Postsynaptic Currents in Deep Cerebellar Nuclei

Davide Anchisi,1 Bibiana Scelfo,1 and Filippo Tempia1,2

 1Department of Neuroscience, University of Turin, I-10125 Turin; and  2Department of Internal Medicine, Section of Human Physiology, University of Perugia, I-06126 Perugia, Italy


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Anchisi, Davide, Bibiana Scelfo, and Filippo Tempia. Postsynaptic Currents in Deep Cerebellar Nuclei. J. Neurophysiol. 85: 323-331, 2001. Postsynaptic currents were studied by whole cell recordings in visually identified large neurons of the deep cerebellar nuclei (DCN) in slices of 4- to 11-day-old mice. Spontaneous postsynaptic currents were abolished by the GABAA receptor antagonist bicuculline and had a single-exponential decay with a mean time constant of 13.6 ± 3.2 (SD) ms. Excitatory postsynaptic currents (EPSCs) were evoked in 48/56 neurons recorded. The addition of AMPA and N-methyl-D-aspartate (NMDA) receptor antagonists together completely abolished all synaptic responses. In 1 mM [Mg2+]o and at a holding potential of -60 mV, the peak amplitude of the NMDA component of the EPSC (NMDA-EPSC) was 83.2 ± 21.2% of the AMPA component (AMPA-EPSC). This indicates that in DCN neurons, at a physiological [Mg2+]o and at the resting membrane potential, NMDA receptors contribute to the synaptic signal. AMPA-EPSCs had a linear current-voltage relationship with a reversal potential of +2.3 ± 0.4 mV and a single-exponential decay with a voltage-dependent time constant that at -60 mV was 7.1 ± 3.3 ms. In 10 µM glycine and 1 mM [Mg2+]o, the I-V relationship of NMDA-EPSCs had a reversal potential of -0.5 ± 3.3 mV and a maximal inward current at -33.4 ± 5.8 mV. The apparent dissociation constant (KD) of Mg2+ for the NMDA receptor-channel at -60 mV, measured by varying [Mg2+]o, was 135.5 ± 55.3 µM, and when measured by fitting the I-V curves with a theoretical function, it was 169.9 ± 119.5 µM. Thus in the DCN, NMDA receptors have a sensitivity to Mg2+ that corresponds to subunits that are weakly blocked by this ion (epsilon 3 and epsilon 4) of which the DCN express epsilon 4. NMDA-EPSCs had a double-exponential decay with voltage-dependent time constants that at -60 mV were 20.2 ± 8.9 and 136.4 ± 62.8 ms. At positive voltages, the time constants were slower and their contributions were about equal, while in the negative slope conductance region of the I-V curve, the faster time constant became predominant, conferring faster kinetics to the EPSC. The weak sensitivity to Mg2+ of NMDA receptors, together with a relatively fast kinetics, provide DCN neurons with strong excitatory inputs in which fast dynamic signals are relatively well preserved.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Deep cerebellar nuclei (DCN) neurons provide the main output of the cerebellum and receive collaterals from the two main cerebellar afferent systems, climbing fibers and mossy fibers. In addition, they receive GABAergic synapses from Purkinje cell axons, which are the only efferents of the cerebellar cortex. Therefore neurons of the DCN perform the final processing of signals leaving thereafter the cerebellum (Ito 1984). DCN contains three main types of neurons: large projection neurons that send their axons outside the cerebellum and are the final cerebellar output element, and smaller inhibitory neurons including GABAergic cells that project to the inferior olive and local interneurons. It is possible that all these types of neurons receive all three main afferents, but the final signal integration takes place in the large projection neurons. The nature of DCN signal processing is still matter of debate. Most of the attention has been devoted recently to the Purkinje cell-DCN neuron synapse, which was the first GABAergic synapse shown to undergo both long-term depression (Aizenman et al. 1998; Morishita and Sastry 1993) and long-term potentiation (Aizenman et al. 1998).

Mossy fibers and climbing fibers, which on cortical neurons act through glutamate receptors, send collateral branches to the DCN. Since DCN neuron axons are the cerebellar efferent fibers, the pathway of mossy and climbing fiber collateral branches-DCN-cerebellar efferent fibers constitutes a cerebellar loop that could in principle work independently of the cortex. It is likely that such a subcortical cerebellar loop adds to all motor commands a basal cerebellar contribution that can be shaped by cortical activity. The contribution of the trans-cortical loop is not fixed but is endowed with several modifiable synapses, including the synapse between Purkinje cell axons and DCN neurons (Aizenman et al. 1998; Morishita and Sastry 1993). It has been shown that DCN neurons have functional glutamate receptors of both AMPA and N-methyl-D-aspartate (NMDA) type (Audinat et al. 1990) and glutamatergic fibers directed to DCN neurons evoke excitatory postsynaptic potentials (EPSPs) with components mediated by both receptors (Audinat et al. 1992). In view of the pivotal role of glutamatergic synapses on DCN neurons, it is very important to throw more light on the relative contributions of the different receptors and on their functional properties.

