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Cocaine-induced endocannabinoid signaling mediated by sigma-1 receptors and extracellular vesicle secretion

  1. Yoki Nakamura
  2. Dilyan I Dryanovski
  3. Yuriko Kimura
  4. Shelley N Jackson
  5. Amina S Woods
  6. Yuko Yasui
  7. Shang-Yi Tsai
  8. Sachin Patel
  9. Daniel P Covey
  10. Tsung-Ping Su  Is a corresponding author
  11. Carl R Lupica  Is a corresponding author
  1. National Institute on Drug Abuse, National Institutes of Health, United States
  2. Vanderbilt University Medical Center, Vanderbilt University, United States
  3. University of Maryland School of Medicine, United States
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Cite this article as: eLife 2019;8:e47209 doi: 10.7554/eLife.47209

Abstract

Cocaine is an addictive drug that acts in brain reward areas. Recent evidence suggests that cocaine stimulates synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) in midbrain, increasing dopamine neuron activity via disinhibition. Although a mechanism for cocaine-stimulated 2-AG synthesis is known, our understanding of 2-AG release is limited. In NG108 cells and mouse midbrain tissue, we find that 2-AG is localized in non-synaptic extracellular vesicles (EVs) that are secreted in the presence of cocaine via interaction with the chaperone protein sigma-1 receptor (Sig-1R). The release of EVs occurs when cocaine causes dissociation of the Sig-1R from ADP-ribosylation factor (ARF6), a G-protein regulating EV trafficking, leading to activation of myosin light chain kinase (MLCK). Blockade of Sig-1R function, or inhibition of ARF6 or MLCK also prevented cocaine-induced EV release and cocaine-stimulated 2-AG-modulation of inhibitory synapses in DA neurons. Our results implicate the Sig-1R-ARF6 complex in control of EV release and demonstrate that cocaine-mediated 2-AG release can occur via EVs.

https://doi.org/10.7554/eLife.47209.001

eLife digest

The cannabis plant contains hundreds of different chemicals, including more than sixty types of cannabinoids. By binding to specific sites on brain cells, cannabinoids change how cells communicate with one another. This in turn triggers widespread alterations in brain activity, which can affect mood, appetite, coordination and perception.

But not all cannabinoids come from plants. The brain also produces its own versions, known as endocannabinoids (or eCBs for short). These bind to the same sites on brain cells as the plant-derived chemicals. Changes in endocannabinoid activity have been implicated in various brain disorders. These include Alzheimer's disease, epilepsy and stress disorders. They may also have a role in drug addiction. Exposing rats to cocaine causes endocannabinoid levels to increase in areas of the brain that process pleasurable sensations. This suggests that the release of endocannabinoids may contribute to cocaine addiction. But how cocaine triggers this release has been unclear.

By studying brain tissues and cells kept alive in petri dishes, Nakamura, Dryanovski et al. show that cocaine drives cells to release endocannabinoids via a process called extracellular vesicle release. In essence, cocaine causes cells to make endocannabinoids that are then enclosed inside membrane-bound packages. These packages – or extracellular vesicles – can then fuse with the cell’s outer membrane. Multiple proteins must interact with each other for cells to assemble and release extracellular vesicles. Nakamura, Dryanovski et al. show that disrupting these interactions prevents vesicles from forming, and also prevents cocaine from triggering endocannabinoid release. Blocking extracellular vesicle release prevents cocaine from altering communication between brain cells.

Cocaine thus drives endocannabinoid release in the brain’s pleasure centers via the assembly of extracellular vesicles. Using other drugs to manipulate the protein interactions that underlie vesicle assembly could provide a new way to counter cocaine addiction.

https://doi.org/10.7554/eLife.47209.002

Introduction

The sigma-1 receptor (Sig-1R) is a small protein that resides at the endoplasmic reticulum (ER)-mitochondrion interface (mitochondrion-associated ER membrane; MAM) (Hayashi and Su, 2007; Hayashi et al., 2009; Mori et al., 2013), where it constrains type-3 inositol 1,4,5-trisphosphate receptors (IP3R3) to facilitate Ca2+ signaling from ER to mitochondria (Hayashi and Su, 2007; Hayashi et al., 2009). In addition, the Sig-1R binds a wide range of molecules, including psychotropic drugs and psychostimulants, such as cocaine and methamphetamine (Largent et al., 1987), and can translocate to other cellular regions to associate with organelles, proteins, plasma membranes, and the nuclear envelope to control trafficking of other molecules, such as ion channels and receptors in neurons (Su et al., 2016; Yasui and Su, 2016). These diverse signaling roles for the Sig-1R highlight a widespread influence on cellular function that is incompletely understood.

Substantial data suggest that the Sig-1R is also a target of the abused psychostimulant cocaine (Hayashi and Su, 2007; Sharkey et al., 1988; Hayashi and Su, 2003; Kourrich et al., 2013; Tsai et al., 2015; Chen et al., 2007). In the mouse nucleus accumbens (NAc), cocaine decreases the excitability of GABAergic medium spiny neurons by strengthening an association between the Sig-1R and Kv1.2 potassium channels, contributing to behavioral sensitization to the drug (Kourrich et al., 2013). Moreover, the Sig-1R is also involved in cocaine reward (Romieu et al., 2002). Given the diverse demonstrated roles for the Sig-1R in cellular signaling, its regulation by cocaine has the potential to affect many unknown cellular properties.

Extracellular vesicles (EVs) are a diverse group of membranous entities of endosomal origin that are secreted from a broad range of cell types (van Niel et al., 2018). The EV classification broadly includes exosomes and microvesicles that range in size from 30 to 150 nm, and 100–1000 nm, respectively. Exosomes are formed by invagination of the endosomal membrane to form multivesicular bodies that are released into the extracellular space via budding of the cellular membrane, whereas microvesicles are formed by budding of plasma membrane (van Niel et al., 2018; Radhakrishna et al., 1996; Huang-Doran et al., 2017). It is increasingly apparent that EV formation occurs through highly regulated cellular processes (Abels and Breakefield, 2016), that permit their participation in intercellular communication via delivery of cargos of RNAs, microRNAs, proteins, and bioactive lipids such as prostaglandins (van Niel et al., 2018; Huang-Doran et al., 2017; EL Andaloussi et al., 2013). This implicates EVs in a wide range of physiological and pathological processes. EV motility can be controlled by signaling molecules such as the guanine-nucleotide binding protein, ADP-ribosylation factor 6 (ARF6) (Abels and Breakefield, 2016; Muralidharan-Chari et al., 2009; D'Souza-Schorey and Chavrier, 2006). As a small GTPase, ARF6 exists in GTP- or GDP-bound forms (ARF6-GTP or ARF6-GDP), and stimulation of ARF6 by neurotransmitters or growth factors recruits guanine nucleotide exchange factors (GEFs) to convert ARF6-GDP to the active ARF6-GTP (EL Andaloussi et al., 2013). Although ARF6 itself has GTPase activity, ARF6-GTP requires GTPase-activating proteins (GAPs) to hydrolyze to its inactive ARF6-GDP form. ARF6-GTP influences a wide variety of cellular events including endocytosis, actin cytoskeleton reorganization and phosphoinositide metabolism in many types of cells. Importantly, ARF6-GTP is involved in EV release from plasma membranes (Muralidharan-Chari et al., 2009; Than et al., 2017), and exosome budding into multivesicular bodies (Ghossoub et al., 2014; Friand et al., 2015; Imjeti et al., 2017). Thus, GEFs and GAPs regulate ARF6 activity to then modulate EV secretion (D'Souza-Schorey and Chavrier, 2006). Also, since ARF6 is a GTPase, it is noteworthy that another GTPase, Rac-GTPase, forms a complex with the Sig-1R (Natsvlishvili et al., 2015), suggesting the possibility that the Sig-1R may interact with other molecules of this class. Collectively, these points of regulation position EVs and ARF6 as important participants in diverse physiological and pathological processes (van Niel et al., 2018).

Endocannabinoids (eCB) are lipid signaling molecules that activate CB1 or CB2 cannabinoid receptors. Of these, CB1Rs are expressed at high levels on neuronal axon terminals where they inhibit fast neurotransmitter release (Misner and Sullivan, 1999; Hoffman and Lupica, 2000; Katona et al., 1999). The eCBs are typically synthesized in postsynaptic structures, such as dendrites, to then retrogradely activate CB1Rs on axon terminals (Wilson and Nicoll, 2001). Moreover, eCBs are not released via canonical mechanisms of calcium-dependent synaptic vesicle exocytosis, but rather through poorly understood processes. Recent evidence gathered using cell cultures suggests that the eCB N-arachidonoylethanolamine (AEA, anandamide) is found in EVs, suggesting a possible mechanism to release these messengers and permit retrograde eCB signaling (Gabrielli et al., 2015a; Gabrielli et al., 2015b). Another eCB, 2-arachidonoylglycerol (2-AG), is released from neurons in an activity-dependent fashion, or via neurotransmitter stimulation of phospholipase-regulating G-protein coupled receptors (GPCRs) (Kano et al., 2009; Maejima et al., 2005; Alger and Kim, 2011). Recent evidence shows that inhibition of catecholamine uptake by cocaine leads to activation of GPCRs that stimulate 2-AG synthesis in the rodent ventral tegmental area (VTA) (Wang et al., 2015). Moreover, as VTA GABAergic axons express CB1Rs, the cocaine-stimulated increase in 2-AG inhibits GABA release via these receptors (Wang et al., 2015; Riegel and Lupica, 2004), and this can be used as a sensitive measure of eCB function. Although measurements like these are used to detect eCBs throughout the CNS, the mechanisms through which these lipids cross the extracellular space to bind to presynaptic CB1Rs remain poorly understood.

Given that cocaine stimulates 2-AG synthesis, can act as a Sig-1R agonist, and that the Sig-1R interacts with Rac-GTPase, we hypothesize that it may also control other GTPases such as ARF6, a known EV release modulator (Muralidharan-Chari et al., 2009; Ghossoub et al., 2014; Natsvlishvili et al., 2015; Tsai et al., 2009), and this might regulate 2-AG release. Through convergent experiments we demonstrate that Sig-1Rs can control EV release via interaction with ARF-6, and that cocaine stimulates this process. Moreover, the cocaine-evoked 2-AG release required intact Sig-1Rs, ARF-6, and cytoskeletal function, implicating EVs as a mechanism for 2-AG release in the VTA.

Results

Cocaine activation of Sig-1Rs stimulates EV release from NG-108 cells

To investigate whether Sig-1Rs are involved in EV function, we first conducted studies in NG-108 cells to permit manipulation of signaling pathways. The integrin β1 (Iβ1; CD29) protein mediates transcellular interaction of EVs with target membranes, and is a useful marker of EVs isolated through differential sequential sucrose-gradient centrifugation (van Niel et al., 2018; EL Andaloussi et al., 2013; Muralidharan-Chari et al., 2009; Imjeti et al., 2017; Benmoussa et al., 2019; Momen-Heravi et al., 2013). We prepared membrane fractions enriched in EVs in effluent from NG-108 cells and measured Iβ1 using western blots. Cocaine (10 µM) caused a time- and concentration-dependent increase in the accumulation of Iβ1 in isolated fractions from these NG-108 cells (Figure 1A and B), suggesting that cocaine increased EV release. Because our previous studies show that cocaine interacts with Sig-1Rs, we next investigated their involvement in cocaine-stimulated EV release. We found that the Sig-1R agonists PRE-084, or fluvoxamine, both increased the Iβ1-marker of EV release from NG-108 cells in the absence of cocaine (Figure 1B), and that pretreatment with either of the Sig-1R antagonists, BD1063 (Figure 1C) or NE100 (Figure 1D), prevented the effect of cocaine. We also found that the knock-down of Sig-1Rs with siRNA alone significantly increased Iβ1 and abolished the stimulatory effect of cocaine (Figure 1—figure supplement 1A), and that overexpression of Halo-tagged Sig-1Rs decreased EV release from NG-108 cells, but also blocked the effect of cocaine (Figure 1—figure supplement 1B). These data support a mechanism in which Sig-1Rs tonically inhibit EV release, and this inhibition is relieved in the presence of cocaine. Having established that Sig-1Rs are involved in the stimulatory effect of cocaine on EV release in NG-108 cells, we next investigated the role of additional other signaling molecules known to also regulate EV secretion (van Niel et al., 2018; Muralidharan-Chari et al., 2009; Imjeti et al., 2017).

Figure 1 with 1 supplement see all
Cocaine stimulates EV release via Sig-1R and ARF6 signaling in NG108 Cells.

(A) Effect of cocaine (10 µM) on integrin β1 (Iβ1) concentration in EV-rich fractions of NG-108 cells at several time points. Western blots also show relative amounts of Iβ1 in cell lysates (CL), and α-tubulin protein (α-tub.) as a control. Bar graph showing the relative change in Iβ1 at 30 and 60 min after cocaine treatment (mean ± S.E.M, F2,9 = 5.7, p=0.026, one-way ANOVA, *=p < 0.05 compared to control, Dunnett’s multiple comparison test). The number of replications of the experiment at left is shown in parentheses for each group in the bar graph. (B) Concentration-dependent effect of cocaine, and effects of the Sig-1R agonists, PRE084 (PRE, 1 µM), and Fluvoxamine (Flv, 10 µM) on Iβ1 concentration in the NG-108 cell culture media, 30 min after treatments (n = 1). (C–D) Sig-1R antagonists prevent cocaine-induced EV release in NG-108 cells. BD1063 (BD, 1 µM) or NE100 (NE, 1 µM) were applied to NG-108 cell cultures 10 min before cocaine treatment (C: means ± S.E.M, F3,15 = 6.2, p=0.006, one-way ANOVA, *=p < 0.05, **=p < 0.01, Dunnett’s multiple comparison test; D: means ± S.E.M, F3,12 = 10.4, p=0.001, one-way ANOVA, *=p < 0.05, **=p < 0.01, ***=p < 0.001, Dunnett’s multiple comparison test). (E) Inhibition of ARF-6 activation by the GEF inhibitor, SH3 (10 µM) blocks the increase in EV release caused by cocaine in NG-108 cells. Cocaine (10 µM) was applied for 30 min, beginning 10 min after SH3 application (n = 4; means ± S.E.M, F3,14 = 6.5, p=0.005, one-way ANOVA, *=p < 0.05, **=p < 0.01, Dunnett’s multiple comparison test). (F) Immunoprecipitation of the Sig-1R/ARF6 complex. Halo-Sig-1R was co-transfected with either cyan-fluorescent protein (CFP) and ARF6 (WT)-CFP into NG108 cells (n = 1). (G) Sig-1R prefers ARF6 inactive form. Halo-Sig-1R was co-transfected with CFP, ARF6 (Q67L: mimicking ARF6-GTP)-CFP, or ARF6 (T27N: mimicking ARF6-GDP)-CFP into NG108 cells and co-immunoprecipitation studies performed (n = 1). The number of replications of each experiment is shown in parentheses for each group in the bar graphs. See Source data 1 for values used in statistical analyses. Figure 1—figure supplement 1 shows that Sig-1R knockdown alters cocaine effects on EV release as well as the identification of the ARF6 binding site in NG108 cells.

https://doi.org/10.7554/eLife.47209.003

Cytohesins are a family of GEFs that activate ARFs by catalyzing a shift from GDP- to GTP-bound forms (D'Souza-Schorey and Chavrier, 2006; Frank et al., 1998; Hafner et al., 2006), and this can trigger EV release from LOX cells (D'Souza-Schorey and Chavrier, 2006; Than et al., 2017). To determine whether ARF6 is similarly involved in cocaine-induced release of EVs in NG108 cells, we used the GEF inhibitor secinH3 (SH3, 10 µM) (Hafner et al., 2006) and found that it prevented the cocaine-stimulated increase in Iβ1 levels in the EV fractions (Figure 1E). To next determine the nature of the association between ARF6 and Sig-1R proteins in NG-108 cells, we overexpressed ARF6 mutants that mimic either the active, GTP-bound (Q67L), or the inactive GDP-bound (T27N) forms of this protein, and performed co-immunoprecipitation experiments with a Halo-tagged Sig-1R (Halo-Sig-1R) (Radhakrishna et al., 1996; Muralidharan-Chari et al., 2009). We found that the Halo-Sig-1R co-immunoprecipitated much more strongly with the GDP-bound form of ARF6 (ARF6-T27N), compared to either wild-type ARF6, or the GTP-bound form (ARF6-Q67L) (Figure 1F, Figure 1G). This suggests that the Sig-1R more strongly binds the inactive GDP-ARF6, rather than the active GTP-ARF6.

