UBC Faculty Research and Publications

Slice orientation and muscarinic acetylcholine receptor activation determine the involvement of N-methyl… Bartlett, Thomas E; Lu, Jie; Wang, Yu T Nov 15, 2011

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
52383-13041_2011_Article_142.pdf [ 403.66kB ]
Metadata
JSON: 52383-1.0216451.json
JSON-LD: 52383-1.0216451-ld.json
RDF/XML (Pretty): 52383-1.0216451-rdf.xml
RDF/JSON: 52383-1.0216451-rdf.json
Turtle: 52383-1.0216451-turtle.txt
N-Triples: 52383-1.0216451-rdf-ntriples.txt
Original Record: 52383-1.0216451-source.json
Full Text
52383-1.0216451-fulltext.txt
Citation
52383-1.0216451.ris

Full Text

RESEARCH Open AccessSlice orientation and muscarinic acetylcholinereceptor activation determine the involvement ofN-methyl D-aspartate receptor subunit GluN2B inhippocampal area CA1 long-term depressionThomas E Bartlett, Jie Lu and Yu Tian Wang*AbstractBackground: The contribution of different GluN2 subunits of the N-methyl D-aspartate (NMDA) receptor to theinduction of bidirectional hippocampal synaptic plasticity is a controversial topic. As both supporting and refutingevidence for the hypothesis of subunit specialization in opposing directions of plasticity has accumulated since itwas first proposed a few years ago, we hypothesize that differences in experimental conditions may have in partcontributed to some of the inconsistent results from these studies. Here we investigate the controversialhypothesis that long-term depression (LTD) is preferentially induced by GluN2B-containing NMDA receptors in areaCA1 of hippocampal slices.Results: We find that brain slices from 2-3 week old rats prepared in the sagittal orientation have GluN2B-independent LTD whereas slices prepared in the coronal orientation have GluN2B-dependent LTD. There was nodifference between the orientations in the fraction of the NMDAR EPSC sensitive to a GluN2B-selective antagonist,leading us to believe that the intracellular signaling properties of the NMDARs were different in the twopreparations. Coronal slices had greater association of LTD-related intracellular signaling protein RasGRF1 withGluN2B relative to sagittal slices. Antagonism of muscarinic acetylcholine receptors (mAChRs) in the sagittal slicesreturned LTD to a GluN2B-dependent form and increased the association of GluN2B with RasGRF1.Conclusions: These results suggest a novel form of NMDAR modulation by mAChRs and clarify somedisagreement in the literature.Keywords: Hippocampus, Long-term depression, N-methyl D-Aspartate receptor, muscarinic acetylcholine receptorBackgroundLong term potentiation (LTP) and long-term depression(LTD) of synaptic transmission are the two best-under-stood mechanisms by which the functional connectivityof neurons is altered [1,2]. In many brain areas,including the most-studied CA3:CA1 synapse of the hip-pocampus, the induction of LTP and LTD depends onactivation of N-methyl D-aspartate receptors(NMDARs). NMDARs are heterotetrameric ligandgated, Ca2+ permeable ion channels comprising twoGluN1 subunits and two GluN2 subunits from type2A-2D [3]. It remains unclear whether different subunitsof the NMDAR are preferentially coupled to LTP orLTD induction, however different GluN2 subunits doconfer different functional properties on the NMDAR.For example the GluN1/GluN2B subtype has slowerchannel deactivation and greater coupling to CaMKIIthan the GluN1/GluN2A subtype [4,5]. Based on theprolonged Ca2+ flux requirements for LTD induction [6]and the developmentally decaying profile of artificiallyinducible LTD [7] matching the early postnatal predo-minance of GluN2B expression [8], it was hypothesizedthat GluN2B is important for LTD induction. In accor-dance with this idea, the predominant extrasynapticlocalization of GluN2B [9] matches the requirement for* Correspondence: ytwang@brain.ubc.caBrain Research Centre, University of British Columbia, 2211 Wesbrook Mall,Room F270, Vancouver, BC, V6T 2B5, CanadaBartlett et al. Molecular Brain 2011, 4:41http://www.molecularbrain.com/content/4/1/41© 2011 Bartlett et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.extrasynaptic NMDAR activation for the induction ofLTD [10]. Indeed, LTD in GluN2B -/- mouse strains islost [11,12]. However, the results of experiments onLTD in-vitro with the GluN2B-selective antagonists Ro25-6981 (Ro) [13] and Ifenprodil have been in disagree-ment, with various groups reporting an enhancement ofLTD at the CA3:CA1 synapse [14], no effect on LTDinduction [15,16] or a complete block of LTD inductionat the same synapse [17]. Outside CA1 the situation iseven more complex, with reports that GluN2B is essen-tial for LTD in the perirhinal cortex [10], but also thatboth GluN2A and GluN2B are required for LTD in theamygdala [18] and anterior cingulate cortex [19].Given the number of different laboratories involved inthese