PFs in the molecular layer of the cerebellar cortex are the principal excitatory input to PNs, driving PNs at frequencies up to 50 Hz. Single PF stimuli evoke fast postsynaptic potentials, acting through glutamate AMPA-type receptors. However, brief tetanic stimulation of PFs produces a slow EPSP seen in the presence of AMPA receptor antagonists (Batchelor & Garthwaite, 1993). The sEPSP is blocked by metabotropic glutamate receptor antagonist MCPG (Batchelor et al. 1994, 1997; Hirono et al. 1998). Application of selective mGluR type 1 agonists also produces an excitation of PNs, resulting in sustained activation of fast spikes, or, in the presence of TTx, slow spiking of dendritic origin (Tempia et al. 1998; Watkins & Ogden, 1999). Approximately 175 000 excitatory synapses are made on postsynaptic spines in the dendritic tree of each PN (Napper & Harvey, 1988). Immunogold labelling has shown mGluR1α receptors located postsynaptically on PN spines at the periphery of the PF synapse where they will be exposed to l-glutamate released from PF terminals (Baude et al. 1993).
A role of mGluR1 receptors in motor coordination is suggested by the observation that mGluR1α-deficient mice are ataxic, with lesions in the cerebellar molecular layer and developmental abnormalities in the innervation of PNs (Aiba et al. 1994; Conquet et al. 1994; Ichise et al. 2000). Also, neoplastic cerebellar ataxia, in which there is a deficit in motor coordination, has been shown to be associated with autoantibodies generated against mGluR1 (Sillevis-Smitt et al. 2000). Thus mGluR1 and the PF/PN sEPSP may have a role in motor coordination. However, the ionic mechanism of the sEPSP is not established and has been attributed to activation of Na+-Ca2+ exchange or a Ca2+-activated channel secondary to Ca2+ release from stores (Vranesic et al. 1991), although contrary evidence has also been reported (Hirono et al. 1998).
Recently we have developed a stable, fast, pharmacologically inert NI-caged glutamate, based on nitroindoline photochemistry (Papageorgiou et al. 1999). This permits the study of kinetics, mechanism and pharmacology of the postsynaptic events during the sEPSP independently of presynaptic processes. The experiments described here were made to determine the kinetics of postsynaptic events with AMPA receptors blocked and to investigate the ion conductance underlying the sEPSP. They provide evidence of a cation channel not directly linked to intracellular Ca2+. Preliminary accounts of some of this work have appeared in abstract form (Watkins & Ogden, 1999; Canepari et al. 2000).
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Wistar rats, 19-22 or 12 days old, were killed by cervical dislocation, decapitated, and the cerebellum placed in ice-cold saline. Parasagittal slices, 200 μm thick, were cut in Hepes-buffered, 0.5 mm Ca2+ saline gassed with O2. External saline contained (mm): NaCl 135, KCl 4, MgSO4 or MgCl2 2, CaCl2 2, glucose 25, NaHCO3 2, Hepes-Na 10, pH 7.3, 305 mosmol kg−1. Experiments were carried out at 32 °C and a continuous stream of hydrated O2 was blown over the solution surface. NI-caged glutamate and antagonists were applied in 1 ml of solution (non-flowing) for 10 min prior to photolysis. Selective mGluR agonists were applied locally by pressure ejection from a patch pipette.
Slices were viewed with a Zeiss Axioskop 1FS, × 40 0.75w Achroplan objective and, to avoid photolysis, 500/40 nm bandpass illumination via a Reichert silica condenser 0.9 NA. A xenon arc flashlamp (Rapp OptoElektronik; Rapp & Güth, 1988) filtered with a UG11 (Schott, bandpass 290-370 nm) was focused into the slice from below, illuminating a spot of 200 μm diameter. The arc image was aligned and focused in the specimen plane with the condenser, optimised visually and by maximising the output of a photodiode. Photolysis calibration was from the fluorescence increase (470 nm excitation, > 530 nm emission) produced by photolysis of the 1-(2-nitrophenyl)ethyl ether of pyranine (NPE-HPTS; Jasuja et al. 1999) contained at 50 μm in 100 mm borate pH 9, in 10-20 μm diameter aqueous vesicles suspended in Sylgard. Conversion of NPE-HPTS is estimated in cuvette experiments as 0.7 times that of NI-caged glutamate. Transmission at 320 nm through 200 μm slices from 20-day-old rats was measured as 0.45 in the molecular layer, 0.4 in the granule cell layer. Flash lamp intensity was set to maximum, converting 7 % of NI-glutamate after correction for attenuation in the slice, and lower intensities were produced by neutral density filters in the condenser light path.
Whole cell patch clamp recordings were made with an Axoclamp-2A and 2.5 MΩ pipettes (Pyrex, 1.5 mm × 1.1 mm) were filled with internal solution (mm): potassium gluconate 110, Hepes 50, KCl 10, MgSO4 4, Na2ATP 4, creatine phosphate 10, GTP 0.05, pH 7.3 with KOH. The junction potential between this solution and external solution was measured as 12 mV, pipette negative.
Data were collected with Spike 2 software via a 1401+ interface (CED, Cambridge, UK; sampled at 10 kHz, lowpass filter 2 kHz, −3 dB). Data are given as means ±s.d. unless specified as s.e.m. Chemicals were Analar grade (BDH, Poole) and biochemicals and drugs obtained from Sigma (Poole), Tocris (Bristol) or RBI (Poole). Experiments with the Ca2+ channel blocker AGA4A (Peptide Institute, Osaka, Japan) were carried out in the presence of 0.1 mg ml−1 cytochrome c.
