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ABSTRACT Drug-induced enhanced dopamine (DA) signaling in the brain is a canonical mechanism that initiates addiction processes. However, indirect evidence suggests that cocaine also

triggers non-canonical, DA-independent, mechanisms that contribute to behavioral responses to cocaine, including psychomotor sensitization and cocaine self-administration. Identifying these

mechanisms and determining how they are initiated is fundamental to further our understanding of addiction processes. Using physiologically relevant in vitro tractable models, we found that

cocaine-induced hypoactivity of nucleus accumbens shell (NAcSh) medium spiny neurons (MSNs), one hallmark of cocaine addiction, is independent of DA signaling. Combining brain slice studies

and site-directed mutagenesis in HEK293T cells, we found that cocaine binding to intracellular sigma-1 receptor (_σ_1) initiates this mechanism. Subsequently, _σ_1 binds to Kv1.2 potassium

channels, followed by accumulation of Kv1.2 in the plasma membrane, thereby depressing NAcSh MSNs firing. This mechanism is specific to D1 receptor-expressing MSNs. Our study uncovers a

mechanism for cocaine that bypasses DA signaling and leads to addiction-relevant neuroadaptations, thereby providing combinatorial strategies for treating stimulant abuse. SIMILAR CONTENT

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access 04 August 2021 INTRODUCTION Enhanced dopamine (DA) signaling is postulated to be a canonical mechanism responsible for drug addiction [1, 2], but also an initial and sufficient event

for the development of drug addiction. Previous studies suggest that activation of the sigma-1 receptor (_σ_1), an endoplasmic reticulum (ER) chaperone [3] that regulates a variety of

proteins through physical protein–protein interactions [4], contributes to addiction processes [5, 6]. Consistent with this role, _σ_1 regulates both DA receptors signaling (DARs) via

protein–protein interactions [7, 8] and DA release in the striatum [9]. In contrast, there is evidence that cocaine and other stimulants may also engage _σ_1 independent of DA signaling and

contribute to cocaine addiction [10], suggesting that redundant or complementary mechanisms exist to shape addiction-related phenotypes. However, no cellular mechanism has been identified so

far. Previous studies showed that repeated in vivo exposure to cocaine leads to persistent neuronal hypoactivity in the nucleus accumbens shell (NAcSh) (i.e., firing rate depression,

FRD)—an adaptation that enhances both psychomotor response to cocaine and cocaine reward [11]. Our previous study shows that cocaine-induced FRD is mediated by the activation of _σ_1 in the

NAcSh and lasts up to 2 weeks after the last cocaine injection. Importantly, prior in vivo blockade of _σ_1 prevents both the development and the maintenance of cocaine-induced FRD in NAcSh

medium spiny neurons (MSNs) [12], suggesting that the effect of _σ_1 blockade during cocaine treatment is enduring. This form of cocaine-driven intrinsic plasticity is now emerging as one of

the hallmarks for cocaine addiction [13, 14]. Here, we demonstrate that cocaine-induced FRD is not prevented by DA receptor antagonists and is unaffected by a non-selective monoamine

reuptake inhibitor, but is blocked by the _σ_1 antagonist BD1063. Combining in vivo pharmacology, biochemical and whole-cell patch-clamp studies on freshly dissected brain slices, with

site-directed mutagenesis of _σ_1 expressed in HEK293T cells, we demonstrate that cocaine-_σ_1 physical interaction initiates the mechanism responsible for cocaine-induced FRD in NAcSh

D1R-expressing MSNs. Further in vitro studies in brain slices demonstrate that, in contrast to typical drug actions on plasma membrane targets, cocaine initiates this mechanism by binding to

_σ_1 intracellularly. Together, our results indicate that in addition to conventional mechanisms, psychostimulant drugs can also bypass DA signaling and lead to addiction-relevant

neuroadaptations, and in particular, cocaine-driven plasticity of neuronal intrinsic excitability in NAcSh D1R-MSNs. MATERIALS AND METHODS ANIMALS Male C57BL/6J mice or male Drd1a-tdTomato

C57BL6J mice (bred on site) (7–12 weeks of age). Mice were group housed and maintained on a 12-h light/dark cycle (light on at 7:00 a.m). See Supplementary Information for details. The

experimental procedures followed the Guide for the Care and Use of Laboratory Animals (eighth edition) and were approved by the Animal Care and Use Committee at the University of Texas

