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Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland
Submitted 24 June 2005; accepted in final form 17 August 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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(PKC), and phosphorylation of ser-880 on the AMPA receptor subunit GluR2. Several lines of evidence have also implicated a complementary phosphatase limb in which N-methyl-D-aspartate (NMDA) receptor-mediated Ca2+ influx activates neuronal nitric oxide synthase (nNOS), the ultimate consequences of which are mediated by nitric oxide (NO), cGMP, and inhibition of postsynaptic protein phosphatases. However, the cellular localization of an NMDA/NO cascade has been complicated by the fact that neither functional NMDA receptors nor nNOS are expressed in Purkinje cells. This has lead to a proposal in which NMDA receptors activate nNOS in parallel fibers. Here, we confirm that pharmacological blockade of NMDA receptor or NO signaling blocks induction of LTD. However, no evidence was found for functional NMDA receptors in parallel fiber terminals: blockade of NMDA receptors did not alter either presynaptic Ca2+ transients or the frequency of miniature excitatory postsynaptic currents. NMDA receptor blockade did abolish a slow depolarization evoked by burst stimulation of parallel fiber-stellate cell synapses. The application of NMDA evoked a Ca2+ transient in stellate cell terminals but not in parallel fiber terminals. These results are consistent with the hypothesis that an NMDA receptor/NO cascade involved in cerebellar LTD is localized to interneurons rather than parallel fibers. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Brasnjo and Otis 2001; Kim et al. 2003). Another mechanism involves N-methyl-D-aspartate (NMDA) receptors in the postsynaptic density, which require both ligation by glutamate and sufficient postsynaptic depolarization to relieve blockade of the ion channel by Mg2+. These NMDA receptors are poorly activated by single afferent volleys but are effectively activated by either brief afferent bursts or by pairing postsynaptic depolarization with single afferent volleys.
Recently, a new cellular mechanism of burst detection has been proposed at the cerebellar parallel fiber-Purkinje cell synapse, where there are no functional postsynaptic NMDA receptors. It has been proposed that an NMDA receptor/neuronal nitric oxide synthase (nNOS) complex is present in parallel fiber terminals and is activated under conditions where the presynaptic terminal is simultaneously depolarized and exposed to glutamate as might occur during high-frequency bursts (Casado et al. 2000, 2002). In this scheme, glutamate released from the first parallel fiber volley must be bound to parallel fiber NMDA receptors when the second pulse arrives (60 ms later), which will briefly relieve the NMDA receptor Mg2+ block. Ca2+ influx through the NMDA receptor then activates nNOS, and the resultant NO may then diffuse across cell membranes. This is computationally interesting because, unlike a burst detector using postsynaptic NMDA receptors, it constitutes a mechanism that is activated by afferent bursts but not low-frequency pairing of pre- and postsynaptic activity.
In many synapses, burst stimulation can be a trigger for persistent changes in synaptic strength. At the parallel fiber-Purkinje cell synapse, LTD is produced when parallel fiber bursts are paired with activation of the massive, excitatory climbing fiber input (or depolarization of the Purkinje cell to mimic climbing fiber activation). This phenomenon has been suggested to constitute a portion of the engram for forms of motor learning such as adaptation of the vestibuloocular reflex and associative eyelid conditioning (Hansel et al. 2001; Linden 2003).
The molecular requirements for cerebellar LTD induction have begun to be defined. LTD is known to be postsynaptically expressed as it may be detected with exogenous pulses of AMPA receptor agonists (Crepel and Krupa 1988; Ito and Kano 1982; Linden et al. 1991). LTD is not associated with changes in AMPA receptor kinetics, agonist affinity, or unitary conductance (Linden 2001) but is associated with a reduction in AMPA receptor number (Matsuda et al. 2000) by clathrin-mediated endocytosis (Wang and Linden 2000). Triggering of AMPA receptor endocytosis is critically dependent on phosphorylation of a particular residue, ser-880, in the carboxy-terminal PDZ ligand of the AMPA receptor subunit GluR2 (Chung et al. 2003) by PKC (Leitges et al. 2004).
