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Function (Oxford, England) 2023Small-conductance calcium-activated potassium (SK) channels show a ubiquitous distribution on neurons, in both somatodendritic and axonal regions. SK channels are...
Small-conductance calcium-activated potassium (SK) channels show a ubiquitous distribution on neurons, in both somatodendritic and axonal regions. SK channels are associated with neuronal activity regulating action potential frequency, dendritic excitability, and synaptic plasticity. Although the physiology of SK channels and the mechanisms that control their surface expression levels have been investigated extensively, little is known about what controls SK channel diffusion in the neuronal plasma membrane. This aspect is important, as the diffusion of SK channels at the surface may control their localization and proximity to calcium channels, hence increasing the likelihood of SK channel activation by calcium. In this study, we successfully investigated the diffusion of SK channels labeled with quantum dots on human embryonic kidney cells and dissociated hippocampal neurons by combining a single-particle tracking method with total internal reflection fluorescence microscopy. We observed that actin filaments interfere with SK mobility, decreasing their diffusion coefficient. We also found that during neuronal maturation, SK channel diffusion was gradually inhibited in somatodendritic compartments. Importantly, we observed that axon barriers formed at approximately days 6 and restricted the diffusion of SK channels on the axon initial segment (AIS). However, after neuron maturation, SK channels on the AIS were strongly immobilized, even after disruption of the actin network, suggesting that crowding may cause this effect. Altogether, our work provides insight into how SK channels diffuse on the neuronal plasma membrane and how actin and membrane crowding impacts SK channel diffusion.
Topics: Humans; Small-Conductance Calcium-Activated Potassium Channels; Actins; Neurons; Axons; Hippocampus
PubMed: 37168495
DOI: 10.1093/function/zqad018 -
The Journal of Neuroscience : the... May 2023
Topics: Axons; Axon Initial Segment; Nerve Regeneration; Motor Neurons; Synapses
PubMed: 37137706
DOI: 10.1523/JNEUROSCI.2326-22.2023 -
PloS One 2023Chandelier (Ch) cells are cortical interneurons with axon terminal structures known as cartridges that synapse on the axon initial segment of excitatory pyramidal...
Chandelier (Ch) cells are cortical interneurons with axon terminal structures known as cartridges that synapse on the axon initial segment of excitatory pyramidal neurons. Previous studies indicate that the number of Ch cells is decreased in autism, and that GABA receptors are decreased in the Ch cell synaptic target in the prefrontal cortex. To further identify Ch cell alterations, we examined whether the length of cartridges, and the number, density, and size of Ch cell synaptic boutons, differed in the prefrontal cortex of cases with autism versus control cases. We collected samples of postmortem human prefrontal cortex (Brodmann Area (BA) 9, 46, and 47) from 20 cases with autism and 20 age- and sex-matched control cases. Ch cells were labeled using an antibody against parvalbumin, a marker that labeles soma, cartridges, and synaptic boutons. We found no significant difference in the average length of cartridges, or in the total number or density of boutons in control subjects vs. subjects with autism. However, we found a significant decrease in the size of Ch cell boutons in those with autism. The reduced size of Ch cell boutons may result in reduced inhibitory signal transmission and impact the balance of excitation to inhibition in the prefrontal cortex in autism.
Topics: Humans; Presynaptic Terminals; Autistic Disorder; Neurons; Axons; Pyramidal Cells; Prefrontal Cortex
PubMed: 37097993
DOI: 10.1371/journal.pone.0281477 -
Frontiers in Neuroanatomy 2023The axon initial segment (AIS) is the site of action potential initiation and important for the integration of synaptic input. Length and localization of the AIS are...
The axon initial segment (AIS) is the site of action potential initiation and important for the integration of synaptic input. Length and localization of the AIS are dynamic, modulated by afferent activity and contribute to the homeostatic control of neuronal excitability. Synaptopodin is a plasticity-related protein expressed by the majority of telencephalic neurons. It is required for the formation of cisternal organelles within the AIS and an excellent marker to identify these enigmatic organelles at the light microscopic level. Here we applied 2 h of high frequency stimulation of the medial perforant path in rats to induce a strong long-term potentiation of dentate gyrus granule cells. Immunolabeling for βIV-spectrin and synaptopodin were performed to study structural changes of the AIS and its cisternal organelles. Three-dimensional analysis of the AIS revealed a shortening of the AIS and a corresponding reduction of the number of synaptopodin clusters. These data demonstrate a rapid structural plasticity of the AIS and its cisternal organelles to strong stimulation, indicating a homeostatic response of the entire AIS compartment.