NMDA receptors of DCN neurons possess an interesting subunit repertoire since they express the subunits NR1, NR2A, NR2B, and NR2D, which in the mouse are called zeta 1, epsilon 1, epsilon 2, and epsilon 4 (Akazawa et al. 1994; Watanabe et al. 1994). For the formation of a functional NMDA receptor channel, it is necessary to have the co-assembly of subunits of both NR1/zeta and NR2/epsilon types, but the functional properties of the channel, like the deactivation rate or the block by Mg2+, seem to depend mainly on the latter type of subunits (Hollmann and Heinemann 1994). In DCN neurons, the presence of NR2D/epsilon 4 is especially significant since this subunit forms, together with NR1/zeta 1, channels that deactivate very slowly and are relatively resistant to the block by Mg2+ while channels containing only NR1/zeta 1 plus the subunits NR2A/epsilon 1 and/or NR2B/epsilon 2 deactivate more rapidly and are more potently blocked by Mg2+ (Kuner and Schoepfer 1996; Monyer et al. 1994; Vicini et al. 1998). The strength of Mg2+ block of NMDA receptor channels with different NR2/epsilon subunits can be distinguished by the degree of rectification of their current-voltage relationship in the presence of a physiological concentration of external Mg2+. Thus in the presence of an external concentration of 1 mM Mg2+, NR2A- or NR2B-containing channels are almost completely blocked at -100 mV and pass maximal current at about -25 mV, while NR2C or NR2D containing channels are incompletely blocked at -100 mV and pass maximal current at about -35 mV (Kuner and Schoepfer 1996). In addition, at membrane voltages close to the resting potential there is a four- to fivefold difference in the apparent dissociation constant of the channels for Mg2+ with values of about 20 µM for NR2A- or NR2B-containing channels and of 80 or 100 µM, respectively, for NR2C- or NR2D-containing channels (Kuner and Schoepfer 1996). This is due to a different voltage dependence of the block, which is expressed by the parameter delta  of the function (see METHODS: Eq. 1) describing the current-voltage relationship of NMDA receptor currents (Kuner and Schoepfer 1996; Woodhull 1973).

A stronger block by Mg2+ confers to the NMDA channel the capability to function as a coincidence detector because at the resting membrane potential almost no inward current is allowed to flow even when the agonist is bound to the receptor/channel (Ishii et al. 1993; Kutsuwada et al. 1992; Monyer et al. 1992). Only the coincidence of agonist binding plus membrane depolarization permits a flow of ions, including Ca2+, through the channel. In contrast, a weak Mg2+ block of NMDA receptor channels formed by NR1/zeta 1 and NR2D/epsilon 4 determines a relevant Ca2+ influx even at resting or at slightly depolarized potentials. Since DCN neurons express NR1/zeta 1, NR2A/epsilon 1, NR2B/epsilon 2, and NR2D/epsilon 4 (Akazawa et al. 1994; Watanabe et al. 1994), in principle they can form both types of NMDA receptors (NMDA-R), respectively, with faster kinetics and a stronger Mg2+ block (with NR2A/epsilon 1, NR2B/epsilon 2) or with slower kinetics and a weaker Mg2+ block (with NR2D/epsilon 4).

The results of our present investigation show that, in DCN neurons, the NMDA-R component of EPSCs has fast kinetics and a weak Mg2+ block, suggesting that NR2A/epsilon 1 and NR2B/epsilon 2 combinations are dominant for the former property while NR2D/epsilon 4 determines the effect of Mg2+. In addition, we show that, in the DCN, EPSCs display a significant NMDA component at the resting membrane potential, indicating that in these neurons NMDA-Rs have a physiological role in normal synaptic signaling instead of working merely as coincidence detectors.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experiments were performed on 4- to 11-day-old CD-1 mice of either sex. The preparation of cerebellar slices was performed according to previously described techniques (Edwards et al. 1989; Llinás and Sugimori 1980). Briefly, the animals were anesthetized with halothane (Fluothane, Zeneca, London, UK) and decapitated. The cerebellar vermis was rapidly removed and placed in an ice-cold extracellular saline solution, that contained (in mM) 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 20 glucose and was bubbled with 95% O2-5% CO2 so that the pH was 7.4. Some experiments were performed with varying concentrations of extracellular Mg2+ ([Mg2+]o) ranging from 0.01 to 3 mM. In these cases, the composition of the extracellular solution was modified only with regard to [Mg2+]o without readjusting the concentration of the other ions. Parasagittal cerebellar slices (300 µm thick) were prepared using a vibrotome (Vibroslice 752, Campden Instruments, Sileby, UK) and kept for the first hour at 35°C and then at 25°C for the rest of the day. After the first hour of incubation, a single slice at a time was transferred to a recording chamber and continuously perfused at room temperature (22-26°C) with the saline solution bubbled with the 95% O2-5% CO2 mixture. A DCN neuron soma was visualized using a ×63 water-immersion objective of an upright microscope (Axioscop, Zeiss, Jena, Germany), and its upper surface was cleaned by gently blowing and sucking saline solution from a cleaning pipette (Edwards et al. 1989). The pipettes used for cleaning and for extracellular stimulation were pulled from sodalime glass to a tip diameter of 10-15 µm. For both cleaning and stimulation, the pipette was filled with extracellular saline solution. For stimulation, the pipette was gently pressed into the tissue surrounding the DCN neuron, and negative current pulses ranging from 3 to 90 µA with durations of 100 µs were delivered. For patch-clamp recording, pipettes of borosilicate glass with tip diameters of 2-3 µm were used. When filled with an intracellular solution the resistance was 1.4-2.5 MOmega . In most experiments, the internal pipette solution had the following composition (in mM): 143 CsCl, 2 MgCl2, 10 HEPES, 4 Na2ATP, 0.4 Na3GTP, and 10 EGTA; in the experiments in which different [Mg2+]o were used, the composition was (in mM) 137 K-gluconate, 2 MgCl2, 10 HEPES, 4 Na2ATP, 0.4 Na3GTP, and 10 EGTA. The pH was adjusted to 7.3 with CsOH, for the CsCl solution, or with KOH, for the K-gluconate solution. In eight cells, the fluorescent dye Lucifer yellow (1 mM) was added to the internal solution and after recording the slice was fixed with 4% paraformaldehyde, dehydrated, and mounted on glass. Then micrographs were taken with an Axiophot (Zeiss) microscope at different planes of focus, digitized, and stored in the hard disk of a Pentium II PC. Then the best focused regions were assembled (Fig. 1). The somatic diameter was measured in 16 neurons: 12 of them were measured in fresh tissue by a graded scale mounted in an ocular of the microscope; another 4 neurons were measured after labeling with Lucifer yellow.