The phosphorylation state of ser-880 can be controlled by both kinases and phosphatases. The kinase limb involves activation of PKC by diacylglycerols and Ca2+, the former derived from parallel fiber activation of mGluR1/phospholipase C and the latter from climbing fiber activation of voltage-sensitive Ca2+ channels. The phosphatase limb is somewhat less defined but appears to involve a cascade in which nNOS produces NO, which activates soluble guanylyl cyclase. The cGMP produced in this fashion activates cGMP-dependent protein kinase (PKG) that has been proposed to phosphorylate G substrate, thereby increasing its potency as an inhibitor of protein phosphatase 1 (PP1) and PP2A (Ito 2001; Launey et al. 2004). In support of this model, cerebellar LTD in brain slices has been reported to be blocked by manipulations that inhibit NO signaling such as bath application of nNOS inhibitors or NO scavengers or genetic deletion of nNOS (Lev-Ram et al. 1997b; Shibuki and Okada 1991). LTD has also been reported to be blocked by inhibitors of guanylyl cyclase or PKG (Boxall and Garthwaite 1996; Hartell 1994) and mimicked by manipulations that inhibit protein phosphatases (Ajima and Ito 1995; Eto et al. 2002; Launey et al. 2004). In this model, the kinase activation and phosphatase inhibition limbs both work to produce phospho-ser-880 GluR2 and thereby, LTD.
What are the cellular compartments where the NO/cGMP/PKG-signaling cascade occurs? nNOS is a Ca2+-sensitive enzyme that is often found in a complex with PSD-95 and NMDA receptors (Christopherson et al. 1999). While both PKG and soluble guanylyl cyclase are expressed in Purkinje cells, nNOS is not (Bredt et al. 1990; El-Husseini et al. 1999). nNOS is also absent from cerebellar climbing fibers (Bredt et al. 1990; Vincent and Kimura 1992). This distribution is reflected in experiments with application of drugs to specific compartments: LTD may be blocked by application in the Purkinje cell of inhibitors of PKG and soluble guanylyl cyclase but not nNOS (Hemart et al. 1995; Lev-Ram et al. 1995, 1997a). Extracellular application of NO scavengers that cannot cross cell membranes can block LTD induction (Ito and Karachot 1990; Lev-Ram et al. 1995), implying that NO must diffuse from some other cellular compartment, through the extracellular space, to activate soluble guanylyl cyclase in the Purkinje cell.
Recently, it was shown that bath application of NMDA/glycine gave rise to a large synaptic depression that was blocked by bath application of an nNOS inhibitor and that occluded subsequent LTD induced by parallel fiber/depolarization pairing (Casado et al. 2000, 2002). Furthermore, bath application of an NMDA receptor antagonist blocked induction of LTD induced by repeated pairing of Purkinje cell depolarization with a stimulus composed of two parallel fiber volleys delivered with a 60-ms interval. These observations have led to the proposal that parallel fiber bursts during LTD induction activate an NMDA receptor/nNOS-signaling complex in the parallel fiber terminal, giving rise to NO production that could then trigger the phosphatase inhibition limb for LTD in the Purkinje cell (Casado et al. 2002). Here, we have sought to test this hypothesis by combining patch-clamp recording with presynaptic confocal Ca2+ imaging in cerebellar slices.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Sagittal (250 µm thick) slices of the cerebellar vermis were prepared from postnatal day 17–19 Sprague-Dawley rats by using a Vibratome 3000 (Vibratome, St. Louis, MO) and standard artificial cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 2.5 KCl, 1.3 MgCl2, 2.5 CaCl2, 1 NaH2PO4, 26.2 NaHCO3, and 20 glucose bubbled with 95% O2-5% CO2 (pH 7.4) at 4°C. Slices were recovered for 30 min in a chamber at 32°C and further recovered for 1 h at room temperature. The slices were placed in a submerged recording chamber that was perfused at a flow rate of 2 ml/min with room temperature ACSF and 5 µM gabazine to block GABAA receptors. Visualized whole cell patch-clamp recording was performed with a Zeiss Axioskop FS and a Multiclamp 700A amplifier (Axon Instruments, Union City, CA). The electrodes for Purkinje cell recording (2–4 M