studies, it is very likely that some of the conflictingdata may have at least in part resulted from differentexperimental conditions employed. To try and resolvethis confusion, we studied the methodologies of twolabs with opposing results from experiments with Roand LTD in area CA1 [15,17]. This led us to test theimportance of slice orientation for the GluN2B-depen-dence of LTD induction. Indeed we found that GluN2B-dependent LTD was a property of the coronal sliceorientation and GluN2B-independent LTD was a prop-erty of the sagittal orientation. There was no significantdifference in the GluN2B-containing fraction of theNMDAR EPSC between the two orientations. However,a muscarinic acetylcholine receptor (mAChR) antago-nist, scopolamine, conferred Ro-sensitivity on sagittalLTD. Furthermore, scopolamine led to an increase inthe association of LTD-related signaling moleculeRasGRF1 with GluN2B in sagittal slices. In the basalstate, coronal slices had higher GluN2B-bound RasGRF1than sagittal slices. This data clarifies some of the exist-ing literature on GluN2B in LTD and hints toward animportant mechanism of NMDAR regulation.ResultsSlice orientation determines the involvement of GluN2Bin LTD inductionFaced with an apparent contradiction between theresults of some groups regarding the involvement ofGluN2B in LTD induction, we sought a resolution tothe disagreement and an understanding of the underly-ing physiological mechanism. In one of the studies thatfound no involvement of GluN2B in LTD a sagittal [15]orientation was used when cutting the slices. In workthat found LTD induction absolutely required GluN2B,the slices were prepared in a coronal orientation [17].Thus we tested the importance of preparing the hippo-campal slice in two different orientations. When weinterleaved experiments preparing the slices in one ofthe two orientations and treating the slices with theGluN2B antagonist Ro 25-6981 (Ro), we found that theLTD induced by 1Hz low frequency stimulation (LFS) inthe sagittal slices was GluN2B-independent (Figure 1A.Control, n = 13, 78.8 ± 2.66% baseline. Ro, n = 9, 80.7 ±2.14% baseline. P = 0.619) consistent with the previousstudy [14], while LTD in the coronal slices wasGluN2B-dependent (Figure 1B. Control, n = 7, 79.5 ±3.62% baseline. Ro, n = 5, 100.2 ± 3.10% baseline. P =0.002) in full agreement with the previous work [16].LTD in the sagittal slices was completely NMDAR-dependent as shown by interleaved experiments wherethe pan-NMDAR antagonist 50 μM D-AP5 blockedLTD induction completely (data not shown).A                          BFigure 1 LTD in sagittal and coronal slices has differentGluN2B-dependence. Hippocampal slices (400 μM) prepared ineither sagittal (A) or coronal (B) orientations, both with CA3 attached,were used for extracellular recordings of field excitatory postsynapticpotentials (fEPSPs) in area CA1. After a stable baseline recordingperiod (at least 30 minutes) the GluN2B-selective antagonist Ro 25-6981 (5 μM, Ro) was applied and stimulation at 0.03 Hz continued foranother 30 minutes until a 1 Hz, 15 minute low frequency stimulation(LFS) at baseline intensity, also in the presence of Ro. Drugapplication was stopped at the end of LFS and the magnitude of LTDwas quantified in the last ten minutes of the hour after LFS. In theslices of either orientation that were not treated with Ro, LFS inducedLTD. Likewise, in the sagittal slices, Ro did not prevent the inductionof LTD (A) although D-AP5 (50 μM) did (data not shown). However, inthe coronal slices treated with Ro, LFS did not induce LTD (B). Scalebars represent 10 ms and 0.5 mV.Bartlett et al. Molecular Brain 2011, 4:41http://www.molecularbrain.com/content/4/1/41Page 2 of 8The simplest explanation for the difference in theGluN2B requirements of LTD induction in the two sliceorientations would be much reduced synaptic expressionof GluN2B in the sagittal slices, leading to GluN2B-independent LTD. To our surprise, we found no signifi-cant difference between the effect of Ro on the NMDAREPSC from CA1 pyramidal cells in slices prepared in thesagittal orientation versus cells from coronal slices(Figure 2, sagittal, n = 8, 39.9 ± 4.72% baseline, Coronal,n = 7, 30.4 ± 5.33% baseline, P = 0.202). The inductionof LTD is dependent not only on NMDAR activationand Ca2+ influx, but on the activation of a cascade ofintracellular processes leading to the expression ofLTD [20]. Thus we supposed that a changedownstream of NMDAR activation was responsible forthe altered subunit dependency of LTD in the twoslice orientations.mAChR antagonist scopolamine increases theinvolvement of GluN2B in LTD inductionThe major cholinergic innervation to area CA1 comesvia the medial septal nucleus and releases acetylcholineonto both principal cells and interneurons [21]. We pro-posed that the sagittal slice may retain a greater