At nicotinic and glutamatergic synapses, the duration of the postsynaptic response depends on the affinity of the receptor for transmitter (Colquhoun et al., 1977;Pan et al., 1993). Affinity is often thought to be determined by the ligand unbinding rate, whereas the binding rate is assumed to be diffusion-limited. In this view, the receptor selects for those ligands that form a stable complex on binding, but binding is uniformly fast and does not itself affect selectivity. We tested these assumptions for the GABAA receptor by dissecting the contributions of microscopic binding and unbinding kinetics for agonists of equal efficacy but of widely differing affinities. Agonist pulses applied to outside-out patches of cultured rat hippocampal neurons revealed that agonist unbinding rates could not account for affinity if diffusion-limited binding was assumed. However, direct measurement of the instantaneous competition between agonists and a competitive antagonist revealed that binding rates were orders of magnitude slower than expected for free diffusion, being more steeply correlated with affinity than were the unbinding rates. The deviation from diffusion-limited binding indicates that a ligand-specific energy barrier between the unbound and bound states determines GABAA receptor selectivity. This barrier and our kinetic observations can be quantitatively modeled by requiring the participation of movable elements within a flexible GABA binding site.

AMPA receptors and glutamate transporters expressed by cerebellar Bergmann glial cells are activated by neurotransmitter released from climbing fibers (). Based on anatomical evidence, this is most likely the result of glutamate diffusing out of the climbing fiber-Purkinje cell synaptic clefts (). We used the change in the EC50 of the Bergmann glia AMPA receptors produced by cyclothiazide (CTZ) to estimate the concentration of glutamate reached at the glial membrane. The decrease of the EC50 gives rise to a concentration-dependent potentiation of the AMPA receptor-mediated responses (). By comparing the increase in amplitude of the AMPA receptor response in the Bergmann glia (840 +/- 240%; n = 8) with the shift in the glutamate dose-response curve measured in excised patches (EC50, 1810 microM in control vs 304 microM in CTZ), we estimate that the extrasynaptic transmitter concentration reaches 160-190 microM. This contrasts with the concentration in the synaptic cleft, thought to rapidly rise above 1 mM, but is still high enough to activate glutamate receptors. These results indicate that the sphere of influence of synaptically released glutamate can extend beyond the synaptic cleft.

Glutamate transporters in the central nervous system are expressed in both neurons and glia, they mediate high affinity, electrogenic uptake of glutamate, and they are associated with an anion conductance that is stoichiometrically uncoupled from glutamate f lux. Although a complete cycle of transport may require 50-100 ms, previous studies suggest that transporters can alter synaptic currents on a much faster time scale. We find that application of L-glutamate to outside-out patches from cerebellar Bergmann glia activates anion-potentiated glutamate transporter currents that activate in <1 ms, suggesting an efficient mechanism for the capture of extrasynaptic glutamate. Stimulation in the granule cell layer in cerebellar slices elicits all or none ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor and glutamate transporter currents in Bergmann glia that have a rapid onset, suggesting that glutamate released from climbing fiber terminals escapes synaptic clefts and reaches glial membranes shortly after release. Comparison of the concentration dependence of both ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor and glutamate transporter kinetics in patches with the time course of climbing fiber-evoked responses indicates that the glutamate transient at Bergmann glial membranes reaches a lower concentration than attained in the synaptic cleft and remains elevated in the extrasynaptic space for many milliseconds.Termination of the actions of synaptically released glutamate requires uptake by high affinity glutamate transporters. These transporters are expressed by both neurons and glia and maintain low extracellular glutamate levels by coupling translocation to the electrochemical gradients for Na ϩ , K ϩ , and H ϩ (1). The importance of these transporters in restricting glutamate neurotoxicity is evidenced by the physiological, behavioral, and anatomical abnormalities that result when their expression is reduced (2) or eliminated (3). On a faster time scale, glutamate transporters appear to be important in limiting the duration of synaptic excitation at some synapses (3, 4-7) by rapidly lowering the concentration of glutamate in the synaptic cleft following exocytosis; however, transporter antagonists do not prolong excitatory postsynaptic currents at all synapses (4,8,9) suggesting that other factors that vary between synapses such as receptor kinetics, location and density of transporters, and diffusional barriers may also be important in shaping the glutamate transient in the cleft. Glutamate transporters located near release sites have also been shown to slow the activation of postsynaptic ionotropic receptors (10, 11) suggesting that glutamate may bind to transporters within a millisecond after release. Such rapid binding kinetics have recently been demonstrated for glutamate transporters expressed in Purkinje cells (12). However, the lack of subtype-selective antagonists has hampered assessment of the relative contribution of neuronal and glial transporters to the uptake of glutamate o...

In the cerebellum, metabotropic glutamate receptors (mGluRs) are required for distinct forms of synaptic plasticity expressed at parallel fiber (PF) and climbing fiber (CF) synapses. At PF synapses, mGluR activation generates a slow synaptic current and triggers intracellular calcium release; at CF synapses, mGluR activation has not been observed. This has led some investigators to propose that mGluR-dependent changes in CF synaptic strength are induced heterosynaptically. Here we describe an mGluR-mediated response to CF stimulation consisting of two parallel signaling pathways: one leading to a slow synaptic conductance and the other leading to internal calcium release. This additional target for glutamate broadens the signaling capabilities of CF synapses and raises the possibility that changes in CF strength are homosynaptically triggered.