Neuropharmacology 55 (2008) 1238–1250 Contents lists available at ScienceDirect Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm A NMDA receptor glycine site partial agonist, GLYX-13, simultaneously enhances LTP and reduces LTD at Schaffer collateral–CA1 synapses in hippocampus Xiao-lei Zhang a, John A. Sullivan a, Joseph R. Moskal b, Patric K. Stanton a,c,* a Department of Cell Biology & Anatomy, New York Medical College, Basic Sciences Building, Room 217, Valhalla, NY 10595, USA b The Falk Center for Molecular Therapeutics, Department of Biomedical Engineering, McCormick School of Engineering and Applied Sciences, Northwestern University, Evanston, IL 60201, USA c Department of Neurology, New York Medical College, Valhalla, NY 10595, USA article info abstract 2þ Article history: N-methyl-D-aspartate glutamate receptors (NMDARs) are a key route for Ca influx into neurons Received 13 June 2008 important to both activity-dependent synaptic plasticity and, when uncontrolled, triggering events that Received in revised form 18 July 2008 cause neuronal degeneration and death. Among regulatory binding sites on the NMDAR complex is Accepted 4 August 2008 a glycine binding site, distinct from the glutamate binding site, which must be co-activated for NMDAR channel opening. We developed a novel glycine site partial agonist, GLYX-13, which is both nootropic and Keywords: neuroprotective in vivo. Here, we assessed the effects of GLYX-13 on long-term synaptic plasticity and Glutamate receptor NMDAR transmission at Schaffer collateral–CA1 synapses in hippocampal slices in vitro.GLYX-13 Learning Memory simultaneously enhanced the magnitude of long-term potentiation (LTP) of synaptic transmission, while N-methyl-D-aspartate receptor reducing long-term depression (LTD). GLYX-13 reduced NMDA receptor-mediated synaptic currents in Partial agonist CA1 pyramidal neurons evoked by low frequency Schaffer collateral stimulation, but enhanced NMDAR Glycine currents during high frequency bursts of activity, and these actions were occluded by a saturating Synaptic plasticity concentration of the glycine site agonist D-serine. Direct two-photon imaging of Schaffer collateral burst- evoked increases in [Ca2þ] in individual dendritic spines revealed that GLYX-13 selectively enhanced burst-induced NMDAR-dependent spine Ca2þ influx. Examining the rate of MK-801 block of synaptic versus extrasynaptic NMDAR-gated channels revealed that GLYX-13 selectively enhanced activation of burst-driven extrasynaptic NMDARs, with an action that was blocked by the NR2B-selective NMDAR antagonist ifenprodil. Our data suggest that GLYX-13 may have unique therapeutic potential as a learning and memory enhancer because of its ability to simultaneously enhance LTP and suppress LTD. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction et al., 1990; Falls et al., 1992), maintaining the epileptic state after kindling (Yeh et al., 1989), reducing the negative symptoms of N-methyl-D-aspartate receptors (NMDARs) have well estab- schizophrenics (Heresco-Levy et al., 2002; Evins et al., 2002), and lished roles in synaptic plasticity, causing the induction of some reducing morphine-induced tolerance (Quartaroli et al., 2001). forms of both LTP and LTD of synaptic strength (Collingridge et al., Although the stoichiometry of native NMDARs is still uncertain, 1983; Dudek and Bear,1992; Huang et al., 2005; Stanton et al., 2003, recombinant NMDARs appear to consist of at least one NR1 and one 2005). This receptor–ionophore complex is functionally involved in NR2 subunit, and most evidence now suggests that NMDARs regulating glutamate-induced excitotoxic neuronal cell death, assemble as tetramers containing 2 NR1 and 2 NR2 subunits attenuating neuropathic pain in rat models measuring mechanical (whenever the NR3 subunit is not expressed; see Prybylowski and allodynia (Dickenson and Aydar, 1991; Laird et al., 1996), promoting Wenthold, 2004; Paoletti and Neyton, 2007). There are multiple the acquisition and extinction of conditioned fear (Miserendino forms of both NR2 (A–D) and NR3 (A and B) subunits, and at least 8 splice variants of NR1. Moreover, NMDAR expression is develop- mentally regulated and differentially distributed in different brain regions. NMDARs are part of a family of ionotropic glutamate * Corresponding author. Department of Cell Biology & Anatomy, New York receptors that include kainate and (AMPA) receptors. Kainate and Medical College, Basic Sciences Building, Room 217, Valhalla, NY 10595, USA. Tel.: þ1 914 594 4883; fax: þ1 914 594 4653. AMPA receptors are each ligand-gated ion channels that, upon þ þ E-mail address: [email protected] (P.K. Stanton). activation, gate the flux of K and Na ions (though some AMPA 0028-3908/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2008.08.018 X.