Southwestern Medical Center. SLICE PREPARATION AND SOLUTIONS Sagittal slices of the NAcSh (250 µm) were prepared as described previously [11, 12, 15,16,17]. Slices recovered in artificial

cerebro-spinal fluid (ACSF) saturated with 95% O2/5% CO2. See Supplementary Materials and methods for details. ELECTROPHYSIOLOGY Whole-cell current-clamp recordings were performed as

previously described [11, 12, 16]. See Supplementary Materials and methods for details. CELL CULTURE AND TRANSFECTION HEK293T cells were cultured at 37 °C and 5% CO2 in Dulbecco’s modified

Eagle’s medium (DMEM, Invitrogen) without sodium pyruvate containing 10% fetal bovine serum with 100 mg/ml streptomycin sulfate, and 100 U/ml penicillin G sodium. Transfection of cells with

expression vectors pCMV6-Kv1.2 (Cat. MC216959 OriGene) and pcDNA3.1-_σ_1-V5-His (kindly provided by Dr. Tsung-Ping Su) was done with Lipofectamine LTX DNA Reagent (Invitrogen) according to

manufacturer’s instructions. Stable cell lines expressing _σ_1 or Kv1.2 were established using G418 selection. Cells stably expressing Kv1.2 were transiently transfected with _σ_1-V5 vector.

SITE-DIRECTED MUTAGENESIS The D188N mutations and the C-terminal 16 amino acids deletion of _σ_1 were introduced sequentially to the pcDNA3.1-_σ_1-V5-His vector using the Phusion

Site-Directed Mutagenesis Kit (ThermoFisher) according to the manufacturer’s instructions. IMMUNOPRECIPITATION FROM TISSUE AND MEMBRANE ISOLATION Medial NAcSh was collected using the same

procedure as described in “Slice preparation and solutions”. After recovery and drug treatment, 2–3 slices at a time were transferred to ice-cold ACSF and NAcSh were microdissected. For

co-immunoprecipitation assays on tissue from in vivo cocaine-treated mice, NAcSh tissues were microdissected directly after slicing (as previously performed in [12]). See Supplementary

Materials and methods for details. STATISTICS Data acquisition and analysis were performed blind to experimental conditions when possible. Results are presented as mean ± S.E.M. Statistical

significance was assessed using two-tailed Student’s _t_-tests, one-way analysis of variance (ANOVA) or two-way repeated-measures ANOVA and Bonferroni post-hoc tests when appropriate.

RESULTS COCAINE DECREASES NACSH NEURONAL INTRINSIC EXCITABILITY IN D1R-EXPRESSING MSNS (D1R-MSNS) In vivo cocaine administration decreases NAcSh MSNs firing (Fig. 1a), consistent with

previous studies that were performed in similar conditions, that is, without distinguishing MSN subtypes [DA 1 vs. DA 2 receptor-expressing MSNs, (D1R- and D2R-MSNs)] ([16, 18,19,20],

reviewed in [13]). To determine its cellular mechanism, we developed a freshly dissected brain slice preparation that mimics in vivo physiological conditions. To this end, we applied cocaine

in vitro at a concentration of 3 μM for 1 h, which corresponds to the concentration and half-life of cocaine in the NAc when injected i.p. at standard doses (10–20 mg/kg) [21, 22]. Using

non-reporter C57BL/6J mice, we found that incubation of brain slices in cocaine (3 μM, 1 h) also depresses firing of NAcSh MSNs (Fig. 1b). Neuronal firing was assessed in cocaine-free ACSF

at least 20’ after transferring slices to the recording chamber, which is sufficient to wash out cocaine [23]. To ensure that decreased firing rate is not due to blockade of voltage-gated

