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1.
Zool Res ; 43(4): 615-633, 2022 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-35758537

RESUMO

Action potentials (APs) in neurons are generated at the axon initial segment (AIS). AP dynamics, including initiation and propagation, are intimately associated with neuronal excitability and neurotransmitter release kinetics. Most learning and memory studies at the single-neuron level have relied on the use of animal models, most notably rodents. Here, we studied AP initiation and propagation in cultured hippocampal neurons from Sprague-Dawley (SD) rats and C57BL/6 (C57) mice with genetically encoded voltage indicator (GEVI)-based voltage imaging. Our data showed that APs traveled bidirectionally in neurons from both species; forward-propagating APs (fpAPs) had a different speed than backpropagating APs (bpAPs). Additionally, we observed distinct AP propagation characteristics in AISs emerging from the somatic envelope compared to those originating from dendrites. Compared with rat neurons, mouse neurons exhibited higher bpAP speed and lower fpAP speed, more distally located ankyrin G (AnkG) in AISs, and longer Nav1.2 lengths in AISs. Moreover, during AIS plasticity, AnkG and Nav1.2 showed distal shifts in location and shorter lengths of labeled AISs in rat neurons; in mouse neurons, however, they showed a longer AnkG-labeled length and more distal Nav1.2 location. Our findings suggest that hippocampal neurons in SD rats and C57 mice may have different AP propagation speeds, different AnkG and Nav1.2 patterns in the AIS, and different AIS plasticity properties, indicating that comparisons between these species must be carefully considered.


Assuntos
Segmento Inicial do Axônio , Potenciais de Ação/fisiologia , Animais , Segmento Inicial do Axônio/fisiologia , Axônios/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios , Ratos , Ratos Sprague-Dawley
2.
Brain ; 145(5): 1632-1640, 2022 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-35661858

RESUMO

The axon initial segment is a specialized compartment of the proximal axon of CNS neurons where action potentials are initiated. However, it remains unknown whether this domain is assembled in sensory dorsal root ganglion neurons, in which spikes are initiated in the peripheral terminals. Here we investigate whether sensory neurons have an axon initial segment and if it contributes to spontaneous activity in neuropathic pain. Our results demonstrate that myelinated dorsal root ganglion neurons assemble an axon initial segment in the proximal region of their stem axon, enriched in the voltage-gated sodium channels Nav1.1 and Nav1.7. Using correlative immunofluorescence and calcium imaging, we demonstrate that the Nav1.7 channels at the axon initial segment are associated with spontaneous activity. Computer simulations further indicate that the axon initial segment plays a key role in the initiation of spontaneous discharges by lowering their voltage threshold. Finally, using a Cre-based mouse model for time-controlled axon initial segment disassembly, we demonstrate that this compartment is a major source of spontaneous discharges causing mechanical allodynia in neuropathic pain. Thus, an axon initial segment domain is present in sensory neurons and facilitates their spontaneous activity. This study provides a new insight in the cellular mechanisms that cause pathological pain and identifies a new potential target for chronic pain management.


Assuntos
Segmento Inicial do Axônio , Neuralgia , Animais , Gânglios Espinais/patologia , Humanos , Hiperalgesia/patologia , Camundongos , Neuralgia/patologia , Células Receptoras Sensoriais
3.
Brain ; 145(5): 1574-1575, 2022 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-35661860
4.
Sci Rep ; 12(1): 8722, 2022 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-35610289