) were filled with a solution containing (in mM) 135 Cs-methanesulfonate, 10 CsCl, 10 HEPES, 0.2 EGTA, 4 Na2-ATP, and 0.4 Na-GTP (pH 7.2). For stable recordings of Purkinje cell miniature excitatory postsynaptic currents (mEPSCs), we used a different solution containing 88 Cs2SO4, 10 EGTA, 4 MgSO4, 4 CaCl2 1.5 MgCl2, 4 Na2-ATP, 0.3 Na3-GTP, and 0.1 D600 (pH 7.2) (Dittman and Regehr 1996). Cells were voltage-clamped at –70 mV unless otherwise indicated. The currents were filtered at 2 kHz and digitized at 10 kHz. For current-clamp recording from stellate cells, electrodes (6–8 M) were filled with a solution containing (in mM) 130 K-methanesulfonate, 10 NaCl, 2 MgCl2, 10 HEPES, 0.2 EGTA, 4 Na2-ATP, and 0.4 Na-GTP (pH 7.2). For extracellular stimulation, standard patch pipettes filled with ACSF were used. Parallel fibers were stimulated in the molecular layer. Test stimulation was given using paired-pulses (100-ms interval) at a frequency of 0.1Hz using 80–120 µA pulses (100-µs duration). Stimulus strength was adjusted so that the first EPSC did not exceed 300 pA for Purkinje cells. For stellate cell recordings, parallel fiber test pulses with similar intensities were used. To induce LTD, parallel fibers were stimulated with a train of five pulses at 100 Hz, which was accompanied by 100-ms-long depolarization of the Purkinje cell to 0 mV (see Fig. 1C). A total of 30 trains were applied at 2-s intervals. For mEPSC analysis, a template was made to detect events in pClamp9 software (Axon Instruments), by averaging 
30 hand-picked mini events. When detecting events, the template match threshold was set to 4. SR 95531 hydrobromide (gabazine), D-2-amino-5-phosphonopentanoic acid (D-AP5), NG-nitro-L-arginine (L-NNA), 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (c-PTIO), MK-801, baclofen, NMDA, and NBQX (disodium salt) were purchased from Tocris Cookson (Ellisville, MO), and TTX from Calbiochem (San Diego, CA). All other chemicals were purchased from Sigma (St. Louis, MO).
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The method of loading the Ca2+ indicator into parallel fibers was adapted from Regehr and Atluri (1995). Briefly, 50 µg of either Fluo-4/AM or Oregon Green BAPTA-1/AM (OGB-1) from Molecular Probes (Eugene, OR) was dissolved in 20 µl of 20% Pluronic in DMSO and was then added to 400 µl of filtered ACSF. The mixture was vortexed and sonicated for 1 min each. The Fluo-4 solution was used to fill a glass pipette with a 6–8 µm tip and was ejected into the molecular layer of a transverse slice (300 µm) by constant positive pressure for 30 min. The direction of ejection was at a right angle to the course of the parallel fibers. A suction pipette with a 15-µm tip was placed near to the ejection site to remove excess dye. Ca2+ transients at areas 400 µm from the delivery site were recorded by a laser scanning confocal microscope (Zeiss LSM 510) with a x40 water-immersion objective lens. Fluo-4 was excited with the 488-nm line of an Argon ion laser. Emitted fluorescence was collected through a 505-nm long-pass filter. Fluo-4 images were recorded in line-scan mode with 512 pixels per line at 500 Hz for 2 s. The scan line was oriented at a right angle to the course of the parallel fibers. Parallel fibers were stimulated with either paired-pulses (60 ms apart) or a train of five pulses at 100 Hz that began 480 ms after the onset of line-scanning. Ca2+ transients are presented as 
F/Fo. Line-scanned images were analyzed off-line with Igor software (WaveMetrics, Lake Oswego, OR) and Origin software (OriginLab, Northhampton, MA).
Stellate cell presynaptic calcium imaging
For stellate cell Ca2+ imaging, 0.2 mM EGTA in the pipette solution was replaced by 0.4 mM Fluo-4, and 1 mM Alexa 594 hydrazide (Molecular Probes, Eugene, OR) was added to visualize axon terminals. To minimize phototoxicity, 0.1 mM Trolox-C was added to ACSF, and the power and exposure time of laser illumination were maintained as low as possible. For the experiments in which NMDA/glycine was applied, a cocktail of blockers were added (in µM): 1 CGP55845, 0.5 DPCPX, and 1 strychnine. Ca2+ transients were elicited by a burst of five brief current injections (2-ms long with 20-ms interval) every 2 min and recorded by a laser scanning confocal microscope (Zeiss Pascal) with a x40 water-immersion objective lens. Fluo-4 was excited with the 488 nm line of an Argon ion laser, and emitted fluorescence was collected through a 505-nm long-pass filter. Alexa 594 hydrazide was excited with the 543-nm line of a He-Ne laser, and the fluorescence was collected through a 560 long-pass filter. Fluo-4 images were recorded in frame-scan mode with 128 x 52 pixels at 20 Hz. For analysis, foreground pixels were determined by thresholding the image, and spatially averaged to calculate F/Fo for each frame.
