Ivan Marchionni

Post‐phosphorylation prolyl isomerisation of gephyrin represents a mechanism to modulate glycine receptors function

The microtubule binding protein gephyrin plays a prominent role in establishing and maintaining a high concentration of inhibitory glycine receptors juxtaposed to presynaptic releasing sites. Here, we show that endogenous gephyrin undergoes proline‐directed phosphorylation, which is followed by the recruitment of the peptidyl‐prolyl isomerase Pin1. The interaction between gephyrin and Pin1 is strictly dependent on gephyrin phosphorylation and requires serine–proline consensus sites encompassing the gephyrin proline‐rich domain. Upon binding, Pin1 triggers conformational changes in the gephyrin molecule, thus enhancing its ability to bind the beta subunit of GlyRs. Consistently, a downregulation of GlyR clusters was detected in hippocampal neurons derived from Pin1 knockout mice, which was paralleled by a reduction in the amplitude of glycine‐evoked currents. Our results suggest that phosphorylation‐dependent prolyl isomerisation of gephyrin represents a mechanism for regulating GlyRs function.

GAT-1 regulates both tonic and phasic GABAA receptor-mediated inhibition in the cerebral cortex

γ‐Aminobutyric acid 1 (GAT‐1) is the most copiously expressed GABA transporter; we studied its role in phasic and tonic inhibition in the neocortex using GAT‐1 knockout (KO) mice. Immunoblotting and immunocytochemical studies showed that GAT‐2 and GAT‐3 levels in KOs were unchanged and that GAT‐3 was not redistributed in KOs. Moreover, the expression of GAD65/67 was increased, whereas that of GABA or VGAT was unchanged. Microdialysis studies showed that in KOs spontaneous extracellular release of GABA and glutamate was comparable in WT and KO mice, whereas KCl‐evoked output of GABA, but not of glutamate, was significantly increased in KOs. Recordings from layer II/III pyramids revealed a significant increase in GABAAR‐mediated tonic conductance in KO mice. The frequency, amplitude and kinetics of spontaneous inhibitory post‐synaptic currents (IPSCs) were unchanged, whereas the decay time of evoked IPSCs was significantly prolonged in KO mice. In KO mice, high frequency stimulation of GABAergic terminals induced large GABAAR‐mediated inward currents associated with a reduction in amplitude and decay time of IPSCs evoked immediately after the train. The recovery process was slower in KO than in WT mice. These studies show that in the cerebral cortex of GAT‐1 KO mice GAT‐3 is not redistributed and GADs are adaptively changed and indicate that GAT‐1 has a prominent role in both tonic and phasic GABAAR‐mediated inhibition, in particular during sustained neuronal activity.

In the developing rat hippocampus a tonic GABAA-mediated conductance selectively enhances the glutamatergic drive of principal cells

In the adult hippocampus, two different forms of GABAA receptor‐mediated inhibition have been identified: phasic and tonic. The first is due to the activation of GABAA receptors facing the presynaptic releasing sites, whereas the second is due to the activation of receptors localized away from the synapses. Because of their high affinity and low desensitization rate, extrasynaptic receptors are persistently able to sense low concentrations of GABA. Here we show that, early in postnatal life, between postnatal day (P) 2 and P6, CA1 and CA3 pyramidal cells but not stratum radiatum interneurons, express a tonic GABAA‐mediated conductance. Block of the neuronal GABA transporter GAT‐1 slightly enhanced the persistent GABA conductance in principal cells but not in GABAergic interneurons. However, in adulthood, a tonic GABAA‐mediated conductance could be revealed in stratum radiatum interneurons, indicating that the ability of these cells to sense ambient GABA levels is developmentally regulated. Pharmacological analysis of the tonic conductance in principal cells demonstrated the involvement of β2/β3, α5 and γ2 GABAA receptor subunits. Removal of the tonic depolarizing action of GABA with picrotoxin, reduced the excitability and the glutamatergic drive of principal cells but did not modify the excitability of stratum radiatum interneurons. The increased cell excitability and synaptic activity following the activation of extrasynaptic GABAA receptors by ambient GABA would facilitate the induction of giant depolarizing potentials.

Quantitative dynamics and spatial profile of perisomatic GABAergic input during epileptiform synchronization in the CA1 hippocampus.