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Fig. 1. Reconstruction of a deep cerebellar nuclei (DCN) neuron filled with Lucifer yellow (1 mM) during recording (see METHODS).

After a tight seal (>5 GOmega ) was formed, the membrane was broken by suction to achieve the whole cell configuration. All recordings were performed in voltage-clamp using an EPC-7 patch-clamp amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany). The holding potential was set at -60 mV and varied during I-V experiments, and the series resistance was compensated. Data were filtered at 3 kHz, digitized at 10 kHz with an A/D converter (ITC 16, Instrutech, Port Washington, NY), and stored on a Macintosh computer (Quadra 650, Apple Computer, Cupertino, CA) using the Pulse Control software kindly provided by Dr. J. D. Herrington and Dr. R. J. Bookman of the University of Miami. Data were analyzed off-line by the commercial program IgorPro (Wavemetrics, Lake Oswego, OR). Drugs were applied by changing the perfusion line (exchange time in the chamber: 10-20 s), at the following concentrations: 40 µM bicuculline, 10 µM 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX), 50 µM D (-)-2-amino-5-phosphonopentanoic acid (D-AP5), 1 µM strychnine, and 10 µM glycine. NBQX and D-AP5 were purchased from Tocris Cookson (Langford, UK); bicuculline, strychnine, and glycine were bought from Sigma Chemical (St. Louis, MO) and Lucifer yellow from Molecular Probes Inc. (Eugene, OR).

The current-voltage relationship of NMDA-EPSCs was fitted by the following equation
<IT>I</IT> = [<IT>ag</IT><SUB>max</SUB>(<IT>V</IT> − <IT>V</IT><SUB>r</SUB>)]&cjs0823;  (<IT>a</IT> + [Mg<SUP>2+</SUP>]<SUB>o</SUB> <IT>e</IT><SUP>−<IT>V</IT>&dgr;<IT>zF</IT>&cjs0823;  <IT>RT</IT></SUP>) (1)
(Perouanski and Yaari 1993) where V is the holding potential, Vr is the reversal potential, gmax the maximal conductance, measured at +40 mV where the I-V relation is linear, a represents the dissociation constant in the absence of transmembrane voltage, delta  indicates the fraction of the membrane voltage at the blocking site and gives the voltage dependence of Mg2+ binding (Woodhull 1973), and R, T, z, and F have their usual meaning. From the a and delta  parameters of the best fitting curve obtained with Eq. 1, it is possible to calculate the dissociation constant (KD) of Mg2+ for the NMDA receptor channel (Woodhull 1973)
<IT>K</IT><SUB>D</SUB> = <IT>ae</IT><SUP><IT>V</IT>&dgr;<IT>zF</IT>&cjs0823;  <IT>RT</IT></SUP> (2)
The effects of different [Mg2+]o on the NMDA-EPSC were analyzed by fitting the data points with the following equation
<IT>I</IT> = (<IT>I</IT><SUB>max</SUB> − <IT>I</IT><SUB>min</SUB>)&cjs0823;  [1 + ([Mg<SUP>2+</SUP>]<SUB>o</SUB>&cjs0823;  IC<SUB>50</SUB>)<SUP><IT>p</IT></SUP>]<IT> + </IT><IT>I</IT><SUB>min</SUB> (3)
This equation was applied to the values normalized to the current at the lowest [Mg2+]o of 10 µM, where the block is negligible or absent. The KD value was considered as the point of the function in which the current was exactly 50% relative to the current at 10 µM [Mg2+]o.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DCN contain different classes of neurons. Neurons projecting to premotor structures have a larger somatic diameter [22.3 ± 4.8 (SD) µm] (De Zeeuw and Berrebi 1995), while GABAergic neurons including those projecting to the inferior olive and local interneurons have smaller soma (11 ± 2.6 µm) (De Zeeuw and Berrebi 1995). The neurons recorded in this study had a somatic size across the major axis ranging from 14.5 to 32.0 µm (mean = 19.8 ± 4.8 SD, n = 16) and across the minor axis between 8.0 and 31.0 µm (mean = 14.7 ± 5.8 SD, n = 16). Therefore the majority of the neurons recorded were projection neurons that send the cerebellar efferent signals to premotor nuclei. It is possible that a few of them were GABAergic neurons projecting to the inferior olive. Since dendrites are not clearly visible in living tissue, in eight cells, the dendritic tree was labeled by Lucifer yellow filling via the recording pipette (Fig. 1). The dendritic morphology of the recorded neurons was characterized by several thin dendrites arising from the soma and with sparse dendritic spines as has been shown previously (Chan-Palay 1977).