PubMed: 37090138
DOI: 10.3389/fnana.2023.1125623 -
Cell and Tissue Research Jul 2023Dorsal root ganglia (DRG) contains thousands of sensory neurons that transmit information about our external and internal environment to the central nervous system. This... (Review)
Review
Dorsal root ganglia (DRG) contains thousands of sensory neurons that transmit information about our external and internal environment to the central nervous system. This includes signals related to proprioception, temperature, and nociception. Our understanding of DRG has increased tremendously over the last 50 years and has established the DRG as an active participant in peripheral processes. This includes interactions between neurons and non-neuronal cells such as satellite glia cells and macrophages that contribute to an increasingly complex cellular environment that modulates neuronal function. Early ultrastructural investigations of the DRG have described subtypes of sensory neurons based on differences in the arrangement of organelles such as the Golgi apparatus and the endoplasmic reticulum. The neuron-satellite cell complex and the composition of the axon hillock in DRG have also been investigated, but, apart from basic descriptions of Schwann cells, ultrastructural investigations of other cell types in DRG are limited. Furthermore, detailed descriptions of key components of DRG, such as blood vessels and the capsule that sits at the intersection of the meninges and the connective tissue covering the peripheral nervous system, are lacking to date. With rising interest in DRG as potential therapeutic targets for aberrant signalling associated with chronic pain conditions, gaining further insights into DRG ultrastructure will be fundamental to understanding cell-cell interactions that modulate DRG function. In this review, we aim to provide a synopsis of the current state of knowledge on the ultrastructure of the DRG and its components, as well as to identify areas of interest for future studies.
Topics: Humans; Ganglia, Spinal; Neuroglia; Schwann Cells; Sensory Receptor Cells; Pain
PubMed: 37079097
DOI: 10.1007/s00441-023-03770-w -
AIMS Neuroscience 2023In the fear memory network, the hippocampus modulates contextual aspects of fear learning while mutual connections between the amygdala and the medial prefrontal cortex...
BACKGROUND
In the fear memory network, the hippocampus modulates contextual aspects of fear learning while mutual connections between the amygdala and the medial prefrontal cortex are widely involved in fear extinction. G-protein-coupled receptors (GPCRs) are involved in the regulation of fear and anxiety, so the regulation of GPCRs in fear signaling pathways can modulate the mechanisms of fear memory acquisition, consolidation and extinction. Various studies suggested a role of M-type K+ channels in modulating fear expression and extinction, although conflicting data prevented drawing of clear conclusions. In the present work, we examined the impact of M-type K+ channel blockade or activation on contextual fear acquisition and extinction. In addition, regarding the pivotal role of the hippocampus in contextual fear conditioning (CFC) and the involvement of the axon initial segment (AIS) in neuronal plasticity, we investigated whether structural alterations of the AIS in hippocampal neurons occurred during contextual fear memory acquisition and short-time extinction in mice in a behaviorally relevant context.
RESULTS
When a single systemic injection of the M-channel blocker XE991 (2 mg/kg, IP) was carried out 15 minutes before the foot shock session, fear expression was significantly reduced. Expression of c-Fos was increased following CFC, mostly in GABAergic neurons at day 1 and day 2 post-fear training in CA1 and dentate gyrus hippocampal regions. A significantly longer AIS segment was observed in GABAergic neurons of the CA1 hippocampal region at day 2.
CONCLUSIONS
Our results underscore the role of M-type K + channels in CFC and the importance of hippocampal GABAergic neurons in fear expression.