RESUMO

The axon initial segment (AIS) is a region of the neuron that is critical for action potential generation as well as for the regulation of neural activity. This specialized structure-characterized by the expression of different types of ion channels as well as adhesion, scaffolding and cytoskeleton proteins-is subjected to morpho-functional plastic changes in length and position upon variations in neural activity or in pathological conditions. In the present study, using immunocytochemistry with the AT8 antibody (phospho-tau S202/T205) and 3D confocal microscopy reconstruction techniques in brain tissue from Alzheimer's disease patients, we found that around half of the cortical pyramidal neurons with hyperphosphorylated tau showed changes in AIS length and position in comparison with AT8-negative neurons from the same cortical layers. We observed a wide variety of AIS alterations in neurons with hyperphosphorylated tau, although the most common changes were a proximal shift or a lengthening of the AISs. Similar results were found in neocortical tissue from non-demented cases with neurons containing hyperphosphorylated tau. These findings support the notion that the accumulation of phospho-tau is associated with structural alterations of the AIS that are likely to have an impact on normal neuronal activity, which might contribute to neuronal dysfunction in AD.


Assuntos
Doença de Alzheimer , Segmento Inicial do Axônio , Doença de Alzheimer/metabolismo , Humanos , Neurônios/metabolismo , Células Piramidais/metabolismo , Proteínas tau/metabolismo
5.
Microsc Res Tech ; 85(7): 2679-2691, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35411984

RESUMO

The axon initial segment (AIS) plays an important role in maintaining neuronal polarity and initiating action potentials (APs). The AIS adapts to its environment by changing its length and distance from the cell body, resulting in modulation of neuronal excitability, which is referred to as AIS plasticity. Previous studies found an ~200 nm single periodic distribution of the key AIS components ankyrinG (AnkG), Nav 1.2, and ßIV-spectrin, while it remains unclear how the lattice structure is altered by AIS plasticity. In this study, we found that the length of the AIS significantly increased, resulting in increased neuronal excitability, with high-concentration glucose treatment. Structured illumination microscopy (SIM) images of the lattice structure showed a dual-spacing periodic distribution (~200 nm and ~260 nm) of AnkG, Nav 1.2, and ßIV-spectrin. Moreover, 480-kDa AnkG was crucial for AIS plasticity and increased lattice structure spacing. The discovery of new regulators for modulating AIS plasticity will help us to understand and manipulate the structure and function of the AIS. Glucose triggers axon initial segment (AIS) plasticity of cultured neurons. AIS lattice structure under glucose treatment shows an increased spacing by structured illumination microscopy imaging. 480-kDa AnkG contributes to AIS plasticity.


Assuntos
Segmento Inicial do Axônio , Potenciais de Ação/fisiologia , Segmento Inicial do Axônio/fisiologia , Axônios , Glucose , Espectrina
6.
Proc Natl Acad Sci U S A ; 119(11): e2114476119, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35263225

RESUMO

SignificanceChandelier cells (ChCs) are a unique type of GABAergic interneuron that form axo-axonic synapses exclusively on the axon initial segment (AIS) of neocortical pyramidal neurons (PyNs), allowing them to exert powerful yet precise control over PyN firing and population output. The importance of proper ChC function is further underscored by the association of ChC connectivity defects with various neurological conditions. Despite this, the cellular mechanisms governing ChC axo-axonic synapse formation remain poorly understood. Here, we identify microglia as key regulators of ChC axonal morphogenesis and AIS synaptogenesis, and show that disease-induced aberrant microglial activation perturbs proper ChC synaptic development/connectivity in the neocortex. In doing so, such findings highlight the therapeutic potential of manipulating microglia to ensure proper brain wiring.


Assuntos
Segmento Inicial do Axônio , Neurônios GABAérgicos , Microglia , Células Piramidais , Sinapses , Animais , Segmento Inicial do Axônio/fisiologia , Neurônios GABAérgicos/fisiologia , Neurônios GABAérgicos/ultraestrutura , Camundongos , Microglia/fisiologia , Células Piramidais/fisiologia , Células Piramidais/ultraestrutura , Sinapses/fisiologia
7.
J Vis Exp ; (180)2022 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-35225255

RESUMO

The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to the maintenance of neuronal polarity by sorting somato-dendritic cargo. A membrane periodic skeleton (MPS) comprising periodic actin rings provides a scaffold for anchoring various AIS proteins, including structural proteins and different ion channels. Although recent proteomic approaches have identified a considerable number of novel AIS components, details of the structure of the MPS and the roles of its individual components are lacking. The distance between individual actin rings in the MPS (~190 nm) necessitates the employment of super-resolution microscopy techniques to resolve the structural details of the MPS. This protocol describes a method for using cultured rat hippocampal neurons to examine the precise localization of an AIS protein in the MPS relative to sub-membranous actin rings using 3D-structured illumination microscopy (3D-SIM). In addition, an analytical approach to quantitively assess the periodicity of individual components and their position relative to actin rings is also described.


