The VGCC auxiliary subunit α2δ1 is an extracellular GluA1 interactor and regulates LTP, spatial memory, and seizure susceptibility.

Activity-dependent synaptic accumulation of AMPA receptors (AMPARs) and subsequent long-term synaptic strengthening underlie different forms of learning and memory. The AMPAR subunit GluA1 amino-terminal domain is essential for synaptic docking of AMPAR during LTP, but the precise mechanisms involved are not fully understood. Using unbiased proteomics, we identified the epilepsy and intellectual disability-associated VGCC auxiliary subunit α2δ1 as a candidate extracellular AMPAR slot.

Evidence for Two Subpopulations of Cerebrospinal Fluid-Contacting Neurons with Opposite GABAergic Signaling in Adult Mouse Spinal Cord.

Spinal cerebrospinal fluid-contacting neurons (CSF-cNs) form an evolutionary conserved bipolar cell population localized around the central canal of all vertebrates. CSF-cNs were shown to express molecular markers of neuronal immaturity into adulthood; however, the impact of their incomplete maturation on the chloride (Cl) homeostasis as well as GABAergic signaling remains unknown. Using adult mice from both sexes, in situ hybridization revealed that a proportion of spinal CSF-cNs (18.

The downregulation of Kv channels in Lgi1mice is accompanied by a profound modification of its interactome and a parallel decrease in Kv channels.

In animal models of LGI1-dependent autosomal dominant lateral temporal lobe epilepsy, Kv channels are downregulated, suggesting their crucial involvement in epileptogenesis. The molecular basis of Kv channel-downregulation in LGI1 knock-out mice has not been elucidated and how the absence of this extracellular protein induces an important modification in the expression of Kv remains unknown. In this study we analyse by immunofluorescence the modifications in neuronal Kv and Kv distribution throughout the hippocampal formation of LGI1 knock-out mice.

Deep learning-based image analysis identifies a DAT-negative subpopulation of dopaminergic neurons in the lateral Substantia nigra.

Here we present a deep learning-based image analysis platform (DLAP), tailored to autonomously quantify cell numbers, and fluorescence signals within cellular compartments, derived from RNAscope or immunohistochemistry. We utilised DLAP to analyse subtypes of tyrosine hydroxylase (TH)-positive dopaminergic midbrain neurons in mouse and human brain-sections. These neurons modulate complex behaviour, and are differentially affected in Parkinson's and other diseases.

Rescue of Normal Excitability in LGI1-Deficient Epileptic Neurons.

Leucine-rich glioma inactivated 1 (LGI1) is a glycoprotein secreted by neurons, the deletion of which leads to autosomal dominant lateral temporal lobe epilepsy. We previously showed that LGI1 deficiency in a mouse model (i.e.

Dysfunctional serotonergic neuron-astrocyte signaling in depressive-like states.

Astrocytes play crucial roles in brain homeostasis and are regulatory elements of neuronal and synaptic physiology. Astrocytic alterations have been found in Major Depressive Disorder (MDD) patients; however, the consequences of astrocyte Ca signaling in MDD are poorly understood. Here, we found that corticosterone-treated juvenile mice (Cort-mice) showed altered astrocytic Ca dynamics in mPFC both in resting conditions and during social interactions, in line with altered mice behavior.

Molecular landscape of BoNT/B bound to a membrane-inserted synaptotagmin/ganglioside complex.

Botulinum neurotoxin serotype B (BoNT/B) uses two separate protein and polysialoglycolipid-binding pockets to interact with synaptotagmin 1/2 and gangliosides. However, an integrated model of BoNT/B bound to its neuronal receptors in a native membrane topology is still lacking. Using a panel of in silico and experimental approaches, we present here a new model for BoNT/B binding to neuronal membranes, in which the toxin binds to a preassembled synaptotagmin-ganglioside GT1b complex and a free ganglioside allowing a lipid-binding loop of BoNT/B to interact with the glycone part of the synaptotagmin-associated GT1b.

Patient-derived antibodies reveal the subcellular distribution and heterogeneous interactome of LGI1.