PubMed: 37077956
DOI: 10.3934/Neuroscience.2023003 -
Anatomical Science International Jul 2023Morphological analysis of organelles is one of the important clues for understanding the cellular conditions and mechanisms occurring in cells. In particular, nanoscale... (Review)
Review
Morphological analysis of organelles is one of the important clues for understanding the cellular conditions and mechanisms occurring in cells. In particular, nanoscale information within crowded intracellular organelles of tissues provide more direct implications when compared to analyses of cells in culture or isolation. However, there are some difficulties in detecting individual shape using light microscopy, including super-resolution microscopy. Transmission electron microscopy (TEM), wherein the ultrastructure can be imaged at the membrane level, cannot determine the whole structure, and analyze it quantitatively. Volume EM, such as focused ion beam/scanning electron microscopy (FIB/SEM), can be a powerful tool to explore the details of three-dimensional ultrastructures even within a certain volume, and to measure several parameters from them. In this review, the advantages of FIB/SEM analysis in organelle studies are highlighted along with the introduction of mitochondrial analysis in injured motor neurons. This would aid in understanding the morphological details of mitochondria, especially those distributed in the cell bodies as well as in the axon initial segment (AIS) in mouse tissues. These regions have not been explored thus far due to the difficulties encountered in accessing their images by conditional microscopies. Some mechanisms of nerve regeneration have also been discussed with reference to the obtained findings. Finally, future perspectives on FIB/SEM are introduced. The combination of biochemical and genetic understanding of organelle structures and a nanoscale understanding of their three-dimensional distribution and morphology will help to match achievements in genomics and structural biology.
Topics: Mice; Animals; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Motor Neurons; Volume Electron Microscopy; Organelles; Imaging, Three-Dimensional
PubMed: 37071350
DOI: 10.1007/s12565-023-00720-y -
Frontiers in Neuroscience 2023Identification of early adaptive and maladaptive neuronal stress responses is an important step in developing targeted neuroprotective therapies for degenerative...
INTRODUCTION
Identification of early adaptive and maladaptive neuronal stress responses is an important step in developing targeted neuroprotective therapies for degenerative disease. In glaucoma, retinal ganglion cells (RGCs) and their axons undergo progressive degeneration resulting from stress driven by sensitivity to intraocular pressure (IOP). Despite therapies that can effectively manage IOP many patients progress to vision loss, necessitating development of neuronal-based therapies. Evidence from experimental models of glaucoma indicates that early in the disease RGCs experience altered excitability and are challenged with dysregulated potassium (K) homeostasis. Previously we demonstrated that certain RGC types have distinct excitability profiles and thresholds for depolarization block, which are associated with sensitivity to extracellular K.
METHODS
Here, we used our inducible mouse model of glaucoma to investigate how RGC sensitivity to K changes with exposure to elevated IOP.
RESULTS
In controls, conditions of increased K enhanced membrane depolarization, reduced action potential generation, and widened action potentials. Consistent with our previous work, 4 weeks of IOP elevation diminished RGC light-and current-evoked responses. Compared to controls, we found that IOP elevation reduced the effects of increased K on depolarization block threshold, with IOP-exposed cells maintaining greater excitability. Finally, IOP elevation did not alter axon initial segment dimensions, suggesting that structural plasticity alone cannot explain decreased K sensitivity.
DISCUSSION
Thus, in response to prolonged IOP elevation RGCs undergo an adaptive process that reduces sensitivity to changes in K while diminishing excitability. These experiments give insight into the RGC response to IOP stress and lay the groundwork for mechanistic investigation into targets for neuroprotective therapy.
PubMed: 37051140
DOI: 10.3389/fnins.2023.1142668 -
Neurology(R) Neuroimmunology &... May 2023Autoantibodies to leucine-rich glioma inactivated protein 1 (LGI1) cause an autoimmune limbic encephalitis with frequent focal seizures and anterograde memory...
BACKGROUND AND OBJECTIVES
Autoantibodies to leucine-rich glioma inactivated protein 1 (LGI1) cause an autoimmune limbic encephalitis with frequent focal seizures and anterograde memory dysfunction. LGI1 is a neuronal secreted linker protein with 2 functional domains: the leucine-rich repeat (LRR) and epitempin (EPTP) regions. LGI1 autoantibodies are known to interfere with presynaptic function and neuronal excitability; however, their epitope-specific mechanisms are incompletely understood.