compo-nent of innervation than the coronal section [22]. Hencewe tested the hypothesis that antagonism of muscarinicacetylcholine receptors (mAChRs) in the sagittal sliceswould recapitulate the phenotype of the coronal slices.In sagittal slices, we found that co-application of themAChR antagonist scopolamine (20 μM) enabled blockof LTD by the GluN2B antagonist Ro. (Figure 3. Scopo-lamine, n = 9, 79.5 ± 3.64% baseline. Ro + Scopolamine,n = 6, 93.0 ± 2.64% baseline. P = 0.018). Scopolaminealone had no significant effect on either the baselinefEPSP or the magnitude of LFS-induced LTD.GluN2B interaction with RasGRF1 is higher in conditionswith GluN2B-dependent LTDWe then asked whether the enhanced involvement ofGluN2B in LTD after scopolamine treatment could bethe result of an increased association with an LTD-related signaling protein. Synaptic plasticity downstreamsignaling can involve MAP kinase activation, most com-monly p38 MAPK for LTD [23,24]. One pathway toMAP kinase signaling is the small GTPase Ras, which isactive in the GTP-bound state and inactive after boundGTP is hydrolyzed to GDP. RasGRF1 is a synaptic Ras-specific guanine nucleotide exchange factor that cata-lyses the release of GDP from Ras and subsequent acti-vation by GTP [25]. RasGRF1 is required for LTDinduction and is a binding partner of the carboxyl-tail ofGluN2B [23,26]. When we immunoprecipitated GluN2Bfrom a lysate of hippocampal slices that had been sub-ject to scopolamine or control treatments we found thatthe ratio of RasGRF1 to GluN2B in the immunoprecipi-tate was significantly increased in the scopolamine con-dition, supporting our hypothesis that increasedassociation of RasGRF1 with GluN2B is part of themechanism by which GluN2B can be included in LTDsignaling (Figure 4A,B, Ratio of normalized RasGRF1 inprecipitate scopolamine/control = 2.04 ± 0.451, P =0.0415, n = 12). No increase in RasGRF1 or GluN2Bwas detected in the crude lysate from the scopolamineslices compared to control slices (Figure 4A, B, Ratio ofnormalized RasGRF1 in lysate: scopolamine/control =1.067 ± 0.0674, P = 0.340, n = 12, Ratio of normalizedGluN2B in lysate: scopolamine/control = 1.11 ± 0.137,P = 0.441, n = 12).ABFigure 2 The GluN2B-mediated fraction of the NMDAR EPSC isnot significantly different between coronal and sagittalorientations. A) Whole-cell patch clamp recordings from CA1pyramidal cells in either sagittal or coronal slices were made andthe NMDAR EPSC was isolated at -40 mV in the presence ofbicuculine (10 μM) and NBQX (5 μM). After a 10 minute stablebaseline recording period at stimulation frequency 0.03 Hz, theGluN2B-selective antagonist Ro was applied for 40 minutes whilestimulation continued. Scale bars represent 200 ms and 200 pA. B)The level of depression in the NMDAR EPSC induced by Ro, whenquantified in the 40-50 minute epoch, was similar in sagittal andcoronal slices. This indicates activation of a similar number ofsynaptic GluN2B-containing NMDARs during baseline synapticstimulation in either slice orientation.Bartlett et al. Molecular Brain 2011, 4:41http://www.molecularbrain.com/content/4/1/41Page 3 of 8When we used recovered but untreated coronal orsagittal hippocampal slices to prepare protein lysates forthe GluN2B coimmunoprecipitation we found that theimmunoprecipitates from the coronal slices were signifi-cantly enriched for GluN2B-bound RasGRF1 relative tothe sagittal slices (Figure 4C and 4D, coronal/sagittalratio of normalized RasGRF1 in GluN2B immunopreci-pitate = 1.330 ± 0.105, P = 0.020, n = 4). Furthermore,we also saw a greater level of GluN2B in the coronalslice lysates relative to slices prepared in the sagittalorientation but no difference in lysate RasGRF1 (Figure4C and 4D, Ratio of normalized RasGRF1 in lysate:coronal/sagittal = 0.909 ± 0.0445, P = 0.086, n = 4,Ratio of lysate GluN2B coronal/sagittal = 1.60 ± 0.229,P = 0.04, n = 4). Previously we did not detect a changein GluN2B at the synapses of coronal versus sagittalslices using electrophysiology to measure NMDAREPSCs (Figure 2).DiscussionThe major findings of this paper are that the GluN2Bsubunit involvement in LTD induction in hippocampalarea CA1 is different according to slice orientation andmAChR activation. The GluN2B-dependence of LTD inthe two orientations is not a consequence of the synap-tic number of GluN2B-containing receptors, but consis-tent with an alteration in GluN2B coupling todownstream signaling molecule RasGRF1 via a mechan-ism regulated by tonic mAchR activation. We show thatdecreased mAchR tone increases the interaction ofGluN2B with RasGRF1 and the involvement of GluN2Bin LTD.The hippocampus receives cholinergic input from themedial septum which passes into all subfields includingCA1 [21,27,28]. Alternate orientations of slice have beenshown to change the properties of other synapses in thebrain, for example, the