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Methods in Molecular Biology (Clifton,... 2024Postsynaptic density (PSD) is a morphologically and functionally specialized postsynaptic membrane structure of excitatory synapses. It contains hundreds of proteins...
Postsynaptic density (PSD) is a morphologically and functionally specialized postsynaptic membrane structure of excitatory synapses. It contains hundreds of proteins such as neurotransmitter receptors, adhesion molecules, cytoskeletal proteins, and signaling enzymes. The study of the molecular architecture of the PSD is one of the most intriguing issues in neuroscience research. The isolation of the PSD from the brain of an animal is necessary for subsequent biochemical and morphological analyses. Many laboratories have developed methods to isolate PSD from the animal brain. In this chapter, we present a simple method to isolate PSD from the mouse brain using sucrose density gradient-based purification of synaptosomes followed by detergent extraction.
Topics: Animals; Mice; Post-Synaptic Density; Synaptic Membranes; Brain; Cytoskeletal Proteins; Laboratories
PubMed: 38630221
DOI: 10.1007/978-1-0716-3810-1_7 -
BioRxiv : the Preprint Server For... Apr 2024Precise connectivity between specific neurons is essential for the formation of the complex neural circuitry necessary for executing intricate motor behaviors and higher...
Precise connectivity between specific neurons is essential for the formation of the complex neural circuitry necessary for executing intricate motor behaviors and higher cognitive functions. While -interactions between synaptic membrane proteins have emerged as crucial elements in orchestrating the assembly of these neural circuits, the synaptic surface proteins involved in neuronal wiring remain largely unknown. Here, using unbiased single-cell transcriptomic and mouse genetic approaches, we uncover that the neurexin family of genes enables olfactory sensory neuron (OSNs) axons to form appropriate synaptic connections with their mitral and tufted (M/T) cell synaptic partners, within the mammalian olfactory system. Neurexin isoforms are differentially expressed within distinct populations of OSNs, resulting in unique pattern of neurexin expression that is specific to each OSN type, and synergistically cooperate to regulate axonal innervation, guiding OSN axons to their designated glomeruli. This process is facilitated through the interactions of neurexins with their postsynaptic partners, including neuroligins, which have distinct expression patterns in M/T cells. Our findings suggest a novel mechanism underpinning the precise assembly of olfactory neural circuits, driven by the -interaction between neurexins and their ligands.
PubMed: 38617205
DOI: 10.1101/2024.04.01.587570 -
International Journal of Molecular... Mar 2024Patients with systemic lupus erythematosus (SLE) frequently experience chronic pain due to the limited effectiveness and safety profiles of current analgesics.... (Review)
Review
Patients with systemic lupus erythematosus (SLE) frequently experience chronic pain due to the limited effectiveness and safety profiles of current analgesics. Understanding the molecular and synaptic mechanisms underlying abnormal neuronal activation along the pain signaling pathway is essential for developing new analgesics to address SLE-induced chronic pain. Recent studies, including those conducted by our team and others using the SLE animal model (/ lupus-prone mice), have unveiled heightened excitability in nociceptive primary sensory neurons within the dorsal root ganglia and increased glutamatergic synaptic activity in spinal dorsal horn neurons, contributing to the development of chronic pain in mice with SLE. Nociceptive primary sensory neurons in lupus animals exhibit elevated resting membrane potentials, and reduced thresholds and rheobases of action potentials. These changes coincide with the elevated production of TNFα and IL-1β, as well as increased ERK activity in the dorsal root ganglion, coupled with decreased AMPK activity in the same region. Dysregulated AMPK activity is linked to heightened excitability in nociceptive sensory neurons in lupus animals. Additionally, the increased glutamatergic synaptic activity in the spinal dorsal horn in lupus mice with chronic pain is characterized by enhanced presynaptic glutamate release and postsynaptic AMPA receptor activation, alongside the reduced activity of glial glutamate transporters. These alterations are caused by the elevated activities of IL-1β, IL-18, CSF-1, and thrombin, and reduced AMPK activities in the dorsal horn. Furthermore, the pharmacological activation of spinal GPR109A receptors in microglia in lupus mice suppresses chronic pain by inhibiting p38 MAPK activity and the production of both IL-1β and IL-18, as well as reducing glutamatergic synaptic activity in the spinal dorsal horn. These findings collectively unveil crucial signaling molecular and synaptic targets for modulating abnormal neuronal activation in both the periphery and spinal dorsal horn, offering insights into the development of analgesics for managing SLE-induced chronic pain.