METHODS
We used patient-derived monoclonal autoantibodies (mAbs), which target either LRR or EPTP domains of LGI1 to investigate long-term antibody-induced alteration of neuronal function. LRR- and EPTP-specific effects were evaluated by patch-clamp recordings in cultured hippocampal neurons and compared with biophysical neuron modeling. K1.1 channel clustering at the axon initial segment (AIS) was quantified by immunocytochemistry and structured illumination microscopy techniques.
RESULTS
Both EPTP and LRR domain-specific mAbs decreased the latency of first somatic action potential firing. However, only the LRR-specific mAbs increased the number of action potential firing together with enhanced initial instantaneous frequency and promoted spike-frequency adaptation, which were less pronounced after the EPTP mAb. This also led to an effective reduction in the slope of ramp-like depolarization in the subthreshold response, suggesting K1 channel dysfunction. A biophysical model of a hippocampal neuron corroborated experimental results and suggests that an isolated reduction of the conductance of K1-mediated K currents largely accounts for the antibody-induced alterations in the initial firing phase and spike-frequency adaptation. Furthermore, K1.1 channel density was spatially redistributed from the distal toward the proximal site of AIS under LRR mAb treatment and, to a lesser extant, under EPTP mAb.
DISCUSSION
These findings indicate an epitope-specific pathophysiology of LGI1 autoantibodies. The pronounced neuronal hyperexcitability and SFA together with dropped slope of ramp-like depolarization after LRR-targeted interference suggest disruption of LGI1-dependent clustering of K channel complexes. Moreover, considering the effective triggering of action potentials at the distal AIS, the altered spatial distribution of K1.1 channel density may contribute to these effects through impairing neuronal control of action potential initiation and synaptic integration.
Topics: Humans; Antibodies, Monoclonal; Autoantibodies; Epitopes; Intracellular Signaling Peptides and Proteins; Leucine; Nerve Tissue Proteins; Neurons
PubMed: 37028941
DOI: 10.1212/NXI.0000000000200096 -
Translational Vision Science &... Apr 2023Axons depend on long-range transport of proteins and organelles which increases susceptibility to metabolic stress in disease. The axon initial segment (AIS) is...
PURPOSE
Axons depend on long-range transport of proteins and organelles which increases susceptibility to metabolic stress in disease. The axon initial segment (AIS) is particularly vulnerable due to the high bioenergetic demand of action potential generation. Here, we prepared retinal ganglion cells derived from human embryonic stem cells (hRGCs) to probe how axonal stress alters AIS morphology.
METHODS
hRGCs were cultured on coverslips or microfluidic platforms. We assayed AIS specification and morphology by immunolabeling against ankyrin G (ankG), an axon-specific protein, and postsynaptic density 95 (PSD-95), a dendrite-specific protein. Using microfluidic platforms that enable fluidic isolation, we added colchicine to the axon compartment to lesion axons. We verified axonopathy by measuring the anterograde axon transport of cholera toxin subunit B and immunolabeling against cleaved caspase 3 (CC3) and phosphorylated neurofilament H (SMI-34). We determined the influence of axon injury on AIS morphology by immunolabeling samples against ankG and measuring AIS distance from soma and length.
RESULTS
Based on measurements of ankG and PSD-95 immunolabeling, microfluidic platforms promote the formation and separation of distinct somatic-dendritic versus axonal compartments in hRGCs compared to coverslip cultures. Chemical lesioning of axons by colchicine reduced hRGC anterograde axon transport, increased varicosity density, and enhanced expression of CC3 and SMI-34. Interestingly, we found that colchicine selectively affected hRGCs with axon-carrying dendrites by reducing AIS distance from somas and increasing length, thus suggesting reduced capacity to maintain excitability.
CONCLUSIONS
Thus, microfluidic platforms promote polarized hRGCs that enable modeling of axonopathy.
TRANSLATIONAL RELEVANCE
Microfluidic platforms may be used to assay compartmentalized degeneration that occurs during glaucoma.
Topics: Humans; Retinal Ganglion Cells; Microfluidics; Axons; Action Potentials
PubMed: 37010860
DOI: 10.1167/tvst.12.4.1