The molecular code that controls synapse formation and maintenance in vivo has remained quite sparse. Here, we identify that the secreted protein Adamtsl3 functions as critical hippocampal synapse ...organizer acting through the transmembrane receptor DCC (deleted in colorectal cancer). Traditionally, DCC function has been associated with glutamatergic synaptogenesis and plasticity in response to Netrin-1 signaling. We demonstrate that early post-natal deletion of Adamtsl3 in neurons impairs DCC protein expression, causing reduced density of both glutamatergic and GABAergic synapses. Adult deletion of Adamtsl3 in either GABAergic or glutamatergic neurons does not interfere with DCC-Netrin-1 function at glutamatergic synapses but controls DCC signaling at GABAergic synapses. The Adamtsl3-DCC signaling unit is further essential for activity-dependent adaptations at GABAergic synapses, involving DCC phosphorylation and Src kinase activation. These findings might be particularly relevant for schizophrenia because genetic variants in Adamtsl3 and DCC have been independently linked with schizophrenia in patients.
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•Adamtsl3 is widely expressed at hippocampal synapses•Adamtsl3 is a ligand for DCC during synaptogenesis and in the mature brain•Presynaptic and postsynaptic Adamtsl3 contribute toward GABAergic synapse maintenance via DCC•Autocrine Adamtsl3-DCC signaling mediates plasticity adaptations at GABAergic postsynapse
The secreted glycoprotein Ce-Punctin clusters neurotransmitter receptors at the C. elegans NMJ. Cramer et al. reveal that Ce-Punctin ortholog Adamtsl3 is a synaptic protein influencing synapse formation via the DCC receptor in the mouse brain. In the adult hippocampus, Adamtsl3-DCC signaling specializes toward GABAergic synapse function with behavioral consequences.
Primary hippocampal cultures grown from genetically modified mice provide a simplified context to study molecular mechanisms underlying neuronal development, synaptogenesis, and synapse plasticity ...in vitro. Here, we describe a simple protocol for culturing hippocampal neurons from P0 to P2 mice and a strategy for inducing alterations in synaptic strength at inhibitory and excitatory synapses in vitro. We also describe approaches for immunofluorescent labeling, image acquisition, and quantification of synaptic proteins.
For complete details on the use and execution of this protocol, please refer to Cramer et al.1
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•Precise culturing and manipulation of primary hippocampal neurons from mice•Induce and quantify synaptic plasticity in cultured mouse hippocampal neurons•Techniques for labeling, image acquisition, and quantification of synaptic proteins•Versatile framework to study synaptic plasticity, neuronal development, or disorders
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Primary hippocampal cultures grown from genetically modified mice provide a simplified context to study molecular mechanisms underlying neuronal development, synaptogenesis, and synapse plasticity in vitro. Here, we describe a simple protocol for culturing hippocampal neurons from P0 to P2 mice and a strategy for inducing alterations in synaptic strength at inhibitory and excitatory synapses in vitro. We also describe approaches for immunofluorescent labeling, image acquisition, and quantification of synaptic proteins.
Studying synapses in vivo presents challenges due to the complexity of accurately targeting and visualizing specific synaptic proteins within the brain. Here, we present a protocol for in vivo ...analysis of pre- and post-synaptic protein function in mice. We describe steps for combining adeno-associated virus (AAV)-mediated gene transfer to manipulate specific neuron subtypes. We also describe immunofluorescence on artificial cerebrospinal fluid (ACSF)-perfused brain sections to enhance the visualization of synaptic proteins.
For complete details on the use and execution of this protocol, please refer to Cramer et al.1
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•ACSF perfusion technique for investigating synaptic proteins in vivo•Steps for AAV gene transfer for broad expression or in specific neurons•Instructions for immunofluorescent labeling of free-floating brain sections•Guidance on improved detection of synaptic proteins using microscopy
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Studying synapses in vivo presents challenges due to the complexity of accurately targeting and visualizing specific synaptic proteins within the brain. Here, we present a protocol for in vivo analysis of pre- and post-synaptic protein function in mice. We describe steps for combining adeno-associated virus (AAV)-mediated gene transfer to manipulate specific neuron subtypes. We also describe immunofluorescence on artificial cerebrospinal fluid (ACSF)-perfused brain sections to enhance the visualization of synaptic proteins.
Gephyrin is the main scaffolding protein at inhibitory postsynaptic sites, and its clusters are the signaling hubs where several molecular pathways converge. Post-translational modifications (PTMs) ...of gephyrin alter GABA
receptor clustering at the synapse, but it is unclear how this affects neuronal activity at the circuit level. We assessed the contribution of gephyrin PTMs to microcircuit activity in the mouse barrel cortex by slice electrophysiology and in vivo two-photon calcium imaging of layer 2/3 (L2/3) pyramidal cells during single-whisker stimulation. Our results suggest that, depending on the type of gephyrin PTM, the neuronal activities of L2/3 pyramidal neurons can be differentially modulated, leading to changes in the size of the neuronal population responding to the single-whisker stimulation. Furthermore, we show that gephyrin PTMs have their preference for selecting synaptic GABA
receptor subunits. Our results identify an important role of gephyrin and GABAergic postsynaptic sites for cortical microcircuit function during sensory stimulation.