As previous studies show that the Sig-1R C-terminus region contains a chaperone domain that interacts with MAM proteins (Hayashi and Su, 2007; Su et al., 2016; Ortega-Roldan et al., 2013), we also performed experiments with mutant Sig-1Rs to determine the regions of interaction with ARF6-GDP (Figure 1—figure supplement 1C). NG-108 cells were transfected with plasmids expressing Halo-tagged N- or C-termini on the full-length Sig-1R (Halo-Sig-1R and Sig-1R-Halo, respectively), or on truncated forms of the Sig-1R (Sig-1R-1–60-Halo or Halo-Sig-1R-61–223) that contained chaperone (Hayashi and Su, 2007), or ligand binding motifs (Chen et al., 2007; Pal et al., 2008), respectively. We then examined whether the Halo-tagged receptors co-immunoprecipitated with either the active or the inactive ARF6 mutants described above. The inactive form of ARF6 (ARF6-T27N) co-precipitated with Sig-1R-61–223-Halo, but not with Sig-1R-1–60-Halo (Figure 1—figure supplement 1C), suggesting that the C-terminus, chaperone region of the Sig-1R interacts with GDP-bound ARF6. Interestingly, co-immunoprecipitation also revealed that ARF6-T27N interacted with the Halo-Sig-1R, but not the Sig-1R-Halo (Figure 1—figure supplement 1C), suggesting that the C-terminus tag interferes with the interaction between Sig-1R and ARF6.

Taken together, our data in NG-108 cells support a model in which the chaperone region of the Sig-1R binds to the inactive form of ARF6 (GDP-ARF6) to tonically inhibit EV release. Therefore, we next examined the co-localization of ARF6 and Sig-1Rs and their ability to regulate EV release in the mouse midbrain to determine the functional relevance of this interaction.

Sig-1Rs mediate effects of cocaine on EV release in mouse midbrain

Mice received single injections of cocaine (15 mg/kg, i.p.), followed by removal and processing of the midbrain for EV content (Figure 2—figure supplement 1). In agreement with previous reports (Perez-Gonzalez et al., 2012; Polanco et al., 2016), a membrane fraction 3 (fr3), obtained by sequential sucrose-gradient centrifugation, was isolated and found to be enriched with several markers of EVs, such as Iβ1, alix, and flotillin-1 (Figure 2A). Moreover, high concentrations of ARF6 and tyrosine hydroxylase (TH) were found in the EV enriched fr3 (Figure 2A). However, because of the stringency of the EV isolation procedure, only a small amount of material could be obtained for analysis from these fractions. Therefore, in several experiments, we also utilized a total EV membrane fraction preparation (tEV) that was not subjected to a stepwise sucrose gradient, but nevertheless contained the same EV markers as fr3 (Figure 2—figure supplement 1). The mean size of the midbrain tEVs was 154 ± 1.41 nm (Figure 2C), and midbrain tEVs contained higher levels of Iβ1, ARF6, and TH, compared to tEVs isolated from cortex and hippocampus (Figure 2B).

Figure 2 with 1 supplement see all
Effect of cocaine on EV secretion in mouse midbrain.

(A) Representative western blots of different sucrose fractions (F1–F6) of EVs isolated from mouse midbrain, showing the markers tyrosine hydroxylase (TH), Iβ1 (Inte. β1), ARF6, Alix, and Flotillin-1. The mitochondrial marker (cytochrome-c: Cyto.-c) was also used as a control, and is western blots from total brain lysates (BL) are also shown (2–3 replicates). (B) Representative western blots from tEVs obtained from midbrain (M), cortex (C), and hippocampus (H) (two replicates). (C) The size distribution of tEVs in mouse midbrain, as measured by NanoSight particle tracking (n = 3 replicates). (D) Proteinase K (PK) treatment of EV preparations from mouse midbrain, with, and without Triton-X (TX) included (two replicates). (E) Effect of cocaine (15 mg/kg, i.p.) on EV markers in preparations from WT mouse midbrain at several 30- and 60 min time points. Bar graphs of the experiments described in E (mean ± S.E.M; TH: F2,12 = 7.3, p=0.0084, one-way ANOVA, *=p < 0.05 compared with naive, Dunnett’s multiple comparison test; Iβ1: F2,9 = 15.2, p=0.001, one-way ANOVA, **=p < 0.01 compared with naive, Dunnett’s multiple comparison test; ARF6: F2,9 = 2.5, p=0.14, one-way ANOVA). (F) Effect of the Sig-1R antagonist (BD1063: BD, 10 mg/kg, s.c.) on cocaine-evoked tEV release in WT mouse midbrain, 30 min after the in vivo cocaine injection. The Bar graphs shows mean effects from these experiments (mean ± S.E.M, TH: F3,12 = 14.2, p=0.0003, one-way ANOVA, **=p < 0.01, ***=p < 0.001, Dunnett’s multiple comparison test; Iβ1: F3,12 = 16.3, p=0.0002, one-way ANOVA, **=p < 0.01, ***=p < 0.001, Dunnett’s multiple comparison test; ARF6: F3,12 = 1.5, p=0.26, n.s., not significant one-way ANOVA). (G) The effect of cocaine on tEV release is absent in Sig-1R knock out mouse midbrain, 30 min after in vivo cocaine injection. The Bar graph shows the means from this experiment (n.s., not significant, unpaired t-test). The number of replications of each experiment is shown in parentheses for each group in the bar graphs. Details All statistical comparisons. See Source data 1 for values used in statistical analyses. Figure 3—figure supplement 1 shows specificity of the Sig-1R antibody. Figure 2—figure supplement 1 shows the protocol for isolation of EVs from mouse midbrain.

https://doi.org/10.7554/eLife.47209.005

The topology of TH, Iβ1, and ARF6 in midbrain tEV preparations was next examined using the broad-spectrum serine protease, proteinase-K (PK) (Wang et al., 2017; de Jong et al., 2016). In tEVs not treated with Triton X detergent, PK decreased only Iβ1 (Figure 2D), which is consistent with its location on the plasma membrane (van Niel et al., 2018; EL Andaloussi et al., 2013; Muralidharan-Chari et al., 2009; Imjeti et al., 2017). In contrast, all three proteins were degraded by PK in tEV preparations treated with Triton X (Figure 2D), suggesting that, unlike Iβ1, TH and ARF6 are located within EVs, rather than on their membranes.

Because they were found in EV-rich preparations of midbrain, TH, Iβ1, and ARF6 were used as markers to evaluate the effect of cocaine on tEVs. Like NG-108 cells, cocaine (15 mg/kg) increased Iβ1 (and TH) levels in midbrain tissue within 30 min of an intraperitoneal (i.p.) injection (Figure 2E), and this returned to control levels 60 min following cocaine treatment (Figure 2E). However, ARF6 levels were not significantly altered by cocaine (Figure 2E). As in NG-108 cells, the cocaine-stimulation of tEV markers in midbrain was also prevented by the Sig-1R antagonist, BD1063 (Figure 2F). Moreover, cocaine failed to increase any of the tEV markers (Figure 2G) in midbrain preparations from mice lacking the Sig-1R gene (Sigmar1), suggesting that Sig-1Rs are essential for cocaine-induced tEV release in mouse midbrain.

The Sig-1R associates with the inactive form of ARF6 in mouse midbrain

To determine cellular locations of the Sig-1R we used immunofluorescence confocal microscopy in the mouse ventral midbrain. We found that Sig-1R (Mavlyutov et al., 2016) and TH fluorescence signals were colocalized (Figure 3A), and as TH is a marker for DA neurons in the ventral midbrain, the data suggest that Sig-1Rs are found in DA neurons. However, the Sig-1R signal was also found associated with the vesicular GABA transporter (vGAT), a marker of GABA neurons in the mouse ventral midbrain (Figure 3A). Therefore, the Sig-1R is likely expressed in both DA and GABA neurons in the midbrain. Immunofluorescence confocal microscopy also revealed co-localization of Sig-1R and ARF6 in TH-positive neurons in the mouse ventral midbrain (Figure 3C), and these proteins co-immunoprecipitated in midbrain samples from wild-type, but not Sig-1R knockout mice (Figure 3D). Also, the Sig-1R immunohistochemical signal was absent in Sig-1R knockout mice (Figure 3—figure supplement 1).

Figure 3 with 1 supplement see all
The Sig-1R interacts with ARF6 at the MAM in mouse midbrain.

(A) Confocal microscopy shows Sig-1R fluorescence immunostaining (Red) in association with either TH (Blue)-, or vGAT (Green)-positive neurons in the wildtype mouse VTA. Scale bar = 50 µm. (B) The subcellular distribution of proteins in wildtype mouse midbrain (P1: nuclear fraction; Mito: mitochondrial fraction; P3: microsomal fraction, containing plasma membrane and ER; Cyt: cytosolic fraction; NucleoP: nucleoporin p62; Cyto-c: cytochrome-c; TH: tyrosine hydroxylase; HSP90: heat-shock protein 90). (C) Confocal microscopic images showing co-localization of fluorescence immunostaining of the Sig-1R (Red) and ARF6 (Blue) in TH (Green)-positive neurons in the wildtype mouse VTA (scale bar = 20 µm). (D) Immunoprecipitation (IP) of the Sig-1R/ARF6 complex. Brain lysates were prepared from wildtype or Sig-1R KO mouse midbrain, immunoprecipitated with anti-ARF6 antibody, and then probed with anti-Sig1R, ARF6, and GAPDH antibody. (E) Schematic drawing of the interaction between Sig1R and ARF6 in mouse midbrain. Each experiment was replicated twice. Figure 3—figure supplement 1 shows the absence of Sig-1R immunofluorescence in the Sig-1R knockout mouse brain. Also see Figure 8—figure supplement 1 for proposed interaction between the Sig-1R and ARF6.

https://doi.org/10.7554/eLife.47209.007

The subcellular distribution of ARF6 in the mouse midbrain was next compared with Sig-1Rs in a fractionation assay allowing detection of the MAM (Figure 3B), where Sig-1Rs are abundant (Hayashi and Su, 2007; Lewis et al., 2016). Both the Sig-1R and ARF6 were found in this MAM fraction (Figure 3B), but another ARF GTPase, ARF-1, was not detected (Figure 3B). Together, our results indicate that Sig-1Rs and ARF6 colocalize with GABA and DA neuron markers and are associated with the MAM in the mouse midbrain.

Involvement of Sig-1Rs, ARF6, and myosin light chain kinase in cocaine-induced EV release

To determine whether, like in NG-108 cells, cocaine-stimulation of EV secretion occurred through Sig-1R- and ARF6-dependent mechanisms, we manipulated signaling by these proteins, followed by preparation of midbrain tEV fractions. We found that an injection of cocaine (15 mg/kg, i.p.) significantly attenuated the co-immunoprecipitation of ARF6 and Sig1R in the mouse midbrain (Figure 4A), and this was prevented by a preceding subcutaneous (s.c.) injection of the Sig-1R antagonist, BD1063 (10 mg/kg) (Figure 4B). This suggests that the cocaine facilitates activation of the Sig-1R, and this triggers Sig-1R dissociation from ARF6. Next, we determined whether in vivo cocaine treatment altered the intracellular localization of ARF6, using the MAM fractionation assay. We found that, unlike that observed in the P3 fraction where ARF6 levels remained unchanged, 10 min after cocaine injection the level of MAM-associated ARF6 was decreased (Figure 4C). Moreover, Sig-1R levels were not significantly altered in either the P3 or the MAM fractions (Figure 4C). These results suggest that the Sig-1R is activated by cocaine while associated with the MAM and this facilitates dissociation of the Sig-1R from ARF6. As ARF6-GTP modulation by the GEF inhibitor SH3 altered EV secretion in NG-108 cells (Figure 1E), we measured its effect (s.c., 10 mg/kg) on cocaine-stimulated tEV secretion in mouse midbrain. Consistent with NG-108 cell data, SH3 significantly inhibited the cocaine-induced increase of TH and Iβ1 in mouse midbrain (Figure 4D). Existing data also support the involvement of cytoskeletal myosin and actin in EV release and show that ARF6 exerts its effects on EV release through phosphorylation of myosin light-chain kinase (MLCK) (van Niel et al., 2018; Muralidharan-Chari et al., 2009). Therefore, we examined MLCK involvement in the cocaine-simulated EV release in midbrain tissue and found that the MLCK inhibitor ML7 (2 µM) prevented the increase in EV release, as measured by Iβ1, or TH in EV-rich fractions (Figure 4D).

Cocaine causes translocation of ARF6 via its dissociation from the Sig-1R in mouse midbrain.

(A) Western blots showing that cocaine reduces the interaction between ARF6 and the Sig-1R in a time-dependent manner in mouse midbrain. The graph shows mean (± s.e.m.) of co-IP of ARF6 and Sig-1R, before, and 10, 20 and 30 min after in vivo cocaine injection (n = 4; (F3,12 = 4.3, p=0.028, one-way ANOVA, *=p < 0.05 compared with naive, Dunnett’s multiple comparison test). *p<0.05, **p<0.01; one-way ANOVA followed by Dunnett post-hoc test). (B) Effect of the Sig-1R antagonist BD1063 (BD, 10 mg/kg, s.c.) on the dissociation of the ARF6-Sig-1R complex in mouse midbrain, 10 min after i.p. cocaine injection. BD1063 was injected 20 min before cocaine. The bar graph represent mean ± s.e.m. (n = 7; F3,23 = 5.3, p=0.006, one-way ANOVA, *=p < 0.05, **=p < 0.01, Dunnett’s multiple comparison test (C) Western blots showing the effect of cocaine versus saline injection on ARF6 concentration associated with the MAM, or P3 in mouse midbrain at 10 min post-i.p. injection. Bar graphs show mean (± S.E.M., n = 3) expression of ARF6 or Sig-1R as a proportion of GAPDH protein in MAM or P3 preparations, for all conditions, expressed as the percent response observed following saline injection (*=p < 0.001, unpaired t-test). (D) Western blots showing the effect of the ARF6 GEF inhibitor (SecinH3: SH3, 10 µmol/kg, s.c.) or the MLCK inhibitor, ML7 (2 µM, s.c.) on cocaine-evoked EV marker release in mouse midbrain, 30 min after i.p. cocaine or saline injection. SH3, ML7, or vehicle was injected 20 min prior to cocaine or saline injection. ERK1 is used as a control protein. The bar graphs represent the mean (± S.E.M) concentration of TH or Iβ1 expressed as a percentage of the level seen following vehicle-saline control injections(n = 4–7, TH: F3,12 = 7.9, p=0.004, one-way ANOVA, *=p < 0.05, **=p < 0.01, Dunnett’s multiple comparison test; Iβ1: F3,12 = 7.0, p=0.006, one-way ANOVA, *=p < 0.05, **=p < 0.01, Dunnett’s multiple comparison test). (E) Schematic illustrating of the effect of cocaine on the Sig1R-ARF6 interaction in mouse midbrain. See Source data 1 for values used in statistical analyses. Also see Figure 8—figure supplement 1 for proposed interaction between the Sig-1R and ARF6 and cocaine.

https://doi.org/10.7554/eLife.47209.009

In consideration of these data, we propose the following model; 1) the Sig-1R forms a stable complex with the inactive ARF6-GDP at the MAM, 2) cocaine, through interaction with the Sig-1R, causes dissociation of the ARF6-GDP/Sig-1R complex, 3) free ARF6-GDP is then converted to the active ARF6-GTP by GEFs, and 4) ARF6-GTP translocates to the plasma membrane where it stimulates EV release into the extracellular space (Figure 4E) by activating MLCK, and permitting EV mobility. Using this model of EV secretion, we next sought to determine its functional relevance to synaptic modulation by eCBs in the mouse midbrain.