synaptic transmission and plasti-city of the amygdala is different between coronal andhorizontal slices [29]. It is possible that more cholinergicfiber tracts are spared in the sagittal orientation than inthe coronal orientation, thus changing the properties ofLTD according to the level of basal mAChR activation.All of the mAChR subtypes (M1-M5) are present inCA1 and may contribute to the phenomenon weobserve here [30,31]. M2/4 subtypes couple through Gito a reduction in cAMP levels, whereas M1/M3 couplethrough Gq to a rise in intracellular Ca2+ from intracel-lular stores. mAChRs elevate the excitability of CA1 pyr-amidal cells and modulate synaptic transmission inmultiple ways from both a presynaptic and postsynapticlocus.Presynaptic M2 AChRs inhibit ACh release. Scopola-mine could thus facilitate ACh release if these receptorsare tonically active [32], but would also inhibit postsy-naptic receptors. Repetitive stimulation of the StratumOrients (SO) has been shown to evoke a cholinergicslow EPSP that increases CA1 pyramidal cell excitability[33]. Stratum Radiatum (SR) stimulation preceded byrepetitive SO stimulation delivers enhanced LTP relativeto SR stimulation alone, via facilitation of NMDARsafter inhibition of SK channels [34,35]. The effect ofevoking extra ACh release on the subunit-dependencyof LTD is unknown; however mAChRs also regulate thesurface expression of NMDARs. M1 receptors activate apathway involving intracellular Ca2+, hippocalcin andclathrin-dependent endocytosis of NMDARs [36]. ItABFigure 3 GluN2B-dependence of LTD is created in the sagittalslice by a muscarinic acetylcholine receptor antagonist. A)Sagittal slices were again used for extracellular recordings ofsynaptic activity. After a stable baseline stimulation period (0.03 Hz,>30 minutes), the muscarinic receptor antagonist scopolamine(Scop, 20 μM) was added to the aCSF with or without GluN2B-selective antagonist Ro (5 μM) and stimulation continued atbaseline frequency for another 30 minutes. LFS was then begunwhile drug application continued. Drug washout began at thetermination of LFS and the magnitude of LTD was quantified in thefinal 10 minutes of the hour after LFS. B) In the sagittal slices,application of scopolamine did not change the induction of LTD orthe baseline fEPSP. However, when co-applied with GluN2Bantagonist Ro there was a significant inhibition of LTD compared tothe slices treated with scopolamine alone. Scale bars represent 10ms and 0.5 mV.Bartlett et al. Molecular Brain 2011, 4:41http://www.molecularbrain.com/content/4/1/41Page 4 of 8remains to be seen whether this pathway preferentiallymodulates GluN2A or GluN2B.GluN2B-containing receptors have a predominantlyextrasynaptic localization that may only be accessed byglutamate during extended stimulation protocols suchas the LFS used here to induce LTD. As such, our quan-tification of GluN2B via the NMDAR EPSC (Figure 2)may overlook differences in the extrasynaptic NMDARpopulation between the slice orientations. Indeed, weobserved greater GluN2B levels in the lysates of coronalslices relative to sagittal slices (Figure 4). Therefore, cor-onal slices may have greater involvement of GluN2B inLTD because of greater GluN2B levels relative to sagittalslices, as well as greater interaction of these NMDARsubunits with RasGRF1 in the coronal slices (Figure 4).Diffuse, rather than synaptic, transmission by acetyl-choline is suggested by the lack of co-localization ofcholinergic varicosities with glutamatergic synaptic ele-ments and the tonic baseline concentration of ACh inthe extracellular space of slices [37]. Tonic extracellularACh levels in the slice would explain the action of sco-polamine, and may be determined by slice preparationconditions. Higher extracellular Ach in the sagittal slicerelative to the coronal slice would be consistent withthe current study. Ambient ACh would not be restrictedto the synapse and could modulate mAChRs in the vici-nity of extrasynaptic GluN2B-containing receptors toelicit a change in GluN2B downstream signaling.RasGRF1 is coupled to p38 MAPK activation, requiredfor LTD induction [23] and associates with the                                               IP        IP                 IgG  GluN2B     Lysate:         C          S        C     C     S GluN2B   RasGRF1   ȕ-Actin        GluN2B   RasGRF1   ȕ-Actin                                                        IP          IP                                             GluN2B   IgG    Lysate:          C        S        C     S      C Blot Blot A                                                                       C                                        B                                                                      D Figure 4 Greater association of GluN2B with RasGRF1 occurs under conditions that show GluN2B involvement in LTD. A,B) Sagittalhippocampal slices were prepared with the same methodology as for the electrophysiological experiments and treated with control aCSF (C) or20 μM scopolamine in aCSF (S) for 2 hrs, followed by homogenization in RIPA buffer. 