Impaired GABAB-mediated presynaptic inhibition increases excitatory strength and alters short-term plasticity in synapsin knockout mice

Synapsins are a family of synaptic vesicle phosphoproteins regulating synaptic transmission and plasticity. SYN1/2 genes are major epilepsy susceptibility genes in humans. Consistently, synapsin I/II/III triple knockout (TKO) mice are epileptic and exhibit severe impairments in phasic and tonic GABAergic inhibition that precede the appearance of the epileptic phenotype. These changes are associated with an increased strength of excitatory transmission that has never been mechanistically investigated. Here, we observed that an identical effect in excitatory transmission could be induced in wild-type (WT) Schaffer collateral-CA1 pyramidal cell synapses by blockade of GABABreceptors (GABABRs). The same treatment was virtually ineffective in TKO slices, suggesting that the increased strength of the excitatory transmission results from an impairment of GABABpresynaptic inhibition. Exogenous stimulation of GABABRs in excitatory autaptic neurons, where GABA spillover is negligible, demonstrated that GABABRs were effective in inhibiting excitatory transmission in both WT and TKO neurons. These results demonstrate that the decreased GABA release and spillover, previously observed in TKO hippocampal slices, removes the tonic brake of presynaptic GABABRs on glutamate transmission, making the excitation/inhibition imbalance stronger

Characterization of a mice model of human epilepsy with Multi-Electrode Arrays

We applied microelectrode array (MEA) recordings to study the generation and propagation of epileptform activity in various connected regions of cortico-hippocampal slices obtained from SynapsinI/II/III knockout (TKO) mice and the effects of the synaptic vesicle-targeted anti epileptic drug levetiracetam (LEV). Synapsins (SynI, SynII and SynIII) are synaptic vesicle phosphoproteins playing a role in synaptic transmission and plasticity. TKO mice display an epileptic phenotype and mutation of the SYN1 gene is associated with epilepsy in man. We found that both interictal (IIC) and ictal (IC) discharges induced by 4AP were more pronounced and widespread in TKO mice, revealing a state of hyperexcitability of TKO networks. To get insight into the frequencies characterizing the IC seizures, we analyzed the average IC power spectral density (PSD) in the 10-50 Hz range in different cortical regions. TKO slices exhibited an increase of power for frequencies above 20Hz with respect to Wild- Type (TWT). To determine whether the hyperexcitability of TKO slices is also reflected by an increased spread of IC discharges and taking advantage of the spatial resolution of the MEA device, we measured the percentage of electrodes recording IC discharges over the total number of cortical electrodes. The spread of excitation was significantly higher in TKO slices than in TWT ones and treatment with LEV decreased the spread of IC discharges in the entorhinal of TKO slices. In order to better characterize the propagation of the IIC events in the hippocampus, we recently coupled MEA recordings with optical imaging using voltage-sensitive dyes by exploiting the possibility of simultaneous recordings with a high spatial and temporal resolution to reveal more detailed patterns of propagation

Cognitive impairment in Gdi1-deficient mice is associated with altered synaptic vesicle pools and short-term synaptic plasticity, and can be corrected by appropriate learning training

The GDI1 gene, responsible in human for X-linked non-specific mental retardation, encodes αGDI, a regulatory protein common to all GTPases of the Rab family. Its alteration, leading to membrane accumulation of different Rab GTPases, may affect multiple steps in neuronal intracellular traffic. Using electron microscopy and electrophysiology, we now report that lack of αGDI impairs several steps in synaptic vesicle (SV) biogenesis and recycling in the hippocampus. Alteration of the SV reserve pool (RP) and a 50% reduction in the total number of SV in adult synapses may be dependent on a defective endosomal-dependent recycling and may lead to the observed alterations in short-term plasticity. As predicted by the synaptic characteristics of the mutant mice, the short-term memory deficit, observed when using fear-conditioning protocols with short intervals between trials, disappeared when the Gdi1 mutants were allowed to have longer intervals between sessions. Likewise, previously observed deficits in radial maze learning could be corrected by providing less challenging pre-training. This implies that an intact RP of SVs is necessary for memory processing under challenging conditions in mice. The possibility to correct the learning deficit in mice may have clinical implication for future studies in huma

Dominant β-catenin mutations cause intellectual disability with recognizable syndromic features