2-AG is found in EV-enriched midbrain fractions

A recent study found that microvesicle-enriched fractions from primary microglia cultures contained the eCB anandamide (Gabrielli et al., 2015a), and work from our laboratory showed that cocaine promotes the release of eCB 2-AG in the midbrain (Wang et al., 2015). However, the potential involvement of EVs in 2-AG function has not been assessed. To determine whether 2-AG is found in EV fractions from mouse midbrain, we used Fourier transform mass spectrometry (FTMS). We found that the levels of 2-AG were higher in midbrain homogenates than in cerebral cortex, and were approximately fivefold larger than those observed in tEV fractions from these brain regions (Figure 5A). The concentration of 2-AG in midbrain tEV fractions (206.9 ± 70.2 pmol/mg, Figure 5A) was also higher than that measured in the cerebral cortex (121.4 ± 16.1 pmol/mg, Figure 5A), suggesting regional differences in concentrations of 2-AG. We also found that cocaine significantly increased 2-AG levels in midbrain tissue (Figure 5B). However, when cocaine-stimulation of 2-AG levels in tEV fraction were measured using FTMS in pooled samples of mouse midbrain, we observed considerable variability in baseline saline-injected controls (n = 15 mice in three experiments; Figure 5C), and in cocaine-stimulated levels of the eCB (n = 15 mice in three experiments). Thus, although a clear trend toward increased 2-AG in these tEV fractions was observed, and cocaine significantly increase midbrain tissue levels of 2-AG (Figure 5B), the effect of cocaine on 2-AG content in the tEV fractions was not significant (t8 = 1.61, p=0.147, unpaired Student’s t-test; Figure 5C).

Figure 5 with 1 supplement see all
Cocaine-stimulation of 2-AG accumulation in midbrain tEVs and brain slices.

(A) Levels of 2-AG measured in midbrain and cortex tissue homogenates and in tEVs from these same brain regions using Fourier transform mass spectrometry (FTMS; F3,8 = 86.92, p<0.0001; Tukey’s posthoc test, ***=p < 0.05, †††=p < 0.05, tissue midbrain vs. cortex, n = 3). (B) Comparison of the concentration of 2-AG in midbrain homogenates from mice injected with saline or cocaine 15 min prior to dissection (mean ± S.E.M.; *=p < 0.05, unpaired Student’s t-test, n = 3). (C) Levels of 2-AG measured using FTMS in fr3 containing tEVs isolated from mouse midbrain 15 min after in vivo injection with saline or 10 mg/kg cocaine. Each point represents data pooled from three mice (t8 = 1.61, p=0.147, unpaired Student’s t-test; n = 15 mice per group). (D) Western blots detecting fatty acid binding protein-5 (FABP5), TH, Iβ1, Flot-1 and Cyto-C in either whole brain lysate (BL) or in the EV fraction (fr3) obtained via sequential centrifugation and sucrose-gradient separation. Note that all EV marker proteins are detected in the BL preparation and that FABP5 is also found in this EV fraction. (E) Cocaine stimulates 2-AG inhibition of GABA release onto VTA DA neurons in vitro. Cocaine application inhibits GABAB-receptor-mediated synaptic IPSCs in DA neurons from wildtype mice, but not in CB1R knockout (KO) mice. (F) Mean inhibition by cocaine of IPSCs in wildtype and CB1R-KO mice (p=0.0004, unpaired t-test). (G) The inhibition of IPSCs by cocaine is absent in mice lacking the gene (Dagla) encoding the 2-AG synthetic enzyme, DGL-α, in DA neurons. (H) Mean effects of cocaine on IPSCs in the presence and absence of the CB1R antagonist/inverse agonist (AM251, 4 µM) in wildtype and DGL-α-KO mice. Note the reversal of the cocaine inhibition by AM251 in wildtype DA neurons, the absence of inhibition of IPSCs by cocaine, and lack of effect of AM251 in the neurons from DGL-α-KO mice (F3, 40 = 8.3, p=0.0002, one-way ANOVA, p=0.009, Tukey’s multiple comparison post-hoc test). Figure 5—figure supplement 1 shows that blockade of CB1Rs or 2-AG synthesis also prevents inhibition of IPSCs by cocaine. See Source data 1 for values used in statistical analyses.

https://doi.org/10.7554/eLife.47209.010

Recent studies show that fatty acid binding proteins can act as intracellular carriers for 2-AG (Kaczocha et al., 2009), and one of these, fatty acid binding protein 5 (FABP5), was involved in mediating extracellular 2-AG release in the mouse brain (Haj-Dahmane et al., 2018). To determine whether this carrier of 2-AG could also be localized to midbrain EVs, we isolated EV fractions from mouse midbrain and used western blots to measure FABP5 and other EV markers. These EV fractions contained FABP5 as well as the EV markers TH, Iβ1, and flotillin-1 (Figure 5D). This suggests that the FABP5 protein is associated with EVs to perhaps mediate 2-AG signaling in the CNS.

Sig-1R antagonism prevents cocaine-stimulated synaptic 2-AG function in VTA DA neurons

There is strong evidence that 2-AG is synthesized in rodent midbrain VTA neurons, where it can modulate synaptic neurotransmitter release (Riegel and Lupica, 2004; Melis et al., 2004; Parsons and Hurd, 2015; Labouèbe et al., 2013). Moreover, 2-AG function is increased during heightened DA neuron activity (Riegel and Lupica, 2004; Melis et al., 2004), or when phospholipases are activated by certain Gαq11-containing GPCRs, such as the α1-noradrenergic (α1R), or type-I metabotropic glutamate receptors (mGluRIs) (Wang et al., 2015; Haj-Dahmane and Shen, 2014). These data also show that cocaine’s ability to increase VTA 2-AG function occurs via its inhibition of the norepinephrine transporter (NET), causing activation of α1Rs on VTA DA neurons and 2-AG synthesis from membrane phospholipids (Wang et al., 2015). Based on this previous work, and our data showing cocaine interactions with midbrain Sig-1Rs, ARF6 and EV release, we evaluated the possibility that 2-AG function in the VTA occurs via EV- and Sig-1R-dependent mechanisms in mouse midbrain DA neurons.

Local 2-AG function can be measured with high temporal fidelity through its activation of CB1Rs leading to local inhibition of synaptic transmission (Alger, 2002). This functionally relevant endogenous 2-AG reduces inhibitory postsynaptic currents (IPSCs) mediated by synaptic GABA release onto GABAB receptors (GABABRs) located on DA neuron dendrites (Wang et al., 2015; Riegel and Lupica, 2004). Similar to previous data from rat VTA DA neurons (Wang et al., 2015), we found that cocaine (10 µM) inhibited IPSCs recorded in mouse DA neurons (Figure 5E and F). The IPSC inhibition by cocaine was prevented by the CB1R antagonist, AM251 (1 µM; Figure 5H-Figure 5—figure supplement 1) and was absent in mice lacking the CB1R (Zimmer et al., 1999) (Figure 5E and F). The inhibition of IPSCs by cocaine was also reduced by tetrahydrolipostatin (THL, 2 µM), an inhibitor of the enzyme diacylglycerol lipase-α (DGLα), preventing conversion of diacylglycerol (DAG) to 2-AG (Figure 5—figure supplement 1A1, Figure 5—figure supplement 1B). Cocaine-mediated 2-AG release was also absent in mutant mice lacking expression of DGLα in DA neurons (Shonesy et al., 2014) (Daglaflox/flox x DATCre mice; Figure 5G and H). These experiments confirm that inhibition of GABA release onto DA neurons by cocaine occurs via stimulation of 2-AG function in the mouse VTA.

We next examined Sig1-R involvement in cocaine-dependent 2-AG release in mouse VTA DA neurons. Each of two Sig-1R antagonists (BD1063 or NE100; 2 µM) significantly reduced the cocaine (10 µM) simulation of 2-AG release in VTA DA neurons (Figure 6A–C and E). This effect of cocaine was also significantly reduced in DA neurons from Sig-1R knockout mice, particularly 5–10 min after beginning cocaine application (Figure 6D and E). Importantly, the inhibition of IPSCs by the synthetic CB1R agonist, WIN55,212–2 (1 µM), was not reduced by Sig-1R antagonism, or by genetic deletion of this receptor (Figure 6—figure supplement 1). This indicates that Sig-1Rs are linked to cocaine-stimulated 2-AG function in the CNS, and that CB1R signaling is not diminished by altered Sig-1R function or expression.

Figure 6 with 1 supplement see all
Inhibition of IPSCs by cocaine in VTA DA neurons depends upon Sig-1Rs.

(A) Mean waveforms showing the effect of cocaine (10 µM) on GABAB IPSCs in a DA neuron from a wildtype mouse during application of the Sig-1R antagonist NE100 (2 µM, left), or in a cell from a Sig-1R KO mouse (right). (B) Mean time-course showing effect of cocaine on IPSCs in absence (Control) and presence of NE100 in wildtype mice. (C) Mean time-course showing effect of cocaine on IPSCs in absence (Control) and presence of BD1063 (2 µM) in wildtype mice. (D) Time-course of cocaine effects in wildtype and Sig-1R KO mice. (E) Summary of Data shown in A-D. The inhibition of IPSCs by cocaine was significantly reduced by NE100 or BD1063 in wildtype mice and was significantly smaller in Sig-1R KO mice (F3,49 = 10.90, one-way ANOVA, p<0.0001; *=p < 0.0001, #=p = 0.0002, %=p = 0.005, Dunnett’s multiple comparisons post-hoc test. Figure 6—figure supplement 1 shows that antagonism or knockout of the Sig-1R does not change CB1R function in mouse VTA DA neurons. See Source data 1 for values used in statistical analyses.

https://doi.org/10.7554/eLife.47209.012

To examine whether Sig-1Rs are involved in facilitating 2-AG release derived from direct GPCR activation, we determined whether α1R and mGluRI co-activation could stimulate 2-AG function in mouse VTA, and whether this is altered in Sig-1R knockout mice. Consistent with our previous report (Wang et al., 2015), co-application of the α1R agonist phenylephrine (PE, 100 µM) and the mGluRI agonist, DHPG (1 µM) inhibited GABAB IPSCs in wildtype mouse VTA DA neurons, and this was blocked by AM251 (Figure 7B and C). However, it is also important to note that the properties of the IPSC inhibition produced by DHPG+PE differed from that seen with cocaine. Thus, the response to DHPG+PE was much slower to reach maximum and lacked the early fast component observed with cocaine (Figure 7—figure supplement 1) in wildtype mice. Therefore, in comparison, the effect of DHPG+PE primarily consisted of the delayed slow component (Figure 7—figure supplement 1C). Also, in DA neurons from Sig-1R knockout mice, the slow response to DHPG+PE was significantly smaller (Figure 7A–7C, Figure 7—figure supplement 1A), which contrasts with that seen with cocaine where the early fast inhibition was absent, but the later inhibition was less affected in Sig-1R knockout mice (Figure 6D, Figure 7—figure supplement 1B). These differences could indicate reliance upon distinct signaling pathways that convergence upon Sig-1Rs to permit 2-AG release via EVs.

Figure 7 with 1 supplement see all
The Sig-1R is necessary for GPCR-induced but not tonic 2-AG release in the mouse VTA.

(A) Mean GABAB IPSC waveforms collected during baseline (control, black line) and during co-application of DHPG and PE (gray line), in DA neurons from wildtype (WT, left), and Sig-1R knockout (KO, center) mice. Also shown is the effect of DHPG+PE in a representative neuron from a WT mouse following preincubation with AM251 (right). (B) Mean time courses of the effects of DHPG+PE in DA neurons from WT, sig-1R KO mice, and WT mice that had been pre-treated with AM251. The effect of DHPG+PE was significant (one-way repeated measures ANOVA, F1.5, 110 = 133, p<0.0001), and this was significantly reduced in the Sig-1R KO, and by AM251 (Tukey’s post hoc test p<0.0001). (C) Bar graph of data from the last 5 min of application of DGPG+PE as shown in B. The inhibition of IPSCs by DHPG+PE was significant (t9 = 4.5, *=p = 0.0014, and the this was significantly reduced in the Sig-1R KO and AM251 groups (F2,34 = 11.0, p=0.0002, one-way ANOVA; ##=p < 0.0001; #=p = 0.0013, Dunnett’s posthoc test, the number of cells in each condition is indicated in parentheses). (D) Mean time course showing tonic inhibition of GABAB IPSCs by endogenous 2-AG, as revealed by antagonist of CB1Rs with AM251 in neurons from wildtype (WT) and Sig-1R KO mice (n = 15 and 11, respectively). (E) Bar graph of the change in IPSC amplitude during the last 5 min of AM251 application for data shown in D. AM251 caused a significant increase in mean IPSC amplitude in both groups (two-tailed unpaired t-test; **=p < 0.0001, *=p = 0.001), but there was no significant difference in this effect between groups (n.s. = not significant, two-tailed unpaired t-test, p=0.76). These data show that Sig-1Rs are necessary for the GPCR-induced 2-AG release caused by DHPG+PE (A–C), but not for tonic non-GPCR-dependent 2-AG release (D–E), and they suggest that DGLα function is not impaired in Sig-1R KO mice. Figure 7—figure supplement 1 shows kinetic differences between 2-AG function elicited by DHPG+PE and cocaine in the mouse VTA. See Source data 1 for values used in statistical analyses.

https://doi.org/10.7554/eLife.47209.014

To determine whether the effects of 2-AG derived from a non-GPCR source are also altered in the Sig-1R knockout mouse, we measured tonic 2-AG release that is observed without GPCR activation (either indirectly by cocaine or directly by DHPG+PE) (Wang et al., 2015). The tonic inhibition of GABAB IPSCs mediated by this basal level of endogenous 2-AG is revealed when CB1Rs are blocked by antagonists, resulting in an increase in these synaptic currents (Wang et al., 2015; Riegel and Lupica, 2004). We found that DA neurons from both wildtype and Sig-1R knockout mice exhibited similar significant IPSC increases when the CB1R antagonist AM251 was applied (Figure 7A and B). Therefore, the data suggest that only 2-AG derived from GPCR stimulation is dependent upon intact Sig-1R function, and additionally that 2-AG synthesis itself is not disrupted in Sig-1R knockout mice.