Na+ currents (VGSCs) [24] by residual cocaine, we show that the low concentration of cocaine used (3 μM, 10 min) does not decrease Na+ currents (Supplementary Fig. S1a, b), consistent with

previous studies [25]; or the action potential’s amplitude (Supplementary Fig. S1c), which is directly controlled by Na+ current. Given the possible opponent roles of D1R- vs. D2R-MSNs in

psychostimulant reward [26,27,28] and consistent with a previous study [29], we found that cocaine-induced FRD occurs specifically in D1R-MSNs (recorded from MSNs in Drd1a-tdTomato C57 mouse

line, Fig. 1c) but not in D2R-MSNs (Fig. 1d). We also show that this effect is dose dependent (Fig. 1e, Supplementary Fig. S1d). Therefore, in subsequent studies, all recordings were

conducted in D1R-MSNs in brain slices from Drd1a-tdTomato C57 mice. Although cocaine-induced FRD is specific to D1R-MSNs, both our previous and others’ studies reliably obtained

cocaine-induced FRD without distinguishing the two sub-populations of MSNs ([11, 12, 16, 18,19,20], reviewed in [13]). Although this may appear surprising, it is reminiscent of

cocaine-induced synaptic potentiation in NAcSh neurons, that is, cocaine-induced synaptic plasticity in the NAcSh is specific to D1R-MSNs ([28], reviewed in [30]) but it is also obtained

without distinguishing the two sub-populations of MSNs ([15], reviewed in [31, 32]). This effect may be explained by the inhomogeneous distribution of D1R- vs. D2R-MSNs in the NAcSh. Indeed,

while studies have not specifically assessed neuronal subtype’s micro-distribution (potential existence of MSN subtype clusters) of D1R- and D2R-MSNs, both the distribution and the

proportion of MSN subtypes are inhomogeneous. In particular, quantitative localization studies revealed the existence of D2R-MSN-poor zones [33] in the dorsomedial shell, near the subregion

where our recordings were performed. Although qualitative but consistent with these findings, we show that while the total cell density in NAcSh appears homogenous, the rostral part of the

medial shell presents a high density of D1R-MSNs (Supplementary Fig. S2). Another hypothetical and non-exclusive contributing factor is that cocaine also decreases neuronal firing in the 17%

of NAcSh MSNs that co-express D1 and D2 receptors [34], allowing to obtain cocaine-induced FRD in the NAcSh consistently and reliably without distinguishing MSN subtypes. COCAINE-INDUCED

FRD IS INDEPENDENT OF DA OR OTHER MONOAMINE SIGNALING The effect of DARs activation on the regulation of NAc MSNs firing is well documented (e.g., [35, 36]), and overactivation of DARs is a

prominent hypothesis for cocaine-induced changes in the firing rate of NAc MSNs. However, studies show that coactivation of D1-like and D2-like receptors using DA or DAR agonists enhances

MSNs firing in the NAcSh [35], which is opposite to what we have observed using cocaine in our previous and present studies [12, 16, 19, 20, 29] and by other groups [19, 20, 29] (reviewed in

[13]). Altogether, these studies suggest that cocaine-induced FRD in NAcSh D1R-MSNs may involve additional or different mechanisms from DA signaling. In addition to inhibiting DA reuptake

[1, 2], cocaine is also an agonist of _σ_1 [3] (reviewed in [37]), and both DARs [38] and _σ_1 regulate VGICs (reviewed in [39,40,41]), which suggest that cocaine may alter VGICs via both

DAR- and _σ_1-dependent pathways. However, the relative contributions of _σ_1- and DA-dependent pathways in cocaine-induced changes in neuronal intrinsic excitability, and especially in

NAcSh D1R-MSNs, is not known. Therefore, to determine whether cocaine-induced FRD in the NAcSh is underpinned by a non-canonical, DA-independent mechanism, we incubated brain slices in DAR

antagonists prior to cocaine. We show that prior application of D1- (SCH23390, SCH, 2 μM) or D2-like receptor antagonists (Sulpiride, SULP, 10 μM) alone or combined does not prevent

cocaine-induced FRD (Fig. 2a, Supplementary Fig. S3). Application of these antagonists alone or together does not alter basal firing rate (Fig. 2a, Supplementary Fig. S3d). To support these

results further, if DAR-modulated VGICs also participate significantly to cocaine-induced FRD in NAcSh D1R-MSNs, we expect DAR blockade prior to cocaine to dampen the effect of cocaine on

neuronal firing. Here, we demonstrate that cocaine alone or with D1R- and D2R-like antagonists decreases neuronal firing to the same extent (Fig. 2a). However, it is interesting to note that

prior application of D1- (SCH23390, SCH, 2 μM) and D2-like receptor antagonists (SULP, 10 μM) slightly dampens cocaine-induced FRD at low depolarizing current injection (Supplementary Fig. 