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RESULTS |
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Casado et al. (2002) reported that bath application of the NMDA receptor antagonist D-AP5 blocked LTD induction (and revealed a small synaptic potentiation). They used slices prepared in a transverse orientation and a K+-based internal saline. We have examined whether D-AP5 also blocks LTD induction in our experimental conditions (a sagittal slice to maintain the integrity of the Purkinje cell dendrites and a Cs-based internal saline to improve the voltage clamp). As shown in Fig. 1B, bath application of 50 µM D-AP5 blocked the induction of LTD. In fact, the EPSC was transiently potentiated (136.9 ± 4.6% at t = 5 min, n = 8) after the pairing stimulation and returned to baseline later (107.8 ± 8.7% at t = 28 min). The potentiation of EPSCs after the failed LTD induction in the presence of D-AP5 may be partly due to a change in the probability of release as it was mirrored by a small, transient decrease in PPR. We also found that LTD induction was blocked by another NMDA receptor antagonist, MK-801 (50 µM; 94.3 ± 8.5% at t = 28 min; n = 6).
One possible downstream effector of NMDA receptors is nNOS activation and the consequent production of NO. We have tested the involvement of NO in LTD by using an nNOS inhibitor (L-NNA, 30 µM) or an NO scavenger (carboxy-PTIO, 30 µM). When parallel fiber/depolarization pairing was given after bath-application of either of these compounds, LTD was blocked (Fig. 2). After parallel fiber/depolarization pairing, the EPSCs were transiently potentiated (118.8 ± 13.9%, n = 3, and 162.7 ± 10.9%, n = 4, at t = 5 min) and returned to the baseline in L-NNA (93.5 ± 15.7% at t = 28 min) or remained potentiated in c-PTIO (135.0 ± 11.8%). A transient decrease in PPR was observed in both conditions. Thus we have replicated previous work implicating NMDA receptor activation (Casado et al. 2002) and NO production (Casado et al. 2002; Ito and Karachot 1990; Shibuki and Okada 1991) in cerebellar LTD.
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, 2002), we sought to measure parallel fiber Ca2+ transients (Regehr and Atluri 1995). After loading the Ca2+-sensitive dye Fluo-4, into parallel fibers in a transverse slice, Ca2+ transients were recorded by a laser scanning confocal microscope along a line oriented at right angles to the parallel fibers. Figure 3 (A, top) is a representative line-scan image where the x axis is time and the y axis is the line-scan coordinate. The spatial average of the fluorescence (middle) shows a transient paired-pulse Ca2+ response. From each episode, we calculated the integral of F/Fo. To maximize the probability of detecting parallel fiber NMDA receptor-mediated Ca2+ transients, Mg2+ was removed from the ACSF solution.
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F/Fo was 114.6 ± 3.4% of baseline at t = 13 min (n = 6). However, it was reduced by baclofen (57.1 ± 10.7% at t = 23 min), which is known to affect the presynaptic Ca2+ transient (Dittman and Regehr 1996), and nearly abolished by Cd2+ (4.5 ± 0.9% at t = 31 min). Because we have used burst stimulation for LTD induction and NMDA antagonists blocked this form of LTD, we examined whether D-AP5 reduces the parallel fiber terminal Ca2+ transients evoked by these same burst parameters. As shown in Fig. 3B, D-AP5 did not reduce the Ca2+ transients evoked in this configuration (99.3 ± 5.3% of baseline at t = 19 min, n = 8).