Our NG-108 experiments indicated that Sig-1Rs stabilize the inactive GDP-bound form of ARF6, and that cocaine activates GTP-bound ARF6 through an interaction with Sig-1Rs, thereby permitting EV release. Moreover, our FTMS experiments identified 2-AG in midbrain tEV fractions (Figure 5A, Figure 5C). Therefore, involvement of ARF6 in the 2-AG-dependent inhibition of GABA release by cocaine was tested in wild-type mouse VTA DA neurons. Manipulation of ARF6 activation with the GEF inhibitor, SH3 (Figure 8A and E), or, direct inhibition of ARF6 with NAV2729 (both at 10 µM) (Yoo et al., 2016), significantly inhibited cocaine-induced 2-AG function in midbrain DA neurons (Figure 8B and E). Also, like that observed with Sig-1R antagonists or knockouts (Figure 6), the reduction in the cocaine inhibition of IPSCs by both SH3 and NAV2729 was more prominent within the first 10 min of cocaine application (Figure 8A, Figure 8B). As inhibition of MLCK significantly reduced EV release in midbrain tissue experiments, we examined its involvement in the synaptic effects of cocaine-simulated 2-AG function in DA neurons. We found that the MLCK inhibition by ML7 (2 µM) also significantly reduced the effect of cocaine on 2-AG release in this electrophysiological assay of eCB function (Figure 8C and E).

Figure 8 with 1 supplement see all
Cocaine stimulated 2-AG inhibition of GABA release is blocked by ARF6 inhibitors or myosin-light chain kinase (MLCK) inhibition.

(A) Mean time-course of the effect of cocaine on GABAB IPSCs under control conditions, and during incubation with the ARF6 GEF inhibitor SH3 (10 µM). (B) Mean time-course of the effect of cocaine on the GABAB IPSCs under control conditions and during incubation with direct ARF6 inhibitor NAV2729 (10 µM). (C) Mean time-course of the effect of cocaine on the GABAB IPSCs under control conditions and during incubation with the MLCK inhibitor ML7 (2 µM). (D) Mean waveforms of GABAB receptor-mediated IPSCs after addition of cocaine in cells preincubated with ML7 or SH3. (E) Summary of data with ML7, SH3, and NAV2729, shown in A-C. The effect of cocaine is significantly reduced by SH3, NAV2729, and ML7 (F3,39 = 8.7, p=0.0002, one-way ANOVA, **=p < 0.001, Dunnett’s multiple comparison test, *=p = 0.0003, #=p = 0.0005, %=p = 0.001; n for each condition shown in parentheses). Figure 8—figure supplement 1 shows our model of the proposed mechanisms underlying the cocaine-regulated synthesis and release of 2-AG in VTA DA neurons and the involvement of EVs and Sig1R-ARF6 signaling pathway See Source data 1 for values used in statistical analyses.

https://doi.org/10.7554/eLife.47209.016

Together these data demonstrate that EV release is controlled by the Sig-1R, ARF6, and MLCK, and that cocaine’s interaction with the Sig-1R can recruit this signaling cascade. The data further demonstrate that disruption of these signaling mechanisms leads to reduced synaptic 2-AG function in the midbrain, thereby implicating these proteins and EVs in the release of eCBs.

Discussion

Previous studies show that a cocaine binds to Sig-1Rs (Sharkey et al., 1988; Chen et al., 2007; Hiranita et al., 2011), and that blockade of this interaction reduces effects of the psychostimulant (Romieu et al., 2002; Hiranita et al., 2011; Lever et al., 2014; Fritz et al., 2011). Additionally, cocaine’s actions at Sig1-Rs alters its ability to influence voltage-gated potassium channel function, and this can reduce its behavioral effects (Kourrich et al., 2013; Romieu et al., 2002; Lever et al., 2014; Fritz et al., 2011). The present data demonstrate that the Sig-1R also regulates EV secretion in cultured cells and in the mouse midbrain, and that cocaine modulates this process through interaction with the Sig-1R. We also show that the interactions among Sig1-Rs, cocaine, and EVs can regulate synaptic transmission in the brain via the control of 2-AG release and its inhibition of GABAergic input to DA neurons in the mouse VTA. Therefore, our study identifies novel mechanisms for Sig-1R control of EV function and implicates EVs in eCB release in the CNS.

EVs are increasingly recognized as a highly regulated mechanism to permit exchange of signaling molecules, such as lipids, nucleic acids, organelles, and proteins, among cells (van Niel et al., 2018). As such, regulatory control points for EV formation, budding, translocation, and cargo release have been delineated in many cell types during normal cellular function, and in disease states (van Niel et al., 2018; Huang-Doran et al., 2017; EL Andaloussi et al., 2013; Muralidharan-Chari et al., 2009; Wang et al., 2017; Yoo et al., 2016). Here, we show that cocaine treatment of NG108 cells, or of mouse midbrain after in vivo injection, stimulates EV release, and that this is mimicked by agonists of Sig-1Rs, and prevented by antagonists or genetic elimination of these receptors. Moreover, using co-immunoprecipitation assays, we provide evidence for an association between ARF6, an established regulator of EV secretion (D'Souza-Schorey and Chavrier, 2006; Yoo et al., 2016), and the Sig-1R in TH-positive VTA neurons, and find that blockade of ARF6 activation prevents cocaine-induced EV release in both NG-108 cells and midbrain. We also report that in vivo cocaine causes the ARF6/Sig1R complex to dissociate, and this is prevented by Sig-1R antagonism. These data suggest that Sig-1Rs bind ARF6 proteins to hold them in an inactive GDP-bound form, and that cocaine facilitates the dissociation of these proteins to permit conversion of ARF-GDP to the active ARF6-GTP. Our data also suggest that this interaction between ARF6 and Sig-1Rs occurs at the MAM, and that cocaine enables translocation of ARF6-GTP to the plasma membrane. This mechanism is notable because ARF6 is implicated in EV secretion via regulation of cytoskeletal actin function in a wide range of mammalian tissues (D'Souza-Schorey and Chavrier, 2006; Yoo et al., 2016), and this is supported by our observation that inhibition of MLCK also prevents the cocaine-induced increase in EV levels in mouse midbrain.

Previous work shows that anandamide is found in EV-containing membrane fractions of rodent microglia cultures, and that these fractions exhibit cannabinoid agonist properties when applied to hippocampal brain slices (Gabrielli et al., 2015a). Here, we show using FTMS that 2-AG is found in acute mouse midbrain preparations that are enriched in tEVs, and that 2-AG levels are significantly increased in midbrain homogenates after in vivo exposure to cocaine. In contrast, although 2-AG could be measured in tEV fractions using FTMS in mouse midbrain, and tEV markers were significantly increased after in vivo cocaine treatment, the increase in 2-AG levels produced by cocaine in the tEV preparation did not reach statistical significance despite a clear trend. As these preparations are technically demanding and yield small amounts of material, it is possible that the between-groups ex vivo design and variability among samples in both saline control and cocaine injected mice contributed to this outcome. Alternatively, it is possible that cocaine causes an increase in 2-AG-containing EV release, but that the amount of 2-AG per vesicle does not change, and this increase in vesicle release could be sufficient to locally activate CB1Rs on GABAergic axon terminals.

The observation that cocaine increased midbrain levels of 2-AG provides biochemical support for our finding of cocaine-increased 2-AG function in mouse (this study) and rat VTA DA neurons in vitro (Wang et al., 2015). In this regard, we demonstrate that cocaine stimulates a 2-AG-dependent inhibition of GABAB receptor-mediated synaptic responses that is absent in mice lacking the CB1R, or the 2-AG biosynthetic enzyme, DGLα, in DA neurons. Based upon present data and our published work (Wang et al., 2015), we propose that 2-AG synthesis is stimulated when cocaine blocks norepinephrine uptake in the VTA, resulting in activation of G-protein-αq-coupled α1Rs, which, together with Gq-coupled mGluRIs stimulated by endogenous glutamate, activate phospholipases and liberate 2-AG from precursor membrane lipids (Figure 8—figure supplement 1) (Kano et al., 2009; Maejima et al., 2005; Alger and Kim, 2011; Wang et al., 2015; Haj-Dahmane and Shen, 2014; Mátyás et al., 2008). Although this model of 2-AG synthesis is supported by our studies, the mechanism of 2-AG is release is unknown. Here, using this 2-AG-sensitive synaptic response, we find that the same manipulations that blocked EV release in NG-108 cells and midbrain EV assays also reduced or eliminated cocaine-stimulated 2-AG effects on synaptic transmission in the mouse VTA. These manipulations include the disruption of Sig-1R signaling, the inhibition of ARF6 function, and the inhibition of MLCK. Moreover, we also found that the IPSC inhibition produced by a synthetic CB1R agonist was not altered by antagonism or genetic deletion of Sig-1Rs, suggesting that Sig-1Rs regulate 2-AG signaling but not CB1R function.

The involvement of Sig-1Rs in the GPCR-dependent 2-AG release was supported by experiments showing that co-activation of mGluRIs and α1Rs by DHPG+PE could increase the release of this eCB, and that this was significantly reduced in Sig-1R KO mice. Moreover, another form of tonic 2-AG release that occurs under basal conditions in the absence of GPCR stimulation was unaltered in Sig-1R KO mice. Therefore, the data suggest that Sig-1Rs and EVs mediate only GPCR-dependent 2-AG release, and not that generated by other cellular pathways.

Based on our biochemical and electrophysiological data, we propose a model (Figure 8—figure supplement 1) in which cocaine initiates 2-AG synthesis via inhibition of the NET, leading to activation of α1Rs coupled to Gq proteins controlling phospholipases and the liberation of the 2-AG precursor DAG. DAG is then converted to 2-AG via DGLα and then packaged in EVs through an unknown process. 2-AG release from EVs is triggered when cocaine binds to Sig-1Rs to liberate ARF6-GDP and permit its conversion to the active ARF6-GTP, which can then act at MLCK to initiate EV fusion with the cellular membrane and release of 2-AG. Although these mechanisms are supported by the present data, our finding that the inhibition of IPSCs by 2-AG release by DHPG+PE is absent cells from Sig-1R KO mice suggests that cocaine binding to the Sig-1R is not necessary to initiate EV release. However, fundamental differences in the characteristics of the inhibition produced by these methods were noted. Thus, the kinetics of the 2-AG-mediated inhibition of GABA release caused by cocaine differ from DHPG+PE in that the effect onset and the peak response to cocaine occurred more rapidly than that seen with DHPG+PE (Figure 6—figure supplement 1). Also, the cocaine effect reached a maximum within approximately the first 5 min after application, and this early phase was completely blocked when Sig-1R, ARF6 or MLCK function was disrupted (Figure 8), whereas the smaller late phase of inhibition was resistant to these manipulations (Figure 8, Figure 7—figure supplement 1). Despite this, data showing that both the early and late phases of cocaine inhibition are prevented by AM251 (Figure 5—figure supplement 1) and absent in mice lacking the CB1R or DGLα (Figure 5E–H), indicate that both inhibitory phases depend upon 2-AG and CB1Rs. In contrast to the effect of cocaine, DHPG+PE does not produce a robust early phase of IPSC inhibition (Figure 6—figure supplement 1) and the delayed inhibition produced by the agonists is smaller, but not absent in Sig-1R KO mice (Figure 7—figure supplement 1A). These differences suggest that although cocaine and DHPG+PE initiate 2-AG-dependent inhibition of synaptic GABA release, they may involve distinct upstream mechanisms that converge on Sig-1Rs and their control of EV release. Thus, the faster time-course of the cocaine effect may result from its direct binding to Sig-1Rs (Sharkey et al., 1988; Chen et al., 2007; Hiranita et al., 2011) to more rapidly stimulate EV release, resulting in their depletion during the late phase. In contrast, the slower and more sustained effect of DHPG+PE on 2-AG release may reflect coupling of EV release to a signaling pathway that relies upon intracellular release of an endogenous Sig-1R agonist. In support of this, several putative endogenous Sig-1R agonists have been identified (Monnet and Maurice, 2006; Ramachandran et al., 2009; Fontanilla et al., 2009), and a more recent study shows that agonists of Gq-coupled receptors that stimulate phospholipases can increase intracellular levels of choline, which then acts as an agonist at Sig-1Rs to enhance their calcium signaling properties (Brailoiu et al., 2019). Therefore, we speculate that the distinct phases of 2-AG-dependent inhibition are related the ability of the cocaine to act as a direct agonist at Sig-1Rs, compared to potential indirect effects of DHPG+PE that may be mediated by an intracellular signaling molecule having agonist properties at sig-1Rs. Future experiments will test this hypothesis.

Fatty acid binding proteins (FABPs) can bind and transport lipid molecules within and between cells (Kaczocha et al., 2009; Ertunc et al., 2015). One of these, adipocyte fatty-acid binding protein 4 (aP2), is secreted from adipocytes via EVs (Ertunc et al., 2015), and several FABPs are found in brain (Owada et al., 1996). Recent studies show that one of these proteins, FABP5, has high affinity for 2-AG, and its inhibition or genetic deletion impairs 2-AG-mediated signaling and plasticity at glutamate synapses in the dorsal raphe nucleus (Haj-Dahmane et al., 2018; Owada et al., 1996; Kaczocha et al., 2012). Based on these results, and our present observation that FABP5 is co-localized with the EV markers Iβ1 and flotillin-1 in EV fractions from the mouse midbrain, it is possible that 2-AG release may occur via binding to FABPs that are transported to the extracellular space via EVs, and therefore subject to mechanisms regulating EV secretion, such as Sig-1Rs, ARF6, and MLCK. Future studies will more closely examine this possibility to more completely understand the mechanisms of EV-dependent eCB release in the brain.