The presence of a considerable dendritic compartment required an assessment of the passive electrical membrane properties to establish the voltage-clamp conditions for distal dendritic regions. For the measurement of passive membrane properties, DCN neurons were recorded in voltage-clamp mode, and small voltage steps of 5 mV were delivered from a holding potential of -70 mV. With recording conditions including a series resistance (Rs) < 12.5 MOmega with 30-40% compensation, the capacitive transients produced by voltage steps had a decay that was always well fitted by a single-exponential function with a decay time constant of 2.0 ± 0.5 (SD) ms (n = 10). The input resistance calculated from the same voltage steps was 159.9 ± 68.3 (SD) MOmega (n = 10) with the intracellular solution based on CsCl (see METHODS). The relatively fast rate of decay and the single-exponential shape indicate that DCN neurons under these recording conditions have passive electrical properties that can be described by a single-compartment electrical circuit, excluding the presence of a less efficient voltage clamp in distal dendritic compartments.

Spontaneous postsynaptic currents

After entering the whole cell configuration, spontaneous inward currents were present as previously reported (Momiyama and Takahashi 1994). Such spontaneous currents were completely and reversibly blocked by the GABAA receptor antagonist bicuculline (40 µM; n = 56; Fig. 2A). The inward direction of the spontaneous currents is in agreement with the chloride equilibrium potential close to 0 mV due to the composition of the intracellular solution (see METHODS). The decay of such spontaneous inhibitory postsynaptic currents (sIPSCs) was well fitted by a single-exponential function (Fig. 2B). The decay time constant was measured in each DCN neuron for 5-19 sIPSCs and its average value was 13.6 ± 3.2 ms (n = 7), and within each cell, its distribution was unimodal. The decay time constant had no correlation with the amplitude of the current, indicating that also the largest currents, possibly giving rise to larger voltage errors, were under adequate voltage-clamp control to assess the decay kinetics.



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Fig. 2. Spontaneous inhibitory postsynaptic currents (IPSCs). A: sIPSCs in a DCN neuron in the standard saline solution (top) and after addition of the GABAA receptor antagonist bicuculline (40 µM). B: a sIPSC is shown expanded. The best fitting curve obtained with a single-exponential function describing the decay of the current is superimposed on the trace.

Evoked excitatory postsynaptic currents

Monopolar electrical stimuli were delivered to the tissue surrounding the neuron during recording under block of GABAA and glycine receptors (bicuculline, 40 µM; strychnine, 1 µM). In these conditions, no spontaneous activity was present. Stimulation evoked excitatory synaptic currents (EPSCs) in 48/56 neurons (86%). The addition of blockers of AMPA and NMDA receptors together completely abolished all synaptic response (Fig. 3, A and B; NBQX, 10 µM, D-AP5, 50 µM; n = 3). In some cells, to dissect the single components of the evoked EPSCs, initially the NMDA-mediated current was blocked with D-AP5. The NMDA antagonist was then washed out until the EPSC recovered to the control amplitude, and next the AMPA receptor antagonist NBQX was applied (Fig. 3, C and D). The peak amplitude of the two components was compared in each cell in the presence of 1 mM [Mg2+]o and at a holding voltage (VH) of -60 mV, which is close to the resting membrane potential of these neurons (Aizenmann et al. 1998; Jahnsen 1986; Llinás and Muhlethaler 1988). The NMDA component of the EPSC (NMDA-EPSC) was 83.2 ± 21.2% (mean ± SD, n = 5) of the AMPA component (AMPA-EPSC). Since NMDA-EPSCs decay slower than AMPA-EPSCs, their contribution to the charge transfer across the membrane is even larger. The net amount of charge that accumulates in the cell during an EPSC can be calculated by constructing the time integral of the membrane current. From the data shown in Fig. 3C, NMDA receptors allowed the entry of 11.2 pC of net positive charge in the first 100 ms from the stimulation, while at the same time point AMPA receptors contributed only for 3.5 pC. Thus in DCN the NMDA component of the EPSC is responsible for a net charge transfer that is more than threefold relative to the AMPA component. Taken together, these data indicate that in DCN neurons, at a physiological [Mg2+]o and at the resting membrane potential, there is a significant contribution of NMDA receptors to synaptic signals.



