Neuropharmacology 89 (2015) 232e244 Contents lists available at ScienceDirect Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm Modulation of direct pathway striatal projection neurons by muscarinic M4-type receptors Teresa Hernandez-Flores, Omar Hernandez-Gonz alez, María B. Perez-Ramírez, Esther Lara-Gonzalez, Mario A. Arias-García, Mariana Duhne, Azucena Perez-Burgos, * G. Aleph Prieto, Alejandra Figueroa, Elvira Galarraga, Jose Bargas Division de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autonoma de Mexico, PO Box 70-253, Mexico City, DF 04510, Mexico article info abstract Article history: Models of basal ganglia (BG) function posit a dynamic balance between two classes of striatal projection Received 7 July 2014 neurons (SPNs): direct pathway neurons (dSPNs) that facilitate movements, and indirect pathway Received in revised form neurons (iSPNs) that repress movement execution. Two main modulatory transmitters regulate the 12 September 2014 output of these neurons: dopamine (DA) and acetylcholine (ACh). dSPNs express D -type DA, M -and Accepted 23 September 2014 1 1 M -type ACh receptors, while iSPNs express D -type DA and M -type ACh receptors. Actions of M -, D -, Available online 5 October 2014 4 2 1 1 1 and D2-receptors have been extensively reported, but we still ignore most actions of muscarinic M4-type receptors. Here, we used whole-cell recordings in acutely dissociated neurons, pharmacological tools Keywords: Acetylcholine such as mamba-toxins, and BAC D1 or 2-eGFP transgenic mice to show that activation of M4-type re- 2þ Striatum ceptors with bath applied muscarine enhances Ca -currents through CaV1-channels in dSPNs and not in Striatal projection neurons iSPNs. This action increases excitability of dSPNs after both direct current injection and synaptically 2þ Excitability driven stimulation. The increases in Ca -current and excitability were blocked specifically by mamba 2þ CaV1Ca -channels toxin-3, suggesting mediation via M4-type receptors. M4-receptor activation also increased network activity of dSPNs but not of iSPNs as seen with calcium-imaging techniques. Moreover, actions of D1-type and M4-type receptors may add to produce a larger enhancement of excitability of dSPNs or, paradox- ically, oppose each other depending on the order of their activation. Possible implications of these findings are discussed. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction express D2-type DA receptors that decrease their excitability in part þ by reducing the same Ca2 -current (Hernandez-L opez et al., 2000; The basal ganglia (BG) are thought to select motor actions, Salgado et al., 2005). Both dSPNs and iSPNs express M1-type ACh participate in cognition and procedural memory (Gerfen and receptors that increase neuronal excitability in part by depressing þ þ Surmeier, 2011). A model of the BG proposes that direct pathway K -currents directly or indirectly and by enhancing persistent Na - striatal projection neurons (dSPNs) facilitate movement whereas currents (Galarraga et al.,1999; Carrillo-Reid et al., 2009b; Goldberg indirect pathway projection neurons (iSPNs) repress movement et al., 2012; Perez-Rosello et al., 2005; Shen et al., 2005, 2007; (Albin et al., 1989; DeLong, 1990; Kravitz et al., 2010). BG are Vilchis et al., 2002). M1-type receptors modulate high voltage þ regulated by two modulatory transmitters whose higher brain activated (HVA) calcium currents involved in activating Ca2 - þ concentrations are in the striatum (Contant et al., 1996; Prensa and dependent K -currents fixing firing patterns and regulating trans- Parent, 2001; Shultz, 2007; Zhou et al., 2002): dopamine (DA) and mitter release (Dolezal and Tucek, 1999; Galarraga et al., 1999; acetylcholine (ACh). dSPNs express D1-type DA receptors that in- Howe and Surmeier, 1995; Perez-Burgos et al., 2008, 2010; Perez- þ crease their excitability in part by enhancing Ca2 -current through Rosello et al., 2005). CaV1-channels (Galarraga et al., 1997; Hernandez-Lopez et al., 1997; The actions of M1-type receptors have been extensively Surmeier et al., 2011; Tritsch and Sabatini, 2012), while iSPNs explored. However, we do not know much about muscarinic M4- type receptors, preferentially expressed in dSPNs (Goldberg et al., 2012; Ince and Ciliax, 1997; Santiago and Potter, 2001; Yan et al., * Corresponding author. Tel.: þ52 55 5622 5670. E-mail address: [email protected] (J. Bargas). 