Materials and methods

Key resources table
Reagent
type
DesignationSource or
reference
IdentifiersAdditional
information
Mouse: M. musculus (C57BL/6J)C57BL/6J; wildtype, WTCharles River LaboratoriesStrain Code: 027
Mouse: M. musculus (C57BL/6J)sigma1r; Sigma1 receptor: Sig-1R; Sig-1R KO, knockouthttps://doi.org/10.1073/pnas.1518894112
Mouse: M. musculus (C57BL/6J)Dagla fl/fl x Slc6a3-Cre +/-; floxed DGL-α x DATCre heterozygote; DGL-α x DATCre; DGL-α KO, knockoutDagla fl/fl, a gift from Sachin Patel; Dagla fl/fl x Slc6a3-Cre + /- breeders a gift from Daniel P. Covey
Mouse: M. musculus (C57BL/6J)CNR1; CB1R; CB1R -/-; CB1R KO; knockouthttps://doi.org/10.1073/pnas.96.10.5780
Cell Line (M. musculus)Mouse neuroblastoma x Rat glioma: NG108-15 cells; NG108 cellsATCCHB-12317
AntibodyMouse monoclonal (mcl) anti-alpha-tubulinSigma-AldrichCat#: T5168Western Blot (WB); Dilution (1:10,000)
AntibodyRabbit polyclonal (plcl) anti-AlixSigma-AldrichCat#: SAB4200476WB (1:1,000)
AntibodyMouse monoclonal (mcl) anti-ARF6Santa Cruz BiotechnologyCat#: sc-7971Immunohistochemistry (IHC); (1:100), Immunoprecipitation (IP), 1 µg
AntibodyRabbit plcl anti-ARF1Thermo Fisher ScientificCat#: PA1-127WB (1:1,000)
AntibodyRabbit plcl anti-ARF6Cell Signaling TechnologyCat#: 3546WB (1:1,000)
AntibodyMouse mcl anti-Cytochrome cBD BiosciencesCat#: 556433WB (1:1,000)
AntibodyRabbit plcl anti-ERK1Santa Cruz BiotechnologyCat#: sc-94WB (1:500)
AntibodyRabbit mcl anti-FABP5 (D1A7T)Cell Signaling TechnologyCat#: 39926WB (1:1,000)
AntibodyRabbit plcl anti-Flotillin-1Santa Cruz BiotechnologyCat#: sc-25506WB (1:1,000)
AntibodyRabbit mcl anti-GAPDH (D16H11)Cell Signaling TechnologyCat#: 5174WB (1:2000)
AntibodyMouse mcl anti-GFPClonetechCat#: 632381WB (1:10,000)
AntibodyRabbit plcl anti-GFPClonetechCat#: 632592IP (1 µg)
AntibodyMouse mcl anti-HaloPromega CorporationCat#: G9211WB (1:10,000)
AntibodyMouse mcl anti-HSP90Enzo Life SciencesCat#: ADI-SPA-830WB (1:1,000)
AntibodyMouse mcl anti-Integrin β1Thermo Fisher ScientificCat#: MA5-17103WB (1:1,000)
AntibodyMouse mcl anti-Nucleoporin p62BD BiosciencesCat#: 610498WB (1:1,000)
AntibodyRabbit mcl anti-PDICell Signaling TechnologyCat#: 3501WB (1:1,000)
AntibodyRabbit anti-Sigma-1 receptor serumA gift from Arnold RuohoN/AIHC (1:1,000)
AntibodyRabbit anti-Sigma-1 receptor serum #5460In houseN/AWB (1:1,000)
AntibodyMouse anti-sigma-1 receptor B-5 mclSanta Cruz BiotechnologyCat#: Sc-137075IP (1 µg)
AntibodyMouse mcl anti-Tyrosine hydroxylaseMillipore CorporationCat#: MAB318IHC (1:1,000), WB (1:2,000)
AntibodyRabbit plcl anti-Tyrosine hydroxylaseMillipore CorporationCat#: AB152IHC (1:1,000)
AntibodyChicken plcl anti-Tyrosine hydroxylaseAves LabsCat#: THIHC (1:1,000)
AntibodyMouse mcl anti-tsg 101Santa Cruz BiotechnologyCat#: Sc-7964WB (1:500)
AntibodyChicken plcl anti-vGATSynaptic SystemsCat#: 131 006IHC (1:500)
AntibodyIRDye 680RD goat anti-mouse IgGLI-COR BiosciencesCat#: 925–68070WB (1:10,000)
AntibodyIRDye 800CW goat anti-mouse IgGLI-COR BiosciencesCat#: 925–32210WB (1:10,000)
AntibodyIRDye 680RD goat anti-rabbit IgGLI-COR BiosciencesCat#: 925–68071WB (1:10,000)
AntibodyIRDye 800CW goat anti-rabbit IgGLI-COR BiosciencesCat#: 925–32211WB (1:10,000)
AntibodyAlexa Fluor 405 goat anti-mouse IgGThermo Fisher Sci.Cat#: A-31553IHC (1:500)
AntibodyAlexa Fluor 488 anti-chicken IgYThermo Fisher Sci.Cat#: A-11039IHC (1:500)
AntibodyAlexa Fluor 568 anti-rabbit IgGThermo Fisher Sci.Cat#: A-11036IHC (1:500)
Recombinant DNA reagentpcDNA3-CFPA gift from Doug GolenbockAddgene Plasmid # 13030
Recombinant DNA reagentpARF6 (WT)-CFPA gift from Joel Swanson; https://doi.org/10.1371/journal.pbio.0040162Addgene Plasmid # 11382
Recombinant DNA reagentpARF6 (Q67L)-CFPA gift from Joel Swanson; https://doi.org/10.1371/journal.pbio.0040162Addgene Plasmid # 11387
Recombinant DNA reagentpARF6 (T27N)-CFPA gift from Joel Swanson; https://doi.org/10.1371/journal.pbio.0040162Addgene Plasmid # 11386
Recombinant DNA reagentpHTC HaloTagPromegaCat#: G7711
Recombinant DNA reagentpHTN HaloTagPromegaCat#: G7721
Recombinant DNA reagentHalo-Sig1RThis paperN/Acontact for resource: Dr. Tsung-Ping Su; TSU@intra.nida.nih.gov
Recombinant DNA reagentSig1R-HaloThis paperN/Acontact for resource: Dr. Tsung-Ping Su; TSU@intra.nida.nih.gov
Recombinant DNA reagentSig1R (1-60)-HaloThis paperN/Acontact for resource: Dr. Tsung-Ping Su; TSU@intra.nida.nih.gov
Recombinant DNA reagentHalo-Sig1R (61-223)This paperN/Acontact for resource: Dr. Tsung-Ping Su; TSU@intra.nida.nih.gov
Commercial assay or kitNanoSight Particle AnalysisSystem BiosciencesCat#: CSNANO100A-1
Commercial assay or kitDynabeads Protein GThermo Fisher ScientificCat#: 10009D
Commercial assay or kitPolyJet In Vitro DNA TransfectionSignagen LaboratoriesCat#: SL100688
Commercial assay or kitMicro BCA Protein Assay KitThermo Fisher ScientificCat#: 23235
Chemical compound, drugCocaine hydrochlorideNIDA Drug SupplyN/Ahttps://d14rmgtrwzf5a.cloudfront.net/sites/default/files/ndspcat24thedmarch2015.pdf
Chemical compound, drugBD 1063 dihydrochlorideTocris BioscienceCat#: 0883; CAS: 206996-13-6
Chemical compound, drugSecinH3Tocris BioscienceCat#: 2849; CAS: 853625-60-2
Chemical compound, drugAM251Tocris BioscienceCat#: 1117; CAS: 183232-66-8
Chemical compound, drugCGP55845 hydrochlorideTocris BioscienceCat#: 1248; CAS: 149184-22-5
Chemical compound, drugHanks' Balanced Salt SolutionThermo Fisher ScientificCat#: 14175095
Chemical compound, drugNeurobasal MediumThermo Fisher ScientificCat#: 21103049
Chemical compound, drugCollagenaseThermo Fisher ScientificCat#: 17100017
Chemical compound, drugProtease Inhibitor CocktailSigma-AldrichCat#: P8340
Chemical compound, drugBlotting-grade blockerBio-Rad LaboratoriesCat#: 1706404
Chemical compound, drugBovine serum albuminSigma-AldrichCat#: A2153
Chemical compound, drugPercollGE Healthcare Life Sci.Cat#: 17-0891-02
Chemical compound, drugDulbecco's Modified Eagle MediumThermo Fisher ScientificCat#: 11965092
Chemical compound, drugFetalgro Bovine Growth SerumRMBIOCat#: FGR-BBT
Chemical compound, drugHAT Supplement (50X)Thermo Fisher ScientificCat#: 21060017
Chemical compound, drugPenicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCat#: 15140122
Chemical compound, drugLauryl maltose neopentyl glycolAnatraceCat#: NG310
Chemical compound, drugtwo x Laemmli Sample BufferBio-Rad LaboratoriesCat#: 1610737
Chemical compound, drugNonidet P-40Sigma-AldrichCat#: I3021
Chemical compound, drugPhenylmethanesulfonyl fluorideSigma-AldrichCat#: P7626
Chemical compound, drugNAV2729Tocris BioscienceCat#: 5986; CAS: 419547-11-8
Chemical compound, drugML seven hydrochlorideTocris BioscienceCat#: 4310; CAS: 110448-33-4
Chemical compound, drugNE100Tocris BioscienceCat#: 3313; CAS: 149409-57-4
Software, algorithm
GraphPad Prism 7GraphPad Software, San Diego, CA
Image Studio LiteL LI-COR Biosciences, Lincoln, Nebraska
WINLTP 2.30WinLTP Ltd., Bristol, U.K.https://www.winltp.com/
G-Power 3.1.9.4https://doi.org/10.3758/BF03193146http://www.psychologie.hhu.de/arbeitsgruppen/allgemeine-psychologie-und-arbeitspsychologie/gpower.html

Drugs

1-[2-(3,4-Dichlorophenyl)ethyl]−4-methylpiperazine dihydrochloride (BD 1063 dihydrochloride, Cat#: 0883, Tocris), and cocaine hydrochloride were dissolved in 0.9% NaCl. N-[4-[5-(1,3-Benzodioxol-5-yl)−3-methoxy-1H-1,2,4-triazol-1-yl]phenyl]−2-(phenylthio)acetamide (SecinH3, Cat#: 2849, Tocris) was dissolved in DMSO, and then diluted with 25% DMSO/75% glucose solution (5 w/v%).

Animals

Ethics statement

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All animal procedures were conducted in accordance with the principles as indicated by the NIH Guide for the Care and Use of Laboratory Animals. These animal protocols were also reviewed and approved by the NIDA intramural research program Animal Care and Use Committee, which is fully accredited by the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International (approved protocols: 17-CNRB-15, 16-CNRB-128, 16-INB-1, 16-INB-3, 17-INB-5).

Adult (8+ weeks) male mice were housed with food and water available ad libitum. Mice were housed on a 12/12 hr light cycle. Wild-type C57Bl6/J mice were ordered from Charles River Laboratories. Sigma one receptor transgenic mice were bred in house. Sigmar1 mutant (+/−) Sigmar1Gt(IRESBetageo)33Lex litters on a C57BL/6J × 129s/SvEv mixed background were purchased from the Mutant Mouse Regional Resource Center at the University of California, Davis. The sigma-1 receptor (+/−) males were backcrossed for 10 generations to female on C57BL/6J to ensure that animals had a homogenous background. The resulting mice were genotyped to select Sig-1R WT and KO mice. To generate mice lacking diacylglycerol lipase-α (DGL-α) in DA neurons, mice in which the Dagla gene was flanked by LoxP were obtained from the laboratory of Dr. Sachin Patel (Vanderbilt University). These mice were then crossed with dopamine transporter (Slc6a3; DAT) Cre mice (Slc6a3Cre+/-) to generate mice lacking the DGL-α gene (Dagla) in DAT-expressing neurons (Daglafl/fl x Slc6a3-Cre+/-).

Group allocation

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Group membership was determined by genotype where transgenic mice were used. In in vitro electrophysiology studies, recordings from untreated control brain slices were interleaved with recordings from drug pre-incubated brain slices from the same animal. In cell biology experiments, mice were chosen for experiments depending upon date of arrival from the supplier. In this way, mice were assigned to groups according availability and to the experimental procedures to be performed that day. In most cases, brain tissue from each mouse was used in both control and treatment conditions. NG-108 cell culture dishes were selected randomly from those available in the tissue incubator.

Isolation of mouse midbrain slices

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Mice were killed with CO2 gas, and brains were removed, and rinsed in ice-cold Hank’s balanced salt solution (Thermo Fisher Scientific). Midbrain samples were isolated by cutting coronal sections containing the VTA using mouse brain matrices (Roboz), and the cortex and a hippocampus dissected free (Figure 2—figure supplement 1).

Preparation of EV fractions

NG108 cells

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To isolate EVs from NG108 cells we used an established protocol with minor modifications (Gabrielli et al., 2015a). First, conditioned HBSS was collected and pre-cleared from cells and debris by centrifugation at 300 x g for 10 min, and 2000 x g for 10 min. Then, for EV purification, the supernatant was centrifuged at 100,000 x g for 60 min. Pellets obtained from this spin-down were then resuspended in 30 μL of lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton-X and protease inhibitor (Sigma-Aldrich) for western blotting. Cocaine stimulation occurred by adding the drug (1–10 µM) to the cultures in HBSS.

Midbrain

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For vesicle fractions from brain tissue we used an established protocol with minor modifications (Perez-Gonzalez et al., 2012; Polanco et al., 2016). Briefly, following dissection, midbrain slices from two wildtype male C57BL/6J mice were chopped and then incubated in 1.5 ml of 0.125% collagenase (Sigma-Aldrich) in Neurobasal medium (Thermo Fisher Scientific) for 30 min at 37°C (see Figure 2—figure supplement 1 for a graphic summary of Ev isolation procedures). To stop the digestion, 4.5 ml of ice-cold phosphate-buffered saline (PBS) was added and the temperature maintained at 4°C throughout subsequent steps. The tissue was then gently disrupted by multiple passes through a 200 µL pipette tip, followed by a series of differential centrifugations at 300 x g for 10 min, 2000 x g for 10 min, and 7500 x g for 30 min. The pellets resulting from these spins, containing cells, membranes, and cellular debris, respectively, were then discarded. For EV purification, the 7500 x g supernatant was syringe filtered at 1.0 μm (Whatman Puradisc Syringe Filters, GE Healthcare Life Sciences, Cat. #6780–2510) and centrifuged at 100,000 x g for 70 min to obtain a pellet containing EVs. The 100,000 x g pellet was washed with PBS and spun again at 100,000 x g for 60 min to obtain a total EV (tEV) pellet. For EV purification, the tEV sample was resuspended in 0.5 mL of 0.95 M sucrose in 20 mM HEPES (pH 7.4) before addition to a sucrose-step gradient column. The column consisted of 6 × 0.5 mL fraction running from the bottom 2.0 M, 1.65 M, 1.3 M, 0.95M, 0.6 M, to 0.25 M at the top. Similarly, sucrose step gradients were centrifuged for 16 hr at 200,000 x g, after which the six fractions were collected. EVs settled typically at 0.95 M sucrose. The original six 0.5 mL fractions were collected and resuspended in 6 mL of ice-cold PBS, followed by a 100,000 x g centrifugation for 70 min at 4°C. Finally, the pellets were resuspended in 30 μL of filtrated-PBS when EVs were used for cell assays or 15 µl of lysis buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton-X and protease inhibitor (Sigma-Aldrich) when EVs were intended for western blots. For western blotting, EV lysates in lysis buffer were quantified for protein content with a Micro BCA Protein Assay Kit (Thermo Fisher Scientific). We also prepared brain lysate sample (BL) in lysate buffer using the midbrain tissues from the 300 x g pellets obtained in the courses of the EV isolations, which were used as positive controls for the western blots and to normalize tEVs sample amount between each treatment.

Drug treatment regimen

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Drugs were injected i.p. at a volume of 5 ml/kg. Regimen 1 (for Figure 2E): Thirty and 60 min after i.p. injections with cocaine (15 mg/kg), midbrain slices were collected. Regimen 2 (for Figures 2F and 4E): Injections with BD1063 (10 mg/kg, s.c.), SecinH3 (10 µmol/kg, s.c.), ML7 (5 mg/kg), or vehicle (inj 1) were performed 20 min prior to injections with saline or cocaine (15 mg/kg, i.p.; inj 2). Thirty min after inj 2, midbrain slices were collected. Regimen 3 (for Figure 4A): 10, 20 and 30 min after i.p. injections with cocaine (15 mg/kg), midbrain slices were collected. Regimen 4 (for Figure 4B): Injections with BD1063 (10 mg/kg, s.c.), SecinH3 (10 µmol/kg, s.c.), or vehicle (s.c.) (inj 1) were performed 20 min prior to injections with saline or cocaine (15 mg/kg, i.p.; inj 2). Ten min after inj 2, midbrain slices were collected. Regimen 5 (for Figure 4C): 20 min after i.p. injections with cocaine (15 mg/kg) or vehicle, midbrain slices were collected. Regimen 6 (for Figure 5B): 30 min after i.p. injections with cocaine (15 mg/kg) or vehicle, midbrain slices were collected. For western blotting of extracellular vesicles from NG108 cells, the cells on 10 cm dishes were washed with prewarmed Hanks' Balanced Salt Solution (HBSS) twice and incubated in HBSS at 37°C in the presence of cocaine.