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Fig. 3. Pharmacological isolation of excitatory postsynaptic current (EPSC) components. A and B: effect of the simultaneous application of the AMPA receptor antagonist 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX, 10 µM) and of the N-methyl-D-aspartate (NMDA) receptor antagonist D (-)-2-amino-5-phosphonopentanoic acid (D-AP5, 50 µM) on an evoked EPSC at VH of +40 mV (A) and of -60 mV (B). C: evoked EPSC recorded at -60 mV and with 1 mM [Mg2+]o in the absence of glutamate receptor blockers (control), during application of D-AP5, which leaves only the AMPA component (AMPA-EPSC), and during application of the AMPA receptor antagonist NBQX, which leaves only the NMDA component (NMDA-EPSC). The trace labeled "AMPA + NMDA EPSC" has been obtained by summation of the AMPA-EPSC plus the NMDA-EPSC traces. Note the similarity of this trace with the control EPSC. D: average peak amplitude of the control EPSC (), of the AMPA-EPSC (), and of the NMDA-EPSC () recorded in 5 DCN neurons at VH of -60 mV and with 1 mM [Mg2+]o.

AMPA-EPSC properties

The current-voltage relationship (I-V relationship) of AMPA-EPSCs, obtained in the presence of bicuculline, strychnine and D-AP5, was nearly linear with a slight outward rectification and a reversal potential of +2.3 ± 0.4 (SD) mV (n = 3; Fig. 4, A and B). The AMPA-EPSCs had a decay that was fitted by a single exponential with a time constant at the VH of -60 mV of 7.1 ± 3.3 ms (n = 3; Fig. 4, A and C). The time constant was voltage dependent, becoming slower at positive voltages (Fig. 4C).



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Fig. 4. A: AMPA-EPSC at VH of -60, -40, -20, 0, +20, and +40 mV. Best fit single-exponential curves (- - -) are superimposed except for the trace at 0 mV. B: current-voltage relationship of AMPA-EPSC (n = 3, bars are SE). The amplitude has been normalized at +40 mV. ---, a polynomial fitting curve. C: voltage-dependence semilogarithmic plot of the decay time constant of AMPA-EPSC (n = 3, bars are SE). The correlation coefficient is 0.97 (P < 0.007).

NMDA-EPSC properties

NMDA-EPSCs were recorded in the presence of bicuculline and strychnine in the perfusate to block GABAA and glycine receptors, respectively, and NBQX to block AMPA receptors and with 10 µM glycine to saturate the binding site of NMDA receptors for this amino acid (Kew et al. 1998; Wilcox et al. 1996). With 1 mM [Mg2+]o the I-V relationship of NMDA-EPSCs showed the typical outward rectification with a region of negative slope conductance (n = 13; Fig. 5, A and B) as described for NMDA receptors-mediated currents (Mayer et al. 1984; Nowak et al. 1984). When the perfusion was changed to one with nominally zero [Mg2+]o, the I-V relationship of the NMDA-EPSCs tended to become linear (not shown) as expected from the known voltage-dependent block by [Mg2+]o. To characterize the properties of the NMDA-EPSC in these neurons, we fitted the I-V curves obtained in 1 mM [Mg2+]o with Eq. 1 (see METHODS). For all cells, this equation fitted the experimental data points very reliably. In Fig. 5B, the fit on the cumulative data from 13 cells is shown. From the individual fits, it was possible to precisely measure the reversal potential of the NMDA-EPSC, which was -0.5 ± 3.3 mV (n = 13), and the maximal inward current, measured as the negative peak of the function fitting the I-V relationship, which was attained at a membrane potential of -33.4 ± 5.8 mV (n = 13).



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Fig. 5. NMDA-EPSC: current-voltage relationship and Mg2+ sensitivity. A: traces of the NMDA component of evoked EPSC in 1 mM [Mg2+]o at VH from -80 to +40 mV with superimposed double-exponential curves (- - -) fitting the decay of the currents, except for the traces at -80 and at 0 mV. B: current-voltage relationship of NMDA-EPSC (n = 13, bars are SE). ---, the best fit curve obtained with Eq. 1. C: relationship between the amplitude of NMDA-EPSC and [Mg2+]o (n = 4, bars are SE) at VH of -60 mV. Currents were normalized at [Mg2+]o of 10 µM.

The parameters a and delta  of the best fit curve for Eq. 1 allowed us to calculate the apparent dissociation constant (KD) of Mg2+ for the NMDA receptor channel by Eq. 2 (see METHODS). The parameter a, which represents the affinity of the channel for Mg2+ in absence of transmembrane potential, was 5.3 ± 1.3 (SE) mM (n = 14). The parameter delta , which represents the voltage dependence of the block, was 0.69 ± 0.04 (n = 14). The KD value calculated for a membrane potential of -60 mV was 169.9 ± 119.5 (SD) µM (n = 14). Since such KD value was unusually high compared with other types of neurons, to provide an independent assessment of this parameter, the amplitude of the NMDA-EPSC was measured at different extracellular concentrations of Mg2+ keeping the VH constant at -60 mV (Fig. 5C). The results were fitted by Eq. 3 and the Mg2+ concentration that gave an inhibition of 50% of the NMDA-EPSC was taken as the KD for Mg2+ (see METHODS). With this procedure, the value of KD was 135.5± 55.3 µM (n = 4), which is not significantly different from the measurement derived from the current-voltage relationship (Student's t-test: P > 0.05), confirming that in DCN neurons NMDA receptor channels are only weakly blocked by external Mg2+. This result is also in agreement with the presence of a relevant inward current detected in these neurons through NMDA receptors at the resting membrane potential of -60 mV (see Fig. 3).