S3c), however, this effect is not significant. As application of these antagonists together does not alter basal firing rate (Fig. 2a, Supplementary Fig. S3d), these data suggest that while

cocaine-induced DA modulation of VGICs may occur, these VGICs do not seem to impact cocaine-induced FRD in NAcSh D1R-MSNs significantly. As cocaine selectively decreases neuronal firing in

D1R-MSNs, we also assessed the effect of the D1-like receptor antagonist administered in vivo (SCH, 0.1 mg/kg, i.p.) prior to cocaine (15 mg/kg, five once-daily) and at a dose that also

abolishes acute psychomotor stimulant effects of cocaine. We found that in vivo SCH does not prevent cocaine-induced FRD in NAcSh D1R-MSNs (Fig. 2b). As cocaine also blocks norepinephrine

(NET) and serotonin transporters (SERT) and norepinephrine and serotonin signaling also modulate K+ currents and intrinsic excitability [42,43,44], we assessed the effect of a non-selective

and structurally different general monoamine uptake blocker on NAcSh MSNs firing (indatraline, a.k.a. Lu 19-005). Indatraline (30 nM, 1 h), at a concentration significantly higher than its

IC50 at the monoamine transporters (≈ 0.2–5 nM) [45,46,47], does not induce MSNs FRD on its own, or block cocaine-induced FRD (Fig. 2c). Altogether, these results are of particular

importance as they provide direct evidence that cocaine mediates some of its addiction-relevant neuroadaptations via a non-canonical DA-independent mechanism (and likely NE- and

5-HT-independent). COCAINE BINDING TO _Σ_1 INITIATES THE MECHANISM RESPONSIBLE FOR FRD IN NACSH D1R-MSNS Prior bath application of the prototypical _σ_1 antagonist BD1063 (500 nM), at a

concentration that does not alter basal firing rate, prevents cocaine-induced FRD (Fig. 3a). Cocaine is an agonist of _σ_1 [3] (reviewed in [37]) and has been shown to alter several sodium,

calcium and potassium conductances in NAc MSNs, each of which is consistent with a decrease in depolarization-induced firing [18, 19, 48,49,50]. These data raise the hypothesis that cocaine

binding to _σ_1 is the initial event that leads to the regulation of VGICs responsible for the FRD in NAcSh D1R-MSNs. However, although other conductances are altered upon cocaine exposure,

our previous electrophysiological and pharmacological studies suggest that they do not contribute significantly to cocaine-induced FRD [12]. Furthermore, combining electrophysiological,

pharmacological and biochemical analyses, we show that in vivo cocaine depresses neuronal firing via a mechanism that critically involves the formation of _σ_1-Kv1.2 protein complexes and

upregulation of Kv1.2 K+ channels at the plasma membrane of NAcSh MSNs [12]. Although we do not exclude the relative participation of other conductances in cocaine-induced FRD, to determine

whether cocaine-induced FRD is triggered by cocaine binding to _σ_1, we developed a cell culture model that recapitulates a critical biochemical outcome that underlies cocaine-induced FRD in

vivo, that is, enhanced _σ_1–Kv1.2 interactions (Supplementary Fig. S4a and [12]). First, we show that cocaine enhances co-immunoprecipitation (CoIP) of _σ_1 with Kv1.2 in both freshly

dissected brain slices (Fig. 3b, left) and HEK293T cells (Fig. 3b, right, Supplementary Fig. S4b). To determine whether the increase in _σ_1–Kv1.2 interactions correlates with recruitment of

Kv1.2 to surface, we isolated plasma membranes from NAcSh tissue and HEK293T cells using immobilized Concanavalin A magnetic beads [51,52,53] (Supplementary Fig. S4c), and found that

cocaine enhances surface levels of Kv1.2 (Fig. 3d), whereas total protein levels of Kv1.2 α-subunits and _σ_1 remain unchanged (Fig. 3c). These data also suggest that cocaine-induced

upregulation of _σ_1–Kv1.2 complexes and surface Kv1.2 are a conserved cellular mechanism that extend to non-neuronal heterologous systems. Therefore, we will use cocaine-induced increase in