Western blotting

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In brief, western blotting was performed with protein samples separated using a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto a Immobilon FL Transfer polyvinylidene difluoride (PVDF) membrane (Mollipore) in the Tris/Glycine buffer (Bio-Rad Laboratories) without methanol. After incubation with 5% blotting-grade blocker (Bio-Rad Laboratories) or 5% bovine serum albumin (BSA, Sigma-Aldrich) in TBST buffer (10 mM Tris. pH 8.0, 150 mM NaCl, and 0.5% Tween 20) for 1 hr, membranes were incubated with the primary antibodies at 4°C overnight. Membranes were washed for 10 min four times by using TBST buffer and incubated with a 1:10,000 dilution of secondly antibodies (LI-COR Biosciences) at room temperature for 1 hr. Blots were washed for 10 min four times by using TBST buffer and the signal intensity was determined using Odyssey Imaging System (LI-COR Biosciences). Resultants were analyzed using an Image Studio Lite (LI-COR Biosciences).

Nanoparticle tracking analysis (NTAs) for EVs

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Total EV (tEV) samples were isolated in filtered (at 1 µm)-PBS from WT and Sig1R KO mouse midbrain, 30 min after treatment with either saline or cocaine (15 mg/kg, i.p.), and sent to Systems Biosciences (Palo Alto, CA) for metric analysis of tEVs.

Isolation of MAM from mouse midbrain tissues

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MAM was isolated from mouse mid brain as previously reported (Hayashi and Su, 2007; Kourrich et al., 2013). Briefly, following homogenization of the brain tissue, nuclear, crude mitochondrial, and microsomal fractions were prepared by differential centrifugation. Supernatants were collected as the cytosolic fraction. The crude mitochondrial fraction in the isolation buffer (250 mM mannitol, 5 mM HEPES, 0.5 mM EGTA, pH 7.4) was subjected to a Percoll gradient centrifugation for separation of the MAM from mitochondria.

Immunofluorescence staining

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Immunofluorescence staining was performed as described previously. In brief, after blocking, the sections were incubated with the first antibodies in 5% BSA/0.1% Triton X-100 PBS overnight at 4°C. Bound antibodies were detected with Alexa Fluor 405-conjugated anti-mouse IgG (1:200, Thermo Fisher Scientific), Alexa Fluor 488-conjugated anti-chicken IgG (1:200, Thermo Fisher Scientific), and Alexa Fluor 568-conjugated anti-Rabbit IgG antibodies (1:200, Thermo Fisher Scientific) in 5% BSA PBS. An UltraView confocal microscopic system (PerkinElmer) was used for imaging.

For the immunostaining of Sig-1R, rabbit anti-serum against Sig-1R, a gift from Dr. Arnold Ruoho (University of Wisconsin, USA; Ramachandran et al., 2007), was used. When compared to several commercially available products, the affinity-purified antibody from this antiserum, is very specific for the sigma-1 receptor in the mouse dorsal root ganglia (Mavlyutov et al., 2016). We established the following procedures to allow for the best specific detection of the Sig-1R in mouse brain slices, using the antiserum from Dr. Ruoho. Deeply anesthetized animals were transcardially perfused with filtered 0.1 M Phosphate buffer (PB; pH 7.4) followed by 4% paraformaldehyde (w/v) in 0.1 M PB. After perfusion, whole brains were isolated and post-fixed in the same fixatives overnight at 4°C with rotation. Subsequently, they were dehydrated with 20% sucrose in 0.1 M PB (w/v) and then 30% sucrose in 0.1 M PB (w/v) at 4°C with rotation. The brain samples were then embedded in O.C.T. compound (Sakura Finetek, Torrance, CA) on dry ice and stored in −80°C. Thirty-µm sections were cut on a cryostat and mounted on Tissue Path Superfrost Plus Gold Microscope Slides (Fisher Scientific, Hamilton, NH) dried overnight. Sections were blocked with 5% bovine serum albumin (BSA, w/v) in PBS containing 0.1% Triton-X100 (v/v) for 1 hr at room temperature. The sections were then incubated with the sigma-1 receptor anti-sera diluted at 1:1000 in the blocking solution overnight at 4°C. Following 10 min PBS washing for three times, sections were incubated with Alexa Fluor (488 for green/568 or 594 or 546 for Red)-conjugated goat anti-rabbit IgG (1:500, Invitrogen, Carlsbad, CA) in 5% BSA in PBS for 90 min at room temperature. The sections were washed with PBS for 5 min three times, then counterstained with 4’,6’-diamino-2-phenylindole (DAPI, Invitrogen, 1 µg/mL in MilliQ; Millipore, Billerica, MA) by 10 min incubation at room temperature. Sections were washed with PBS for 5 min three times, mounted on coverslips with Prolong Diamond Antifade Mountant (Life technologies, Carlsbad, CA) for imaging. The specificity of this antiserum in labeling the Sig-1R is demonstrated in brain slices from wildtype mice, where strong staining is shown, and in and Sig-1R knockout mice, where staining is absent (Figure 3—figure supplement 1).

Immunoprecipitation

Brain tissue

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The midbrain slice sample was homogenized in 900 µl of ice-cold IP lysis buffer-1 (50 mM Tris pH7.4, 150 mM NaCl, 0.1% lauryl maltose neopentyl glycol (Anatrace, Maumee, OH) and protease inhibitors (Sigma-Aldrich) with a glass Dounce homogenizer (20 strokes). After centrifugation at 15,000 g for 10 min, protein concentration of cellular extracts was measured using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific). Five hundred µg of protein amount in supernatants were mixed with ice-cold IP lysis buffer-1 with protease inhibitors to adjust total 1000 µl. The samples were incubated and rotated with 5 µg ARF6 (Santa cruz) antibody at 4°C for overnight. Forty µl of prewashed Dynabeads Protein G (Thermo Fisher Scientific) added into the sample, incubated and rotated at 4°C for 90 min. Immunoprecipitants were washed five times with 0.8 ml of ice-cold IP lysis buffer-1 for 5 min. Samples were boiled in 30 µl elution buffer, which is combined between 15 µl of 2 x Laemmli Sample Buffer (Bio-Rad Laboratories) and 15 µl 7 M Urea/1% CHAPS at 37°C for 10 min. Importantly, 2-mercaptoethanol was omitted from the endogenous Sig1R IP assay to prevent degrading antibody disulfide bonds. Proteins were analyzed with a 12% SDS-PAGE.

NG-108 cells

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All processes were performed on ice. The overexpressed NG108 cells in 100 mm dishes were washed twice with cold PBS and then lysed in 1.0 ml of IP lysis buffer-2 (50 mM Tris pH7.4, 150 mM NaCl, 1% Nonidet P-40 (Sigma-Aldrich) and protease inhibitors (Sigma-Aldrich). After centrifugation at 15,000 g for 10 min, protein concentration of cellular extracts was measured using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific). One-hundred fifty µg of supernatants were mixed with PBS in equal volume. The supernatants were incubated and rotated at 4°C overnight with 1 µg of the rabbit anti-EGFP/EYFP/ECFP (Clontech) or 1 µg normal rabbit IgG (Santa Cruz). Thirty ml of prewashed Dynabeads Protein G (Thermo Fisher Scientific) was then applied, and samples were rotated for 90 min at 4°C. Immunoprecipitants were washed 4 times with 0.8 ml of IP lysis buffer-2 for 5 min, and twice with 1 ml of PBS for 5 min. Samples were boiled in 70 µl elution buffer combined between 35 µl of 2 x Laemmli Sample Buffer with 5% 2-ME and 35 µl lysis buffer at 95°C for 5 min. Proteins were analyzed with a 12% SDS-PAGE.

Cell culture and transfection

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NG108 cells were cultured at 37°C and 5% CO2 in High glucose Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific) containing L-glutamine, 10% Fetalgro Bovine Growth Serum (RMBIO), HAT supplement (Thermo Fisher Scientific), 100 mg/ml Penicillin-Streptomycin (Thermo Fisher Scientific). Transfection of cells with expression vectors was done by using PolyJet DNA In Vitro Transfection Reagent (Signagen Laboratories, Rockville, MD) according to manufacturer’s instructions. Sources of vectors are provided above.

Measurement of 2-AG in brain tissue

2-AG extraction

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2-Arachidonoyl glycerol (2-AG) was extracted from samples using a modified Folch extraction method. A mixture of chloroform/methanol (2:1 v/v) was added to the sample at a rate of 8 µL for each µg of protein detected. An internal standard 10Z-heptadecenoylethanolamide (HEA, 17:1 ethanolamide, Avanti Polar Lipids, Alabaster, Al) (4 µg/mL) was included in this volume and was added at a rate of 0.05 µL per µg of protein. Samples were homogenized, sonicated and vortexed. Two µL of water was added for each µg of protein in the sample. The mixture was again vortexed and centrifuged. The extraction results in an upper aqueous phase and a lower organic phase (containing 2-AG and the internal standard, HEA 17:1 ethanolamide). The lower phase (organic phase) was evaporated to dryness using nitrogen, re-suspended in 500 µL of chloroform and fractionated. The procedure used for fractionation was similar to one developed previously for eCBs (Schmid et al., 2000). The fractionation was performed with Discovery SPE-Si tubes 1 mL (Sigma-Aldrich, St. Louis, MO). The samples were loaded on the columns in 500 µL chloroform and then washed with 3 mL of chloroform. Next, the 2-AG was eluted with 3 mL of chloroform/methanol (98/2%). Finally, the elute was evaporated to dryness using nitrogen and re-suspended in 100 µL of acetonitrile.

Mass spectrometry analysis

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Samples were diluted 1:1 (v/v) in 400 µm silver acetate in acetonitrile prior to mass analysis. A previous study has demonstrated the advantages to adding silver cations into the sample mixture for detecting 2-AG (Kingsley and Marnett, 2003). Samples were analyzed on an Oribtrap Velos (Thermo Fisher) in positive ion mode with a static nanospray source with 4 µm spray tips and a capillary temperature of 200°C. The Fourier transform mass spectrometry (FTMS) mode with a mass resolution of 100K was employed for all samples. The mass error for 2-AG assignment was ±3 ppm and MS/MS analyses were also conducted to confirm the identification of 2-AG.

In vitro electrophysiology

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Twelve-week-old WT C57BL6, Cnr1-/- (CB1R knockout), or Sigmar1+/- Sig-1R KO mice were decapitated, and their brains rapidly removed and transferred to an oxygenated (95% O2, 5% CO2) ice-cold solution containing (in mM) 93 N-Methyl-D-glucamine (NMDG), 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 Glucose, 3 Sodium pyruvate, 10 MgCl2, 0.5 CaCl2, 5.6 Ascorbic acid. Horizontal slices (220 µm) containing the VTA were sectioned using a Leica VT1200S vibratome (Leica Biosystems) and transferred to a holding chamber at room temperature (RT) filled with oxygenated solution containing (in mM) 109 NaCl, 4.5 KCl, 1.2 NaH2PO4, 35 NaHCO3, 20 HEPES, 11 Glucose, 1 MgCl2, 2.5 CaCl2, 0.4 Ascorbic acid. After incubation for at least 1 hr in the holding chamber at RT, slices were transferred to a recording chamber perfused with oxygenated aCSF containing (in mM) 126 NaCl, 3 KCl, 1.2 NaH2PO4, 26 NaHCO3, 11 Glucose, 1.5 MgCl2, 2.4 CaCl2, maintained at 35–36°C using an inline solution heater (Warner Instruments, Hamden, CT). Cells were visualized with an upright microscope (Olympus BX51WI) equipped with infrared interference-contrast optics. Recorded neurons identified in the lateral VTA, medial to the terminal nucleus of the accessory optic track (MT) and anterior to the third cranial nerve. Dopamine neurons were identified in the lateral VTA using electrophysiological criteria in cell-attached mode. Only cell demonstrating regular pacemaker firing (>3 Hz) and action potential widths > 2.5 ms were chosen for further recording (Ungless and Grace, 2012). Whole-cell voltage-clamp recordings from DA neurons were acquired using an Axopatch 200B amplifier (Molecular Devices, San Jose, CA). Recording pipettes (3.5–5 MΩ) were pulled with a P-97 horizontal micropipette puller (Sutter Instruments, Novato, CA) and filled with internal solution containing (in mM) 140 K-gluconate, 2 NaCl, 1.5 MgCl2, 10 HEPES, 10 Tris-phosphocreatine, 4 Mg-ATP, 0.3 Na-GTP, 0.1 EGTA (pH 7.2, 290 mOSM). DNQX (20 µM), DL-AP5 (40 µM), picrotoxin (100 µM) and strychnine (1 µM) were present in the aCSF to block AMPA, NMDA, GABAA and glycine receptors, respectively. Electrophysiological identification of DA neurons was performed in cell-attached mode to select only cells exhibiting pacemaker firing and action potential widths < GABAB IPSCs were evoked using electrical stimulation with bipolar tungsten stimulating electrodes with tip separation of 300–400 µm. A train of 6 stimuli of 100µs duration were delivered at 50 Hz every 30 s. Stimulation protocols were generated, and signals acquired using the electrophysiology software WinLTP. Control GABAB currents were recorded for 10 min before the appropriate drug was applied for an additional 30 min. Data was analyzed using WinWCP software (Courtesy of Dr. John Dempster, Strathclyde University, Glasgow, UK). Figures were generated, and statistics analyzed using GraphPad Prism6 (v6.07; LaJolla, CA). Data are presented as the change in percent from control.

Quantification, statistical analysis and reporting

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The experiments were designed using estimates of effect size and standard error derived from prior experience and pilot experiments. These values were then used in power analysis calculations using the program G-Power (version 3.1.9.4, University of Dusseldorf, Germany) to determine sample sizes. Means ± s.e.m. are used throughout to report measures of centricity and dispersion. A spreadsheet (Source data 1) describing means, significance levels and 95% confidence intervals for each experiment is included with this report. Statistical tests were determined by the number of groups and treatments to be compared. An omnibus test was used when necessary statistical assumptions could be met. Thus, in experiments where repeated measures could be obtained from the same subjects, samples, or cells (e.g. time course data), a repeated-measures ANOVA was used. When repeated measures were not performed, and group size was >2, a one-way ANOVA was used. Post-hoc analyses (Tukey’s, Dunnett’s, or Bonferroni’s multiple comparison tests) were determined by the type of omnibus test, as well as the nature of the multiple comparisons (pairwise rows and columns, comparison to control columns, main effects versus interactions). When only two groups of data were compared, a Student’s t-test was used. In all cases, a two-tailed p value of 0.05 was considered the minimum for significance. Actual p values are reported for all omnibus tests, unless p<0.0001, and the statistical information is reported in the figure captions. In immunoprecipitation experiments, co-localization was determined from observed association on Western blots, and therefore, statistical tests were not used (Figure 1F and G; Figure 3B and D; Figure 5D; Figure 1—figure supplement 1C).