Autoantibodies against leucine-rich glioma-inactivated 1 (LGI1) occur in patients with encephalitis who present with frequent focal seizures and a pattern of amnesia consistent with focal hippocampal damage. To investigate whether the cellular and subcellular distribution of LGI1 may explain the localization of these features, and hence gain broader insights into LGI1's neurobiology, we analysed the detailed localization of LGI1 and the diversity of its protein interactome, in mouse brains using patient-derived recombinant monoclonal LGI1 antibodies. Combined immunofluorescence and mass spectrometry analyses showed that LGI1 is enriched in excitatory and inhibitory synaptic contact sites, most densely within CA3 regions of the hippocampus.

Refining the Identity and Role of Kv4 Channels in Mouse Substantia Nigra Dopaminergic Neurons.

Substantia nigra pars compacta (SNc) dopaminergic (DA) neurons display a peculiar electrical phenotype characterized by a spontaneous tonic regular activity (pacemaking activity), a broad action potential (AP) and a biphasic postinhibitory response. The transient A-type current (I) is known to play a crucial role in this electrical phenotype, and so far, this current was considered to be carried exclusively by Kv4.3 potassium channels.

Distinctive binding properties of human monoclonal LGI1 autoantibodies determine pathogenic mechanisms.

Autoantibodies against leucine-rich glioma inactivated 1 (LGI1) are found in patients with limbic encephalitis and focal seizures. Here, we generate patient-derived monoclonal antibodies (mAbs) against LGI1. We explore their sequences and binding characteristics, plus their pathogenic potential using transfected HEK293T cells, rodent neuronal preparations, and behavioural and electrophysiological assessments in vivo after mAb injections into the rodent hippocampus.

Effects of chronic immobilization stress on biokinetics and dosimetry of Ga in a murine model.

The aim of this work is to determine the effect of chronic immobilization stress on kinetics and dosimetry of Ga in a mouse model. A control group (CG) and a stress group (SG), each with 15 mice, were included in the study, and the latter group was subjected to a chronic immobilization stress model 2 h daily for 14 consecutive days. At day 13, Ga-citrate was administered intraperitoneally (11.

Gangliosides interact with synaptotagmin to form the high-affinity receptor complex for botulinum neurotoxin B.

Botulinum neurotoxin type B (BoNT/B) recognizes nerve terminals by binding to 2 receptor components: a polysialoganglioside, predominantly GT1b, and synaptotagmin 1/2. It is widely thought that BoNT/B initially binds to GT1b then diffuses in the plane of the membrane to interact with synaptotagmin. We have addressed the hypothesis that a GT1b-synaptotagmin complex forms the BoNT/B receptor.

Melanopsin for precise optogenetic activation of astrocyte-neuron networks.

Optogenetics has been widely expanded to enhance or suppress neuronal activity and it has been recently applied to glial cells. Here, we have used a new approach based on selective expression of melanopsin, a G-protein-coupled photopigment, in astrocytes to trigger Ca signaling. Using the genetically encoded Ca indicator GCaMP6f and two-photon imaging, we show that melanopsin is both competent to stimulate robust IP3-dependent Ca signals in astrocyte fine processes, and to evoke an ATP/Adenosine-dependent transient boost of hippocampal excitatory synaptic transmission.

CB1 receptors down-regulate a cAMP/Epac2/PLC pathway to silence the nerve terminals of cerebellar granule cells.

Cannabinoid receptors mediate short-term retrograde inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at excitatory synapses. The responses of individual nerve terminals in VGLUT1-pHluorin transfected cerebellar granule cells to cannabinoids have shown that prolonged activation of cannabinoid type 1 receptors (CB1Rs) silences a subpopulation of previously active synaptic boutons. Adopting a combined pharmacological and genetic approach to study the molecular mechanisms of CB1R-induced silencing, we found that adenylyl cyclase inhibition decreases cAMP levels while it increases the number of silent synaptic boutons and occludes the induction of further silencing by the cannabinoid agonist HU-210.

Cross-talk between metabotropic glutamate receptor 7 and beta adrenergic receptor signaling at cerebrocortical nerve terminals.

