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MS 0932 Received 5 April 2000; accepted 14 April 2000.
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ABSTRACT |
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INTRODUCTION |
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Fast excitatory synaptic transmission in the mammalian central nervous system (CNS) usually involves the activation of postsynaptic NMDARs by the neurotransmitter L-glutamate. NMDARs are heteromultimeric assemblies formed from NR1 subunits in combination with at least one type of NR2 subunit. The identity of the NR2 subunit is important in determining both pharmacological and biophysical properties of the receptor (for reviews see Feldmeyer & Cull-Candy, 1996; Dingledine et al. 1999).
Studies of both recombinant and native systems suggest that NMDARs containing NR1/NR2D subunits give rise to receptors with unique properties (Monyer et al. 1994; Momiyama et al. 1996; Wyllie et al. 1996, 1998; Vicini et al. 1998). These include low single-channel conductance, low sensitivity to block by Mg2+ and a low EC50 for glutamate (Momiyama et al. 1996; Wyllie et al. 1996; Kuner & Schoepfer, 1996). Additionally, recombinant NR1/NR2D-containing NMDARs possess unusually slow deactivation kinetics (Monyer et al. 1994; Vicini et al. 1998; Wyllie et al. 1998). If present at the synapse, receptors with these properties would give rise to long-lasting NMDAR activation, and hence prolonged Ca2+ entry during synaptic transmission. However, to date, even those neurones that have been shown to express functional NR1/NR2D-containing NMDARs in their extrasynaptic membrane do not display slow deactivation kinetics in their synaptic responses (Momiyama et al. 1996; Bardoni et al. 1998; Clark & Cull-Candy, 1999; Misra et al. 2000; Momiyama, 2000). There are two possible explanations for this observation. First, native NR1/NR2D-containing NMDARs do not exhibit slow deactivation, and this behaviour is peculiar to recombinant receptors. Alternatively, native and recombinant NR1/NR2D-containing NMDARs exhibit similar properties, but this NMDAR subtype is absent from those central synapses that have been examined so far.
To distinguish between these possibilities we have made recordings from rat cerebellar Purkinje cells. These are known to contain mRNA for the NR1 and NR2D subunits during the early postnatal period (postnatal days (P) 0-8; Akazawa et al. 1994), and express a homogeneous population of low-conductance NMDARs (Momiyama et al. 1996). These appear to be restricted to the extrasynaptic membrane as NMDAR-mediated synaptic currents have not been detected in Purkinje cells (Perkel et al. 1990; Llano et al. 1991). We have characterized, for the first time, the deactivation kinetics of this pure population of native NR1/NR2D-containing NMDARs. These receptors, in outside-out patches from P6 Purkinje cells, do indeed exhibit unusually slow deactivation kinetics following a brief application of glutamate.
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METHODS |
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Slice preparation
Parasagittal cerebellar slices were obtained from Sprague-Dawley rats (P6). Following decapitation, the brain was removed rapidly and maintained in cold 'slicing' solution (2-4°C) oxygenated with 95 % O2-5 % CO2. The bisected cerebellum was glued onto the stage of a vibrating microslicer (DTK-1000 Dosaka Co. Ltd, Kyoto, Japan) and 200-250 µm slices were prepared. These were incubated at room temperature (22-25°C) and subsequently maintained at room temperature for up to 8 h (Farrant et al. 1994). Slices were then transferred to a recording chamber and the cells visualised with a Zeiss Axioskop FS microscope.
Solutions and drugs
The composition of the slicing and incubating solutions was (mM): NaCl, 125; KCl, 2·5; CaCl2, 1; MgCl2, 4; NaHCO3, 26; NaH2PO4, 1·25; glucose, 25; this was supplemented with 10 µM 5-amino phosphonate pentanoic acid (AP5). The external solution used during electrophysiological experiments was of similar composition, except that MgCl2 and AP5 were omitted. All external solutions were prepared using HPLC water (BDH). Recordings were made in the presence of 10 µM bicuculline methobromide (RBI, Natick, MA, USA), 0·5 µM strychnine hydrochloride (Sigma, UK), 20 µM 6-cyano-7-dinitroquinoxaline (CNQX; Tocris Cookson, UK) and 300 nM tetrodotoxin (TTX, Sigma). The external solution was supplemented with 50 µM glycine (BDH) to overcome the action of CNQX at the glycine site on the NMDAR (Lester et al. 1989).
