1. Whole-cell patch-clamp recordings of excitatory postsynaptic currents (EPSCs) were made from guinea pig hippocampal CA1 pyramidal cells. The sensitivity of paired pulse facilitation (PPF) and EPSC variance to changes in synaptic transmission was investigated and the results were compared with the changes in these parameters evoked by long-term potentiation (LTP). 2. Presynaptic manipulations, such as activation of presynaptic gamma-aminobutyric acid-B receptors by baclofen, blockade of presynaptic adenosine receptors by theophylline, blockade of presynaptic potassium channels by cesium, and increasing the Ca(2+)-Mg2+ ratio in the external recording solution, each reliably changed PPF in a fashion reciprocal to the change in the EPSC amplitude. However, recruitment of additional synaptic release sites by increasing stimulus strength and antagonism of non-N-methyl-D-aspartate (NMDA) glutamate receptors by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) failed to alter PPF. 3. Presynaptic manipulations including increased stimulus strength gave the predicted changes in the value of mean 2/variance (M2/sigma 2). Moreover, postsynaptic manipulations that altered EPSC amplitude, including blockade of non-NMDA receptors by CNQX, or changing the holding potential of the postsynaptic cell, gave little change in M2/sigma 2, as would be predicted for manipulations resulting in a uniform postsynaptic change. 4. LTP caused no change in PPF, whereas the presynaptic manipulations, which caused a similar amount of potentiation to that induced by LTP, significantly decreased PPF. On the other hand, LTP did increase M2/sigma 2, although the increase was less than that predicted for a purely presynaptic mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

The NMDA type of ligand-gated glutamate receptor requires the presence of both glutamate and glycine for gating. These receptors are hetero-oligomers of NR1 and NR2 subunits. Previously it was thought that the binding sites for glycine and glutamate were formed by residues on the NR1 subunit. Indeed, it has been shown that the effects of glycine are controlled by residues on the NR1 subunit, and a "Venus flytrap" model for the glycine binding site has been suggested by analogy with bacterial periplasmic amino acid binding proteins. By analysis of 10 mutant NMDA receptors, we now show that residues on the NR2A subunit control glutamate potency in recombinant NR1/NR2A receptors, without affecting glycine potency. Furthermore, we provide evidence that, at least for some mutated residues, the reduced potency of glutamate cannot be explained by alteration of gating but has to be caused primarily by impairing the binding of the agonist to the resting state of the receptor. One NR2A mutant, NR2A(T671A), had an EC50 for glutamate 1000-fold greater than wild type and a 255-fold reduced affinity for APV, yet it had single-channel openings very similar to those of wild type. Therefore we propose that the glutamate binding site is located on NR2 subunits and (taking our data together with previous work) is not on the NR1 subunit. Our data further imply that each NMDA receptor subunit possesses a binding site for an agonist (glutamate or glycine).

SUMMARY1. Patch-clamp methods have been used to examine the action of excitatory amino acids on three types of glial cell in cultures of rat cerebellum, namely type-ilike astrocytes, type-2 astrocytes and oligodendrocytes. In addition we have examined glutamate sensitivity of the precursor cell (the O-2A progenitor) that gives rise to type-2 astrocytes and oligodendrocytes.2. Glutamate (30 ftM), quisqualate (3-100 ,tM), (S)-a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA, 10-30 /SM) and kainate (10-500 ,uM) were applied to cerebellar type-2 astrocytes examined under whole-cell voltage clamp. Each of these agonists induced inward currents in cells held at negative membrane potentials. The currents reversed direction near 0 mV holding potential. N-Methyl-D-aspartate (NMDA, 30-100 /,M) or aspartate (30 ,tM) in the presence of glycine (1 /1M) did not evoke any whole-cell current changes in type-2 astrocytes.3. The distribution of glutamate receptors in type-2 astrocytes was mapped with single-or double-barrelled ionophoretic pipettes containing quisqualate or kainate. Application of these agonists (current pulses 100 ms, 50-100 nA) to cells held at -60 mV evoked inward currents of 20-120 pA in the cell soma and 10-80 pA in the processes. Responses could also be obtained at the extremities of processes (-% 60 ,um from the soma). D. J. A. WYLLIE AND OTHERS type-1-like astrocytes were inhibited when external Na+ was replaced by Li', although Li+ was found to pass through the glutamate channel in type-2 astrocytes.6. Oligodendrocytes in cerebellar cultures did not respond to glutamate, quisqualate or kainate indicating a lack both of a detectable electrogenic glutamate uptake mechanism and of glutamate receptor ion channels in these cells.7. We conclude that, of the various macroglial cells, only the type-2 astrocyte and its progenitor cell possess 'fast' glutamate receptor channels in short-term ( < 4 day) cultures. Detectable uptake currents were confined to type-i-like astrocytes. However, in older cultures (> 7 day) type-I-like astrocytes also developed glutamate receptor channels, in addition to their uptake currents. The possible involvement of glial glutamate receptors and glutamate uptake in neuronal-glial interaction is considered.

Fragile X Syndrome (FXS) is one of the most common monogenic forms of autism and intellectual disability. Preclinical studies in animal models have highlighted the potential of pharmaceutical intervention strategies for alleviating the symptoms of FXS. However, whether treatment strategies can be tailored to developmental time windows that define the emergence of particular phenotypes is unknown. Similarly, whether a brief, early intervention can have long-lasting beneficial effects, even after treatment cessation, is also unknown. To address these questions, we first examined the developmental profile for the acquisition of associative learning in a rat model of FXS. Associative memory was tested using a range of behavioral paradigms that rely on an animal’s innate tendency to explore novelty. Fmr1 knockout (KO) rats showed a developmental delay in their acquisition of object-place recognition and did not demonstrate object-place-context recognition paradigm at any age tested (up to 23 weeks of age). Treatment of Fmr1 KO rats with lovastatin between 5 and 9 weeks of age, during the normal developmental period that this associative memory capability is established, prevents the emergence of deficits but has no effect in wild-type animals. Moreover, we observe no regression of cognitive performance in the FXS rats over several months after treatment. This restoration of the normal developmental trajectory of cognitive function is associated with the sustained rescue of both synaptic plasticity and altered protein synthesis. The findings provide proof of concept that the impaired emergence of the cognitive repertoire in neurodevelopmental disorders may be prevented by brief, early pharmacological intervention.