Microglia interact with neurons to facilitate synapse plasticity; however, signal(s) contributing to microglia activation for synapse elimination in pathology are not fully understood. Here, using in ...vitro organotypic hippocampal slice cultures and transient middle cerebral artery occlusion (MCAO) in genetically engineered mice in vivo, we report that at 24 hours after ischemia, microglia release brain-derived neurotrophic factor (BDNF) to downregulate glutamatergic and GABAergic synapses within the peri-infarct area. Analysis of the cornu ammonis 1 (CA1) in vitro shows that proBDNF and mBDNF downregulate glutamatergic dendritic spines and gephyrin scaffold stability through p75 neurotrophin receptor (p75
) and tropomyosin receptor kinase B (TrkB) receptors, respectively. After MCAO, we report that in the peri-infarct area and in the corresponding contralateral hemisphere, similar neuroplasticity occurs through microglia activation and gephyrin phosphorylation at serine-268 and serine-270 in vivo. Targeted deletion of the
gene in microglia or
S268A/S270A (phospho-null) point mutations protects against ischemic brain damage, neuroinflammation, and synapse downregulation after MCAO.
Abstract
Glycine receptors (GlyRs), together with GABA
A
receptors, mediate postsynaptic inhibition in most spinal cord and hindbrain neurons. In several CNS regions, GlyRs are also expressed in ...presynaptic terminals. Here, we analysed the effects of a phospho‐deficient mutation (S346A) in GlyR α3 subunits on inhibitory synaptic transmission in superficial spinal dorsal horn neurons, where this subunit is abundantly expressed. Unexpectedly, we found that not only were the amplitudes of evoked glycinergic inhibitory postsynaptic currents (IPSCs) significantly larger in GlyRα3(S346A) mice than in mice expressing wild‐type α3GlyRs (GlyRα3(WT) mice), but so were those of GABAergic IPSCs. Decreased frequencies of spontaneously occurring glycinergic and GABAergic miniature IPSCs (mIPSCs) with no accompanying change in mIPSC amplitudes suggested a change in presynaptic transmitter release. Paired‐pulse experiments on glycinergic IPSCs revealed an increased paired‐pulse ratio and a smaller coefficient of variation in GlyRα3(S346A) mice, which together indicate a reduction in transmitter release probability and an increase in the number of releasable vesicles. Paired‐pulse ratios of GABAergic IPSCs recorded in the presence of strychnine were not different between genotypes, while the coefficient of variation was smaller in GlyRα3(S346A) mice, demonstrating that the decrease in release probability was readily reversible by GlyR blockade, while the difference in the size of the pool of releasable vesicles remained. Taken together, our results suggest that presynaptic α3 GlyRs regulate synaptic glycine and GABA release in superficial dorsal horn neurons, and that this effect is potentially regulated by their phosphorylation status.
image
Key points
A serine‐to‐alanine point mutation was introduced into the glycine receptor α3 subunit of mice.
This point mutation renders α3 glycine receptors resistant to protein kinase A mediated phosphorylation but has otherwise only small effects on receptor function.
Patch‐clamp recordings from neurons in mouse spinal cord slices revealed an unexpected increase in the amplitudes of both glycinergic and GABAergic evoked inhibitory postsynaptic currents (IPSCs).
Miniature IPSCs, paired‐pulse ratios and synaptic variation analyses indicate a change in synaptic glycine and GABA release.
The results strongly suggest that α3 subunit‐containing glycine receptors are expressed on presynaptic terminals of inhibitory dorsal horn neurons where they regulate transmitter release.
In this manuscript, we investigate the "stones best left unturned" of sample storage and preparation and their implications for the next-generation sequencing of infant faecal microbial communities ...by the 16S ribosomal ribonucleic acid (rRNA) gene. We present a number of experiments that investigate the potential effects of often overlooked methodology factors, establishing a "normal" degree of variation expected between replica sequenced samples. Sources of excess variation are then identified, as measured by observation of alpha diversity, taxonomic group counts and beta diversity magnitudes between microbial communities.
Extraction of DNA from samples on different dates, by different people and even using varied sample weights results in little significant difference in downstream sequencing data. A key assumption in many studies is the stability of samples stored long term at -80 °C prior to extraction. After 2 years, we see relatively few changes: increased abundances of lactobacilli and bacilli and a reduction in the overall OTU count. Where samples cannot be frozen, we find that storing samples at room temperature does lead to significant changes in the microbial community after 2 days. Mailing of samples during this time period (a common form of sample collection from outpatients for example) does not lead to any additional variation.
Important methodological standards can be drawn from these results; painstakingly created archives of infant faecal samples stored at -80 °C are still largely representative of the original community and varying factors in DNA extraction methodology have comparatively little effect on overall results. Samples taken should ideally be either frozen at -80 °C or extracted within 2 days if stored at room temperature, with mail samples being mailed on the day of collection.
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