_σ_1–Kv1.2 complexes as readout for cocaine-induced activation of _σ_1. Site-directed mutagenesis studies showed that cocaine binding to _σ_1 requires an aspartate residue at AA 188 located

near the C-terminus, and the last 16 amino-acid residues of _σ_1 [54, 55]. Therefore, we truncated _σ_1 from the last 16 residues and replaced Asp188 by Asn188 (Δ_σ_1-V5), then

overexpressed Δ_σ_1-V5 in HEK293T cell line along with Kv1.2 subunits. To verify otherwise competent chaperone capability of Δ_σ_1-V5, we first tested whether Δ_σ_1-V5 can associate with one

of _σ_1’s targets IP3 receptors (IP3Rs) [56], and whether _σ_1–IP3R protein complexes are dynamically regulated by ER stress, a known mechanism for _σ_1 [3]. We found that

tunicamycin-induced ER stress enhances associations of IP3R1 with both wt_σ_1 (native form of _σ_1) and Δ_σ_1-V5 (Fig. 3e). As _σ_1 exhibits protective properties against cell death

[57,58,59], we also ensured that the Δ_σ_1-V5 protective effect is preserved. We found that while tunicamycin-induced ER stress leads to 50% cell death in cells overexpressing Kv1.2 alone,

Δ_σ_1-V5 protected cells from tunicamycin-induced apoptosis to the same extent as wt_σ_1 (Fig. 3f). Although _σ_1 is an inter-organelle signaling modulator that exerts several distinct

functions (e.g., ER lipid metabolisms/transports [60], and indirectly regulating the transcription of genes [4]), these data demonstrate that the two necessary functions to test our

hypothesis, chaperone activity and protection against cell death, are preserved in Δ_σ_1-V5. Significantly, although cocaine upregulates wt_σ_1–Kv1.2 complex levels, it fails to upregulate

the formation of Δ_σ_1-Kv1.2 protein complexes with the mutant _σ_1 (Fig. 3g), similar to the effect of _σ_1 blockade with BD1063 (500 nM). This suggests that cocaine binding to _σ_1 is a

necessary mechanism for the recruitment of additional _σ_1–Kv1.2 protein complexes. COCAINE-INDUCED FRD IS MEDIATED VIA ACTIVATION OF INTRACELLULAR _Σ_1 _σ_1 is enriched in intracellular

organelles, and especially at the ER level. Protonated cocaine, like several drugs including antidepressants and abused substances [61], coexists with their deprotonated form in the

physiological milieu (membrane permeant), indicating that cocaine can cross the plasma membrane. In addition, although it is still a subject of research, it is thought that _σ_1 can also be

inserted in the plasma membrane. Although the bulk of the C-terminus (containing the binding pocket) may be located either in the cytosol (crystal study in micelles) [62] or in the

extracellular space (in vivo study in dorsal root ganglions) [63], Ruoho and colleagues demonstrate that inactive intracellular oligomeric states can bind the _σ_1 agonist (+)-pentazocine in

vitro, and not monomer/dimer states that may exist at the membrane ([64], reviewed in [65]). Altogether, this suggests that cocaine targets intracellular _σ_1. To determine whether cocaine

activating intracellular _σ_1 is the initiating mechanism for FRD in NAcSh D1R-MSNs, we introduced cocaine directly into the recording pipette. Beforehand, and to make accurate predictions

on the onset of cocaine-induced FRD in D1R-MSNs, we perfomed a between-cell analysis with cocaine applied extracellularly and found that neuronal firing is decreased within ≈30 min from

cocaine application (Fig. 4a, Supplementary Fig. S5a). Second, we introduced cocaine into the recording patch pipette and performed a within-neuron comparison. We analyzed spike trains

elicited at a nonsaturating current injection of 200 pA or 240 pA, and found that intracellular cocaine decreases NAcSh D1R-MSNs firing within ~30 min after establishing whole-cell

configuration (Fig. 4b, Supplementary Fig. S5a). Next, using (–)-cocaine methiodide (Coc-M), a chemical analog of cocaine with a stable positive charge at physiological pH that prevents free

diffusion through membranes [66], we found that extracellular Coc-M at an equimolar concentration (3.9 μM) fails to decrease NAcSh D1R-MSNs firing (Fig. 4c, Supplementary Fig. S5b). Taken