The 'internal' (pipette) solution was composed of (mM): CsF, 110; CsCl, 30; Cs-Hepes, 10; Cs-EGTA, 5; NaCl, 4; CaCl2, 0·5; Mg-ATP, 2. Internal solutions were adjusted to pH 7·3 with CsOH. Patch pipettes were pulled from thick-walled glass tubing (GC150F-7·5; Clark Electromedical), coated with Sylgard resin (Dow Corning 184) and fire-polished to a final resistance of 5-10 M
. Recordings were made at room temperature using an Axopatch 200A patch-clamp amplifier (Axon Instruments) and stored on digital audiotape for subsequent analysis (Biologic DTR 1204).
Neurobiotin-filling procedure
N-(2-Aminoethyl) biotinamide hydrochloride (Neurobiotin; Vector Laboratories Inc., USA) was added to the pipette solution at a concentration of 5 mg ml-1. This solution was used during whole-cell recording. The slices were then washed and stored overnight in a cold solution of phosphate-buffered saline (PBS; Sigma: 0·1 M) containing 3 % paraformaldehyde. Slices were rinsed in 0·1 M PBS and incubated for 1 h in 4 % Triton X-100 before incubating for a further 1 h in fluorescein streptavidin (Vector Laboratories: 30 µg ml-1 in 0·1 M PBS). The slices were mounted in VectaShield mounting medium (Mowiol; Vector Laboratories) and viewed under a confocal microscope (LEICA TCS SP; Leica Microsystems UK Ltd).
Concentration jump experiments
Rapid concentration jumps into 1 mM glutamate were performed on outside-out patches as previously described (Wyllie et al. 1998). Briefly, patch pipettes were placed at the interface of control and 1 mM glutamate-containing solutions, flowing from either side of a theta-glass partition (Hilgenberg, Germany). The theta-glass was moved by a piezo translator (Burleigh Instruments, UK). Concentration jumps (1-10 ms in duration) were performed at 1 min intervals. To confirm the jump profile, liquid junction potentials were measured at the end of the experiment. Individual sweeps were averaged and the resulting decay of the mean current fitted (least squares) with a single exponential curve using the CJFIT program (David Colquhoun, University College London; see http://www.ucl.ac.uk/Pharmacology/dc.html). To be included in the average, the sweeps had to fulfil the following criteria: (a) display no channel openings in the 500 ms baseline preceding the jump, (b) contain single-channel openings that had the conductance typical of NR2D-containing NMDARs, and (c) show no 'breakdown' during the sweep.
Single-channel analysis
The amplitudes of single-channel events occurring towards the end of a concentration jump were analysed. Currents were replayed from tape, filtered at 1 kHz and digitised at 10 kHz (1401 plus interface; CED, UK) and individual openings and closings were fitted using the SCAN program. Single-channel amplitude distributions were fitted with the sum of two Gaussian components with the EKDIST program, using the method of maximum likelihood (Colquhoun & Sigworth, 1995; http://www.ucl.ac.uk/Pharmacology). In addition, we also examined the frequency of transitions occurring between the main- and sub-conductance levels to check that the single-channel events evoked by brief concentration jumps displayed the temporal asymmetry characteristic of NR2D-containing NMDARs.