Assuntos
Segmento Inicial do Axônio , Animais , Segmento Inicial do Axônio/metabolismo , Axônios/fisiologia , Iluminação , Microscopia , Proteômica , Ratos , Esqueleto
8.
Handb Clin Neurol ; 184: 481-495, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35034756

RESUMO

To adapt to the sustained demands of chronic stress, discrete brain circuits undergo structural and functional changes often resulting in anxiety disorders. In some individuals, anxiety disorders precede the development of motor symptoms of Parkinson's disease (PD) caused by degeneration of neurons in the substantia nigra (SN). Here, we present a circuit framework for probing a causal link between chronic stress, anxiety, and PD, which postulates a central role of abnormal neuromodulation of the SN's axon initial segment by brainstem inputs. It is grounded in findings demonstrating that the earliest PD pathologies occur in the stress-responsive, emotion regulation network of the brainstem, which provides the SN with dense aminergic and cholinergic innervation. SN's axon initial segment (AIS) has unique features that support the sustained and bidirectional propagation of activity in response to synaptic inputs. It is therefore, especially sensitive to circuit-mediated stress-induced imbalance of neuromodulation, and thus a plausible initiating site of neurodegeneration. This could explain why, although secondary to pathophysiologies in other brainstem nuclei, SN degeneration is the most extensive. Consequently, the cardinal symptom of PD, severe motor deficits, arise from degeneration of the nigrostriatal pathway rather than other brainstem nuclei. Understanding when and how circuit dysfunctions underlying anxiety can progress to neurodegeneration, raises the prospect of timed interventions for reversing, or at least impeding, the early pathophysiologies that lead to PD and possibly other neurodegenerative disorders.


Assuntos
Segmento Inicial do Axônio , Doença de Parkinson , Ansiedade , Transtornos de Ansiedade , Humanos , Substância Negra
9.
Neurobiol Dis ; 164: 105609, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34990802

RESUMO

We recently described new pathogenic variants in VRK1, in patients affected with distal Hereditary Motor Neuropathy associated with upper motor neurons signs. Specifically, we provided evidences that hiPSC-derived Motor Neurons (hiPSC-MN) from these patients display Cajal Bodies (CBs) disassembly and defects in neurite outgrowth and branching. We here focused on the Axonal Initial Segment (AIS) and the related firing properties of hiPSC-MNs from these patients. We found that the patient's Action Potential (AP) was smaller in amplitude, larger in duration, and displayed a more depolarized threshold while the firing patterns were not altered. These alterations were accompanied by a decrease in the AIS length measured in patients' hiPSC-MNs. These data indicate that mutations in VRK1 impact the AP waveform and the AIS organization in MNs and may ultimately lead to the related motor neuron disease.


Assuntos
Potenciais de Ação/fisiologia , Segmento Inicial do Axônio/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neurônios Motores/fisiologia , /genética , Linhagem Celular , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas , Doença dos Neurônios Motores/genética , Doença dos Neurônios Motores/metabolismo , Doença dos Neurônios Motores/fisiopatologia , Mutação , Mioblastos/metabolismo
10.
Neurochem Int ; 153: 105273, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34971749

