If it is so widespread in the neocortex why has the impact of CaSR signaling been overlooked? Previous studies examining external Ca2+ on synaptic transmission may have attenuated the impact of the CaSR-NSCC pathway by using non-physiological high resting [Ca2+]o and by increasing bath [Mg2+] as bath [Ca2+] was decreased [8,67,68]. The use of physiological [Ca2+]o has unmasked other, previously overlooked signaling pathways that modulate synaptic transmission [69]. In fact, both recurrent rhythmic activity and repetitive synaptic transmission in brain slices more closely resembled findings in vivo when slice [Ca2+]o was reduced from the commonly used 2 mM to the more physiological 1.0–1.2 mM [70,71]. It remains to be determined how much of the change in neuronal function associated with use of physiological [Ca2+]o arises from a reduction in CaSR activation. GPCR promiscuity [72,73], the large number of downstream CaSR signaling pathways [74], and its presence in non-neuronal brain tissue [50,75] all point to CaSR potentially having additional actions in the brain. Further experiments are required to elucidate these other actions. Conclusions Decreases in [Ca2+]o will depress synaptic transmission because of the exquisite sensitivity of transmitter release to [Ca2+]o[8,44]. Our experiments identify CaSR as a key component in the [Ca2+]o-sensor-NSCC signaling pathway in neocortical terminals that may alter terminal excitability. The prevalence of CaSR in neocortex indicates that this signaling pathway may have wide- ranging influence on synaptic transmission during normal function and disease states. Materials and Methods Synaptosome All animal procedures were approved by OHSU I.A.C.U.C. in accordance with the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals and the N.I.H. Guide for the Care and Use of Laboratory Animals. Male or female Sprague-Dawley rats or 129S6/SvEv mice (six to eight weeks old) were deeply anesthetized with isoflurane and decapitated. As described previously [28], the neocortex was submerged in ice-cold 320 mM sucrose solution in a Teflon-glass tissue homogenizer driven at 400–500 RPM, then centrifuged at 3000 g for 3 minutes. The supernatant was centrifuged at 14600 g for 12 minutes and the upper layer of the resulting pellet resuspended in approximately 2 ml ice-cold sucrose. Before use the synaptosomes were washed with Tyrode solution. Genotyping CaSR Mutant Mice DNA from mouse tail samples was released by treatment with 50 mM NaOH at 95uC for 15 minutes, followed by the addition of 1 M Tris buffer, pH 8.0, containing 10 mM EDTA. Polymerase chain reaction was then performed using DNA solution and three primers: CaSR 59: TCTCTTCTCTTTAGGTCCTGAAAGA, CaSR 39: TCATTGATGAACAGTCTTTCTCCCT, and r- neo2: TCTTGATTCCCACTTTG TCCT TG TA. The samples were run on a 1% agarose gel and the sample identified as CaSR+/+, CaSR+/2, or CaSR2/2. HEK Cell Culture and Transient Transfection HEK 293 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum (FBS). For intracellular [Ca2+] ([Ca2+]i) imaging experiments, ,105 HEK cells were plated in 500 ml medium onto poly-D-lysine-coated glass CaSR Regulates Nerve Terminals coverslips in 24-well plate. Cells were transfected with pEGFP/ CaSR plasmid DNA using Lipofectamine one day after plating. Briefly, 0.8 mg pEGFP/CaSR plasmid DNA was mixed in 50 ml Opti-MEMH I Reduced Serum Medium and 10 ml Lipofectamine 2000 (Invitrogen) mixed in 50 ml Opti-MEMH I Reduced Serum Medium. The mixtures were incubated for 20 minutes and then added to each well containing cells and medium. The transfection medium was replaced with DMEM containing 5% FBS, six hours later. Cells were kept in culture for another 18 to 24 hours before use. Untransfected cells were used in control experiments 42– 48 hours after plating. Stable lines of HEK cells containing mGluR1 were used once confluent. Immunofluorescence Synaptosomes were fixed for 30 minutes at 4uC with 4% formaldehyde, washed with PBS, and then placed in blocking solution containing 2% goat serum, 1% BSA and 0.4% saponin for 30 mins at 25uC. Overnight incubation in primary antibody- containing solution (4641, polyclonal antibody kindly provided by Dr E. Nemeth of NPS Pharmaceuticals [31] or a monoclonal antibody against synaptophysin, MAB5258 Chemicon) at 4uC was followed by a wash and then 30 minute incubation with secondary antibodies at 37uC. Coverslips were then washed and mounted, together with a quenching agent (Citifluor), and viewed by fluorescence microscopy. Western Blots Brains were harvested, flash frozen, and homogenized in a protease inhibitor-containing lysis buffer (10 mg/mL aprotinin, 10 mg/mL pepstatin A, 10 mg/mL leupeptin, 10 mg/mL benza- midine, and 1 mM PMSF) on ice. Protein concentration was determined by the Bradford technique. Membrane fragments were run on a 7% polyacrylamide gel and then transferred overnight. Blots were blocked with 3% albumin and then stained with a polyclonal anti-CaSR antibody [76] at 1 in 1000 dilution (kindly provided by Dr K Rodland) and with secondary antibodies conjugated with horseradish peroxidase at 1 in 50,000. In control experiments the primary antibody was preincubated with 50 mg/ mL blocking peptide. Bands were visualized with chemilumines- cence. Electrophysiology Recordings were made from acutely isolated single nerve terminals (synaptosomes) visualized using an inverted microscope (IX70, Olympus) as previously described [6]. Electrodes with resistances of 15–40 MV were used to make cell-attached recordings from terminals. Test solutions were continuously applied from a nearby capillary tube and manifold at 1–3 ml/ min (23–25uC). We employed a Tyrode solution (in mM; 150 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2 10 HEPES, and 10 glucose, at pH 7.35 with NaOH) in the bath and electrode unless otherwise stated. In the mouse synaptosome experiments, when [Ca2+] was greater than 0.6 mM, NaCl was decreased isotonically for CaCl2. Voltage-clamp recordings were obtained using Pulse software and an EPC- amplifier 9 (Heka Instruments Inc, MA). Leak currents were subtracted with a p/-4 protocol. Currents were prefiltered with a 2 or 5 kHz Bessel filter and digitized at 20–100 ms per point. Calindol stock solution was made up at 10 mM in ethanol and stored as aliquots at 220uC. Measurement of [Ca2+]i Response [Ca2+]i was measured in HEK cells transfected with CaSR- EGFP or mGluR1 using the fluorescent indicators, X-rhod-1 or PLoS ONE | www.plosone.org 9 January 2010 | Volume 5 | Issue 1 | e8563