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RESULTS |
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Morphology and whole-cell NMDAR-mediated responses of P6 Purkinje cells
Purkinje cells were initially distinguished from the numerous surrounding granule cells by their large soma size (cell capacitance, 29·3 ± 1·7 pF (mean ±S.E.M.); n = 22). In addition, Purkinje cells fire spontaneous action potentials that are detectable in the cell-attached configuration, a characteristic shared only by GABAergic interneurones. However, due to the disorganised nature of the Purkinje cell layer at this age, we also felt it necessary to confirm Purkinje cell identity using morphological criteria. At this stage of development Purkinje cells do not exhibit the 'elegant, regular, complex arborisation of the adult cell' (Ramón Y Cajal, 1995). As shown in Fig. 1A, the Purkinje cells had a large number of short, stubby, irregularly-shaped dendrites. In addition, we observed a long axon, projecting towards the white matter. As is characteristic of developing Purkinje neurones, all the cells contained exuberant axon collaterals that formed a tangled plexus in the granular layer.
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A, Neurobiotin-filled P6 Purkinje cell. Note the large soma, the many short irregularly shaped dendrites and the long axon extending into the white matter. Characteristically, this young Purkinje cell also possesses numerous axon collaterals that form a tangled plexus in the internal granular layer. B, whole-cell response of a P6 Purkinje cell to 20 µM NMDA at -60 mV. C, example of a response from an outside-out patch to a 1 ms application of 1 mM glutamate. 'c' indicates the closed state and 1-4 the number of channels open. Dotted lines indicate the amplitudes of the underlying single-channel events. Note the long duration of the response. The lower panel shows selected examples of the single-channel events present during the final portion of this type of response. The main- and sub-conductance levels are indicated and direct transitions between these levels are evident. D, amplitude distribution of glutamate-evoked single-channel events from a patch held at -60 mV. E, asymmetry plot showing that transitions from the main- to the sub-conductance (lower right quadrant) level are more prevalent than those from the sub- to the main-conductance level (upper left quadrant), where i is the transition number. | ||
In the presence of CNQX, bicuculline, strychnine and TTX, application of NMDA (20 µM) to whole cells evoked an inward current with an associated increase in membrane 'noise' (Fig. 1B). The mean current from four cells was -45 ± 10·7 pA (at -60 mV), corresponding to a mean conductance of 750 ± 91 pS. This is estimated to represent the synchronous opening of approximately 20 NMDAR channels. However, given the very low open probability of these channels (Wyllie et al. 1998) this level of activity suggests that several hundred NR2D-containing NMDARs are present in Purkinje cells.
Single-channel properties of NMDARs in P6 Purkinje cells
Single-channel NMDAR activity was also recorded in the presence of blockers of non-NMDA, GABAA and glycine receptors. Outside-out patches exposed to a brief application of 1 mM glutamate gave rise to responses lasting several seconds (as shown in Figs 1C and 2A). Our previous work has demonstrated that a homogeneous population of NR1/NR2D-containing NMDARs is activated during steady-state application of NMDA to Purkinje cell patches (Momiyama et al. 1996). To examine whether the single-channel events activated by rapid glutamate application also arose solely from these receptors, we analysed the individual openings underlying these currents.
Figure 1C indicates that the channel openings activated throughout the response, including those generating the initial peak, appear to be integer multiples of the unitary conductance (
33 pS) of low-conductance NMDAR channels (compared with a mean single-channel conductance of AMPAR channels of approximately 5-8 pS in these cells; Momiyama et al. 1996; Häusser & Roth, 1997). In this example, at least four channels were active in the patch. We also analysed individual events recorded during the tail of the jump response, where the single-channel openings were well separated. Examples of these are shown in the lower part of Fig. 1C. As is apparent from these single-channel currents and from the amplitude histogram (Fig. 1D), two conductance levels were present. The mean chord conductance was 34·4 ± 0·6 pS for the main-conductance state, and 21·2 ± 1·3 pS for the sub-conductance state (n = 3 patches). Low-conductance NMDA channel openings of this type are known to be characteristic of recombinant and native receptors containing NR1/NR2C or NR1/NR2D subunit combinations (Stern et al. 1992; Farrant et al. 1994; Momiyama et al. 1996; Wyllie et al. 1996).