RESUMO

The axon initial segment (AIS) is a structural neuronal compartment of the proximal axon that plays key roles in sodium channel clustering, action potential initiation, and signal propagation of neuronal outputs. Mutations in constitutive genes of the AIS, such as ANK3, have been identified in patients with neurodevelopmental disorders. Nevertheless, morphological changes in the AIS in neurodevelopmental disorders have not been characterized. In this study, we investigated the length of the AIS in rodent models of attention-deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD). We observed abnormalities in AIS length in both animal models. In ADHD model rodents, we observed shorter AIS length in layer 2/3 (L2/3) neurons of the medial prefrontal cortex (mPFC) and primary somatosensory barrel field (S1BF). Further, we observed shorter AIS length in S1BF L5 neurons. In ASD model mice, we observed shorter AIS length in L2/3 and L5 neurons of the S1BF. These results suggest that impairments in AIS length are common phenomena in neurodevelopmental disorders such as ADHD and ASD and may be conserved across species. Our findings provide novel insight into the potential contribution of the AIS to the pathophysiology and pathogenesis of neurodevelopmental disorders.


Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Transtorno do Espectro Autista , Segmento Inicial do Axônio , Transtornos do Neurodesenvolvimento , Animais , Transtorno do Deficit de Atenção com Hiperatividade/genética , Transtorno do Espectro Autista/genética , Humanos , Camundongos , Roedores
11.
PLoS One ; 16(11): e0259918, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34797870

RESUMO

The axon initial segment (AIS) responsible for action potential initiation is a dynamic structure that varies and changes together with neuronal excitability. Like other neuron types, alpha motoneurons in the mammalian spinal cord express heterogeneity and plasticity in AIS geometry, including length (AISl) and distance from soma (AISd). The present study aimed to establish the relationship of AIS geometry with a measure of intrinsic excitability, rheobase current, that varies by 20-fold or more among normal motoneurons. We began by determining whether AIS length or distance differed for motoneurons in motor pools that exhibit different activity profiles. Motoneurons sampled from the medial gastrocnemius (MG) motor pool exhibited values for average AISd that were significantly greater than that for motoneurons from the soleus (SOL) motor pool, which is more readily recruited in low-level activities. Next, we tested whether AISd covaried with intrinsic excitability of individual motoneurons. In anesthetized rats, we measured rheobase current intracellularly from MG motoneurons in vivo before labeling them for immunohistochemical study of AIS structure. For 16 motoneurons sampled from the MG motor pool, this combinatory approach revealed that AISd, but not AISl, was significantly related to rheobase, as AIS tended to be located further from the soma on motoneurons that were less excitable. Although a causal relation with excitability seems unlikely, AISd falls among a constellation of properties related to the recruitability of motor units and their parent motoneurons.


Assuntos
Segmento Inicial do Axônio/metabolismo , Segmento Inicial do Axônio/fisiologia , Neurônios Motores/fisiologia , Potenciais de Ação/fisiologia , Animais , Segmento Inicial do Axônio/patologia , Axônios/metabolismo , Axônios/patologia , Eletrofisiologia , Masculino , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Músculos/fisiologia , Condução Nervosa , Neurônios Eferentes/fisiologia , Ratos , Ratos Wistar , Medula Espinal/fisiologia
12.
eNeuro ; 8(5)2021.
Artigo em Inglês | MEDLINE | ID: mdl-34531281