It has been reported that D-AP5 reversibly reduced the frequency of mEPSCs recorded from pyramidal neurons of the visual (Sjostrom et al. 2003) or entorhinal (Berretta and Jones 1996) cortex. These findings suggests that Ca2+ influx through presynaptic NMDA receptors can elevate mEPSC frequency by contributing to resting Ca2+ levels in the presynaptic terminal. To assess whether this action of presynaptic NMDA receptors exists in parallel fiber-Purkinje cell synapses, mEPSCs were recorded from Purkinje cells in the presence of 500 nM tetrodotoxin (TTX). When D-AP5 (50 µM) was applied, the frequency of mEPSCs did not significantly change (106.5 ± 5.1% baseline at t = 14 min, n = 6, Fig. 4A). However, when the GABAB receptor agonist baclofen (5 µM) was applied as a positive control, mEPSC frequency decreased (77.6 ± 8.4% baseline at t = 24 min). We then tried more permissive conditions in an attempt to reveal an effect of NMDA receptor antagonists on mEPSC frequency: the external concentration of Mg2+ was reduced to 0.1 mM. However, even with the potential Mg2+ block of NMDA receptors greatly reduced, neither D-AP5 nor (R)-CPP (20 µM) produced significant changes in mEPSC frequency [96.5 ± 6.2% (n = 6) and 108.9 ± 8.8% (n = 6) of baseline at t = 15 min, respectively, Fig. 4B]. In these experiments, 5 µM CGP55845, GABAB-receptor antagonist, was added to eliminate potential indirect effects from GABAergic interneurons.
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; Clark and Cull-Candy 2002).
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; Glitsch and Marty 1999; Huang and Bordey 2004), presumably through activation of interneuronal NMDA receptors. As these recordings were made in TTX, it is presumed that this effect mostly results from activation of NMDA receptors in the interneuron terminals. However, it has been suggested that a portion of this effect on miniature inhibitory postsynaptic current (mIPSC) frequency results from activation of NMDA receptors in the somato-dendritic region of interneurons as well (Glitsch and Marty 1999).
One report (Huang and Bordey 2004) found a reversible decrease in mIPSC amplitude with D-AP5 (and 1 mM external Mg2+), suggesting that, at least in their conditions, there was a tonic level of NMDA receptor activation in interneurons. To address this possibility, we measured internal Ca2+ from the presynaptic terminals of stellate cells loaded with Fluo-4 and bathed in the GABAB receptor antagonist CGP 55845 (5 µM). Under current-clamp mode, the cells were injected with a train of five current steps (2-ms long with an interval of 20 ms) to elicit a reproducible burst of five action potentials (Fig. 6B, left). As shown in Fig. 6B (middle), this evoked a substantial Ca2+ transient that slowly returned to the baseline. After 10 min of recording, 50 µM D-AP5 was applied, and this treatment affected neither the evoked calcium transients (107.4 ± 10.1% baseline at t = 20 min, n = 4, Fig. 6B, right) nor the basal Ca2+ concentration (94.7 ± 6.0% baseline at t = 20 min). These results suggest that, in our hands, tonic NMDA receptor activation on interneuron presynaptic terminals was minimal. However, the presence of interneuron terminal NMDA receptors was confirmed by external application of NMDA (30 µM) in the presence of 1.3 mM Mg2+ which evoked a small but significant increase in basal Ca2+ in stellate cell terminals (117.8 ± 7.8% baseline, n = 7; P < 0.05 by t-test, Fig. 6C). Thus functional NMDA receptors appear to be present in both somato-dendritic and axonal compartments of stellate cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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These results are also consistent with some previous immunocytochemical and electrophysiological findings. NMDA receptor subunit immunoreactivity is expressed weakly and only in a small subset of parallel fibers (Petralia et al. 1994) but strongly in interneurons (Akazawa et al. 1994). nNOS immunoreactivity is weak or absent in parallel fibers but is strong in both stellate and basket cells (in both somata and terminals) (Rodrigo et al. 2001). Furthermore, bath application of NMDA did not produce an alteration in the probability of glutamate release as indexed by the PPR of parallel fiber-Purkinje cell EPSCs (Casado et al. 2000).
Is it possible to reconcile the present results with the parallel fiber localization of an NMDA receptor/nNOS cascade by assuming compartmentalization of the parallel fiber terminals? For this to hold, Ca2+ influx through NMDA receptors would have to be able to activate nNOS but be invisible to both the release machinery (as indexed by both mEPSC frequency and paired-pulse facilitation) and confocal Ca2+ imaging (even when evoked by a burst of five pulses at 100 Hz in Mg2+-free saline or bath NMDA application). While this is formally possible, we believe that it is unlikely and would require an unprecedented degree of cytosolic compartmentalization beyond what we (Fig. 6) and others (Duguid and Smart 2004; Glitsch and Marty 1999; Huang and Bordey 2004) have observed for NMDA receptors in the terminals of cerebellar interneurons. Furthermore, other manipulations which attenuate Ca2+ channels in parallel fiber terminals, produce reductions in parallel fiber-evoked presynaptic Ca2+ transients. These include agonists of adenosine A1 receptors (Dittman and Regehr 1996), GABAB receptors (Dittman and Regehr 1996), and CB1 cannabinoid receptors (Kreitzer and Regehr 2001).