To distinguish between the low-conductance NMDAR subtypes, we examined the transitions occurring between the conductance levels. As is apparent from the individual openings in Fig. 1C (lower part) and from the asymmetry plot shown in Fig. 1E, steps down from the main- to the sub-conductance state were more prevalent (62·5 ± 7·3 %; n = 3 patches) than steps up from the sub to the main state. Hence more events were present in the lower right quadrant of asymmetry plots (Fig. 1E). This pattern of transitions is typical of NMDARs present in Purkinje cells (Momiyama et al. 1996) which are known to contain mRNA for NR1 and NR2D subunits (Akazawa et al. 1994) and of recombinant NR1/NR2D channels (Wyllie et al. 1996).
Deactivation kinetics of Purkinje cell NMDARs
Figure 2A shows examples of single-channel activity recorded from three outside-out patches following exposure to 1 mM glutamate for 1 ms. Single-channel openings still occurred many seconds after glutamate application. Due to the low open probability and low density of NR2D-containing NMDARs in Purkinje cells, superimposed channel openings were rare following the initial peak. Figure 2B shows the average of 18 sweeps recorded from a single patch. The most striking feature of this average is its remarkably slow decay time. In all cases, this slow-component was well described by a single exponential decay. In the average current shown in Fig. 2B, this single exponential function decayed with a time constant (
decay) of 3292 ms. The NMDAR-mediated responses evoked by brief (1-10 ms) applications of 1 mM glutamate gave a mean decay of 2895 ± 701 ms (range, 978-4329 ms; n = 4 patches; 3 animals).
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A, three sweeps (from different patches) showing examples of single-channel activity evoked in response to a 1 ms pulse of 1 mM glutamate. Note the prolonged duration of the single-channel activations persistent after removal of glutamate. B, average of 18 individual sweeps from a single patch fitted with a single exponential component with a | ||
In the example shown in Fig. 2B an additional fast component was present, with a time constant of approximately 60 ms (compared with a time constant of 1·2 ms for AMPAR-mediated glutamate currents in these cells; Häusser & Roth, 1997). This is in good agreement with previous observations on the kinetic properties of recombinant NR1/NR2D NMDARs (Wyllie et al. 1998), and suggests a close similarity in their channel behaviour. A minimum of six exponential components was required to describe satisfactorily the distribution of activation lengths of recombinant NR1/NR2D-containing single channels. Theoretically, the time constants describing the low concentration limit of the burst length probability function should also describe the deactivation of the macroscopic response (for detailed discussion of this see Appendix in Wyllie et al. 1998). The dashed line shown in Fig. 2B represents a fit of six exponential components with time constants (but not amplitudes) constrained to equal the time constants obtained from this previous study. This fit includes both the rising and falling phase of the macroscopic response (the rising phase is expanded in the inset of Fig. 2B). The fact that this fit provides a good description of the entire macroscopic response is consistent with our proposal that this response is mediated solely by NR2D-containing NMDARs.
Figure 2C illustrates the difference in the rates of decay of the NMDARs found in two types of cerebellar neurone. Granule cells from P6 animals contain NR1, NR2B and possibly NR2A NMDAR subunits (Akazawa et al. 1994; Takahashi et al. 1996). The recording in Fig. 2C is from a nucleated granule cell patch (P6) exposed to 1 mM glutamate for 1 ms. The decay of this current was well described by the sum of two exponential components with a fast of 69·8 ms (80 % of the relative amplitude) and a slow of 327 ms. The decay of the granule cell response is considerably faster than the decay of the main component of the NMDAR-mediated currents in Purkinje cells indicated by the dashed line (decay = 3292 ms) scaled to the peak of the granule cell response.