500 μg of each lysate was used for immunoprecipitationwith a polyclonal GluN2B antibody bound to protein A sepharose. After washing in RIPA, the coIPs were subjected to SDS PAGE and Westernblot, alongside 30 μg per lane of the crude lysates. The blots were sequentially probed with RasGRF1 and GluN2B antibodies using HRP- and AP-conjugated secondary antibodies respectively. In each coimmunoprecipitate the level of RasGRF1 was normalized to GluN2B and then ascopolamine/control ratio was calculated and averaged across experiments. C, D) Coronal (C) or sagittal (S) slices were prepared and recoveredin the absence of scopolamine. The lysates were processed and taken forward into immunoprecipitation as described above.Bartlett et al. Molecular Brain 2011, 4:41http://www.molecularbrain.com/content/4/1/41Page 5 of 8C-terminal domain of GluN2B [26]. The increased asso-ciation of GluN2B and RasGRF1 seen in this study afterscopolamine treatment of slices suggests a mechanismby which mAChR activation usually restricts the invol-vement of GluN2B in the signaling for LTD. Whentonic activation of mAChRs is prevented in the sagittalslice by mAChR antagonism there is greater involve-ment of GluN2B in LTD induction (Figure 3). In theuntreated condition there is greater association ofGluN2B with RasGRF1 in the coronal slices relative tothe sagittal slices (Figure 4), suggesting a mechanism toaccount for the sensitivity of coronal LTD to theGluN2B-selective antagonist Ro 25-6981.Loss of cholinergic brain function is a factor in normalaging and is especially prominent in several neurodegen-erative disorders [38,39]. Our data suggests that reduc-tion in acetylcholine occupation of muscarinic receptorsleads to increased LTD-related- and RasGRF1/Ras/MAPK signaling from GluN2B-containing NMDAreceptors. Considering that GluN2B-containing, extrasy-naptic NMDA receptors promote excitotoxic cell deathwhile synaptic GluN2A-containing receptors promotecell survival [40-45], it is tempting to speculate that thismechanism is important in glutamate-receptor relatedpathology.ConclusionsWe conclude that the difference between some labsdoing experiments on GluN2B involvement in LTD[15-17] can be explained at least in part by slice orienta-tion. This highlights the need for more physiologicallyrelevant preparations such as behaving animals [46].Furthermore, we have found that there is flexibility inthe coupling of GluN2B to LTD induction, such thatreduced signaling from mAChRs increases the associa-tion of GluN2B with RasGRF1 and the sensitivity ofLTD to a GluN2B antagonist. Thus, the NMDAR subu-nit rules in regulating synaptic plasticity may be moredynamic then previously envisaged, and subject todynamic regulation under different physiological andpathological conditions.MethodsSlice preparationMale Sprague-Dawley rats aged 14-21 days were eutha-nized in accordance with UBC animal care policy andthe brain rapidly removed into ice-cold (2-4°C) ‘slicingaCSF’ comprising (in mM) NaCl 124, KCl 3, NaH2PO41.25, NaHCO3 26, Glucose 15, CaCl2 1, MgSO4 10, 310mOsm and pH 7.35 when gassed with 95% O2 5% CO2.For sagittal slices, the brain was trimmed to give flat lat-eral surfaces and then hemisected along the sagittalplane before being glued onto a vibratome stage withthe medial surface uppermost. For coronal slices therostral end of the brain was chopped with a coronal cutto create a flat surface upon which to glue the brainwith the caudal end uppermost. Slices (400 μM thick)were prepared from the dorsal hippocampus and recov-ered at room temperature (23-25°C) for at least 2 hoursin ‘recording’ aCSF with CaCl2 2 mM, MgSO4 1 mM.Patch-clamp electrophysiologySlices were superfused with room temperature (23-25°C)‘recording’ aCSF flowing at 2.5 ml/minute with compo-sition as above, except including bicuculine methobro-mide (10 μM) and NBQX (5 μM). Pyramidal neurons inarea CA1 were patched under visual guidance with glassmicropipettes of resistance 4-6 MΩ when filled with asolution containing (in mM) CsMeSO4 130, NaCl 8,EGTA 0.5, HEPES 10, QX-314 bromide 5, Mg.ATP 8,Na.GTP 4, pH 7.25, 295 mOsm. Data acquisition hard-ware was via Axon Instruments amplifier Multiclamp700A and analog-digital converter Digidata 1322a. Dataacquisition and analysis software was WinLTP, [47-49].Cells were voltage clamped at -40 mV and the Schaffercollateral/commissural pathway was stimulated with atwisted NiCr bipolar electrode at 0.03 Hz to evokeNMDAR-mediated EPSCs. After a stable baseline for 20minutes, the application of Ro 25-6981 began. The mag-nitude of Ro-induced depression of the NMDA EPSCwas quantified as the average of