Perisomatic GABAergic input appears spared or even increased in intractable temporal lobe epilepsy, and has been suggested to contribute to the generation of pathological discharges. Nevertheless, its degree of functional activity during epileptiform synchronization has not been thoroughly investigated. Thus, it remains unclear how structural preservation or loss of domain‐specific GABAergic input may affect the network. Here, we have taken advantage of a model of epileptiform activity in vitro to quantify the charge transfer provided by perisomatic GABAA receptor‐mediated input to CA1 pyramidal neurons during interictal‐like bursts. By recording both firing in GABAergic interneurons and the charge transfer generated by unitary postsynaptic currents to target pyramidal cells, we have estimated the charge transfer that would be dynamically generated by the recruitment of the entire pool of perisomatic‐targeting interneurons and the number of perisomatic‐targeting interneurons that would be required to generate the experimentally observed GABAergic input. In addition, we have recorded and compared the dynamics and charge density of GABAergic input recorded at different membrane compartments such as the soma vs. the proximal dendrite. Our results suggest that GABAA receptor‐mediated perisomatic input is massively activated during burst synchronization and that its kinetic properties and charge density are similar at the soma and proximal dendrite. These functional results match structural data published by other laboratories very well and strengthen the hypothesis that the potential preservation of perisomatic GABAergic input in intractable epilepsies may be a key factor in the generation of pathological network activity.

Distinctive properties of CXC chemokine receptor 4-expressing Cajal–Retzius cells versus GABAergic interneurons of the postnatal hippocampus

The CXC chemokine receptor 4 (CXCR4) for the chemokine (C‐X‐C motif) ligand 12/stromal cell‐derived factor‐1 α (CXCL12/SDF‐1 α) is highly expressed in the postnatal CA1 stratum lacunosum‐moleculare. However, both the network events triggered by SDF‐1 α in this microcircuit and the cellular targets of this chemokine remain virtually unexplored. Here, we have studied SDF‐1 α‐mediated neuromodulation of the stratum lacunosum‐moleculare by directly comparing the properties of CXCR4‐expressing Cajal–Retzius cells vs. CXCR4‐non‐expressing interneurons, and by recording the electrophysiological effects caused by application of SDF‐1 α on either cell type. We demonstrate that SDF‐1 α dramatically reduces spontaneous firing in Cajal–Retzius cells via hyerpolarization, and that cessation of firing is prevented by the CXCR4‐specific antagonist AMD3100. In contrast, no effects on the excitability of interneurons of the same layer were observed following exposure to the chemokine. We also provide evidence that, despite the expression of functional glutamate receptors, Cajal–Retzius cells are integrated in the synaptic network of the stratum lacunosum‐moleculare via excitatory GABAergic input. Furthermore, we show that the axons of Cajal–Retzius cells target specifically the stratum lacunosum‐moleculare and the dentate gyrus, but lack postsynaptic specializations opposite to their axonal varicosities. These results, taken together with our observation that SDF‐1 α reduces evoked field responses at the entorhinal cortex–CA1 synapse, suggest that Cajal–Retzius cells produce a diffuse output that may impact information processing of stratum lacunosum‐moleculare. We propose that pathological alterations of local levels of SDF‐1 α or CXCR4 expression may affect the functions of an important hippocampal microcircuit.

NEW INSIGHTS ON THE ROLE OF GEPHYRIN IN REGULATING BOTH PHASIC AND TONIC GABAergic INHIBITION IN RAT HIPPOCAMPAL NEURONS IN CULTURE