The rise time of NMDA-EPSCs was measured as the interval between the beginning of the stimulation and the time at which the current reached its peak amplitude. NMDA-EPSCs rise time was on average 12.5 ± 3.0 ms (n = 9). The decay of NMDA-EPSCs was fitted by a double-exponential curve (Figs. 5A and 6A) with, at the VH of -60 mV, a faster time constant of 20.2 ± 8.9 ms (n = 9) and a slower one of 136.4 ± 62.8 ms (n = 9). Both exponential components had a time constant that was voltage dependent, becoming slower toward positive values (Fig. 6, A and B). To analyze the contribution of each component, the amplitudes extrapolated to the time origin (beginning of the stimulus artifact) were plotted as a function of the holding voltage (Fig. 6C). While at positive voltages, the contributions of the slow and of the fast component are about equal: at -40 and -60 mV, the amplitude of the faster component becomes larger than the slower one, so that at negative potentials the slow component contributes less to the decay (Fig. 6, A and C). This relative prevalence of the faster time constant at negative potentials accounts for a further increase of the decay velocity at such potentials. In fact, this effect adds to the faster decay of each component at negative potentials (Fig. 6B).



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Fig. 6. NMDA-EPSC: decay time constants. A: NMDA-EPSCs at VH of -40 mV (inward trace) and of +20 mV (outward trace) with superimposed double-exponential fitting curves (dashed lines). The solid and the dotted lines are the single-exponential functions representing separately the 2 components of the double-exponential fittings. Note that at -40 mV, the faster exponential function is at the same time faster and larger than at +20 mV. B: semi-logarithmic plot of the voltage dependence of the decay time constants of NMDA-EPSC, derived from double-exponential fittings (n = 9, bars are SE). Correlation coefficients are 0.98 (P < 0.004) for the faster time constant and 0.96 (P < 0.01) for the slower one. C: absolute initial amplitude of the faster and slower time constant components of NMDA-EPSC as a function of VH. The amplitudes for each exponential function have been obtained by extrapolation to the time origin defined as the beginning of the stimulation artifact (see A).

The presence of a stronger rectification of the slow component at negative voltages suggests that this component could be more sensitive to [Mg2+]o than the fast one. To test this hypothesis, we compared the amplitude of the two components at a VH of -60 mV, in 0 [Mg2+]o relative to 1 mM [Mg2+]o. In the former condition, of Mg2+-free extracellular solution, the slow component accounted for 44.7% of the amplitude while the remaining 55.3% was due to the fast component (n = 5). The addition of 1 mM [Mg2+]o significantly decreased the contribution of the slow component to 29.8% while the fast component increased to 70.2% (Student's paired t-test, P < 0.05).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we show that in large neurons of the DCN fast synaptic transmission is mediated by GABAergic synaptic currents with properties very similar to other neuronal types and by glutamatergic currents that, in contrast, present some unusual features.

The sIPSCs were completely abolished by the addition of bicuculline, indicating that they were exclusively mediated by GABAA receptors and therefore that DCN neurons do not receive a significant number of spontaneously active glycinergic inhibitory synapses. This result is in agreement with previous studies in which the addition of bicuculline abolished miniature IPSCs (Momiyama and Takahashi 1994) or spontaneous and evoked inhibitory postsynaptic potentials (Aizenmann et al. 1998; Audinat et al. 1992; Mouginot and Gahwiler 1995).

The single-exponential decay of sIPSCs with relatively fast kinetics is comparable to the characteristics of IPSCs of principal neurons in slices of other regions of the CNS, including Purkinje cells (Vincent et al. 1992) pyramidal neurons of the hippocampus (Collingridge et al. 1984) and neocortex (Salin and Prince 1996). This contrasts with the double-exponential decay described for granule cells of the dentate gyrus (Edwards et al. 1990) and for cerebellar granule cells (Puia et al. 1994). It is interesting to note that, as in the principal neurons of other regions, the principal neurons of DCN, investigated in this study, also have sIPSCs with a single-exponential decay.

A main finding of our experiments is a significant NMDA component in EPSCs even at a membrane voltage corresponding to the resting potential. Therefore in these neurons, NMDA receptors must be involved in normal synaptic signaling and are responsible for a significant portion of the EPSC. This NMDA contribution results in EPSCs that last longer than those mediated by AMPA receptors as can be appreciated by comparing the AMPA component with the total glutamatergic EPSC in the same cell (see Fig. 3). This finding is in agreement with a previous study in organotypic cultures, where, in the DCN, EPSPs recorded in 0.5 mM [Mg2+]o had AMPA and NMDA components of comparable amplitude (Audinat et al. 1992). An explanation for the presence of a relevant NMDA-mediated current at the resting potential is a relatively weak effect of Mg2+ at negative potentials. Our results indicate that this is the case: in fact, in DCN neurons, we observed a maximal inward current at about -35 mV and the KD for Mg2+ at -60 mV was 135.5 µM measured by the dose-response curve for [Mg2+]o. Such a high KD value is confirmed by the KD, calculated by Eq. 2, of 169.9 µM at the same VH, derived from the data obtained by fitting Eq. 1 to the I-V relationship. Both the maximal inward current at about -35 mV and the KD for Mg2+ at -60 mV larger than 100 µM correspond to the values of NMDA receptor channels with a weak Mg2+ block (Kuner and Schoepfer 1996). The weaker effect of Mg2+ on the channels containing either NR2C/epsilon 3 or NR2D/epsilon 4 is due to a lower voltage dependence of the block (Kuner and Schoepfer 1996), which is described by the parameter delta  of Eq. 1. The channels containing either NR2A/epsilon 1 or NR2B/epsilon 2 have a strong voltage dependence of the block by Mg2+ with a value of delta  of about 1, while the channels containing either NR2C/epsilon 3 or NR2D/epsilon 4 have a delta  of about 0.7 (Kuner and Schoepfer 1996). In DCN, the value of delta  of 0.69 indicates that the NMDA receptor channels of these neurons have the same voltage-dependent block by Mg2+ as NR2C/epsilon 3- or NR2D/epsilon 4-contaning channels.

