Choline-Sigma-1R as an Additional Mechanism for Potentiation of Orexin by Cocaine

Orexin A, an endogenous peptide involved in several functions including reward, acts via activation of orexin receptors OX1 and OX2, Gq-coupled GPCRs. We examined the effect of a selective OX1 agonist, OXA (17-33) on cytosolic calcium concentration, [Ca2+]i, in neurons of nucleus accumbens, an important area in the reward circuit. OXA (17-33) increased [Ca2+]i in a dose-dependent manner; the effect was prevented by SB-334867, a selective OX1 receptors antagonist. In Ca2+-free saline, the OXA (17-33)-induced increase in [Ca2+]i was not affected by pretreatment with bafilomycin A1, an endo-lysosomal calcium disrupter, but was blocked by 2-APB and xestospongin C, antagonists of inositol-1,4,5-trisphosphate (IP3) receptors. Pretreatment with VU0155056, PLD inhibitor, or BD-1047 and NE-100, Sigma-1R antagonists, reduced the [Ca2+]i response elicited by OXA (17-33). Cocaine potentiated the increase in [Ca2+]i by OXA (17-33); the potentiation was abolished by Sigma-1R antagonists. Our results support an additional signaling mechanism for orexin A-OX1 via choline-Sigma-1R and a critical role for Sigma-1R in the cocaine–orexin A interaction in nucleus accumbens neurons.

OX 1 receptors were identified in brain nuclei from the reward circuit, including nucleus accumbens [14,15], and OX 1 -selective antagonists have been evaluated as potential therapeutic agents for addiction treatment [16][17][18]. Previous studies indicate that orexins via OX 1 receptor activation are involved in the response to cocaine and play multiple roles in cocaine addiction-related behaviors [7,[19][20][21]. Orexin-OX 1 signaling is required for stimulant locomotor sensitization and cocaine seeking when it is driven by highly 2 of 9 motivated states [3]. OX 1 receptors in the nucleus accumbens mediate chronic cocaineinduced locomotor sensitization [22]. Other studies indicate that SB-334867, a selective OX 1 receptor antagonist, prevents cocaine seeking and is a potential treatment target for cocaine relapse prevention [23].
Cocaine acts primarily by blocking the dopamine transporter, thus increasing dopamine transmission in the nucleus accumbens [24], an important area in the reward circuit [25]. In addition, cocaine binds to and activates Sigma-1R [26]. Since nucleus accumbens neurons express Sigma-1R [27][28][29] and OX 1 receptors [14,15] and behavioral studies support the cocaine-orexin interaction at this level [3,22], in this work, we examined the underlying mechanisms and role of Sigma-1R in the cocaine-orexin interaction in nucleus accumbens neurons.  Figure 1C (n = 6 neurons/each concentration). Of note, 20-30 neurons were tested for each condition, and an increase in [Ca 2+ ] i was identified in about 25% of neurons tested; the amplitude of [Ca 2+ ] i from the response of six neurons was used for analysis. stimulant locomotor sensitization and cocaine seeking when it is driven by highly motivated states [3]. OX1 receptors in the nucleus accumbens mediate chronic cocaine-induced locomotor sensitization [22]. Other studies indicate that SB-334867, a selective OX1 receptor antagonist, prevents cocaine seeking and is a potential treatment target for cocaine relapse prevention [23]. Cocaine acts primarily by blocking the dopamine transporter, thus increasing dopamine transmission in the nucleus accumbens [24], an important area in the reward circuit [25]. In addition, cocaine binds to and activates Sigma-1R [26]. Since nucleus accumbens neurons express Sigma-1R [27][28][29] and OX1 receptors [14,15] and behavioral studies support the cocaine-orexin interaction at this level [3,22], in this work, we examined the underlying mechanisms and role of Sigma-1R in the cocaine-orexin interaction in nucleus accumbens neurons.
A diagram summarizing the proposed mechanism of potentiation of orexin by cocaine via Sigma-1R activation in nucleus accumbens neurons is illustrated in Figure 5.

Discussion
Orexin A, via activation of OX1 receptor, can activate both phospholipase C (PLC) and phospholipase D (PLD) in various cell models [9,10] including neurons [37]. PLC activation leads to hydrolysis of phosphoinositides and formation of inositol-1,4,5-trisphosphate (IP3), the Ca 2+ -releasing second messenger that releases Ca 2+ from endoplasmic reticulum (ER) through IP3 receptors [38]. PLD activation promotes the hydrolysis of phosphatidylcholine to choline and phosphatidic acid [11]. Whereas phosphatidic acid was considered the main effector downstream to PLD activation, we recently identified choline as a second messenger that activates Sigma-1R [12].
We next demonstrated that the OXA (17-33)-induced increase in [Ca 2+ ]i was mediated by IP3-dependent Ca 2+ release from ER, as previously reported [6]; the effect was abolished by IP3 receptor antagonists, but not affected by disruption of lysosomal Ca 2+ stores.