-lei Zhang et al. / Neuropharmacology 55 (2008) 1238–1250 1239 receptor subunits confer significant Ca2þ permeability; Hollmann transferred to an interface holding chamber for incubation at room temperature for et al., 1991). In contrast, the NMDA receptor–ion channel complex a minimum of 1 h before commencing recording. opens a channel with significant Ca2þ permeability. The NMDA receptor–ionophore complex is unique among 2.2. Extracellular recordings glutamate receptors in requiring binding of both glutamate and Slices were transferred to an interface recording chamber and continuously glycine for activation. While glutamate binding during synaptic perfused at 3 ml/min with oxygenated ACSF at 32 Æ 0.5 C. Low resistance recording transmission causes channel opening, tonic binding of endogenous electrodes were made from thin-walled borosilicate glass (1–2 MU after filling with agonists of the glycine site is also necessary for glutamate-driven ACSF) and inserted into the apical dendritic region of the Schaffer collateral termi- nation field in stratum radiatum of the CA1 region to record field excitatory post- opening to occur (Forsythe et al., 1988; Kleckner and Dingledine, synaptic potentials (fEPSPs). A bipolar stainless steel stimulating electrode (FHC Co.) 1988). Moreover, there are various modulatory sites on the complex was placed on Schaffer collateral–commissural fibers in CA3 stratum radiatum, and that can modulate function, including a Mg2þ site located within constant current stimulus intensity adjusted to evoke approximately half-maximal the channel that causes the voltage-dependence of NMDARs, and fEPSPs once each 30 s (50–100 pA; 100 ms duration). fEPSP slope was measured by proton sensor, polyamine and Zn2þ sites found on various subunits linear interpolation from 20–80% of maximum negative deflection, and slopes confirmed to be stable to within Æ10% for at least 10 min before commencing an (Mayer et al., 1992). experiment. Signals were recorded using a Multiclamp 700B amplifier and digitized While there are many glutamate receptors found in the central with a Digidata 1322 (Axon Instruments, Foster City, CA). Data were analyzed using nervous system, the strychnine-insensitive glycine site is unique to pClamp software (version 9, Axon Instruments) on an IBM-compatible personal the NMDA receptor. This, together with the number of clinically computer. relevant functions that NMDA receptors play, has made the glycine site an important target for drug development (see Wood, 2005 for 2.3. Intracellular recordings review). For example, the glycine site partial agonist, D-cycloserine Patch pipettes were pulled from borosilicate glass (1B150F-4, World Precision (DCS), has been shown to have cognitive-enhancing properties in Instruments) using a flaming/brown micropipette puller (P-97, Sutter Instruments). vivo (Hood et al., 1989; Monaghan et al., 1988; Flood et al., 1992; The composition of the patch pipette solution for NMDA current recordings was (in Schuster and Schmidt, 1992; Thompson et al., 1992) as has the mM): 135 mM CsMeSO3, 8 mM NaCl, 10 HEPES, 2 Mg-ATP, 0.3 Na-GTP, 0.5 EGTA, 1 QX-314. The patch pipette solution pH was adjusted to 7.25 with CsOH, and had an glycine prodrug, milacemide (Handelmann et al., 1989; Quarter- osmolarity of 280 Æ 10 mOsm. When filled with this solution, patch pipettes had tip main et al.,1991; Schwartz et al.,1991,1992; Finkelstein et al.,1994). resistances of 5–6 MU. Both of these compounds, however, appear to result in desensiti- Whole cell patch clamp recordings were obtained from CA1 pyramidal neurons zation with chronic administration (Herting, 1991; Quartermain in slices in a fully submerged recording chamber at room temperature, and 95% O2/ et al.,1994). Recently, Tuominen et al. (2005) found that glycine and 5% CO2 was passed over the perfusate to ensure an oxygenated local environment. The submerged recording chamber was mounted on a Zeiss Axioskop 2 FS upright D-serine, but not the partial agonist DCS, are effective at reducing microscope equipped with infrared differential interference contrast (DIC) optics. some of the negative symptoms of schizophrenia when used to Pyramidal neurons of the hippocampal CA1 region were visualized with a 63Â water augment antipsychotic therapeutics. immersion lens, and patched in the voltage-clamp configuration. Excitatory post- synaptic currents (EPSCs) were recorded using a MultiClamp 700B (Axon Instru- Glyxins are a new family of glycine site-specific, N-methyl-D- ments, Foster City, CA), with the low-pass filter setting at 1–3 kHz, series resistance aspartate receptor modulators. They were generated from an amino was compensated in the voltage-clamp