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    Mechanisms of cannabinoid inhibition of GABA(A) synaptic transmission in the Hippocampus
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Decision letter

  1. Gary L Westbrook
    Senior and Reviewing Editor; Oregon Health and Science University, United States
  2. Ken Mackie
    Reviewer; INSERM-Indiana University, United States
  3. Christopher Ford
    Reviewer; University of Colorado Anschutz Medical Campus, United States
  4. Nephi Stella
    Reviewer

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Cocaine-induced endocannabinoid signaling mediated by sigma-1 receptors and extracellular vesicle secretion" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Gary Westbrook as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Ken Mackie (Reviewer #1); Christopher Ford (Reviewer #2); Nephi Stella (Reviewer #3). The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

This study examines the hypothesis that cocaine stimulates 2-AG secretion through its interactions with the sigma-1 receptor (Sig-1R) leading to release of EVs (enriched) in 2-AG, which then engage CB1 receptors.

Essential revisions:

The revised version of this study must fully address the main concerns of reviewer #1. I want to emphasize that this is indispensable and understand that additional experiments may need to be carried on. Thus, experimental evidence showing that EVs are secreted by cocaine stimulated NG108-15 and that the cocaine stimulated 2-AG increase is specific for EVs and not general for all midbrain membranes must be provided (see verbatims of the reviewer's comments below). The editors and reviewers during their discussion were in full agreement on these essential revisions.

Reviewer #1:

In this interesting and provocative study, Nakamura and colleagues examine the hypothesis that cocaine stimulates 2-AG secretion through its interactions with the sigma-1 receptor (Sig-1R) leading to release of EVs (enriched) in 2-AG, which then engage CB1 receptors.

1) Central to their hypothesis is that cocaine causes release of EVs containing 2-AG. To demonstrate release, it is necessary to isolate them from the media of the NG108-15 cells (as was done in the Gabrielli study). From a careful reading of the Materials and methods, it does not seem that this was done. Rather, NG108-15 cells were washed, scraped, homogenized and membranes with EV markers enriched. Thus, what is established by the NG108-15 studies is that the various signature of EVs are enriched within NG108-15 cells by cocaine, but not that the EVs are secreted.

2) Indirect support for 2-AG being secreted by EVs is that 2-AG concentration, when normalized to membrane protein content, is increased in EVs compared to 2-AG present in total midbrain membranes. It is preferable to normalize lipids to total lipid content or mole% of lipids in the preparation to control for different protein content in EVs vs. total membrane. In support of the central hypothesis, it is important to demonstrate that the increase in 2-AG following cocaine is specific for EVs and not generally for all midbrain membranes.

3) Figure 5 (in combination from earlier work by this group) nicely shows that GABA IPSC inhibition likely results from 2-AG produced by DA neuronal DAGL-α. Is the rather large increase in midbrain 2-AG all due to increase in 2-AG from DA neurons? This should be evaluated by measuring 2-AG in midbrain in the DAGL-α conditional KOs after cocaine treatment.

4) That inhibition or genetic deletion of Sig-1R slows, but does not block cocaine inhibition of IPSCs is quite interesting. The authors establish that CB1 responses are not altered by Sig-1R manipulations, but do Sig-1R manipulations affect IPSC inhibition when 2-AG is synthesized by other routes? For example, will the combination of DHPG/phenylephrine still produce 2-AG to reduce IPSCs after inhibition of Sig-1Rs? Establishing whether Sig-1Rs are needed for all synaptic release of 2-AG would greatly increase the impact of the findings. Similarly, does this synapse show DSI? If so, will manipulations of Sig-1R prevent DSI? (The phenomenon shown here is reminiscent of inhibition of DSI, but not LTD.)

5) While the authors interpret the inhibition of the rapid, but not the delayed, phase of cocaine IPSC inhibition as potential support for a "readily releasable pool" of 2-AG, an alternative interpretation is that manipulating Sig-1R affects DAGL localization to the synapse. This should be examined in the Sig-1R KO.

Reviewer #2:

In the present manuscript by Nakamura et al., the authors examine a novel pathway by which cocaine regulates the release of 2-AG. Over a series of experiments, the authors outline a pathway by which cocaine activates the Sig-1R, which releases a tonic inhibition on ARF6. This small G-protein when in the GTP bound state activates MLCK which in turn increases non-synaptic extracellular vesicle release. In the first part of the manuscript the authors utilize NG-108 cells as well as midbrain homogenates to elucidate this pathway. They find that cocaine can increase markers of EVs suggesting that cocaine increases EV release in a Sig-1R dependent manner. Utilizing Western blots, co-IPs and immunostaining they then go on to show that in the midbrain cocaine decreases the association of Sig1R and ARF6 and that the GEF inhibitor SH3 inhibits cocaine-induced EV release. Finally to examine the functional consequence of cocaine on EV release they utilize slice electrophysiology. The authors find that the mechanism by which cocaine leads to the increase in 2AG release (as measured by presynaptic inhibition of GABAB IPSCs) may partially involve this pathway as the effects were blocked by sigma-1 receptor antagonists as well as ARF6 and MLCK blockers.

The experiments are thorough, have been done well and interpreted appropriately. Furthermore, the combination of approaches come together nicely to clearly define this new mechanism by which cocaine may regulate endocannabinoid signaling. Based on volume of work presented I have no further suggestions for other experiments as the results and conclusions are clear. A potential caveat is that the ARF6 and MLCK blockers could have additional effects, not dependent on EV release, which could also be driving 2AG release. However further confirming this pathway and true EV release would likely involve a significant amount of further work, beyond the scope of this manuscript.

A minor point, however is that in many of the figures, summary data are only presented for some experiments. It is unclear at least from the results and figure legends if the other experiments are an n = 1 or if they are only representative blots from experiments that have been replicated several times. A second point is that in Figure 4D, the authors show that cocaine increases GTP-bound ARF6 in the midbrain. Presumably this relies on a specific antibody that only recognizes GTP-ARF6 however it was not clear that this antibody can distinguish between GDP and GTP bound forms.

Reviewer #3:

This study demonstrates the involvement of a novel cellular compartment (non-synaptic extracellular vesicle (EVs) and its intracellular machinery (Sig-1R > ARF6) in 2-AG-mediated activation of CB1R. The experimental design is thorough and uses validated pharmacological and genetic tools in established model systems. The technical approaches are sound, the manuscript clearly written, and conclusions and interpretations based on convincing results. I believe that this study will have an important impact on the field of cannabinoid research and neuroscience in general, and provide an important foundation for future studies.

1) Figure 1—figure supplement 1A and B. The authors should provide the fold change in expression of Sig-1R to help the reader better understand the impact of this result.

2) Figure 2—figure supplement 1: Mention in the text the time between stimulation with cocaine and EV harvesting to help the reader understand this result.

3) Figure 3A and 3C: the magnified images are not convincing and should be improved. Their scale bars are missing, and it appears that the magnification is merely 2X. Higher quality and magnification images should be provided here.

4) Figure 3E: the diagram could be increased in size and its clarity improved (e.g. reduce size/thickness of the X that overlays ARF6).

5) Figure 4D: The qualities of the images should be greatly improved, and the authors should provide magnifications that clearly illustrate TH versus ARF6-GTP locations.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Cocaine-induced endocannabinoid signaling mediated by sigma-1 receptors and extracellular vesicle secretion" for further consideration at eLife. Your revised article has been favorably evaluated by Gary Westbrook as the Senior and Reviewing Editor, and three reviewers. The manuscript has been improved but there a few remaining issues that we would like addressed before acceptance, as outlined below: We expect to handle the revised manuscript at the editorial level.

Summary:

The reviewers were generally satisfied with the revisions of your manuscript and agreed that it provides a significant conceptual advance in understanding the mechanisms by which cocaine regulates 2AG release via the sigma-1 receptor. The authors clarified that EVs were purified from NG108-15 culture medium. They were also able to demonstrate the requirement of Sig-1R in phenylephrine/DHPG 2-AG-mediated IPSC inhibition, while there appears to be no role for Sig-1R in "basal" 2-AG-mediated CB1 activation. However the reviewers had a few additional comments that should be addressed in a final revision.

1) The authors were not able to demonstrate an increase in midbrain EV 2-AG levels following cocaine administration (possibly due to the sensitivity of their 2-AG measurement technique coupled with the small number of EVs that can be purified from midbrain), which undermines a major premise of the study-cocaine stimulates release of 2-AG enriched EVs to inhibit IPSCs. This limitation needs to be appropriately integrated into their Results and Discussion. Perhaps an alternative explanation that should be considered is that there is not more 2-AG per EV, but there are more EVs produced, following cocaine or phenylephrine/DHPG and this is sufficient to activate CB1 to inhibit GABA release.

2) Emphasis in the Discussion. a) There needs to be a significant shift in the emphasis of the paper's Discussion. Because Sig-1R is required for phenylephrine/DHPG 2-AG-mediated IPSC inhibition (Figure 7), the emphasis of cocaine binding to Sig-1R as a causal mechanism in mid-brain eCB IPSC inhibition appears misplaced. If anything, in the cocaine treated slices, Sig-1R is only necessary for the short term (<20 minutes) cocaine/eCB-mediated depression of IPSCs (Figure 6D) as IPSC inhibition by cocaine seems similar in the wildtype and Sig-1R KOs by 30 minutes (Figure 6D). In contrast (Figure 7B), for phenylephrine/DHPG 2-AG-mediated IPSC inhibition, Sig-1R KO reduces IPSC inhibition over the entire 30 minutes of drug application. An alternative to the mechanism proposed in the Discussion (cocaine binds to the Sig-1R…, and Figure 8—figure supplement 1) is that cocaine increases NE, activating α1Rs, which then require Sig-1R, ARF6, etc., to release EVs (or stimulate DAGLA activity). The experimental data in midbrain slices that cocaine needs to bind to sig1-Rs for IPSC inhibition does not seem conclusive, only that Sig-1R and ARF6 are required at some point in the pathway.

b) Similarly, the difference in the role of Sig-1R, ARF6, MLCK between cocaine and phenylephrine/DHPG inhibition of IPSCs needs to be briefly discussed. As mentioned in the Discussion, these proteins seem to play a role in early IPSC inhibition by cocaine, but not late inhibition. On the other hand, at least Sig-1Rs are required for both early and late IPSC inhibition by phenylephrine/DHPG. Can the authors integrate these findings into their model?

3) Figure 5C: The WB results showing FLOT-1 in EV has a "dark dot" in its lane which limits the interpretation of the result. The authors should provide another representative result.

https://doi.org/10.7554/eLife.47209.021

Author response

Reviewer #1:

[…] 1) Central to their hypothesis is that cocaine causes release of EVs containing 2-AG. To demonstrate release, it is necessary to isolate them from the media of the NG108-15 cells (as was done in the Gabrielli study). From a careful reading of the Materials and methods, it does not seem that this was done. Rather, NG108-15 cells were washed, scraped, homogenized and membranes with EV markers enriched. Thus, what is established by the NG108-15 studies is that the various signature of EVs are enriched within NG108-15 cells by cocaine, but not that the EVs are secreted.

We appreciate the reviewer mentioning this oversight on our part. We neglected to state in the original Materials and methods that we followed the protocol published in the Gabrielli et al. paper, with only a few modifications. Thus, we collected HBSS from the cultures after treatment with cocaine (and other manipulations) and then subjected this effluent to EV isolation procedures (sequential centrifugation and sucrose gradients to yield fr3). Therefore, the EV markers were isolated from fractions collected from the cells after stimulation, and not from homogenized cells. We now include a more complete description of these methods in the manuscript (subsection “Midbrain”).

2) Indirect support for 2-AG being secreted by EVs is that 2-AG concentration, when normalized to membrane protein content, is increased in EVs compared to 2-AG present in total midbrain membranes. It is preferable to normalize lipids to total lipid content or mole% of lipids in the preparation to control for different protein content in EVs vs. total membrane. In support of the central hypothesis, it is important to demonstrate that the increase in 2-AG following cocaine is specific for EVs and not generally for all midbrain membranes.

Total lipids were not measured in the mass spectrometry assay as we only measured 2-AG and our internal standard. This is because it is not possible to measure total lipid content using MS. It is possible to record a larger m/z range which permits detection of a broader range of lipid species, but this would still only be a small fraction of the total lipid content. Also, it is worth noting here that the methods in the Gabrielli et al. paper indicate that anandamide levels were also normalized to total protein content.

We also agree that measuring cocaine-induced changes in 2-AG in EV fractions using FTMS would provide specific and direct evidence of our hypothesis. However, after conducting this experiment several times we have encountered several limitations. First and foremost, the amount of EV material obtained in fr3 from ventral midbrain and available for analysis using FTMS is extremely small, necessitating the pooling of fr3 samples from several mice. To achieve this, we must first kill the mice 15 min after injection of saline or cocaine (3 mice per sample), then rapidly dissect the ventral midbrain from each mouse and pool samples across mice to quickly perform centrifugation and sucrose gradient procedures. We have now performed these experiments on three separate occasions in three different cohorts of mice (n = 30 mice), and we observe a high degree of variability in both baseline levels of 2-AG using FTMS, and in the level of the cocaine-induced increase in 2-AG levels. In general, there is a trend toward increased 2-AG in fr3 in the cocaine-injected mice (see Author response image 1), but this does not reach statistical significance because of the high degree of variability across cohorts. It is likely that some of this variability results from the difficulty of the dissection procedure (identifying and dissecting only ventral midbrain from a whole a mouse brain) and to other factors that we have not yet identified. Therefore, at present we are confident that we can identify 2-AG in the EV-containing fr3 from mouse midbrain, but we do not have the statistical power to demonstrate a significant increase in 2-AG content of these fractions after cocaine stimulation. We defer to the discretion of the reviewer and the editors as to whether these data should be included in the manuscript.

Author response image 1

3) Figure 5 (in combination from earlier work by this group) nicely shows that GABA IPSC inhibition likely results from 2-AG produced by DA neuronal DAGL-α. Is the rather large increase in midbrain 2-AG all due to increase in 2-AG from DA neurons? This should be evaluated by measuring 2-AG in midbrain in the DAGL-α conditional KOs after cocaine treatment.

A previous study, that we now cite in the manuscript, used in-situ hybridization, immunohistochemistry and electron microscopy to localize DGLα in most neurons in the VTA, including DA and non-DA neurons (Mátyás et al., 2008, Identification of the sites of 2-arachidonoylglycerol synthesis and action imply retrograde endocannabinoid signaling at both GABAergic and glutamatergic synapses in the ventral tegmental area. Neuropharmacology, 54:95-107). Therefore, it is likely that 2-AG is synthesized in several cell types in the VTA. However, we reason that because we measure the inhibition of IPSCs in only identified DA neurons, 2-AG release from these other sources may not be detected at synapses onto these cells because of the limited spatial range over which 2-AG can act before its uptake or metabolism occurs. Alternatively, it is possible that the non-DA neurons that express DGLα do not release 2-AG when exposed to cocaine because they lack α1-noradrenergic receptors, or some other necessary component.