Gephyrin is a tubulin-binding protein that acts as a scaffold for clustering glycine and GABA(A) receptors at postsynaptic sites. In this study, the role of gephyrin on GABA(A) receptor function was assessed at the post-translational level, using gephyrin-specific single chain antibody fragments (scFv-gephyrin). When expressed in cultured rat hippocampal neurons as a fusion protein containing a nuclear localization signal, scFv-gephyrin were able to remove endogenous gephyrin from GABA(A) receptor clusters. Immunocytochemical experiments revealed a significant reduction in the number of synaptic gamma2-subunit containing GABA(A) receptors and a significant decrease in the density of the GABAergic presynaptic marker vesicular GABA transporter (VGAT). These effects were associated with a slow down of the onset kinetics, a reduction in the amplitude and in the frequency of miniature inhibitory postsynaptic currents (mIPSCs). The quantitative analysis of current responses to ultrafast application of GABA suggested that changes in onset kinetics resulted from modifications in the microscopic gating of GABA(A) receptors and in particular from a reduced entry into the desensitized state. In addition, hampering gephyrin function with scFv-gephyrin induced a significant reduction in GABA(A) receptor-mediated tonic conductance. This effect was probably dependent on the decrease in GABAergic innervation and in GABA release from presynaptic nerve terminals. These results indicate that gephyrin is essential not only for maintaining synaptic GABA(A) receptor clusters in the right position but also for regulating both phasic and tonic inhibition.

Developmental Profile, Morphology, and Synaptic Connectivity of Cajal–Retzius Cells in the Postnatal Mouse Hippocampus

Cajal–Retzius (CR) cells are early generated neurons, involved in the assembly of developing neocortical and hippocampal circuits. However, their roles in networks of the postnatal brain remain poorly understood. In order to get insights into these latter functions, we have studied their morphological and synaptic properties in the postnatal hippocampus of the CXCR4-EGFP mouse, where CR cells are easily identifiable. Our data indicate that CR cells are nonuniformly distributed along different subfields of the hippocampal formation, and that their postnatal decline is regulated in a region-specific manner. In fact, CR cells persist in distinct areas of fully mature animals. Subclasses of CR cells project and target either local (molecular layers) or distant regions [subicular complex and entorhinal cortex (EC)] of the hippocampal formation, but have similar firing patterns. Lastly, CR cells are biased toward targeting dendritic shafts compared with spines, and produce large-amplitude glutamatergic unitary postsynaptic potentials on γ-aminobutyric acid (GABA) containing interneurons. Taken together, our results suggest that CR cells are involved in a novel excitatory loop of the postnatal hippocampal formation, which potentially contributes to shaping the flow of information between the hippocampus, parahippocampal regions and entorhinal cortex, and to the low seizure threshold of these brain areas.

The chemokine CXCL12 and the HIV‐1 envelope protein gp120 regulate spontaneous activity of Cajal–Retzius cells in opposite directions

•  The CXC chemokine ligand 12 (CXCL12) modulates spontaneous firing of Cajal–Retzius cells via the CXC chemokine receptor 4 (CXCR4). However, the underlying mechanism(s) are poorly understood. CXCR4 also binds the human immunodeficiency virus type 1 (HIV‐1) envelope glycoprotein 120 (gp120), but the functional effects of this interaction on Cajal–Retzius cell excitability remain unknown. •  We show that CXCL12 reduces spontaneous firing in Cajal–Retzius cells by opening a BK‐type calcium‐activated potassium conductance, whereas gp120 increases their excitability via a calcium‐ and chloride‐dependent mechanism. •  Our data suggest that, depending on the use of CXCL12 or gp120 as ligands, partial agonism at the CXCR4 receptor generates calcium responses of different strengths, which lead to the recruitment of either calcium‐activated potassium or chloride channels. •  We propose that HIV infection disrupts a signalling pathway important for the regulation of the excitability of Cajal–Retzius cells, and alters their functions.

Gephyrin selective intrabodies as a new strategy for studying inhibitory receptor clustering.

The microtubule-binding protein gephyrin is known to play a pivotal role in targeting and clustering postsynaptic inhibitory receptors. Here, the Intracellular Antibodies Capture Technology (IATC) was used to select two single-chain antibody fragments or intrabodies, which, fused to nuclear localization signals (NLS), were able to efficiently and selectively remove gephyrin from glycine receptor (GlyR) clusters. Co-transfection of NLS-tagged individual intrabodies with gephyrin-enhanced green fluorescent protein (EGFP) in HEK 293 cells revealed a partial relocalization of gephyrin aggregates onto the nucleus or in the perinuclear area. When expressed in cultured neurons, these intrabodies caused a significant reduction in the number of immunoreactive GlyR clusters, which was associated with a decrease in the peak amplitude of glycine-evoked whole cell currents as assessed with electrophysiological experiments. Hampering protein function at a posttranslational level may represent an attractive alternative for interfering with gephyrin function in a more spatially localized manner.