the 30-40 minute per-iod after beginning application of Ro. Statistical compar-ison between the orientations were by t-test.Extracellular electrophysiologySlices were superfused with room temperature (23-25°C) ‘recording’ aCSF at 2.5 ml/minute and field excitatorypostsynaptic potentials (fEPSPs) were measured in stra-tum radiatum of CA1 using a glass micropipette ofresistance 2 MΩ when filled with aCSF and data acqui-sition hardware/software as described above. The Schaf-fer Collateral/Commissural pathway was stimulated asabove to evoke baseline fEPSPs at 50% of the slope atwhich population spikes were first visible. BaselinefEPSPs were recorded for at least 30 minutes and sliceswith an unstable baseline were discarded. LTD wasinduced by low frequency stimulation (LFS) of 900pulses at 1Hz, baseline intensity. The magnitude of LTDwas quantified as the average slope of the fEPSP in thelast ten minutes of the hour after LFS. Ro 25-6981 andscopolamine were from Tocris and both dissolved inwater and then aCSF just before the experiment. Drugswere applied at times indicated in the figures by thefilled bars. Statistical comparisons were by t-test.Co-immunoprecipitationSagittal or coronal slices were prepared as above and leftto recover at room temperature (23-25°C ) for at least 2Bartlett et al. Molecular Brain 2011, 4:41http://www.molecularbrain.com/content/4/1/41Page 6 of 8hours. Slices were split into coronal, sagittal, control(sagittal) and scopolamine (sagittal) groups and the lat-ter treated with 20 μM scopolamine for 2 hrs. Sliceswere homogenized in ice-cold RIPA buffer comprised of(in mM unless indicated) NaCl 150, Sodium deoxycho-late 0.3%, Tris 50, EDTA 1, Triton X-100 1%, SDS 0.1%,protease inhibitor cocktail (Roche), pH 7.4. All followingsteps were conducted at 4°C or on ice. The homogenatewas triturated and cleared by centrifugation at 14,000rpm for 30 minutes. The soluble fraction was quantifiedand 500 μg of protein was loaded onto protein A beads(GE) that had been incubated for 1 hr with rabbit poly-clonal anti-GluN2B C-terminal (YTW lab) or with nor-mal rabbit IgG (SantaCruz, #sc2027). Beads wereincubated in protein lysate with rotation overnightbefore four 5 minute washes in RIPA buffer and resus-pension in PBS and sample buffer.SDS PAGE and Western BlotHalf of each immunoprecipitation was loaded per laneof an SDS PAGE gel, alongside 30 μg per lane of crudelysate. Gels were blotted onto PVDF and probed withmouse monoclonal anti-GluN2B C-terminal (Millipore,#ab28373) or rabbit polyclonal anti-RasGRF1 (Santa-Cruz, #sc224). Secondary antibodies were HRP-conju-gated anti-rabbit (Perkin Elmer, NEF812001), or AP-conjugated anti-mouse (Promega #S372B). Luminescentsubstrates were Pierce ECL substrate for HRP (#32106)or Thermo Scientific Lumi Phos WB for AP (#34150).Signals were quantified with Quantity One software(BioRad) and the level of RasGRF1 in each immunopre-cipitation was normalized to the respective level ofGluN2B. The ratio of scopolamine to control, or of cor-onal to sagittal in each experiment (n) was calculated,then averaged across the experiments and analyzed in aone sample t-test.List of abbreviationsEPSC: Excitatory Postsynaptic Current; fEPSP: Field Excitatory PostsynapticPotential; LFS: Low Frequency Stimulation; LTD: Long-Term Depression; LTP:Long-Term Potentiation; mAchR: Muscarinic Acetylcholine Receptor; NMDA(R): N-methyl D-aspartate (receptor); Scop: Scopolamine; RasGRF1: RasGuanine nucleotide Release Factor 1; Ro: Ro 25-6981 (R-(R*,S*)-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidine propanol).AcknowledgementsThis work is supported by a grant from the Canadian Institutes of HealthResearch (YTW). YTW is the holder of the Heart and Stroke Foundation of BCand Yukon Chair of Stroke Research and is also a Howard Hughes MedicalInstitute International Scholar. We would like to thank Grigory Krapivinsky foradvice on RasGRF1.Authors’ contributionsTEB completed the experiments and wrote the manuscript. JL made theGluN2B antibody used for the co-immunoprecipitation. YTW helped writethe manuscript and coordinated the project. All authors read and approvedthe final manuscript.Competing interestsThe authors declare that they have no competing interests.Received: 16 July 2011 Accepted: 15 November 2011Published: 15 November 2011References1. Bliss TV, Collingridge GL: A synaptic model of memory: long-termpotentiation in the hippocampus. Nature 1993, 361:31-39.2. Massey PV, Bashir ZI: Long-term depression: multiple forms andimplications for brain function. Trends Neurosci 2007, 30:176-184.3. Cull-Candy S, Brickley S, Farrant M: NMDA receptor subunits: diversity,development and disease. Curr Opin Neurobiol 2001, 11:327-335.4. Monyer H, Sprengel R, Schoepfer R, Herb A, Higuchi M, Lomeli H,Burnashev N, Sakmann B, Seeburg PH: Heteromeric NMDA receptors:molecular