The recent identification of multiple dominant mutations in the gene encoding β-catenin in both humans and mice has enabled exploration of the molecular and cellular basis of β-catenin function in cognitive impairment. In humans, β-catenin mutations that cause a spectrum of neurodevelopmental disorders have been identified. We identified de novo β-catenin mutations in patients with intellectual disability, carefully characterized their phenotypes, and were able to define a recognizable intellectual disability syndrome. In parallel, characterization of a chemically mutagenized mouse line that displays features similar to those of human patients with β-catenin mutations enabled us to investigate the consequences of β-catenin dysfunction through development and into adulthood. The mouse mutant, designated batface (Bfc), carries a Thr653Lys substitution in the C-terminal armadillo repeat of β-catenin and displayed a reduced affinity for membrane-associated cadherins. In association with this decreased cadherin interaction, we found that the mutation results in decreased intrahemispheric connections, with deficits in dendritic branching, long-term potentiation, and cognitive function. Our study provides in vivo evidence that dominant mutations in β-catenin underlie losses in its adhesion-related functions, which leads to severe consequences, including intellectual disability, childhood hypotonia, progressive spasticity of lower limbs, and abnormal craniofacial features in adult

A three-dimensional micro-electrode array for in-vitro neuronal interfacing

Objective. In this paper, we report on the development of an easy-to-fabricate three-dimensional Micro-Electrode Array (3D-MEA) specifically designed for brain-on-a-dish applications. Approach. The proposed device consists of pillar-shaped gold microelectrodes realized by electroplating directly on top of a standard MEA, making this approach highly versatile and convenient for batch fabrication. Moreover, with this simple technique, it is possible to obtain electrodes with a height of more than 100 µm onto different kind of substrates, ranging from glass to flexible plastic ones. Main results. This novel 3D-MEA structure has been validated with acute brain slices, successfully recording both epileptiform-like discharges (upon the administration of 4-AP), and electrically-evoked neuronal activity. The preliminary validation showed a substantial improvement in the signals amplitude with respect to both commercial and custom planar electrodes thanks to a better coupling offered by the peculiar shape of the three-dimensional electrodes. Significance. Beside the versatility of the fabrication approach, which allows to obtain 3D MEA devices onto both rigid and flexible substrates, the reported validation showed how the pillar approach can outperform standard planar MEA recordings in terms of signal amplitude. Moreover, thanks to the possibility of obtaining multi-level 3D structures within the same device, the proposed fabrication technique offers an interesting and flexible approach for the development of a new family of electrophysiological tools for 3D in vitro electrophysiology, in particular for acute brain slices and 3D neuronal cultures for brain-on-a-dish applications

Acute stress increases depolarization-evoked glutamate release in the rat prefrontal/frontal cortex: the dampening action of antidepressants

Background: Behavioral stress is recognized as a main risk factor for neuropsychiatric diseases. Converging evidence suggested that acute stress is associated with increase of excitatory transmission in certain forebrain areas. Aim of this work was to investigate the mechanism whereby acute stress increases glutamate release, and if therapeutic drugs prevent the effect of stress on glutamate release. Methodology/findings: Rats were chronically treated with vehicle or drugs employed for therapy of mood/anxiety disorders (fluoxetine, desipramine, venlafaxine, agomelatine) and then subjected to unpredictable footshock stress. Acute stress induced marked increase in depolarization-evoked release of glutamate from synaptosomes of prefrontal/frontal cortex in superfusion, and the chronic drug treatments prevented the increase of glutamate release. Stress induced rapid increase in the circulating levels of corticosterone in all rats (both vehicle- and drug-treated), and glutamate release increase was blocked by previous administration of selective antagonist of glucocorticoid receptor (RU 486). On the molecular level, stress induced accumulation of presynaptic SNARE complexes in synaptic membranes (both in vehicle- and drug-treated rats). Patch-clamp recordings of pyramidal neurons in the prefrontal cortex revealed that stress increased glutamatergic transmission through both pre- and postsynaptic mechanisms, and that antidepressants may normalize it by reducing release probability. Conclusions/significance: Acute footshock stress up-regulated depolarization-evoked release of glutamate from synaptosomes of prefrontal/frontal cortex. Stress-induced increase of glutamate release was dependent on stimulation of glucocorticoid receptor by corticosterone. Because all drugs employed did not block either elevation of corticosterone or accumulation of SNARE complexes, the dampening action of the drugs on glutamate release must be downstream of these processes. This novel effect of antidepressants on the response to stress, shown here for the first time, could be related to the therapeutic action of these drugs