Fragile X syndrome (FXS) is caused by a failure of neuronal cells to express the gene encoding the fragile mental retardation protein (FMRP). Clinical features of the syndrome include intellectual disability, learning impairment, hyperactivity, seizures and anxiety. Fmr1 knockout (KO) mice do not express FMRP and, as a result, reproduce some FXS behavioral abnormalities. While intrinsic and synaptic properties of excitatory cells in various part of the brain have been studied in Fmr1 KO mice, a thorough analysis of action potential characteristics and input-output function of CA1 pyramidal cells in this model is lacking. With a view to determining the effects of the absence of FMRP on cell excitability, we studied rheobase, action potential duration, firing frequency–current intensity relationship and action potential after-hyperpolarization (AHP) in CA1 pyramidal cells of the hippocampus of wild type (WT) and Fmr1 KO male mice. Brain slices were prepared from 8- to 12-week-old mice and the electrophysiological properties of cells recorded. Cells from both groups had similar resting membrane potentials. In the absence of FMRP expression, cells had a significantly higher input resistance, while voltage threshold and depolarization voltage were similar in WT and Fmr1 KO cell groups. No changes were observed in rheobase. The action potential duration was longer in the Fmr1 KO cell group, and the action potential firing frequency evoked by current steps of the same intensity was higher. Moreover, the gain (slope) of the relationship between firing frequency and injected current was 1.25-fold higher in the Fmr1 KO cell group. Finally, AHP amplitude was significantly reduced in the Fmr1 KO cell group. According to these data, FMRP absence increases excitability in hippocampal CA1 pyramidal cells.

The NMDA receptors (NMDARs) expressed by AII and A17 amacrine cells, the two main inhibitory interneurons of the rod pathway microcircuit in the mammalian retina, are exclusively extrasynaptic, activated by ambient levels of glutamate, and molecularly distinct, with AII and A17 amacrines expressing GluN2B-and GluN2A-containing receptors, respectively. This important sensory microcircuit thus provides a unique model to study the activation and function of extrasynaptic NMDARs. Here, we investigated the sources of glutamate and the endogenous coagonists (d-serine or glycine) that activate these distinct populations of NMDARs.With acute slices from rat retina, we used whole-cell voltage-clamp recording and measurement of current noise to monitor levels of NMDAR activity. Pre-incubation of retina with bafilomycin A1 (an inhibitor of neurotransmitter uptake into synaptic vesicles) abolished NMDAR-mediated noise in AII, but not A17 amacrines, suggesting a vesicular source of glutamate activates AII NMDARs, whereas a non-vesicular source activates A17 NMDARs. Pre-incubation of retina with l-methionine sulfoximine (an inhibitor of glutamine synthetase) also abolished NMDAR-mediated noise in AII, but not A17 amacrines, suggesting a neuronal source of glutamate activates AII NMDARs, whereas a glial source activates A17 NMDARs. Enzymatic breakdown of d-serine reduced NMDAR-mediated noise in AII, but not A17 amacrines, suggesting d-serine is the endogenous co-agonist at AII, but not A17 NMDARs.Our results reveal unique characteristics of these two populations of extrasynaptic NMDARs. The differential and independent activation of these receptors is likely to provide specific contributions to the signal processing and plasticity of the cellular components of the rod pathway microcircuit.

Amacrine cells are a highly diverse group of inhibitory retinal interneurons that sculpt the responses of bipolar cells, ganglion cells, and other amacrine cells. They integrate excitatory inputs from bipolar cells and inhibitory inputs from other amacrine cells, but for most amacrine cells, little is known about the specificity and functional properties of their inhibitory inputs. Here, we have investigated GABAA receptors of the AII amacrine, a critical neuron in the rod pathway microcircuit, using patch-clamp recording in rat retinal slices. Puffer application of GABA evoked robust responses, but, surprisingly, spontaneous GABAA receptor-mediated postsynaptic currents were not observed, neither under control conditions nor following application of high-K+ solution to facilitate release. To investigate the biophysical and pharmacological properties of GABAA receptors in AIIs, we therefore used nucleated patches and a fast application system. Both brief and long pulses of GABA (3 mM) evoked GABAA receptor-mediated currents with slow, multi-exponential decay kinetics. The average weighted time constant (τw) of deactivation was ~163 ms. Desensitization was even slower, with τw ~330 ms. Non-stationary noise analysis of patch responses and directly observed channel gating yielded a single-channel conductance of ~23 pS. Pharmacological investigation suggested the presence of α2 and/or α3 subunits, as well as the γ2 subunit. Such subunit combinations are typical of GABAA receptors with slow kinetics. If synaptic GABAA receptors of AII amacrines have similar functional properties, the slow deactivation and desensitization kinetics will facilitate temporal summation of GABAergic inputs, allowing effective summation and synaptic integration to occur even for relatively low frequencies of inhibitory inputs.

Amacrine cells constitute a large and heterogeneous group of inhibitory interneurons in the retina. The A17 amacrine plays an important role for visual signalling in the rod pathway microcircuit of the mammalian retina. It receives excitatory input from rod bipolar cells and provides feedback inhibition to the same cells. However, from ultrastructural investigations, there is evidence for input to A17s from other types of amacrine cells, presumably inhibitory, but there is a lack of information about the identity and functional properties of the synaptic receptors and how inhibition contributes to the integrative properties of A17s. Here, we studied the biophysical and pharmacological properties of GABAergic spontaneous inhibitory postsynaptic currents (spIPSCs) and GABAA receptors of A17 amacrines using whole‐cell and outside‐out patch recordings from rat retinal slices. The spIPSCs displayed fast onsets (10%–90% rise time ~740 μs) and double‐exponential decays (τfast ~4.5 ms [43% of amplitude]; τslow ~22 ms). Ultra‐fast application of brief pulses of GABA (3 mM) to patches evoked responses with deactivation kinetics best fitted by a triple‐exponential function (τ1 ~5.3 ms [55% of amplitude]; τ2 ~48 ms [32% of amplitude]; τ3 ~187 ms). Non‐stationary noise analysis of spIPSCs and patch responses yielded single‐channel conductances of ~21 and ~25 pS, respectively. Pharmacological analysis suggested that the spIPSCs are mediated by receptors with an α1βγ2 subunit composition and the somatic receptors have an α2βγ2 and/or α3βγ2 composition. These results demonstrate the presence of synaptic GABAA receptors on A17s, which may play an important role in signal integration in these cells.

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