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DISCUSSION |
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Our experiments demonstrate that native NR1/NR2D-containing NMDARs display very slow deactivation kinetics following brief exposure to glutamate. The decay time constant of around 3 s is similar to values reported for recombinant NR1/NR2D-containing NMDARs expressed in human embryonic kidney (HEK 293) cells (Monyer et al. 1994; Vicini et al. 1998) and oocytes (Wyllie et al. 1998). Our results, therefore, imply that a contribution from NR1/NR2D NMDARs has not so far been demonstrated at any central synapse.
Despite the presence of functional NR1/NR2D-containing NMDARs in many central neurones, there are no reports of synaptic responses with decay times comparable to the deactivation rate that we find for native NR1/NR2D-containing NMDARs. For example, it has been demonstrated that cerebellar Purkinje (Momiyama et al. 1996), Golgi (Misra et al. 2000) and stellate cells (Clark & Cull-Candy, 1999) all express a population of low-conductance NMDARs in their extrasynaptic membrane. In Golgi and stellate cells these occur along with high-conductance NMDAR channels. The low-conductance events exhibit properties characteristic of NR1/NR2D-containing NMDARs. However, the deactivation kinetics of the synaptic responses in these cells is fast - either because they lack an NMDAR-mediated EPSC component (in Purkinje and stellate cells: Perkel et al. 1990; Llano et al. 1991; Clark & Cull-Candy, 1999) or because the NMDA EPSC is mediated entirely by the NMDAR subtypes that exhibit fast deactivation kinetics (Misra et al. 2000). A similar situation occurs in dorsal horn spinal cord neurones, which express a mixed population of low- (NR2D-containing) and high-conductance NMDARs in their extrasynaptic membrane (Momiyama et al. 1996) but nevertheless, display rapidly decaying NMDAR EPSCs (Bardoni et al. 1998; Momiyama, 2000).
In an earlier study we obtained preliminary data which suggested that NR1/NR2D NMDARs in Purkinje cells may deactivate rapidly, in contrast with their recombinant counterpart (see Discussion in Momiyama et al. 1996). Re-examination of our original experimental protocol indicates that these currents were measured during the rapid removal of both glutamate and glycine. Hence the fast deactivation time almost certainly reflected the off-rate for glycine at these receptors. In the present study, with 50 µM glycine included in all control and drug solutions, we obtained consistently slow deactivation times. However, it was sometimes possible to detect an initial fast component in the response, prior to the slowly deactivating component. The fact that the amplitude of the channel openings underlying this fast component were integer-multiples of the NMDAR channel openings that underlie the slow component is consistent with the idea that both the fast and slow components were mediated by NR1/NR2D-containing NMDARs. Indeed, the presence of a fast component is expected on theoretical grounds, if native and recombinant NR1/NR2D receptors show quantitative similarity in their burst length probability functions.
It remains to be seen whether slowly deactivating NR1/NR2D-mediated currents are present at any central synapse. There is currently no functional evidence that NR2D subunits are targeted to synaptic sites, although most cell types within the cerebellum express a population of extrasynaptic NR2D-containing NMDARs. This raises the possibility that such receptors perform a specific function that is not associated with transmission. Alternatively, the NR2D subunit may occur at some synapses, co-assembled with other NR2 subunits, to form receptors with distinct deactivation kinetics. Indeed, immunoprecipitation studies have suggested that NR2D subunits can co-assemble with the NR2A and NR2B subunits (Dunah et al. 1997). Furthermore, it has been demonstrated that co-expression of NR1, NR2A and NR2D can result in the formation of some recombinant NMDAR assemblies with a single-channel conductance that is distinct from either NR1/NR2A or NR1/NR2D assemblies (Cheffings & Colquhoun, 1999). However, to date, there is no functional evidence for such assemblies in vivo, and the deactivation kinetics of the recombinant channel containing this subunit combination is unknown. Clearly it would be useful to determine the deactivation times of recombinant receptors containing NR2D along with other NR2 subunits. In this context, it is of interest that dendritic patches from CA1 and CA3 pyramidal cells exhibit a component of their NMDAR-mediated currents with a slow of 3 s in response to brief glutamate applications (Spruston et al. 1995). It is of note that mRNA for NR2D, NR2A and NR2B subunits is present in hippocampal cells, although low-conductance NMDAR channel openings were not observed.