together, these results obtained from complementary strategies provide direct evidence that cocaine depresses D1R-MSNs firing rate via its action on intracellular _σ_1. DISCUSSION Using in

vivo and in vitro models, we provide convergent evidence that cocaine-induced hypoactivity of NAcSh D1R-MSNs is mediated by a DA-independent mechanism and is neither induced nor blocked by

accumulation of monoamines in the synaptic cleft (Fig. 2 and Supplementary Fig. S3). Second, using site-directed mutagenesis of _σ_1 cocaine binding site in HEK293T cells, and both

extracellular cocaine methiodide (non-permeant cocaine) and intracellular application of cocaine in brain slices, we demonstrate that cocaine-induced FRD is triggered by cocaine binding to

intracellular _σ_1 (Fig. 3g and Fig. 4). Determining the mechanism by which cocaine crosses the plasma membrane, free diffusion or transported via an unidentified target, is beyond the scope

of this study. Altogether, our study provides direct evidence that besides actions mediated through conventionally studied mechanisms, cocaine also engages a mechanism that is DA-

independent, but _σ_1 binding-dependent. This mechanism of action leads to neuronal hypoactivity of NAcSh MSNs firing—an adaptation that promotes behavioral responses to cocaine (reviewed in

[13]). Although a previous study [29] and the present found that cocaine-induced FRD occurs specifically in D1R- but not in D2R-MSNs (Fig. 1c, d), these findings are unexpected. This

suggests that other neuronal subtype-specific factors may control _σ_1-dependent functions (reviewed in [40, 41]). Further investigations are warranted to identify these factors. Although we

hypothesize that a differential expression of targets of interests (i.e., _σ_1 and Kv1.2) is a factor, there is no study to date suggesting different levels of _σ_1 and Kv1.2 proteins in

D1R- vs. D2R-MSNs. NON-CANONICAL DA-INDEPENDENT MECHANISM TRIGGERED BY PSYCHOSTIMULANT DRUGS Earlier studies show a clear implication of DA signaling in the acute locomotor stimulatory

effects of cocaine, the gradual increase in cocaine-induced locomotion upon repeated cocaine treatment [67,68,69,70], and in cocaine self-administration (reviewed in [71]). However, several

studies using various experimental designs to assess behavioral sensitization to cocaine in mice and rats showed that in vivo blockade of D1- or D2-like receptors, at doses that abolish

acute psychomotor stimulant effects of cocaine, just attenuate or fail to prevent the induction of behavioral sensitization to cocaine, that is, enhanced psychomotor behavior when animals

are challenged during withdrawal [72,73,74]. In addition, other studies suggest that the effect of systemic D1-like receptor antagonists on behavioral sensitization may also depend on

cocaine doses used when animals are challenged [75]. Together, these studies imply that psychostimulant-induced DA signaling is important in the development of addiction-related behaviors;

however, DA signaling may be differentially involved as a function of experimental conditions, the behavioral paradigm used, or the behavioral stage under consideration (e.g., induction or

expression of psychomotor sensitization). Therefore, cocaine and other stimulants may engage additional mechanisms that would participate in specific addiction-related phenotypes. In that

regard, a stream of studies demonstrates that cocaine also engages mechanisms that are dependent on _σ_1, but independent of DA signaling, and which contribute to cocaine addiction [10]. For

example, animals with cocaine experience, but not after experience with food reinforcement, self-administer _σ_1 agonists (e.g., PRE-084 and (+)-Pentazocine) [76] at doses that do not

induce DA release in the NAcSh [77] (reviewed in [10]). Furthermore, self-administration of PRE-084 is blocked by _σ_1 antagonists (e.g., BD1063), but not blocked by the D1R antagonist SCH

39166 effective against cocaine ([76], reviewed in [10]). These data demonstrate that the role of DA in behavioral response to cocaine or the development of addiction-relevant behaviors is

complex and that cocaine engages additional mechanisms that also participate in the development of addiction-relevant phenotypes. Thus, the role of _σ_1 vs. DARs in cocaine’s behavioral

effects remains elusive. Their contributions are likely synergetic, and teasing apart their relative contributions in cocaine-related behaviors and in specific stages of the addiction cycle