Topics: Humans; Animals; Mice; Mice, Inbred MRL lpr; Chronic Pain; Interleukin-18; AMP-Activated Protein Kinases; Glutamic Acid; Interleukin-1beta; Lupus Erythematosus, Systemic; Analgesics
PubMed: 38612414
DOI: 10.3390/ijms25073602 -
Journal of Neurophysiology May 2024Two subtypes of striatal spiny projection neurons, iSPNs and dSPNs, whose axons form the "indirect" and "direct" pathways of the basal ganglia, respectively, both make... (Comparative Study)
Comparative Study
Two subtypes of striatal spiny projection neurons, iSPNs and dSPNs, whose axons form the "indirect" and "direct" pathways of the basal ganglia, respectively, both make synaptic connections in the external globus pallidus (GPe) but are usually found to have different effects on behavior. Activation of the terminal fields of iSPNs or dSPNs generated compound currents in almost all GPe neurons. To determine whether iSPNs and dSPNs have the same or different effects on pallidal neurons, we studied the unitary synaptic currents generated in GPe neurons by action potentials in single striatal neurons. We used optogenetic excitation to elicit repetitive firing in a small number of nearby SPNs, producing sparse barrages of inhibitory postsynaptic currents (IPSCs) in GPe neurons. From these barrages, we isolated sequences of IPSCs with similar time courses and amplitudes, which presumably arose from the same SPN. There was no difference between the amplitudes of unitary IPSCs generated by the indirect and direct pathways. Most unitary IPSCs were small, but a subset from each pathway were much larger. To determine the effects of these unitary synaptic currents on the action potential firing of GPe neurons, we drove SPNs to fire as before and recorded the membrane potential of GPe neurons. Large unitary potentials from iSPNs and dSPNs perturbed the spike timing of GPe neurons in a similar way. Most SPN-GPe neuron pairs are weakly connected, but a subset of pairs in both pathways are strongly connected. This is the first study to record the synaptic currents generated by single identified direct or indirect pathway striatal neurons on single pallidal neurons. Each GPe neuron receives synaptic inputs from both pathways. Most striatal neurons generate small synaptic currents that become influential when occurring together, but a few are powerful enough to be individually influential.
Topics: Animals; Mice; Neurons; Inhibitory Postsynaptic Potentials; Optogenetics; Corpus Striatum; Globus Pallidus; Action Potentials; Male; Mice, Inbred C57BL; Female; Neural Pathways; Synapses
PubMed: 38596834
DOI: 10.1152/jn.00066.2024 -
The Journal of Physiology Apr 2024We used whole-cell patch clamp to estimate the stationary voltage dependence of persistent sodium-current density (i) in rat hippocampal mossy fibre boutons. Cox's...