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The excitatory slow EPSP produced by brief tetanic stimulation of PFs was described by Batchelor & Garthwaite (1993) and shown to be mediated by mGluR1 receptors (Batchelor et al. 1994; Hirono et al. 1998). Here a response mediated by mGluR1 was elicited by photorelease of l-glutamate in the presence of NBQX. It was blocked by the antagonists MCPG and CPCCOEt. Furthermore, selective mGluR group 1 agonists also produced an excitation similar to the sEPSP. This excitation is shown to be due to the mGluR1-mediated opening of Na+- and K+-permeable cation channels through a pathway not requiring the activation of PLC. In view of the evidence of a role of mGluR1 in cerebellar function it is likely that the mGluR1-mediated conductance investigated here with photolytic l-glutamate release has a role in cerebellar motor coordination.
The rapid photolytic release of glutamate from NI-caged glutamate permits the kinetic distinction of fast and slowly developing postsynaptic currents. With AMPA receptors blocked two components were distinguished, a transient current peaking in 7-8 ms with pharmacological and electrophysiological properties characteristic of an electrogenic glutamate transporter, followed by a slow rising current, peaking in 0.7 s with the pharmacology of type 1 mGluR. Photolytically released glutamate persists in the slice with a half-time of 200 ms, implying that the mGluR1-activated current rises after the glutamate concentration has fallen substantially. This is consistent with the observation that the sEPSP rises with a delay (Batchelor & Garthwaite, 1993; present study, data not shown) at a time when the synaptic glutamate has declined following PF stimulation (Barbour et al. 1994). This delay indicates the involvement of intracellular messengers or slow, membrane-associated coupling steps.
The initial transient component was not affected by the AMPA receptor antagonist NBQX applied at concentrations up to 1 mm. The competitive glutamate transport inhibitor THA, at 100-300 μm, modified the time course as might be expected if the number of transporters initially available is reduced by bound THA and the glutamate persisting after photorelease competes with THA for transport. The inward rectification and failure to reverse the initial current at holding potentials as positive as 50 mV are consistent with concerted coupled transport of extracellular glutamate and Na+ into the cell immediately on glutamate release. A fast-rising, transient transporter current has been seen with climbing fibre stimulation in the presence of NBQX (Auger & Attwell, 2000) and with PF stimulation. Integrating the current in response to 70 μm l-glutamate with NBQX, bicuculline and MCPG present gave an estimate for the charge carried of 1.6 pC, corresponding to 16 × 10−18 mol univalent ions. If this is due to one cycle of transport (2 charges are translocated in each cycle; Takahashi et al. 1996; Auger & Attwell, 2000) it corresponds to 5 × 106 sites. This number is likely to be an underestimate of the number of sites because of the lower concentration of glutamate released here than synaptically (70 μmvs. 1 mm) and because of cable losses. However, the magnitude is not large when compared to the number of parallel fibre synapses, approximately 175 000, estimated anatomically to be present on a rat PN (Napper & Harvey, 1988).
The current underlying the sEPSP has previously been attributed to a Ca2+-activated non-selective cation conductance or to Na+-Ca2+ exchange, based on the effects of Na+ replacement with Li+ and block by high internal BAPTA concentrations (Vranesic et al. 1991). Recently mGluR activation has been shown to result in a dendritic increase of [Na+] (Knopfel et al. 2000) and in another study, inhibitors of Na+-Ca2+ exchange have been shown to be ineffective against the sEPSP (Hirono et al. 1998). Moreover, the effect of increasing intracellular Ca2+ photolytically, either with caged InsP3 or Ca2+-DM-nitrophen, is to activate an outward, inhibitory current (Khodakhah & Ogden, 1993, 1995; Watkins & Ogden, 1999) and not the excitatory slow current seen with mGluR receptor activation. This indicates that the sEPSP is not secondary to a rise of intracellular Ca2+ concentration, but is activated via another pathway. The results presented here show a conductance increase with reversal potential near 0 mV, indicating permeability to Na+, K+ and possibly Ca2+ but not Cl− (ECl=−85 mV). There was an increase of membrane current noise attributable to the stochastic opening of channels, with maximum variance at the peak of the mGluR1-activated inward current. Preliminary experiments to investigate the nature of the channels activated show no block by Cd2+, Co2+, Mg2+, Mn2+, Gd3+ or Ba2+, the inhibitor of the cyclic-nucleotide-gated conductance IH, ZD7288, or by the purinoceptor antagonist PPADS. The rate of activation of the conductance increased at positive potential, about 8-fold in 120 mV, indicating that membrane-delimited interactions are involved.
Thus, we have shown that metabotropic type 1 glutamate receptors slowly activate a cation conductance in PNs that underlies the slow EPSP at PF-PN synapses. The coupling between mGluR1 at parallel fibre synapses and the excitatory conductance underlying the sEPSP shows characteristics of indirect activation, rising after photolytic or synaptically generated glutamate concentrations have declined, and evidence presented here and in other studies (Hirono et al. 1998) indicates that the coupling is not via the phosphoinositide pathway. A non-selective cation conductance activated by mGluR1 independently of phosphoinositide and G-protein coupling has been demonstrated in hippocampal CA3 neurones (Heuss et al. 1999). In this case the GABAB receptors served as an internal control for G-protein inactivation, and activation of the conductance was via protein tyrosine kinases.
The most striking consequence of mGluR activation in these experiments was a large dendritic excitation, resulting in regenerative Ca2+ spikes within the dendritic tree. Further experiments need to be done to identify the ion channel, its potential role, directly or indirectly, in Ca2+ influx, and the coupling mechanism to mGluR1 receptors.