RESUMO

Recent evidence suggests that alteration of axon initial segment (AIS) geometry (i.e., length or location along the axon) contributes to CNS dysfunction in neurological diseases. For example, AIS length is shorter in the prefrontal cortex of type 2 diabetic mice with cognitive impairment. To determine the key type 2 diabetes-related factor that produces AIS shortening we modified levels of insulin, glucose, or the reactive glucose metabolite methylglyoxal in cultures of dissociated cortices from male and female mice and quantified AIS geometry using immunofluorescent imaging of the AIS proteins AnkyrinG and ßIV spectrin. Neither insulin nor glucose modification altered AIS length. Exposure to 100 but not 1 or 10 µm methylglyoxal for 24 h resulted in accumulation of the methylglyoxal-derived advanced glycation end-product hydroimidazolone and produced reversible AIS shortening without cell death. Methylglyoxal-evoked AIS shortening occurred in both excitatory and putative inhibitory neuron populations and in the presence of tetrodotoxin (TTX). In single-cell recordings resting membrane potential was depolarized at 0.5-3 h and returned to normal at 24 h. In multielectrode array (MEA) recordings methylglyoxal produced an immediate ∼300% increase in spiking and bursting rates that returned to normal within 2 min, followed by a ∼20% reduction of network activity at 0.5-3 h and restoration of activity to baseline levels at 24 h. AIS length was unchanged at 0.5-3 h despite the presence of depolarization and network activity reduction. Nevertheless, these results suggest that methylglyoxal could be a key mediator of AIS shortening and disruptor of neuronal function during type 2 diabetes.


Assuntos
Segmento Inicial do Axônio , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Animais , Feminino , Masculino , Camundongos , Neurônios , Aldeído Pirúvico
13.
Mol Neurobiol ; 58(12): 6153-6169, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34458961

RESUMO

The axon initial segment (AIS) is essential for maintaining neuronal polarity, modulating protein transport into the axon, and action potential generation. These functions are supported by a distinctive actin and microtubule cytoskeleton that controls axonal trafficking and maintains a high density of voltage-gated ion channels linked by scaffold proteins to the AIS cytoskeleton. However, our knowledge of the mechanisms and proteins involved in AIS cytoskeleton regulation to maintain or modulate AIS structure is limited. In this context, formins play a significant role in the modulation of actin and microtubules. We show that pharmacological inhibition of formins modifies AIS actin and microtubule characteristics in cultured hippocampal neurons, reducing F-actin density and decreasing microtubule acetylation. Moreover, formin inhibition diminishes sodium channels, ankyrinG and ßIV-spectrin AIS density, and AIS length, in cultured neurons and brain slices, accompanied by decreased neuronal excitability. We show that genetic downregulation of the mDia1 formin by interference RNAs also decreases AIS protein density and shortens AIS length. The ankyrinG decrease and AIS shortening observed in pharmacologically inhibited neurons and neuron-expressing mDia1 shRNAs were impaired by HDAC6 downregulation or EB1-GFP expression, known to increase microtubule acetylation or stability. However, actin stabilization only partially prevented AIS shortening without affecting AIS protein density loss. These results suggest that mDia1 maintain AIS composition and length contributing to the stability of AIS microtubules.


Assuntos
Segmento Inicial do Axônio/metabolismo , Citoesqueleto/metabolismo , Forminas/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Animais , Axônios/metabolismo , Células Cultivadas , Camundongos , Microtúbulos/metabolismo
14.
Cells ; 10(8)2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34440880

RESUMO

The 20-60 µm axon initial segment (AIS) is proximally located at the interface between the axon and cell body. AIS has characteristic molecular and structural properties regulated by the crucial protein, ankyrin-G. The AIS contains a high density of Na+ channels relative to the cell body, which allows low thresholds for the initiation of action potential (AP). Molecular and physiological studies have shown that the AIS is also a key domain for the control of neuronal excitability by homeostatic mechanisms. The AIS has high plasticity in normal developmental processes and pathological activities, such as injury, neurodegeneration, and neurodevelopmental disorders (NDDs). In the first half of this review, we provide an overview of the molecular, structural, and ion-channel characteristics of AIS, AIS regulation through axo-axonic synapses, and axo-glial interactions. In the second half, to understand the relationship between NDDs and AIS, we discuss the activity-dependent plasticity of AIS, the human mutation of AIS regulatory genes, and the pathophysiological role of an abnormal AIS in NDD model animals and patients. We propose that the AIS may provide a potentially valuable structural biomarker in response to abnormal network activity in vivo as well as a new treatment concept at the neural circuit level.


