Low frequency stimulation (LFS, 1 Hz for 15 min) has been shown to produce an NMDA receptor dependent homosynaptic long-term depression (LTD) of synaptic potentials in the CA1 area of the rat hippocampus. Here we describe experiments aimed at characterizing EPSP/spike (E/S) coupling associated with this form of LTD. Our data show that following LFS neurons have a higher probability of synchronous discharge in response to a population EPSP of fixed slope (E/S potentiation). This E/S potentiation was not significantly enhanced by a tetanic stimulation. When the protocol was reversed, that is, starting with a tetanic stimulus, E/S potentiation was observed which was unaffected by a subsequent LFS. Saturating these synaptic responses to either a maximal or a minimal value produced similar effects on E/S coupling. E/S depression was never encountered. Finally, we found that the expression of E/S potentiation did not require the activation of NMDA receptors. These data suggest that at the level of a local neuronal network in the CA1 area, LFS is not a very powerful tool since the synaptic depression is associated with a potentiation of the population response of these neurons. Furthermore, the expression of E/S dissociation seems different from that of homosynaptic long-term potentiation and LTD.

1. Stimulation of the surviving afferents in the stratum radiatum of the CAl area in kainic acid-lesioned hippocampal slices produced graded epileptiform activity, part of which (> 20 %) involved the activation of N-methyl-D-aspartate (NMDA) receptors. There was also a failure of synaptic inhibition in this region. In this preparation, we have tested the effects of low-frequency stimulation (LFS; 1 Hz for 15 min) on synaptic responses and epileptiform activity.2. LFS resulted in long-term depression (LTD) of excitatory synaptic potentials (EPSPs), longterm decrease of population spike amplitudes (PSAs) and EPSP-spike (E-S) potentiation. Evoked epileptiform activity was reduced but neurons had a higher probability of discharge. LTD could be reversed by subsequent tetanic stimulation whereas E-S dissociation remained unchanged. Synaptic and network responses could be saturated towards either potentiation or depression. However, E-S potentiation was maximal following the first conditioning stimulus. 3. NMDA receptor-mediated responses were pharmacologically isolated. LFS resulted in LTD of synaptic responses, long-term decrease of PSAs and E-S depression. These depressions could not be reversed by subsequent tetanic stimulation. a-Amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) and NMDA receptor-mediated responses were then measured in isolation before and following conditioning stimuli. LFS was shown to simultaneously produce LTD of AMPA and NMDA receptor-mediated responses. E-S potentiation of the AMPA component and E-S depression of the NMDA component occurred coincidentally. 4. LTD of AMPA and NMDA receptor-mediated responses were shown to be NMDA dependent. In contrast, E-S potentiation and depression occurred even when NMDA receptors were pharmacologically blocked. 5. These findings indicate that synaptic responses could be modified bidirectionally in the CAI area of kainic acid-lesioned rat hippocampus in an NMDA receptor-dependent manner.However, E-S dissociations were independent of the activation of NMDA receptors, hinting at mechanisms different from those of synaptic LTD. We suggest that changes in E-S coupling were caused by a modification of the firing threshold of the CAl pyramidal neurons.Furthermore, the firing mechanisms controlling NMDA and AMPA receptor-mediated network activity appeared to be different. The possible use of LFS applied to the hippocampus as a clinical intervention to suppress epileptiform activity is discussed.The understanding of the plasticity of synaptic and network common mechanisms through the activation of N-methylresponses in the hippocampus may provide some insight D-aspartate (NMDA) receptors. As a consequence, the into mechanisms that can be used to control epileptiform manipulation of one phenomenon may also strongly affect activity and the generation of epileptiform discharges. the other. On the one hand, if long-term potentiation (LTP) However, epileptiform activity and synaptic plasticity share of synaptic responses is induced, it is likely that the

1. Graded N-methyl-D-aspartate receptor (NMDAR)-dependent epileptiform discharges were recorded from ex vivo hippocampal slices obtained from rats injected a week earlier with an intracerebroventricular dose of kainic acid. Intracellular recordings from pyramidal cells of the CA1 area showed that glutamate NMDAR actively participated in synaptic transmission, even at resting membrane potential. When NMDAR were pharmacologically isolated, graded burst discharges could still be evoked. 2. The oxidizing reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, 200 microM, 15 min) suppressed the late part of the epileptiform burst that did not recover after wash but could be reinstated by the reducing agent tris (2-carboxyethyl) phosphine (TCEP, 200 microM, 15 min) and again abolished with the NMDA antagonist D-2-amino-5-phosphonovaleric acid (D-APV). 3. Pharmacologically isolated NMDAR-mediated responses were decreased by DTNB (56 +/- 10%, mean +/- SD, n = 6), an effect reversed by TCEP. 4. When only the fast glutamateric synaptic component was blocked, NMDA-dependent excitatory postsynaptic potentials (EPSPs) could be evoked despite the presence of underlying fast and slow inhibitory postsynaptic potentials (IPSPs). DTNB decreased EPSPs to 48 +/- 12% (n = 5) of control. 5. Since a decrease of the NMDAR-mediated response by +/- 50% is sufficient to suppress the late part of the burst, we suggest that epileptiform activity can be controlled by manipulation of the redox sites of NMDAR. Our observations raise the possibility of developing new anticonvulsant drugs that would spare alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-R (AMPAR)-mediated synaptic responses and decrease NMDAR-mediated synaptic transmission without blocking it completely.

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In this communication, we describe the design of an online multi-chromatographic approach to the routine NMR analyses of low-level components ( approximately 0.1%) in complex mixtures. The technique, termed LC(2)-SPE-NMR, optimally combines multi-dimensional liquid chromatography with SPE technology for isolating, enriching and delivering trace analytes to the NMR probe. The fully automated LC(2)-SPE-NMR system allows for maximal loading capacity (in the first, preparative LC dimension), close to optimal peak resolution (in the second, analytical LC dimension) and enhanced sample concentration (through SPE). Using this system, it is feasible to conveniently conduct a wide range of NMR experiments on, for example, drug impurities at the low microgram per milliliter level, even for components poorly resolved in the first dimension. Such a sensitivity gain significantly elevates the analytical power of online NMR technology in terms of the level at which substances of pharmaceutical significance can be structurally characterized.