The distinct expression pattern of the NR2D subunit in early development suggests that it may play a role in the early stages of neuronal development or synapse formation. In this respect it may be relevant that the NR1/NR2D receptor exhibits an unusually low EC50 for glutamate compared with other NMDAR subtypes, allowing activation by low ambient levels of glutamate. Recently, Watanabe et al. (1999) demonstrated that the gene encoding the NR2D subunit contains an oestrogen-responsive element not seen in other NR2 subunits. This untranslated region is involved in the upregulation by oestrogen of NR2D mRNA in the hypothalamus, and could suggest a novel role for NR2D-containing NMDARs in sexual dimorphism of the brain.
While techniques such as in situ hybridization and RT-PCR provide valuable information as to the NMDAR subunit mRNA present in a particular neurone, the localisation and possible co-assembly of the subunits remain much less clear. In the absence of immunocytochemical data, functional methods offer us the best opportunity for determining the subunit composition of synaptic NMDARs. Direct resolution of single-channel currents in the tail of the EPSC can give information about the NMDA receptor subtype present. However, this is possible only in those cells that are small and electrically compact (Silver et al. 1992; Clark et al. 1997; Brickley et al. 1999). Unfortunately, this approach is not readily applicable to the majority of neurones in the CNS. The slow deactivation kinetics of native NR1/NR2D-containing NMDARs therefore provides us with a valuable alternative 'signature' for identifying this receptor subtype at synapses.
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We thank Beverley Clark, Mark Farrant, Akiko Momiyama and David Colquhoun for helpful discussions. This work was supported by the Wellcome Trust and the Royal Society. C.M. held a Wellcome Prize Fellowship. D.J.A.W. held a Royal Society University Research Fellowship in the Department of Pharmacology, UCL during part of this work.
Corresponding authors
S. G. Cull-Candy: Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK.
Email: s.cull-candy{at}ucl.ac.uk
D. J. A. Wyllie: Department of Neuroscience, University of Edinburgh, 1 George Square, Edinburgh EH8 9JZ, UK.
Email: david.j.a.wyllie{at}ed.ac.uk
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D. J. A. Wyllie 2B or 2B and 2D? - That is the question J. Physiol., February 1, 2008; 586(3): 693 - 693. [Full Text] [PDF]
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D. C. Wrighton, E. J. Baker, P. E. Chen, and D. J. A. Wyllie Mg2+ and memantine block of rat recombinant NMDA receptors containing chimeric NR2A/2D subunits expressed in Xenopus laevis oocytes J. Physiol., January 1, 2008; 586(1): 211 - 225. [Abstract] [Full Text] [PDF]
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S. M. Logan, J. G. Partridge, J. A. Matta, A. Buonanno, and S. Vicini Long-Lasting NMDA Receptor-Mediated EPSCs in Mouse Striatal Medium Spiny Neurons J Neurophysiol, November 1, 2007; 98(5): 2693 - 2704. [Abstract] [Full Text] [PDF]
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 C. Piochon, T. Irinopoulou, D. Brusciano, Y. Bailly, J. Mariani, and C. Levenes NMDA Receptor Contribution to the Climbing Fiber Response in the Adult Mouse Purkinje Cell J. Neurosci., October 3, 2007; 27(40): 10797 - 10809. [Abstract] [Full Text] [PDF]
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A. M. Binshtok, I. A. Fleidervish, R. Sprengel, and M. J. Gutnick NMDA Receptors in Layer 4 Spiny Stellate Cells of the Mouse Barrel Cortex Contain the NR2C Subunit J. Neurosci., January 11, 2006; 26(2): 708 - 715. [Abstract] [Full Text] [PDF]
