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.

SUMMARY1. Application of non-NMDA (non-N-methyl-D-aspartate) receptor agonists onto outside-out patches of cerebellar granule cells gave two characteristic types of response (in different patches) which we have referred to as 'high conductance' and 'low conductance' responses. At a qualitative level these patches could be readily distinguished by the size of the noise increase accompanying their membrane currents.2. In high conductance patches both AMPA (oc-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate gave discrete single-channel conductances (10-30 pS), while in low conductance patches, AMPA produced small discrete events (6-10 pS), and kainate opened channels with conductances too small to be directly resolved. All patches examined contained NMDA receptor channels with characteristic 50 and 40 pS conductance levels.3. Despite the marked differences in single-channel conductances, kainate dose-response curves constructed for high and low conductance patches had similar EC50 values of approximately 150 /bM.4. Spectral analysis of low conductance kainate responses gave an estimated channel conductance of -1X5 pS. In these same low conductance patches AMPA produced discrete openings with two conductance levels; their mean conductances (and relative proportions) were 6 (87 %) and 10 pS (13 %). $ To whom all correspondence should be addressed.MS 1361 D. J. A. WYLLIE AND OTHERS the relative proportion of levels wa's constant between patches, and all three levels were invariably present. These observations are all consistent with the idea that the three multiple conductances originate from a single receptor channel, activated by AMPA, kainate and glutamate. 6. Non-NMDA single-channel current-voltage (I-V) plots showed outward rectification in high conductance patches. For all three multiple conductance levels the ratio of outward to inward single-channel slope conductance was 1-8+0-1 and this rectification remained present in symmetrical Na+ solutions.7. In high conductance patches, the events produced by a rapid application of 20-50 ,CM glutamate were compared with those activated during steady-state application. There was no significant difference either in the amplitudes of the various multiple conductance levels opened, or in the relative proportions of these levels, under the two conditions. 8. Kinetic analysis of single channels in high conductance patches indicated that the mean apparent open times were brief (-0-5 ms for all agonists). In low conductance patches, AMPA opened channels with a mean apparent open time of 11 ims.9. Distributions of channel shut times could be described by the sum of three or four exponential components; the mean time constants (and areas) were not significantly different for glutamate, AMPA or kainate. Pooling data for these agonists gave mean values for the time constants (and areas) of T, = 147 jts, (22%), T2= 539 Iss (3%), TS = 27-3 ms (35%) and T4 = 127 ms (40%).10. Burst length distributions of non-NMDA channels in high conductance patches, contained a main...

1. Patch-clamp recording methods have been used to compare the pharmacological properties and single-channel characteristics of non-NMDA receptor channels in cerebellar type-2 astrocytes and granule cells. 2. In type-2 astrocytes whole-cell concentration-response curves for glutamate, quisqualate, AMPA and kainate gave EC50 values of 5-8, 3-8, 7-6 and 160 uM and Hill slopes of P-65, 1P18, 164 and 1-65, respectively, resembling estimates for granule cell receptors. 3. The non-NMDA receptor antagonists CNQX and diCl-HQC (see Methods) inhibited wholecell kainate currents in both cell types. The IC50 for CNQX antagonism of the kainate response was 536 nM in type-2 astrocytes, and 500 nm in granule cells. The IC50 for diCl-HQC was 3'5SM in astrocytes and 37 /uM in granule cells. 4. CNQX acted as a competitive antagonist of whole-cell kainate responses in type-2 astrocytes and granule cells giving Schild plots with a slope near 1. The equilibrium constant, K, for CNQX binding was 524 nm in astrocytes and 489 nm in granule cells. 5. Quisqualate and AMPA responses showed rapid desensitization in type-2 astrocytes with a ratio of steady-state to peak response of 0'09. Concanavalin A reduced this desensitization. 6. Non-NMDA channels in type-2 astrocytes and granule cells showed a low permeability to Ca2+ ions with a reversal potential, for kainate-activated whole-cell currents in isotonic Ca2+, of approximately -25 mV for astrocytes and -45 mV for granule cells. 7. Outside-out patches from type-2 astrocytes exhibited a range of single-channel conductances that were superficially similar to the glutamate-activated conductances in granule cells. However, the type-2 astrocytes were devoid of NMDA receptors, hence all of these conductances originated from non-NMDA channels. Their slope conductances were approximately 11, 21, 32, 42 and 52 pS. Amplitudes were verified with mean lowvariance plots and single-channel current-voltage curves, which were linear. 8. There was also evidence of lower conductance kainate-activated channels in astrocyte patches. From noise analysis their estimated mean conductance was 1P9 pS, as described for the 'low-conductance' type kainate responses in cerebellar neurones. 9. Apparent open times, shut times and burst lengths of AMPA-activated (3-10 /M) channels were examined in patches from type-2 astrocytes, and kinetic properties of the 40 and 50 pS levels were compared with the lower levels. 10. Our results indicate some marked pharmacological similarities between non-NMDA receptor channels in type-2 astrocytes and granule cells. Furthermore, in astrocyte patches the 10, 20 and 30 pS single-channel conductance levels, as well as the 1 9 pS kainate conductance, were similar in amplitude to non-NMDA channels in granule cells. Clear differences were, however, apparent in higher conductance levels. In particular, 40 and 50 pS openings arose from non-NMDA channels in astrocytes while in granule cells conductances of this range arise exclusively from NMDA channels. These higher conductance levels can t...

Although the underlying neurobiology of major mental illness (MMI) remains unknown, emerging evidence implicates a role for oligodendrocyte-myelin abnormalities. Here, we took advantage of a large family carrying a balanced t(1;11) translocation, which substantially increases risk of MMI, to undertake both diffusion tensor imaging and cellular studies to evaluate the consequences of the t(1;11) translocation on white matter structural integrity and oligodendrocyte-myelin biology. This translocation disrupts among others the DISC1 gene which plays a crucial role in brain development. We show that translocation-carrying patients display significant disruption of white matter integrity compared with familial controls. At a cellular level, we observe dysregulation of key pathways controlling oligodendrocyte development and morphogenesis in induced pluripotent stem cell (iPSC) derived case oligodendrocytes. This is associated with reduced proliferation and a stunted morphology in vitro. Further, myelin internodes in a humanized mouse model that recapitulates the human translocation as well as after transplantation of t(1;11) oligodendrocyte progenitors were significantly reduced when compared with controls. Thus we provide evidence that the t(1;11) translocation has biological effects at both the systems and cellular level that together suggest oligodendrocyte-myelin dysfunction.

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