Assuntos
Segmento Inicial do Axônio/patologia , Transtornos do Neurodesenvolvimento/fisiopatologia , Potenciais de Ação , Anquirinas/genética , Anquirinas/metabolismo , Segmento Inicial do Axônio/metabolismo , Humanos , Canais Iônicos/metabolismo , Canais Iônicos/fisiologia , Mutação , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Neuroglia/metabolismo , Plasticidade Neuronal , Espectrina/genética , Espectrina/metabolismo , Sinapses/metabolismo
15.
Prog Neurobiol ; 205: 102123, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34302896

RESUMO

The axon initial segment (AIS) is a specialized region in neurons that encompasses two essential functions, the generation of action potentials and the regulation of the axodendritic polarity. The mechanism controlling the position of the axon initial segment to allow plasticity and regulation of neuron excitability is unclear. Here we demonstrate that plasmalogens, the most abundant ether-phospholipid, are essential for the homeostatic positioning of the AIS. Plasmalogen deficiency is a hallmark of Rhizomelic Chondrodysplasia Punctata (RCDP) and Zellweger spectrum disorders, but Alzheimer's and Parkinson's disease, are also characterized by plasmalogen defects. Neurons lacking plasmalogens displaced the AIS to more distal positions and were characterized by reduced excitability. Treatment with a short-chain alkyl glycerol was able to rescue AIS positioning. Plasmalogen deficiency impaired AKT activation, and we show that inhibition of AKT phosphorylation at Ser473 and Thr308 is sufficient to induce a distal relocation of the AIS. Pathway analysis revealed that downstream of AKT, overtly active ULK1 mediates AIS repositioning. Rescuing the impaired AKT signaling pathway was able to normalize AIS position independently of the biochemical defect. These results unveil a previously unknown mechanism that couples the phospholipid composition of the neuronal membrane to the positional assembly of the AIS.


Assuntos
Segmento Inicial do Axônio , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Condrodisplasia Punctata Rizomélica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Plasmalogênios , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais
16.
J Comp Neurol ; 529(16): 3621-3632, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34235750

RESUMO

The axon initial segment (AIS) is structurally and functionally distinct from other regions of the axon, yet alterations in the milieu of the AIS after brain injury have not been well characterized. In this study, we have examined extracellular and intracellular changes in the AIS after hypoglossal nerve injury. Microglial adhesions to the AIS were rarely observed in healthy controls, whereas microglial adhesions to the AIS became apparent in the axonal injury model. Regarding intra-AIS morphology, we focused on mitochondria because mitochondrial flow into the injured axon appears critical for axonal regeneration. To visualize mitochondria specifically in injured axons, we used Atf3:BAC transgenic mice whose mitochondria were labeled with GFP in response to nerve injury. These mice clearly showed mitochondrial localization in the AIS after nerve injury. To precisely confirm the light microscopic observations, we performed three-dimensional ultrastructural analysis using focused ion beam/scanning electron microscopy (FIB/SEM). Although the healthy AIS was not surrounded by microglia, tight microglial adhesions with thick processes adhering to the AIS were observed after injury. FIB/SEM simultaneously allowed the observation of mitochondrial localization in the AIS. In the AIS of non-injured neurons, few mitochondria were observed, whereas mitochondria were abundantly localized in the cell body, axon hillock, and axon. Intriguingly, in the injured AIS, numerous mitochondria were observed throughout the AIS. Taken together, axonal injury changes the extracellular glial environment surrounding the AIS and intracellular mitochondrial localization in the AIS. These changes would be crucial responses, perhaps for injured neurons to regenerate after axonal injury.