Our observation of a weak sensitivity to [Mg2+]o of NMDA receptors in DCN neurons is unexpected because these neurons express the epsilon 1 subunit and from P0 to P7 also the epsilon 2 subunit, which are both strongly blocked by Mg2+ (Ishii et al. 1993; Monyer et al. 1994). A possible explanation is an involvement of the other epsilon  subunit expressed by DCN neurons, epsilon 4, which has a lower affinity for Mg2+ (Ishii et al. 1993; Monyer et al. 1994). Although in adult animals, NR2D/epsilon 4 expression is restricted to a few types of neurons, during development, especially in embryonic and neonatal age, this subunit is strongly expressed throughout the CNS (Monyer et al. 1994). In addition to a low sensitivity to Mg2+, the NR2D/epsilon 4 subunit generates currents that decay slowly with a time course of several seconds (Ishii et al. 1993; Kutsuwada et al. 1992; Monyer et al. 1992). For these reasons, it has been suggested that such a long-lasting current is involved in the generation of Ca2+ signals important for the development of the CNS (Momiyama et al. 1996; Monyer et al. 1994). However, in DCN neurons we find that NMDA-EPSCs are weakly blocked by Mg2+, but at the same time, they are relatively rapid, with kinetics that are typical of the epsilon 1 and epsilon 2 subunits. However, the latter subunits are potently blocked by Mg2+ so that normally they do not contribute significantly to synaptic signaling unless the postsynaptic cell is at the same time depolarized by another excitatory synapse. Thus if we consider together the Mg2+ dependence and the decay rate of NMDA-EPSCs in DCN neurons, they do not correspond to any single subunit known at present. It should be emphasized that all our measurements of Mg2+ effects on NMDA-EPSCs have been obtained by taking the peak amplitude of the current, when possible slow channels, like those formed by NR1/zeta 1 with NR2D/epsilon 4, would not yet be significantly activated. In fact, while NMDA receptor channels formed by NR1/zeta 1 plus NR2A/epsilon 1 activate with a rise time constant of about 13 ms (Wyllie et al. 1998), those formed by NR1/zeta 1 plus NR2D/epsilon 4 have a slower activation rate, with a time constant of about 45 ms (Wyllie et al. 1998). Since in DCN the peak of the NMDA-EPSC is reached 12.5 ms from the beginning of the stimulation of presynaptic fibers, the activation kinetics better corresponds to NMDA receptor channels containing the NR2A/epsilon 1 subunit. As a consequence, our finding of a weak sensitivity to Mg2+ really refers to currents with fast kinetic properties. Thus NMDA-EPSCs in DCN possess novel physiological properties that cannot be explained simply by the sum of the properties of the cloned subunits as studied by expression in heterologous systems.

Recent evidence (Buller and Monaghan 1997; Dunah et al. 1998) indicates that NR2D/epsilon 4 can assemble with NR2A/epsilon 1 or NR2B/epsilon 2 subunits but without affecting the conductance levels of individual receptor channels, which are dominated by the latter subunits (Cull-Candy et al. 1998). However, the kinetics and/or the degree of block by Mg2+ of such heterotrimeric channels (zeta 1, epsilon 1/2, epsilon 4) are not known. They could have intermediate properties, or the features of one NR2/epsilon subunit could dominate over the others. Our results of NMDA EPSCs, which are at the same time rapid but weakly blocked by Mg2+, suggest that NMDA-Rs in DCN neurons are formed by the co-assembly of both kinds of epsilon  subunits (1 or 2 together with 4). The possible formation of such a subunit composition has been suggested by immunoprecipitation experiments (Dunah et al. 1998). If this is the case, our study points to the possibility that in these neurons the epsilon 4 subunit is dominant for the determination of the Mg2+ block with the result of allowing more current flow at negative potentials, while the epsilon 1/2 subunits are dominant regarding the decay rate, keeping the kinetics fast. More studies of functional expression are needed to test this hypothesis. However, our results cannot exclude other explanations, such as the involvement of a novel subunit, like for instance NR3A (Das et al. 1998; Sun et al. 1998) or an as yet undiscovered one, or posttranslational modifications altering the functional properties of NMDA receptor channels.