Discussion
Orexin A, via activation of OX 1 receptor, can activate both phospholipase C (PLC) and phospholipase D (PLD) in various cell models [9,10] including neurons [37]. PLC activation leads to hydrolysis of phosphoinositides and formation of inositol-1,4,5-trisphosphate (IP3), the Ca 2+ -releasing second messenger that releases Ca 2+ from endoplasmic reticulum (ER) through IP 3 receptors [38]. PLD activation promotes the hydrolysis of phosphatidylcholine to choline and phosphatidic acid [11]. Whereas phosphatidic acid was considered the main effector downstream to PLD activation, we recently identified choline as a second messenger that activates Sigma-1R [12].
Sigma-1 receptor is a chaperone protein expressed in the endoplasmic reticulum (ER), mainly at the mitochondria-associated ER membrane domains (MAMs) [13]. Sigma-1Rs interact with many different signaling proteins. At the ER, Sigma-1Rs potentiate the Ca 2+ release via IP 3 receptors [13]; they also interact with STIM1, the Ca 2+ sensor for storeoperated Ca 2+ entry [39]. Sigma-1R ligands include antidepressants, antipsychotics, and drugs of abuse [40]. Cocaine, in addition to its canonical target that elevates synaptic dopamine levels, binds to and activates Sigma-1Rs [41,42]. Neurons in the nucleus accumbens, a key area involved in the reward circuit [25], express Sigma-1R [27][28][29] and OX 1 receptors [14,15]. Behavioral studies supported the cocaine-orexin interaction in nucleus accumbens [3,22], but the underlying mechanisms remained unclear; this prompted us to investigate the mechanisms of cocaine-orexin interaction at this level.
We next demonstrated that the OXA (17-33)-induced increase in [Ca 2+ ] i was mediated by IP 3 -dependent Ca 2+ release from ER, as previously reported [6]; the effect was abolished by IP 3 receptor antagonists, but not affected by disruption of lysosomal Ca 2+ stores.

Neuronal Cell Culture
Nucleus accumbens neurons were dissociated from neonatal Sprague Dawley rats (Ace Animal Inc., Boyertown, PA, USA) of both sexes as previously described [29,45]. Newborn rats were decapitated, and the brains quickly removed surgically and immersed in ice-cold Hanks balanced salt solution (HBSS). The nucleus accumbens was identified, removed, minced, and subjected to enzymatic (papain, 37 • C) and mechanical dissociation. Cells were cultured in Neurobasal A medium (Life Technologies, ThermoFisher Scientific, Carlsbad, CA, USA) containing 10% fetal bovine serum, 1% GlutaMax, and 1% penicillinstreptomycin-amphotericin B solution at 37 • C in a humidified atmosphere with 5% CO 2 .The mitotic inhibitor cytosine β-arabinofuranoside (1 µM) was added to the culture to inhibit glial cell proliferation. For calcium imaging, neurons were cultured on round 25 mm diameter glass coverslips coated with poly-L-lysine, in six-well plates.

Measurement of Cytosolic Ca 2+ Concentration
Cytosolic Ca 2+ concentration, [Ca 2+ ] i , was measured by calcium imaging methods in nucleus accumbens neurons loaded with Fura-2AM, as previously described [29,45]. Cells were incubated with 5 µM Fura-2AM (Invitrogen) in HBSS at room temperature for 45 min, in the dark, and then incubated for another 45 min in HBSS to allow for complete de-esterification of the dye. Coverslips (25 mm diameter) were subsequently mounted in an open bath chamber (Warner Instruments, Hamden, CT, USA) on the stage of an inverted microscope Nikon Eclipse TiE (Nikon Inc., Melville, NY, USA), equipped with a Perfect Focus System and a Photometrics CoolSnap HQ2 CCD camera (Photometrics, Tucson, AZ, USA). During the experiments, the Perfect Focus System was activated. Fura-2AM fluorescence (emission = 510 nm), following alternate excitation at 340 and 380 nm, was acquired at a frequency of 0.25 Hz. Images were acquired and analyzed using NIS-Elements AR software (Nikon Inc.). After appropriate calibration with ionomycin and CaCl 2 and