and functional distinction of subtypes. Science 1992,256:1217-1221.5. Bayer KU, De Koninck P, Leonard AS, Hell JW, Schulman H: Interaction withthe NMDA receptor locks CaMKII in an active conformation. Nature 2001,411:801-805.6. Cummings JA, Mulkey RM, Nicoll RA, Malenka RC: Ca2+ signalingrequirements for long-term depression in the hippocampus. Neuron1996, 16:825-833.7. Kemp N, Bashir ZI: NMDA receptor-dependent and -independent long-term depression in the CA1 region of the adult rat hippocampus invitro. Neuropharmacology 1997, 36:397-399.8. Monyer H, Burnashev N, Laurie DJ, Sakmann B, Seeburg PH: Developmentaland regional expression in the rat brain and functional properties offour NMDA receptors. Neuron 1994, 12:529-540.9. Tovar KR, Westbrook GL: The incorporation of NMDA receptors with adistinct subunit composition at nascent hippocampal synapses in vitro. JNeurosci 1999, 19:4180-4188.10. Massey PV, Johnson BE, Moult PR, Auberson YP, Brown MW, Molnar E,Collingridge GL, Bashir ZI: Differential roles of NR2A and NR2B-containingNMDA receptors in cortical long-term potentiation and long-termdepression. J Neurosci 2004, 24:7821-7828.11. Kutsuwada T, Sakimura K, Manabe T, Takayama C, Katakura N, Kushiya E,Natsume R, Watanabe M, Inoue Y, Yagi T, et al: Impairment of sucklingresponse, trigeminal neuronal pattern formation, and hippocampal LTDin NMDA receptor epsilon 2 subunit mutant mice. Neuron 1996,16:333-344.12. Brigman JL, Wright T, Talani G, Prasad-Mulcare S, Jinde S, Seabold GK,Mathur P, Davis MI, Bock R, Gustin RM, et al: Loss of GluN2B-containingNMDA receptors in CA1 hippocampus and cortex impairs long-termdepression, reduces dendritic spine density, and disrupts learning. JNeurosci 2010, 30:4590-4600.13. Fischer G, Mutel V, Trube G, Malherbe P, Kew JN, Mohacsi E, Heitz MP,Kemp JA: Ro 25-6981, a highly potent and selective blocker of N-methyl-D-aspartate receptors containing the NR2B subunit. Characterization invitro. J Pharmacol Exp Ther 1997, 283:1285-1292.14. Hendricson AW, Miao CL, Lippmann MJ, Morrisett RA: Ifenprodil andethanol enhance NMDA receptor-dependent long-term depression. JPharmacol Exp Ther 2002, 301:938-944.15. Bartlett TE, Bannister NJ, Collett VJ, Dargan SL, Massey PV, Bortolotto ZA,Fitzjohn SM, Bashir ZI, Collingridge GL, Lodge D: Differential roles of NR2Aand NR2B-containing NMDA receptors in LTP and LTD in the CA1 regionof two-week old rat hippocampus. Neuropharmacology 2007, 52:60-70.16. Morishita W, Lu W, Smith GB, Nicoll RA, Bear MF, Malenka RC: Activation ofNR2B-containing NMDA receptors is not required for NMDA receptor-dependent long-term depression. Neuropharmacology 2007, 52:71-76.17. Liu L, Wong TP, Pozza MF, Lingenhoehl K, Wang Y, Sheng M, Auberson YP,Wang YT: Role of NMDA receptor subtypes in governing the direction ofhippocampal synaptic plasticity. Science 2004, 304:1021-1024.18. Muller T, Albrecht D, Gebhardt C: Both NR2A and NR2B subunits of theNMDA receptor are critical for long-term potentiation and long-termdepression in the lateral amygdala of horizontal slices of adult mice.Learn Mem 2009, 16:395-405.19. Toyoda H, Zhao MG, Zhuo M: Roles of NMDA receptor NR2A and NR2Bsubtypes for long-term depression in the anterior cingulate cortex. Eur JNeurosci 2005, 22:485-494.Bartlett et al. Molecular Brain 2011, 4:41http://www.molecularbrain.com/content/4/1/41Page 7 of 820. Man HY, Lin JW, Ju WH, Ahmadian G, Liu L, Becker LE, Sheng M, Wang YT:Regulation of AMPA receptor-mediated synaptic transmission byclathrin-dependent receptor internalization. Neuron 2000, 25:649-662.21. Dutar P, Bassant MH, Senut MC, Lamour Y: The septohippocampalpathway: structure and function of a central cholinergic system. PhysiolRev 1995, 75:393-427.22. Yoshida K, Oka H: Topographical projections from the medial septum-diagonal band complex to the hippocampus: a retrograde tracing studywith multiple fluorescent dyes in rats. Neurosci Res 1995, 21:199-209.23. Li S, Tian X, Hartley DM, Feig LA: Distinct roles for Ras-guaninenucleotide-releasing factor 1 (Ras-GRF1) and Ras-GRF2 in the inductionof long-term potentiation and long-term depression. J Neurosci 2006,26:1721-1729.24. Sweatt JD: Mitogen-activated protein kinases in synaptic plasticity andmemory. Curr Opin Neurobiol 2004, 14:311-317.25. Sturani E, Abbondio A, Branduardi P, Ferrari C, Zippel R, Martegani E,Vanoni M, Denis-Donini S: The Ras Guanine nucleotide Exchange FactorCDC25Mm is present at the synaptic junction. Exp Cell Res 1997,235:117-123.26. Krapivinsky G, Krapivinsky L, Manasian Y, Ivanov A, Tyzio R, Pellegrino C,Ben-Ari Y, Clapham DE, Medina I: The NMDA receptor is coupled to theERK pathway by a direct interaction between NR2B and RasGRF1.Neuron 2003, 40:775-784.27. Aznavour N, Watkins KC, Descarries L: Postnatal development of thecholinergic innervation in the dorsal hippocampus of rat: Quantitativelight