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Y. Wu, R. Kawakami, Y. Shinohara, M. Fukaya, K. Sakimura, M. Mishina, M. Watanabe, I. Ito, and R. Shigemoto Target-Cell-Specific Left-Right Asymmetry of NMDA Receptor Content in Schaffer Collateral Synapses in {epsilon}1/NR2A Knock-Out Mice J. Neurosci., October 5, 2005; 25(40): 9213 - 9226. [Abstract] [Full Text] [PDF]
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 S. H. Ahmed, R. Lutjens, L. D. van der Stap, D. Lekic, V. Romano-Spica, M. Morales, G. F. Koob, V. Repunte-Canonigo, and P. P. Sanna Gene expression evidence for remodeling of lateral hypothalamic circuitry in cocaine addiction PNAS, August 9, 2005; 102(32): 11533 - 11538. [Abstract] [Full Text] [PDF]
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P. Lachamp, B. Balland, F. Tell, A. Baude, C. Strube, M. Crest, and J.-P. Kessler Early expression of AMPA receptors and lack of NMDA receptors in developing rat climbing fibre synapses J. Physiol., May 1, 2005; 564(3): 751 - 763. [Abstract] [Full Text] [PDF]
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 B. Zucker, R. Luthi-Carter, J. A. Kama, A. W. Dunah, E. A. Stern, J. H. Fox, D. G. Standaert, A. B. Young, and S. J. Augood Transcriptional dysregulation in striatal projection- and interneurons in a mouse model of Huntington's disease: neuronal selectivity and potential neuroprotective role of HAP1 Hum. Mol. Genet., January 15, 2005; 14(2): 179 - 189. [Abstract] [Full Text] [PDF]
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A. Qian, A. L. Buller, and J. W. Johnson NR2 subunit-dependence of NMDA receptor channel block by external Mg2+ J. Physiol., January 15, 2005; 562(2): 319 - 331. [Abstract] [Full Text] [PDF]
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A. Bradaia, R. Schlichter, and J. Trouslard Role of glial and neuronal glycine transporters in the control of glycinergic and glutamatergic synaptic transmission in lamina X of the rat spinal cord J. Physiol., August 15, 2004; 559(1): 169 - 186. [Abstract] [Full Text] [PDF]
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P. E. Chen, A. R. Johnston, M. H. S. Mok, R. Schoepfer, and D. J. A. Wyllie Influence of a threonine residue in the S2 ligand binding domain in determining agonist potency and deactivation rate of recombinant NR1a/NR2D NMDA receptors J. Physiol., July 1, 2004; 558(1): 45 - 58. [Abstract] [Full Text] [PDF]
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 T. A. Simeone, R. M. Sanchez, and J. M. Rho Molecular Biology and Ontogeny of Glutamate Receptors in the Mammalian Central Nervous System J Child Neurol, May 1, 2004; 19(5): 343 - 360. [Abstract] [PDF]
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S. G. Brickley, C. Misra, M. H. S. Mok, M. Mishina, and S. G. Cull-Candy NR2B and NR2D Subunits Coassemble in Cerebellar Golgi Cells to Form a Distinct NMDA Receptor Subtype Restricted to Extrasynaptic Sites J. Neurosci., June 15, 2003; 23(12): 4958 - 4966. [Abstract] [Full Text] [PDF]
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B. Tabakoff, S. V. Bhave, and P. L. Hoffman Selective Breeding, Quantitative Trait Locus Analysis, and Gene Arrays Identify Candidate Genes for Complex Drug-Related Behaviors J. Neurosci., June 1, 2003; 23(11): 4491 - 4498. [Abstract] [Full Text] [PDF]
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L. Cathala, C. Misra, and S. Cull-Candy Developmental Profile of the Changing Properties of NMDA Receptors at Cerebellar Mossy Fiber-Granule Cell Synapses J. Neurosci., August 15, 2000; 20(16): 5899 - 5905. [Abstract] [Full Text] [PDF]
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