(acquisition, extinction, relapse) has been an intense subject of research in Dr. Katz’s laboratory (reviewed in [10]). In the present study, we identify a DA-independent cellular mechanism

by which cocaine alters neuronal intrinsic excitability (i.e., neuronal firing) of NAcSh MSNs. Non-canonical, intracellular, actions of psychiatric drugs or abused substances are emerging as

intriguing complementary mechanisms that contribute to their pernicious addictive properties [61]. For example, besides conventional action of nicotine on membrane nicotinic acetylcholine

receptors (nAChRs), nicotine exhibits pharmacological chaperoning activity upon binding to specific intracellular nAChRs located at the ER level, which underlies some initial events of

nicotine addiction (reviewed in [61]). Directly pertinent to the present study, intravenous self-administration of methamphetamine (METH) in rats, a psychostimulant drug that is chemically

different from cocaine, decreases NAcSh MSNs firing rate [78]. METH also binds to _σ_1 [79], consistent with the capability of the _σ_1 ligand-binding cavity to bind structurally different

compounds [37, 62]. Future studies are warranted to determine whether NAcSh neuronal hypoactivity is a unifying _σ_1-dependent, DA-independent, mechanism among abused psychostimulant drugs

that bind _σ_1. CONCLUSION Present and future information obtained on DA-independent, but _σ_1-dependent, mechanisms will have the potential to pave the way to novel and combinatorial

pharmacotherapies to specifically treat stimulant abuse or provide alternatives for treatment-resistant stimulant abuse. Furthermore, because this mechanism of action occurs in cell

type-specific manner (D1R- vs. D2R-MSNs), it suggests that the diversity of _σ_1’s effects on cellular physiology is influenced by _σ_1’s differential engagement of multiple signaling

pathways that may depend on several biological factors. The translational implication of such findings is important; it suggests that directly targeting _σ_1 may have less unwanted side

effects than originally expected. More fundamentally, the present findings also further our understanding of the mechanisms through which _σ_1 regulates K+ channel trafficking—a topic of

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studies. Neuropharmacology. 2005;49:638–45. Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Drs. Tsung-Ping Su (National Institute on Drug Abuse, NIDA)

for kindly providing pcDNA3.1-_σ_1-V5-His, Mark J. Thomas (University of Minnesota) for providing Drd1a-tdTomato C57BL6J mouse line, and Ilya Bezprozvanny for providing the IP3R1 construct

and antibody against IP3R1. We also thank Dr. Hau-Jie Yau (NIDA) for advising on recording Na+ currents, Deena Sajitharan for technical assistance and Dr. Steven J. Shabel for careful

reading of the manuscript. This work was supported by the start-up funding from the University of Texas Southwestern Medical Center (UTSW) and by support from the UTSW President’s Research

Council. AUTHOR CONTRIBUTIONS Conceptualization, SK; Methodology, ID-R, AS and SK; Formal analysis, ID-R and SK; Investigation, ID-R., FG-O., AS, and SK.; Writing—Original draft, SK;

Writing-review and editing, ID-R, AS and SK; Visualization, SK.; Funding acquisition, SK; Supervision, SK. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Psychiatry, University

of Texas Southwestern Medical Center, Dallas, TX, 75390, USA Ilse Delint-Ramirez, Francisco Garcia-Oscos, Amir Segev & Saïd Kourrich Authors * Ilse Delint-Ramirez View author

publications You can also search for this author inPubMed Google Scholar * Francisco Garcia-Oscos View author publications You can also search for this author inPubMed Google Scholar * Amir

Segev View author publications You can also search for this author inPubMed Google Scholar * Saïd Kourrich View author publications You can also search for this author inPubMed Google

Scholar CORRESPONDING AUTHOR Correspondence to Saïd Kourrich. ETHICS DECLARATIONS CONFLICT OF INTEREST The authors declare that they have no conflict of interest. ELECTRONIC SUPPLEMENTARY

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THIS ARTICLE CITE THIS ARTICLE Delint-Ramirez, I., Garcia-Oscos, F., Segev, A. _et al._ Cocaine engages a non-canonical, dopamine-independent, mechanism that controls neuronal excitability

in the nucleus accumbens. _Mol Psychiatry_ 25, 680–691 (2020). https://doi.org/10.1038/s41380-018-0092-7 Download citation * Received: 10 August 2017 * Revised: 03 April 2018 * Accepted: 13

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