Parallel fiber stimulation does not appear to activate presynaptic parallel fiber NMDA receptors but does activate NMDA receptors on stellate cells. However, it is unclear whether an interneuronal NMDA receptor/nNOS complex would function in the somato-dendritic membrane of the interneuron as driven by synapses from parallel fibers or in the presynaptic terminals of the interneuron-Purkinje cell synapse or both. In support of the latter possibility, several laboratories have found that bath application of NMDA produced an increase in the frequency of mIPSCs recorded in Purkinje cells that could be blocked by an NMDA receptor antagonist (Duguid and Smart 2004; Glitsch and Marty 1999; Huang and Bordey 2004). nNOS (Rodrigo et al. 2001) and PSD-95 (McGee et al. 2001) immunoreactivity have also been reported in interneuron presynaptic terminals and could therefore potentially form a signaling complex with NMDA receptors at this location. The recordings illustrated in Fig. 6 confirm the presence of functional NMDA receptors in both somato-dendritic and axonal compartments of stellate cells but do not allow us to determine which of these receptors are important in induction of parallel fiber-Purkinje cell LTD.
To this point, we have assumed that the source of glutamate for activating interneuron NMDA receptors during LTD induction is the parallel fiber. However, it should be mentioned that there are lines of evidence suggesting that depolarization-evoked release of glutamate from Purkinje cell dendrites may function as a retrograde signal to activate interneuron NMDA receptors. For example, Duguid and Smart (2004) report that the interneuronal presynaptic NMDA receptors are involved in a transient enhancement of GABA release triggered by Purkinje cell depolarization and a subsequent postsynaptic Ca2+ transient.
At present, the argument that interneuronal NMDA receptors drive the NO signal that contributes to cerebellar LTD is one of exclusion. We have no mechanism at hand for interfering with the NO cascade specifically in stellate and/or basket cells that would provide a definitive test of this hypothesis. Optical measurement of NO levels in cerebellar slices would be useful to address the question of where NO is produced, but in pilot experiments, we found that the NO dyes currently available have technical issues that preclude their use in these experiments.
A consequence of a model for LTD induction with complementary kinase activation and phosphatase inhibition limbs (Fig. 7) is that in conditions of low basal phosphatase activity, kinase activation may be sufficient to produce LTD, whereas in high basal phosphatase activity, both kinase activation and phosphatase inhibition may be required for LTD. The former may be the case in cultured Purkinje cells, where inhibition of NO/cGMP/PKG signaling fails to block LTD (Linden et al. 1995), possibly due to reduced activation of interneurons. In cultured Purkinje cells, PKC itself has the ability to inhibit the myosin/moesin phosphatase form of PP1 through phosphorylation of the inducible inhibitor CPI-17 (Eto et al. 2002). It will be interesting to determine whether the PKC/CPI-17 mechanism is also operative in cerebellar slices.
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). Thus activation of an NMDA receptor/nNOS module in cerebellar interneurons may ultimately serve to persistently attenuate Purkinje cell activity in two different ways: through LTD of parallel fiber-Purkinje cell synapses and by LTP of parallel fiber-interneuron synapses, the latter of which will result in increased Purkinje cell inhibition by interneurons. |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. J. Linden, Dept. of Neuroscience, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., 916 Hunterian Bldg., Baltimore, MD 21205 (E-mail: dlinden{at}jhmi.edu)
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) and PPRs (
) from representative cells (middle), and normalized average amplitude from populations (bottom, n = 4 cells/group).
10 min before the recording. D-AP5 (50 µM) was applied, and later baclofen (5 µM) was further applied as a positive control. The number of events (black) was plotted each minute. The series resistance (gray) was monitored every minute as well. Right: normalized mean ± SE (n = 6) of the frequency was plotted as a function of time. B: similar experiments were done in 0.1 mM Mg2+ and 5 µM CGP55845. The concentration of (R)-CPP was 20 µM. C: representative traces before and after the addition of NMDA receptor antagonists (2 s long).