We used whole-cell patch clamp to estimate the stationary voltage dependence of persistent sodium-current density (i) in rat hippocampal mossy fibre boutons. Cox's method for correcting space-clamp errors was extended to the case of an isopotential compartment with attached neurites. The method was applied to voltage-ramp experiments, in which i is assumed to gate instantaneously. The raw estimates of i led to predicted clamp currents that were at variance with observation, hence an algorithm was devised to improve these estimates. Optionally, the method also allows an estimate of the membrane specific capacitance, although values of the axial resistivity and seal resistance must be provided. Assuming that membrane specific capacitance and axial resistivity were constant, we conclude that seal resistance continued to fall after adding TTX to the bath. This might have been attributable to a further deterioration of the seal after baseline rather than an unlikely effect of TTX. There was an increase in the membrane specific resistance in TTX. The reason for this is unknown, but it meant that i could not be determined by simple subtraction. Attempts to account for i with a Hodgkin-Huxley model of the transient sodium conductance met with mixed results. One thing to emerge was the importance of voltage shifts. Also, a large variability in previously reported values of transient sodium conductance in mossy fibre boutons made comparisons with our results difficult. Various other possible sources of error are discussed. Simulations suggest a role for i in modulating the axonal attenuation of EPSPs. KEY POINTS: We used whole-cell patch clamp to estimate the stationary voltage dependence of persistent sodium-current density (i) in rat hippocampal mossy fibre boutons, using a KCl-based internal (pipette) solution and correcting for the liquid junction potential (2 mV). Space-clamp errors and deterioration of the patch-clamp seal during the experiment were corrected for by compartmental modelling. Attempts to account for i in terms of the transient sodium conductance met with mixed results. One possibility is that the transient sodium conductance is higher in mossy fibre boutons than in the axon shaft. The analysis illustrates the need to account for various voltage shifts (Donnan potentials, liquid junction potentials and, possibly, other voltage shifts). Simulations suggest a role for i in modulating the axonal attenuation of excitatory postsynaptic potentials, hence analog signalling by dentate granule cells.
Topics: Rats; Animals; Mossy Fibers, Hippocampal; Sodium; Presynaptic Terminals
PubMed: 38594842
DOI: 10.1113/JP284657 -
The Journal of Biological Chemistry May 2024Synapse formation depends on the coordinated expression and regulation of scaffold proteins. The JNK family kinases play a role in scaffold protein regulation, but the...
Synapse formation depends on the coordinated expression and regulation of scaffold proteins. The JNK family kinases play a role in scaffold protein regulation, but the nature of this functional interaction in dendritic spines requires further investigation. Here, using a combination of biochemical methods and live-cell imaging strategies, we show that the dynamics of the synaptic scaffold molecule SAP102 are negatively regulated by JNK inhibition, that SAP102 is a direct phosphorylation target of JNK3, and that SAP102 regulation by JNK is restricted to neurons that harbor mature synapses. We further demonstrate that SAP102 and JNK3 cooperate in the regulated trafficking of kainate receptors to the cell membrane. Specifically, we observe that SAP102, JNK3, and the kainate receptor subunit GluK2 exhibit overlapping expression at synaptic sites and that modulating JNK activity influences the surface expression of the kainate receptor subunit GluK2 in a neuronal context. We also show that SAP102 participates in this process in a JNK-dependent fashion. In summary, our data support a model in which JNK-mediated regulation of SAP102 influences the dynamic trafficking of kainate receptors to postsynaptic sites, and thus shed light on common pathophysiological mechanisms underlying the cognitive developmental defects associated with diverse mutations.
Topics: Animals; Humans; Rats; Cell Membrane; Dendritic Spines; GluK2 Kainate Receptor; Hippocampus; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mitogen-Activated Protein Kinase 10; Neurons; Neuropeptides; Phosphorylation; Protein Transport; Receptors, Kainic Acid; Synapses; Cells, Cultured
PubMed: 38582451
DOI: 10.1016/j.jbc.2024.107263 -
Frontiers in Synaptic Neuroscience 2024Epileptiform activity is the most striking result of hyperexcitation of a group of neurons that can occur in different brain regions and then spread to other sites....