Although these are undoubtedly interesting issues, the observation that the 2-AG effect on synaptic transmission is absent in the DGLα KOs, as well as in the brain slices treated with the DGLα inhibitor tetrahydrolipostatin (Figure 5—figure supplement 1), strongly suggest that the endocannabinoid involved in the inhibition is 2-AG. Since the identification of the endocannabinoid was the primary goal of these experiments, we feel that this has been achieved. Moreover, these transgenic mice are not yet freely available, and were included in this study under a material transfer agreement restricting their use to only DA neurons.

4) That inhibition or genetic deletion of Sig-1R slows, but does not block cocaine inhibition of IPSCs is quite interesting. The authors establish that CB1 responses are not altered by Sig-1R manipulations, but do Sig-1R manipulations affect IPSC inhibition when 2-AG is synthesized by other routes? For example, will the combination of DHPG/phenylephrine still produce 2-AG to reduce IPSCs after inhibition of Sig-1Rs? Establishing whether Sig-1Rs are needed for all synaptic release of 2-AG would greatly increase the impact of the findings. Similarly, does this synapse show DSI? If so, will manipulations of Sig-1R prevent DSI? (The phenomenon shown here is reminiscent of inhibition of DSI, but not LTD.)

These are excellent suggestions, and we have conducted additional experiments to address some of these issues. First, as indicated by the reviewer, our prior work in rat VTA shows that the increase in 2-AG function produced by cocaine could be replicated by combined application of DHPG and phenylephrine (PE) to activate type-I metabotropic glutamate receptors (mGluRI) and α1-noradrenergic receptors, respectively. Therefore, if this effect can be replicated in mice, we would predict that this effect of DHPG+PE should, like that of cocaine, be inhibited when Sig-1R function is impaired or absent, and this would provide a strong case that Sig-1Rs and the EV signaling cascade is important for GPCR-dependent 2-AG release in the VTA. In these new experiments we found that the inhibition of IPSCs produced by DHPG+PE was very similar to that produced by cocaine alone, and that the effect of DHPG+PE was significantly reduced in DA neurons from Sig-1R knockout mice. Moreover, the effects of DHPG+PE were blocked in mouse DA neurons, as reported in rats. Therefore, this supports the involvement of Sig-1Rs in GPCR-dependent 2-AG release. We have now included these data in a new figure (Figure 7A-C) and discuss them in the paper (subsection “Sig-1R antagonism prevents cocaine-stimulated 2-AG function in VTA DA neurons” and Discussion, last paragraph).

Regarding the second part of the reviewer’s question, we have only established that DSI occurs at GABA inputs to VTA DA neurons in THCre mutant rats, using cre-driven channel rhodopsin to initiate DSI in the DA neuron (see Wenzel et al., Curr Biol., 28:1392-1404, 2018). However, we have not performed these experiments in mice because of limitations in obtaining THCre mice. However, we have also reported that tonic inhibition of IPSCs by 2-AG is observed in both rat and muse DA neurons, and this is uncovered when CB1Rs are blocked by AM251, or when DGLα is blocked by THL (Riegel and Lupica, 2004; Wang et al., 2015). Although we have not verified that this source of 2-AG is 100% independent of GPCR stimulation, it is present under baseline conditions in the absence of cocaine, or other exogenously applied GPCR agonists. Therefore, in new experiments we measured this tonic 2-AG response in the mouse VTA and observed the expected robust increase in IPSCs during AM251 application. Importantly, this level of tonic 2-AG inhibition did not differ between wildtype and Sig-1R knockout mice. This, together with the DHPG+PE data, suggests that Sig-1Rs and EVs are primarily involved in promoting 2-AG release derived from GPCR stimulation-dependent synthesis. We have compiled these data obtained with the Sig-1R KO and WT mice and have added them to a new Figure 7D-E, describe them the Results section, and expanded the Discussion a bit to deal with the distinction in Sig-1R modulation of GPCR-dependent 2-AG function.

5) While the authors interpret the inhibition of the rapid, but not the delayed, phase of cocaine IPSC inhibition as potential support for a "readily releasable pool" of 2-AG, an alternative interpretation is that manipulating Sig-1R affects DAGL localization to the synapse. This should be examined in the Sig-1R KO.

Although this is an interesting possibility, it would probably best be addressed using electron microscopy to examine the localization of DGLα in the VTA of Sig-1R KO mice. Alternatively, evidence that DGLα function is intact in the Sig-1R KOs would suggest that the enzyme is not altered in these mutant mice. Prior work from our lab shows that 2-AG is tonically present at GABAergic synapses on DA neurons in the rat VTA, and its influence on IPSCS can be observed when CB1Rs are antagonized by AM215, resulting in an increase in the IPSC response (Riegel and Lupica, 2004; Wang et al., 2015, cited in the manuscript). If this tonic 2-AG release was not altered in the Sig-1R KO mouse VTA, then this would provide support for intact DGLα function. Therefore, we have performed additional experiments comparing the effect of AM251 on IPSCs in wildtype and Sig-1R KO mouse VTA and find no difference. Therefore, it appears that functionally, with respect to tonic 2-AG, there is no change in DGLα function in the mutant mice. We have added an additional figure (Figure 7) to the manuscript showing the impaired DHPG+PE-induced 2-AG effect (comment #4), and intact tonic 2-AG function in the Sig-1R KO mice. We have also added this information to the Results and Discussion sections.

Reviewer #2:

[…] The experiments are thorough, have been done well and interpreted appropriately. Furthermore, the combination of approaches come together nicely to clearly define this new mechanism by which cocaine may regulate endocannabinoid signaling. Based on volume of work presented I have no further suggestions for other experiments as the results and conclusions are clear. A potential caveat is that the ARF6 and MLCK blockers could have additional effects, not dependent on EV release, which could also be driving 2AG release. However further confirming this pathway and true EV release would likely involve a significant amount of further work, beyond the scope of this manuscript.

A minor point, however is that in many of the figures, summary data are only presented for some experiments. It is unclear at least from the results and figure legends if the other experiments are an n = 1 or if they are only representative blots from experiments that have been replicated several times.

This information is included in the metadata file that has been revised to add newly-included experiments. We have also now added this information to the relevant figure legends.

A second point is that in Figure 4D, the authors show that cocaine increases GTP-bound ARF6 in the midbrain. Presumably this relies on a specific antibody that only recognizes GTP-ARF6 however it was not clear that this antibody can distinguish between GDP and GTP bound forms.

The product description for the antibody (New East Biosciences, Malvern, PA, catalog number 26918) indicates that it can distinguish ARF6-GTP from ARF6-GDP. However, as we are unable to use this antibody for quantitative Western blot analyses of ARF6-GTP, and we have confirmed co-localization of ARF6 with TH and the Sig-1R in the mouse midbrain in Figure 3, we have elected to remove this image (Figure 4D) and descriptions of measurement of ARF6-GTP from the manuscript.

Reviewer #3:

[…] 1) Figure 1—figure supplement 1A and B. The authors should provide the fold change in expression of Sig-1R to help the reader better understand the impact of this result.

We agree with the reviewer that this information is important to include, and we have added it to the Figure 1—figure supplement 1A caption. The siRNA significantly reduced Sig-1R expression by approximately 50% (49 ± 12% of control, p < 0.0001, unpaired t-test). However, a description of the overexpression of the Halo-tagged Sig-1R in Figure 1—figure supplement 1B is somewhat meaningless because this receptor is not natively expressed (see “Halo-”, 3rd row Western blot Figure 1—figure supplement 1B). Therefore, the x-fold change will be deceptively high and is not reported.

2) Figure 2—figure supplement 1: Mention in the text the time between stimulation with cocaine and EV harvesting to help the reader understand this result.

These times vary according to the experiment and are always mentioned in the figure legends and text describing these experiments.

3) Figure 3A and 3C: the magnified images are not convincing and should be improved. Their scale bars are missing, and it appears that the magnification is merely 2X. Higher quality and magnification images should be provided here.

We have performed additional immunohistochemical experiments and have provided new images at higher resolution. The scale bar is now 20 µm, rather than 50 µm as in the original figure.

4) Figure 3E: the diagram could be increased in size and its clarity improved (e.g. reduce size/thickness of the X that overlays ARF6).

We have re-worked this schematic to improve clarity.

5) Figure 4D: The qualities of the images should be greatly improved, and the authors should provide magnifications that clearly illustrate TH versus ARF6-GTP locations.

See response to reviewer 3. We have removed this image from the manuscript.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

1) The authors were not able to demonstrate an increase in midbrain EV 2-AG levels following cocaine administration (possibly due to the sensitivity of their 2-AG measurement technique coupled with the small number of EVs that can be purified from midbrain), which undermines a major premise of the study-cocaine stimulates release of 2-AG enriched EVs to inhibit IPSCs. This limitation needs to be appropriately integrated into their Results and Discussion. Perhaps an alternative explanation that should be considered is that there is not more 2-AG per EV, but there are more EVs produced, following cocaine or phenylephrine/DHPG and this is sufficient to activate CB1 to inhibit GABA release.

We have expanded the Results section to include these data and have added them to a revised Figure 5 (Figure 5C). In addition, we now consider this information in the Discussion and have added the alternative explanation provided by the reviewer.

2) Emphasis in the Discussion. a) There needs to be a significant shift in the emphasis of the paper's Discussion. Because Sig-1R is required for phenylephrine/DHPG 2-AG-mediated IPSC inhibition (Figure 7), the emphasis of cocaine binding to Sig-1R as a causal mechanism in mid-brain eCB IPSC inhibition appears misplaced. If anything, in the cocaine treated slices, Sig-1R is only necessary for the short term (<20 minutes) cocaine/eCB-mediated depression of IPSCs (Figure 6D) as IPSC inhibition by cocaine seems similar in the wildtype and Sig-1R KOs by 30 minutes (Figure 6D). In contrast (Figure 7B), for phenylephrine/DHPG 2-AG-mediated IPSC inhibition, Sig-1R KO reduces IPSC inhibition over the entire 30 minutes of drug application. An alternative to the mechanism proposed in the Discussion (cocaine binds to the Sig-1R…, and Figure 8—figure supplement 1) is that cocaine increases NE, activating α1Rs, which then require Sig-1R, ARF6, etc., to release EVs (or stimulate DAGLA activity). The experimental data in midbrain slices that cocaine needs to bind to sig1-Rs for IPSC inhibition does not seem conclusive, only that Sig-1R and ARF6 are required at some point in the pathway.

In re-considering these data, we noted differences between the inhibition of IPSCs produced by DHPG+PE and cocaine that we feel might explain this and we have included this in the manuscript in the form of new information in the Results and Discussion, and a new Figure 7—figure supplement 1. Specifically, we noted that the kinetics of the response to DHPG+PE differed from that of cocaine, and we reason that the faster onset and offset of the cocaine effect could reflect direct activation of Sig-1Rs that initiates rapid EV release and depletions. On the other hand, we suggest that the slower kinetics of the DHPG+PE effect could reflect delayed recruitment of EVs by an intracellular messenger release by GPCR activation, and we cite a recent study showing that choline can act as a Sig-1R agonist to perhaps fulfill this role.

b) Similarly, the difference in the role of Sig-1R, ARF6, MLCK between cocaine and phenylephrine/DHPG inhibition of IPSCs needs to be briefly discussed. As mentioned in the Discussion, these proteins seem to play a role in early IPSC inhibition by cocaine, but not late inhibition. On the other hand, at least Sig-1Rs are required for both early and late IPSC inhibition by phenylephrine/DHPG. Can the authors integrate these findings into their model?

See response to part “a” above.

3) Figure 5C: The WB results showing FLOT-1 in EV has a "dark dot" in its lane which limits the interpretation of the result. The authors should provide another representative result.

This has been remedied by providing another WB from another similar experiment.

https://doi.org/10.7554/eLife.47209.022

Article and author information

Author details

  1. Yoki Nakamura

    Cellular Pathobiology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, United States
    Present address
    Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan
    Contribution
    Conceptualization, Data curation, Formal analysis, Validation, Investigation, Methodology, Writing—original draft, Writing—review and editing
    Contributed equally with
    Dilyan I Dryanovski
    Competing interests
    No competing interests declared
  2. Dilyan I Dryanovski

    Electrophysiology Research Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, United States
    Contribution
    Conceptualization, Validation, Investigation, Methodology, Project administration, Writing—review and editing
    Contributed equally with
    Yoki Nakamura
    Competing interests
    No competing interests declared
  3. Yuriko Kimura

    Cellular Pathobiology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, United States
    Contribution
    Data curation, Formal analysis, Investigation
    Competing interests
    No competing interests declared
  4. Shelley N Jackson

    Structural Biology Unit, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, United States
    Contribution
    Resources, Formal analysis, Validation, Investigation, Visualization, Methodology
    Competing interests
    No competing interests declared
  5. Amina S Woods

    Structural Biology Unit, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, United States
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Investigation, Methodology, Project administration
    Competing interests
    No competing interests declared
  6. Yuko Yasui

    Cellular Pathobiology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, United States
    Contribution
    Data curation, Validation, Investigation, Methodology
    Competing interests
    No competing interests declared
  7. Shang-Yi Tsai

    Cellular Pathobiology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, United States
    Contribution
    Formal analysis, Investigation, Visualization, Methodology
    Competing interests
    No competing interests declared
  8. Sachin Patel

    1. Cellular Pathobiology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, United States
    2. Department of Psychiatry and Behavioral Sciences, Vanderbilt Brain Institute, Vanderbilt University Medical Center, Vanderbilt University, Nashville, United States
    Contribution
    Resources, Funding acquisition, Validation, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8052-520X
  9. Daniel P Covey

    Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, United States
    Contribution
    Resources, Data curation, Validation, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9596-108X
  10. Tsung-Ping Su

    Cellular Pathobiology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, United States
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Project administration, Writing—review and editing
    For correspondence
    TSU@intra.nida.nih.gov
    Competing interests
    No competing interests declared
  11. Carl R Lupica

    Electrophysiology Research Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, United States
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    clupica@mail.nih.gov
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5375-3263

Funding

National Institute on Drug Abuse (1ZIADA000487-14)

  • Carl R Lupica

National Institute on Drug Abuse (1ZIADA000206-33)

  • Tsung-Ping Su

Japan Society for the Promotion of Science

  • Yoki Nakamura

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work is supported by the US Department of Health and Human Services, National Institutes of Health, and National Institute on Drug Abuse, Intramural Research Program. YN was supported in part by the Japanese Society for Promotion of Sciences. We would like to acknowledge expert assistance of Dr. Shiliang Zhang of the NIDA-IRP Confocal and Electron Microscopy Core, as well as the efforts of Dr. Francois Vautier, Director, of the NIDA-IRP Breeding Facility, and all of NIDA-IRP the transgenic facility staff.

Ethics

Animal experimentation: Ethics Statement: All animal procedures were conducted in accordance with the principles as indicated by the NIH Guide for the Care and Use of Laboratory Animals. These animal protocols were also reviewed and approved by the NIDA intramural research program Animal Care and Use Committee, which is fully accredited by the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International (approved protocols: 17-CNRB-15, 16-CNRB-128, 16-INB-1, 16-INB-3, 17-INB-5).

Senior and Reviewing Editor

  1. Gary L Westbrook, Oregon Health and Science University, United States

Reviewers

  1. Ken Mackie, INSERM-Indiana University, United States
  2. Christopher Ford, University of Colorado Anschutz Medical Campus, United States
  3. Nephi Stella

Publication history

  1. Received: March 28, 2019
  2. Accepted: October 3, 2019
  3. Accepted Manuscript published: October 9, 2019 (version 1)
  4. Version of Record published: November 12, 2019 (version 2)

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

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