and electron microscopic immunocytochemical study. J CompNeurol 2005, 486:61-75.28. Frotscher M, Leranth C: Cholinergic innervation of the rat hippocampusas revealed by choline acetyltransferase immunocytochemistry: acombined light and electron microscopic study. J Comp Neurol 1985,239:237-246.29. Drephal C, Schubert M, Albrecht D: Input-specific long-term potentiationin the rat lateral amygdala of horizontal slices. Neurobiol Learn Mem 2006,85:272-282.30. Dutar P, Nicoll RA: Classification of muscarinic responses in hippocampusin terms of receptor subtypes and second-messenger systems:electrophysiological studies in vitro. J Neurosci 1988, 8:4214-4224.31. Levey AI, Edmunds SM, Koliatsos V, Wiley RG, Heilman CJ: Expression ofm1-m4 muscarinic acetylcholine receptor proteins in rat hippocampusand regulation by cholinergic innervation. J Neurosci 1995, 15:4077-4092.32. Johnson DE, Nedza FM, Spracklin DK, Ward KM, Schmidt AW, Iredale PA,Godek DM, Rollema H: The role of muscarinic receptor antagonism inantipsychotic-induced hippocampal acetylcholine release. Eur JPharmacol 2005, 506:209-219.33. Cole AE, Nicoll RA: Characterization of a slow cholinergic post-synapticpotential recorded in vitro from rat hippocampal pyramidal cells. JPhysiol 1984, 352:173-188.34. Buchanan KA, Petrovic MM, Chamberlain SE, Marrion NV, Mellor JR:Facilitation of long-term potentiation by muscarinic M(1) receptors ismediated by inhibition of SK channels. Neuron 2010, 68:948-963.35. Shinoe T, Matsui M, Taketo MM, Manabe T: Modulation of synapticplasticity by physiological activation of M1 muscarinic acetylcholinereceptors in the mouse hippocampus. J Neurosci 2005, 25:11194-11200.36. Jo J, Son GH, Winters BL, Kim MJ, Whitcomb DJ, Dickinson BA, Lee YB,Futai K, Amici M, Sheng M, et al: Muscarinic receptors induce LTD ofNMDAR EPSCs via a mechanism involving hippocalcin, AP2 and PSD-95.Nat Neurosci 2010, 13:1216-1224.37. Descarries L, Gisiger V, Steriade M: Diffuse transmission by acetylcholine inthe CNS. Prog Neurobiol 1997, 53:603-625.38. Bierer LM, Haroutunian V, Gabriel S, Knott PJ, Carlin LS, Purohit DP, Perl DP,Schmeidler J, Kanof P, Davis KL: Neurochemical correlates of dementiaseverity in Alzheimer’s disease: relative importance of the cholinergicdeficits. J Neurochem 1995, 64:749-760.39. Schliebs R, Arendt T: The cholinergic system in aging and neuronaldegeneration. Behav Brain Res 2011, 221:555-563.40. Hardingham GE, Fukunaga Y, Bading H: Extrasynaptic NMDARs opposesynaptic NMDARs by triggering CREB shut-off and cell death pathways.Nat Neurosci 2002, 5:405-414.41. Liu Y, Wong TP, Aarts M, Rooyakkers A, Liu L, Lai TW, Wu DC, Lu J,Tymianski M, Craig AM, Wang YT: NMDA receptor subunits havedifferential roles in mediating excitotoxic neuronal death both in vitroand in vivo. J Neurosci 2007, 27:2846-2857.42. Taghibiglou C, Martin HG, Lai TW, Cho T, Prasad S, Kojic L, Lu J, Liu Y, Lo E,Zhang S, et al: Role of NMDA receptor-dependent activation of SREBP1in excitotoxic and ischemic neuronal injuries. Nat Med 2009,15:1399-1406.43. Tu W, Xu X, Peng L, Zhong X, Zhang W, Soundarapandian MM, Balel C,Wang M, Jia N, Lew F, et al: DAPK1 interaction with NMDA receptor NR2Bsubunits mediates brain damage in stroke. Cell 2010, 140:222-234.44. Leveille F, Papadia S, Fricker M, Bell KF, Soriano FX, Martel MA, Puddifoot C,Habel M, Wyllie DJ, Ikonomidou C, et al: Suppression of the intrinsicapoptosis pathway by synaptic activity. J Neurosci 2010, 30:2623-2635.45. Soriano FX, Martel MA, Papadia S, Vaslin A, Baxter P, Rickman C, Forder J,Tymianski M, Duncan R, Aarts M, et al: Specific targeting of pro-deathNMDA receptor signals with differing reliance on the NR2B PDZ ligand. JNeurosci 2008, 28:10696-10710.46. Ge Y, Dong Z, Bagot RC, Howland JG, Phillips AG, Wong TP, Wang YT:Hippocampal long-term depression is required for the consolidation ofspatial memory. Proc Natl Acad Sci USA 2010, 107:16697-16702.47. Anderson W: 2011 [http://www.winLTP.com].48. Anderson WW, Collingridge GL: The LTP Program: a data acquisitionprogram for on-line analysis of long-term potentiation and othersynaptic events. J Neurosci Methods 2001, 108:71-83.49. Anderson WW, Collingridge GL: Capabilities of the WinLTP dataacquisition program extending beyond basic LTP experimentalfunctions. J Neurosci Methods 2007, 162:346-356.doi:10.1186/1756-6606-4-41Cite this article as: Bartlett et al.: Slice orientation and muscarinicacetylcholine receptor activation determine the involvement of N-methyl D-aspartate receptor subunit GluN2B in hippocampal area CA1long-term depression. Molecular Brain 2011 4:41.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitBartlett et al. Molecular Brain 2011, 4:41http://www.molecularbrain.com/content/4/1/41Page 8 of 8

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.52383.1-0216451/manifest

Comment

Related Items