INTRODUCTION
Epileptiform activity is the most striking result of hyperexcitation of a group of neurons that can occur in different brain regions and then spread to other sites. Later it was shown that these rhythms have a cellular correlate called paroxysmal depolarization shift (PDS). In 13-15 DIV neuron-glial cell culture, inhibition of the GABA(A) receptors induces bursts of action potential in the form of clasters PDS and oscillations of intracellular Ca concentration ([Ca]). We demonstrate that GABAergic neurons expressing calcium-permeable AMPA receptors (CP-AMPARs) as well as Kv7-type potassium channels regulate hippocampal glutamatergic neurons' excitability during epileptiform activity in culture.
METHODS
A combination of whole-cell patch-clamp in current clamp mode and calcium imaging microscopy was used to simultaneously register membrane potential and [Ca] level. To identify GABAergic cell cultures were fixed and stained with antibodies against glutamate decarboxylase GAD 65/67 and neuron-specific enolase (NSE) after vital [Ca] imaging.
RESULTS AND DISCUSSION
It was shown that CP-AMPARs are involved in the regulation of the PDS clusters and [Ca] pulses accompanied them. Activation of CP-AMPARs of GABAergic neurons is thought to cause the release of GABA, which activates the GABA(B) receptors of other GABAergic interneurons. It is assumed that activation of these GABA(B) receptors leads to the release of beta-gamma subunits of Gi protein, which activate potassium channels, resulting in hyperpolarization and inhibition of these interneurons. The latter causes disinhibition of glutamatergic neurons, the targets of these interneurons. In turn, the CP-AMPAR antagonist, NASPM, has the opposite effect. Measurement of membrane potential in GABAergic neurons by the patch-clamp method in whole-cell configuration demonstrated that NASPM suppresses hyperpolarization in clusters and individual PDSs. It is believed that Kv7-type potassium channels are involved in the control of hyperpolarization during epileptiform activity. The blocker of Kv7 channels, XE 991, mimicked the effect of the CP-AMPARs antagonist on PDS clusters. Both drugs increased the duration of the PDS cluster. In turn, the Kv7 activator, retigabine, decreased the duration of the PDS cluster and Ca pulse. In addition, retigabine led to deep posthyperpolarization at the end of the PDS cluster. The Kv7 channel is believed to be involved in the formation of PDS, as the channel blocker reduced the rate of hyperpolarization in the PDS almost three times. Thus, GABAergic neurons expressing CP-AMPARs, regulate the membrane potential of innervated glutamatergic neurons by modulating the activity of postsynaptic potassium channels of other GABAergic neurons.
PubMed: 38577639
DOI: 10.3389/fnsyn.2024.1349984 -
The European Journal of Neuroscience Jun 2024The postsynaptic density (PSD) is a collection of specialized proteins assembled beneath the postsynaptic membrane of dendritic spines. The PSD proteome comprises ~1000... (Review)
Review
The postsynaptic density (PSD) is a collection of specialized proteins assembled beneath the postsynaptic membrane of dendritic spines. The PSD proteome comprises ~1000 proteins, including neurotransmitter receptors, scaffolding proteins and signalling enzymes. Many of these proteins have essential roles in synaptic function and plasticity. During brain development, changes are observed in synapse density and in the stability and shape of spines, reflecting the underlying molecular maturation of synapses. Synaptic protein composition changes in terms of protein abundance and the assembly of protein complexes, supercomplexes and the physical organization of the PSD. Here, we summarize the developmental alterations of postsynaptic protein composition during synapse maturation. We describe major PSD proteins involved in postsynaptic signalling that regulates synaptic plasticity and discuss the effect of altered expression of these proteins during development. We consider the abnormality of synaptic profiles and synaptic protein composition in the brain in neurodevelopmental disorders such as autism spectrum disorders. We also explain differences in synapse development between rodents and primates in terms of synaptic profiles and protein composition. Finally, we introduce recent findings related to synaptic diversity and nanoarchitecture and discuss their impact on future research. Synaptic protein composition can be considered a major determinant and marker of synapse maturation in normality and disease.