In cerebellar Purkinje cells of young rats, NR2D is the only NR2 subunit expressed (Akazawa et al. 1994). In these cells, recordings from outside-out patches in young animals revealed a single channel activity that was attributed to NR2D (Cull-Candy et al. 1998; Momiyama et al. 1996) and NMDA application to Purkinje cells from newborn rats evoked an inward current clearly attributable to NMDA-R (Krupa and Crepel 1990; Rosenmund et al. 1992). However, whole cell recordings in older animals failed to detect NMDA-R-mediated EPSCs (Llano et al. 1991; Perkel et al. 1990). Taken together, these data suggest the possibility that in Purkinje cells NR2D/epsilon 4 is located extrasynaptically and that, as a consequence, it does not contribute to the EPSC. In contrast, in DCN neurons that also express other NR2/epsilon subunits in addition to NR2D/epsilon 4, our data suggest that this subunit contributes to the generation of EPSCs.

CA1 pyramidal neurons in the first postnatal week express NR2A, NR2B, and NR2D, like DCN neurons (Kirson et al. 1999): as can be calculated from the data of Kirson et al. (1999), in these neurons the KD for Mg2+ of NMDA-evoked currents at -60 mV is 65.6 µM, which is intermediate between the value of the strongly blocked subunits (20 µM) and that of NR2D (100 µM) (Kuner and Schoepfer 1997), but only about half of the KD that we find for DCN. A difference relative to our study is that NMDA application also activates extrasynaptic receptors, which could have a different subunit composition, like a larger proportion of NR2B subunits (Tovar and Westbrook 1999).

Another study on DCN (Cull-Candy et al. 1998), using single-channel recordings, described a mixed population of single-channel openings: "high conductance," which can be attributed to NR2A or NR2B subunits, and "low conductance," which can be attributed to NR2D subunits. But the latter behavior was always found to be associated with high-conductance openings, so that it was not possible to assess the [Mg2+]o sensitivity of low-conductance channels. However, in some recordings, high-conductance channels could be observed alone: the percentage of block of the single-channel charge transfer exerted by 0.1 mM [Mg2+]o at -60 mV was 81%. This value corresponds to NR2A and NR2B (Kuner and Schoepfer 1997), as can be expected by the fact that high-conductance openings are due to these subunits.

The [Mg2+]o sensitivity of the NR2D subunit in native neurons has been measured by single-channel recording in P3 Purkinje cells, which only express this subunit (Momiyama et al. 1996). In this study, low-conductance channels are blocked only by about 34% by 0.1 mM [Mg2+]o (Momiyama et al. 1996), which is in agreement with the value of 45% obtained for the same subunit expressed in Xenopus oocytes (Kuner and Schoepfer 1997) and with the value of 46% found in the present study in DCN neurons.

Our study has been conducted on DCN neurons from 4- to 11-day-old mice, an age at which the cerebellum is still developing. Actually, the expression of the epsilon 2 subunit in DCN is strongly reduced between P7 and P14 (Watanabe et al. 1994). In contrast, the expression of epsilon 1 and epsilon 4 remains constant in the DCN throughout postnatal life (Watanabe et al. 1994). Therefore our conclusions on the physiological properties of NMDA-EPSCs are not likely to change at later stages of development. Over the short range of age in which our study has been conducted, we did not find any correlation with age for any of the parameters measured. However, it is possible that at older ages the diminished expression of epsilon 2 could cause some degree of reduction of the NMDA-EPSC relative to the AMPA component.

The physiological meaning of keeping the EPSCs faster could be related to the nature of the dynamic signals that they represent in which a duration of more than a few hundred milliseconds would reduce the resolution of temporal information. Why do DCN neurons need an NMDA component in their EPSC? A possible explanation could be the extremely powerful GABAergic inhibition exerted by Purkinje cells on DCN neurons. Adding an NMDA component to excitatory synaptic signals could be a mechanism to obtain a stronger excitation in spite of the relatively small number of excitatory synapses. In the medial vestibular nucleus, the block of NMDA receptors decreases the spontaneous firing rate, suggesting that in this structure NMDA receptors may contribute to the resting activity of neurons (Smith et al. 1990). Our results suggest that, in the DCN, NMDA receptors could have a similar role and that they can contribute to synaptic signals.

A recent report (Aizenman and Linden 2000) has shown that in these synapses the influx of Ca2+ through NMDA-R channels triggers a novel form of plasticity resulting in a long-term increase of intrinsic excitability not accompanied by changes in synaptic efficacy. This form of plasticity can be induced also at hyperpolarized potentials and therefore requires NMDA-Rs with a weak sensitivity to Mg2+. Our results of a relatively weak effect of Mg2+ on NMDA-R channels help to explain how they can trigger such a form of plasticity also without a concurrent membrane depolarization.


    ACKNOWLEDGMENTS

We thank Dr. Piergiorgio Strata for valuable help and advice and Dr. Nick Hartell for revising the manuscript.

The financial support of Telethon-Italy (Grant 1129) is gratefully acknowledged.


    FOOTNOTES

Address for reprint requests: F. Tempia, Dept. of Neuroscience, University of Turin, C.so Raffaello 30, I-10125 Turin, Italy (E-mail: tempia{at}medfarm.unito.it).

Received 1 March 2000; accepted in final form 21 September 2000.


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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society