The co-existence of presynaptic G protein coupled receptors, GPCRs, has received little attention, despite the fact that interplay between the signaling pathways activated by such receptors may affect the neurotransmitter release. Using immunocytochemistry and immuhistochemistry we show that mGlu7 and β-adrenergic receptors are co-expressed in a sub-population of cerebrocortical nerve terminals. mGlu7 receptors readily couple to pathways that inhibit glutamate release.

The regulation of synaptic vesicle recycling by cGMP-dependent protein kinase type II in cerebellar granule cells under strong and sustained stimulation.

From the early periods of neurogenesis and migration, up until synaptogenesis, both nitric oxide (NO) and its downstream messenger, cGMP, are thought to influence the development of neurons. The NO/cGMP/cGMP-dependent protein kinase (cGK) pathway regulates the clustering and recruitment of synaptic proteins and vesicles to the synapse, adjusting the exoendocytic cycle to the intensity of activity and accelerating endocytosis following large-scale exocytosis. Here, we show that blockage of the N-methyl-D-aspartate receptor impairs the cycling of synaptic vesicles in a subset of boutons on cerebellar granule cells, an effect that was reversed by increasing cGMP.

Cannabinoid type 1 receptors transiently silence glutamatergic nerve terminals of cultured cerebellar granule cells.

Cannabinoid receptors are the most abundant G protein-coupled receptors in the brain and they mediate retrograde short-term inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at many excitatory synapses. The induction of presynaptically silent synapses is a means of modulating synaptic strength, which is important for synaptic plasticity. Persistent activation of cannabinoid type 1 receptors (CB1Rs) mutes GABAergic terminals, although it is unclear if CB1Rs can also induce silencing at glutamatergic synapses.

Studying synaptic efficiency by post-hoc immunolabelling.

Background: In terms of vesicular recycling, synaptic efficiency is a key determinant of the fidelity of synaptic transmission. The ability of a presynaptic terminal to reuse its vesicular content is thought to be a signature of synaptic maturity and this process depends on the activity of several proteins that govern exo/endocytosis. Upon stimulation, individual terminals in networks of cultured cerebellar granule neurons exhibit heterogeneous exocytic responses, which reflect the distinct states of maturity and plasticity intrinsic to individual synaptic terminals.

β-Adrenergic receptors activate exchange protein directly activated by cAMP (Epac), translocate Munc13-1, and enhance the Rab3A-RIM1α interaction to potentiate glutamate release at cerebrocortical nerve terminals.

The adenylyl cyclase activator forskolin facilitates synaptic transmission presynaptically via cAMP-dependent protein kinase (PKA). In addition, cAMP also increases glutamate release via PKA-independent mechanisms, although the downstream presynaptic targets remain largely unknown. Here, we describe the isolation of a PKA-independent component of glutamate release in cerebrocortical nerve terminals after blocking Na(+) channels with tetrodotoxin.

Efficient synaptic vesicle recycling after intense exocytosis concomitant with the accumulation of non-releasable endosomes at early developmental stages.

Following the exocytosis of neurotransmitter-containing synaptic vesicles, endocytosis is fundamental to re-establishing conditions for synaptic transmission. As there are distinct endocytotic pathways that each differ in their efficiency to generate releasable synaptic vesicles, we used the dye FM1-43 to track vesicle recycling, and to determine whether nerve terminals use multiple pathways of endocytosis. We identified two types of synaptic boutons in cultured cerebellar granule cells that were characterized by weak or strong FM1-43-unloading profiles.

Membrane depolarization regulates AMPA receptor subunit expression in cerebellar granule cells in culture.

The physiological responses of AMPA receptors can be modulated through the differential expression of their subunits and by modifying their number at the cell surface. Here we have studied the expression of AMPA receptor subunits (GluR1-4) mRNAs in cerebellar granule cells grown in depolarizing (25mMK(+)) medium, and we have evaluated the effect of decreasing the [K(+)] in the culture medium for 24 h on both GluR1-4 expression (both mRNA and protein) and their presence at the plasma membrane. The expression of the four AMPAR subunits increases as the [K(+)] decreases, although the increase in GluR2 and GluR3 was only observed in the cell soma but not in the dendrites.