Meanwhile, zinc metabolism can be important in the context of SUDs. Further research is needed to explore the relationship between aging, zinc metabolism, and substance use disorders.

The evidence discussed highlights the potential modulatory role of Zn²⁺ in circuits relevant to substance use disorders, much of it is based on correlations, receptor-binding studies, or indirect associations. It remains a need for studies using causal approaches by using pharmacological manipulations, optogenetics, or circuit-level interventions in models of drug self-administration or relapse. These approaches will be necessary to determine whether Zn²⁺ directly contributes to drug-seeking behaviors and the underlying neuroadaptations that occur with chronic drug exposure.

Although research on zinc supplementation for SUDs is still in its early stages. Evidence suggests that zinc could play a key role in reducing relapse and supporting recovery. A better understanding of the role of zinc in SUD may help unravel new ways to treat addiction more effectively. In parallel, the development of novel chemical agents that selectively modulate ZnT3 activity or enhance synaptic Zn²⁺ signaling represents a promising pharmacological direction. These pharmacological agents could allow for more precise control of synaptic Zn²⁺ signaling and its interaction with dopamine and glutamate systems, thereby offering targeted interventions to correct the neurobiological alterations associated with chronic drug exposure. In addition, there is also a need to explore how natural variation in dietary zinc intake may be associated with psychological traits relevant to substance use vulnerability. While supplementation studies are beginning to provide evidence for zinc’s therapeutic potential, observational studies in humans that assess zinc intake under normal dietary conditions and its correlation with psychological traits related to addiction could help identify populations at risk and guide prevention strategies.

Zinc supplementation has shown promise in modulating mechanisms relevant to substance use disorders, but it also carries medical risks that should not be overlooked. Chronic or excessive intake can lead to zinc-induced copper deficiency (ZICD), a condition primarily associated with anemia and neurological symptoms like sensory deficits and gait disturbances (Halfdanarson et al., 2008). Mechanistically, high Zn²⁺ levels upregulate metallothioneins in enterocytes, which preferentially bind copper, leading to its excretion and systemic copper deficiency (Stiles et al., 2024). ZICD often develops slowly and is underdiagnosed, partly due to limited monitoring of serum zinc and copper in clinical settings and overprescription has been flagged as a key contributor (Duncan et al., 2023). While hematologic abnormalities are often reversible with copper supplementation, some neurological impairments may persist (Duncan et al., 2023). These risks underscore the need for careful dosing, proper monitoring, and clinical guidelines when considering zinc as a potential therapeutic or prophylactic approach.

9. Conclusion

Zn²⁺ interacts with proteins that mediate the reinforcing effects of drugs of abuse and that are dysregulated in individuals with SUD. Furthermore, Zn²⁺-releasing neurons are advantageously distributed across brain circuits impacted in SUD. Therefore, Zn²⁺ has a potential key role in modulating the physiological and behavioral processes relevant to SUDs. In the striatum specifically, Zn⁺² may act to modulate the activity of pathways involved with the affective and reinforcing properties of drugs of abuse. Nevertheless, many questions remain regarding the precise role of Zn²⁺ in reinforcement and SUDs. For instance, the specific circuits that could be participating in SUDs are still unknown. In addition, the dynamics of synaptic Zn²⁺ release following drug-taking and the regional changes in synaptic Zn²⁺ as a function of drug exposure are unknown. Such observations could be invaluable for understanding the role of synaptic Zn²⁺ in the neurobiological adaptations seen in SUDs. Detecting synaptic Zn²⁺ under physiological conditions, particularly in vivo, remains a major technical challenge and has limited the investigation of its role in SUDs. Traditional methods provide only static information and cannot track Zn²⁺ dynamics during behavior. However, recent advances such as genetically encoded sensors like the far-red fluorescent indicator for monitoring synaptic Zn²⁺ (FRISZ) now allow real-time detection of Zn²⁺ in living systems (Wu et al., 2022). When combined with circuit-specific tools, pharmacological approaches, and SUD relevant behavioral models, these technologies offer new opportunities to clarify the contribution of Zn²⁺ to the neurobiology of addiction. Finally, the manipulation of Zn²⁺ transmission, by developing pharmaceutical agents targeting ZnT3 function may serve as a promising avenue of research for the treatment of SUDs.

Abbreviations:

AMPAR

AMPA receptors

BLA

basolateral amygdala

CPP

conditioned place preference

D1R

dopamine D1 receptor

D2R

dopamine D2 receptor

DAT

dopamine transporter

GABA-R

GABA receptor

Gly-R

glycine receptor

MSNs

medium spiny neurons

KO

knockout

NAcc

nucleus accumbens

PFC

prefrontal cortex

NMDAR

NMDA receptors

OUD

opioid use disorders

PVT

paraventricular thalamus

WT

wild-type

SUD

substance use disorder

ZnT3

Zn²⁺ transporter 3