2001). http://dx.doi.org/10.1016/j.neuropharm.2014.09.028 0028-3908/© 2014 Elsevier Ltd. All rights reserved. T. Hernandez-Flores et al. / Neuropharmacology 89 (2015) 232e244 233 Here, we chose to use potent pharmacological tools, mamba- voltage ramps (Hernandez-Gonz alez et al., 2014; Prieto et al., 2009, 2011; Perez- toxins, on isolated neurons obtained from bacterial artificial chro- Burgos et al., 2008, 2010; Perez-Rosello et al., 2005). mosome (BAC) transgenic mice expressing enhanced green fluo- rescent protein (eGFP) associated with dopamine (DA) receptors 2.3. Current clamp recordings In addition, we recorded from brain slices of BAC D or D eGFP transgenic mice. promoters: D1 and D2 receptors (Gerfen and Surmeier, 2011)to 1 2 Slices were submerged in an iced saline solution containing (in mM): 124 NaCl, 2.5 show that activation of M4-type receptors increase the excitability KCl, 1.3 MgCl2, 2 CaCl2, 26 NaHCO3, 1.2 NaH2PO4 and 15 glucose (pH 7.4, 300 mOsm/l, of dSPNs and that the mechanism of action involves the enhance- saturated with 95% O2 and 5% CO2; temperature was maintained around 34 C) and 2þ ment of Ca -currents through CaV1-channels. were left for equilibration in this oxygenated saline at room temperature for 1h. Main experiments were done in dissociated neurons from BAC- Single slices were transferred to a submerged recording chamber and super- e mice to avoid indirect actions. However, complementary experi- fused continuously with oxygenated saline (4 5 ml/min; 25 C). Current-clamp recordings were performed with the patch clamp technique in the whole cell ments in slices supported the conclusion that M4-receptors configuration in SPNs from the dorsal striatum. The slices were visualized using modulate synaptically driven and network activities in dSPNs. infrared differential interference contrast (IR-DIC) microscopy with an upright mi- croscope and a digital camera. Data acquisition used software designed in the 2. Materials and methods LabVIEW environment (National Instruments, Austin TX). Patch pipettes (3e6MU) were filled with internal saline containing (in mM): 115 2.1. Preparation of slices and dissociated cells KH2PO4, 2 MgCl2, 10 HEPES, 1.1 EGTA, 0.2 ATP, 0.2 GTP, and 5% biocytin (pH ¼ 7.2; The protocols followed the National University of Mexico (UNAM) and the Na- 285 mOsm/l). Nominally corticostriatal suprathreshold responses were evoked and tional Institutes of Health guide for the care and use of laboratory animals (NIH recorded by stimulating sensory-motor cortical areas with concentric bipolar elec- Publications No. 8023, revised 1996) including minimizing the number of animals to trodes (50 mm at the tip; FHC, Bowdoinham, ME). The distance between recording achieve statistical significance and the avoidance of animal suffering. Brain slices and stimulating electrodes was about 1 mm. Synaptic responses were evoked by a and acutely dissociated neurons were obtained as described in previous work series of current pulses of increasing intensities until eliciting suprathreshold (Bargas et al., 1994; Perez-Burgos et al., 2008, 2010). BAC D1 or D2-eGFP transgenic male mice (BAC D1-eGFP mice, https://www.mmrrc.org/catalog/sds.php?mmrrc_ id¼297, RRID:IMSR_MMRRC:000297; BAC D2-eGFP mice, https://www.mmrrc.org/ catalog/sds.php?mmrrc_id¼230, RRID:IMSR_MMRRC:000230; postnatal days 60e90) were anesthetized with a mixture of ketamine (85 mg/kg ip) and xylazine (15 mg/kg ip), decapitated, their brains removed and submerged in iced saline so- lution containing (in mM): 126 NaCl, 3 KCl, 26 NaHCO3, 2 CaCl2, 1 MgCl2, 11 glucose, 0.2 thiourea and 0.2 of ascorbic acid (25 C; pH ¼ 7.4 with HCl, 300 ± 5 mOsm/l with glucose; saturated with 95% O2 and 5% CO2). When using the other preparations described below, animals were anesthetized in a similar way. Sagittal brain slices, 300 mm thick, were cut on a vibratome (Pelco 102, Ted Pella. INC) and placed for 1 h at 34 C in the same saline solution. To obtain dissociated cells the dorsal neo- striatum was dissected from the slices and then returned into the saline solution containing 10 mM HEPES plus 1 mg/ml of pronase E type XIV (SigmaeAldrich-RBI, Cat # P5147; St. Louis, MO, USA) at 34 C. After about 20 min of digestion, the slices 2þ were transferred to a low Ca (0.4 mM CaCl2) saline solution to obtain the cells by mechanical dissociation with a graded series of fire-polished Pasteur pipettes. The cell suspension (1 ml) was plated into a Petri dish mounted on the stage of an inverted microscope. Neurons adhered to the bottom of the dish within 10e15 min. The dish contained 1 ml of the whole-cell recording saline (in mM): 0.001 tetro- dotoxin (TTX), 140 NaCl, 3 KCl, 5 BaCl2, 2 MgCl2, 10 HEPES, and 10 glucose (pH ¼ 7.4 with NaOH; 300 ± 5 mOsm/l with glucose). Thereafter, the cells were superfused at 1 ml/min with saline of the same composition at room temperature (around 25 C). eGFP-positive neurons were visualized using a UV lamp (X-Cite; EXFO, Ontario, Canada). 2.2. Voltage clamp recordings of calcium currents Voltage-clamp recordings were performed on eGFPþ SPNs obtained from BAC D1 or D2 mice. Cells had 10e12 mm of main diameter, whole-cell capacitance of 6e7 pF and with short dendritic trunks (Perez-Burgos et al., 2010; Yan and Surmeier, 1996). Patch pipettes of borosilicate glass (WPI, Sarasota, FL, USA) were pulled in a Flaming-Brown puller (Sutter Instrument Corporation, Novato, CA, USA) and fire polished prior to use.