Topics: Animals; Humans; Synapses; Nerve Tissue Proteins; Neuronal Plasticity; Post-Synaptic Density; Brain
PubMed: 38571321
DOI: 10.1111/ejn.16304 -
Proceedings of the National Academy of... Apr 2024Stable matching of neurotransmitters with their receptors is fundamental to synapse function and reliable communication in neural circuits. Presynaptic neurotransmitters...
Stable matching of neurotransmitters with their receptors is fundamental to synapse function and reliable communication in neural circuits. Presynaptic neurotransmitters regulate the stabilization of postsynaptic transmitter receptors. Whether postsynaptic receptors regulate stabilization of presynaptic transmitters has received less attention. Here, we show that blockade of endogenous postsynaptic acetylcholine receptors (AChR) at the neuromuscular junction destabilizes the cholinergic phenotype in motor neurons and stabilizes an earlier, developmentally transient glutamatergic phenotype. Further, expression of exogenous postsynaptic gamma-aminobutyric acid type A receptors (GABA receptors) in muscle cells stabilizes an earlier, developmentally transient GABAergic motor neuron phenotype. Both AChR and GABA receptors are linked to presynaptic neurons through transsynaptic bridges. Knockdown of specific components of these transsynaptic bridges prevents stabilization of the cholinergic or GABAergic phenotypes. Bidirectional communication can enforce a match between transmitter and receptor and ensure the fidelity of synaptic transmission. Our findings suggest a potential role of dysfunctional transmitter receptors in neurological disorders that involve the loss of the presynaptic transmitter.
Topics: Synapses; Receptors, Cholinergic; Synaptic Transmission; Motor Neurons; Receptors, GABA-A; gamma-Aminobutyric Acid; Neurotransmitter Agents; Cholinergic Agents; Receptors, Presynaptic
PubMed: 38568976
DOI: 10.1073/pnas.2318041121 -
Cellular and Molecular Neurobiology Apr 2024The phenomenon of ischemic postconditioning (PostC) is known to be neuroprotective against ischemic reperfusion (I/R) injury. One of the key processes in PostC is the...
The phenomenon of ischemic postconditioning (PostC) is known to be neuroprotective against ischemic reperfusion (I/R) injury. One of the key processes in PostC is the opening of the mitochondrial ATP-dependent potassium (mito-K) channel and depolarization of the mitochondrial membrane, triggering the release of calcium ions from mitochondria through low-conductance opening of the mitochondrial permeability transition pore. Mitochondrial calcium uniporter (MCU) is known as a highly sensitive transporter for the uptake of Ca present on the inner mitochondrial membrane. The MCU has attracted attention as a new target for treatment in diseases, such as neurodegenerative diseases, cancer, and ischemic stroke. We considered that the MCU may be involved in PostC and trigger its mechanisms. This research used the whole-cell patch-clamp technique on hippocampal CA1 pyramidal cells from C57BL mice and measured changes in spontaneous excitatory post-synaptic currents (sEPSCs), intracellular Ca concentration, mitochondrial membrane potential, and N-methyl-D-aspartate receptor (NMDAR) currents under inhibition of MCU by ruthenium red 265 (Ru265) in PostC. Inhibition of MCU increased the occurrence of sEPSCs (p = 0.014), NMDAR currents (p < 0.001), intracellular Ca concentration (p < 0.001), and dead cells (p < 0.001) significantly after reperfusion, reflecting removal of the neuroprotective effects in PostC. Moreover, mitochondrial depolarization in PostC with Ru265 was weakened, compared to PostC (p = 0.004). These results suggest that MCU affects mitochondrial depolarization in PostC to suppress NMDAR over-activation and prevent elevation of intracellular Ca concentrations against I/R injury.
Topics: Animals; Mice; Mice, Inbred C57BL; Ischemic Postconditioning; Receptors, N-Methyl-D-Aspartate; Brain Injuries; Adenosine Triphosphate; Calcium Channels; Ruthenium Compounds
PubMed: 38568450
DOI: 10.1007/s10571-024-01464-7