Assuntos
Segmento Inicial do Axônio/fisiologia , Axônios/fisiologia , Espaço Extracelular/fisiologia , Mitocôndrias/fisiologia , Neuroglia/fisiologia , Fator 3 Ativador da Transcrição/genética , Animais , Segmento Inicial do Axônio/ultraestrutura , Axônios/ultraestrutura , Adesão Celular , Feminino , Humanos , Imageamento Tridimensional , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/ultraestrutura , Compressão Nervosa , Neuroglia/ultraestrutura
17.
J Comp Neurol ; 529(16): 3593-3620, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34219229

RESUMO

Signal processing within the retina is generally mediated by graded potentials, whereas output is conveyed by action potentials transmitted along optic nerve axons. Among retinal neurons, amacrine cells seem to be an exception to this general rule, as several types generate voltage-gated Na+ (Nav ) channel-dependent action potentials. The AII, a narrow-field, bistratified axon-less amacrine cell found in mammalian retinas, displays a unique process that resembles an axon initial segment (AIS), with expression of Nav channels colocalized with the cytoskeletal protein ankyrin-G, and generates action potentials. As the role of spiking in AIIs is uncertain, we hypothesized that the morphological properties of the AIS-like process could provide information relevant for its functional importance, including potential pre- and/or postsynaptic connectivity. For morphological analysis, we injected AII amacrine cells in slices with fluorescent dye and immunolabeled the slices for ankyrin-G. Subsequently, this enabled us to reliably identify AII-type processes among ankyrin-G-labeled processes in wholemount retina. We systematically analyzed the laminar localization, spatial orientation, and distribution of the AIS-like processes as a function of retinal eccentricity. In the horizontal plane, the processes displayed no preferred orientation and terminal endings were randomly distributed. In the vertical plane, the processes displayed a horizontal preference, but also ascended and descended into the inner nuclear layer and proximal inner plexiform layer, respectively. These results suggest that the AII amacrine AIS-like process is unlikely to take part in conventional synaptic connections, but may instead be adapted to respond to volume neurotransmission by means of extrasynaptic receptors.


Assuntos
Células Amácrinas/ultraestrutura , Segmento Inicial do Axônio/ultraestrutura , Axônios/ultraestrutura , Retina/ultraestrutura , Potenciais de Ação/fisiologia , Animais , Anquirinas/fisiologia , Dendritos , Feminino , Masculino , Ratos , Ratos Wistar , Canais de Sódio/fisiologia , Transmissão Sináptica
18.
J Mol Biol ; 433(20): 167176, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34303720

RESUMO

The axon initial segment (AIS) is a distinct neuronal domain, which is responsible for initiating action potentials, and therefore of key importance to neuronal signaling. To determine how it functions, it is necessary to establish which proteins reside there, how they are organized, and what the dynamic features are. Great strides have been made in recent years, and it is now clear that several AIS cytoskeletal and membrane proteins interact to form a higher-order periodic structure. Here we briefly describe AIS function, protein composition and molecular architecture, and discuss perspectives for future structural characterization, and if structure predictions will be able to model complex higher-order assemblies.


Assuntos
Segmento Inicial do Axônio/química , Potenciais de Ação , Animais , Segmento Inicial do Axônio/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Neurônios/química , Neurônios/citologia , Neurônios/metabolismo , Conformação Proteica
19.
J Neuroendocrinol ; 33(7): e12969, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33890333

RESUMO

Axon initial segments (AIS) of dentate granule cells in the hippocampus exhibit prominent spines (AISS) during early development that are associated with microglial contacts. In the present study, we investigated whether developmental changes in AISS could be modified by early-life stress (ELS), specifically neonatal maternal separation (MS), through stress hormones and microglial activation and examined the potential behavioural consequences. We examined AISS at postnatal day (PND)5, 15 and 50, using Golgi-Cox staining and anatomical analysis. Neurone-microglial interaction was assessed using antibodies against ankyrin-G, PSD-95 and Iba1, for AIS, AISS and microglia visualisation, respectively, in normally reared and neonatal maternally separated male and female rats. We observed a higher density of AISS in ELS rats at both PND15 and PND50 compared to controls. Effects were more pronounced in females than males. AIS-associated microglia in ELS rats showed a hyper-ramified morphology and less co-localisation with PSD-95 compared to controls at PND15. ELS-associated alteration in microglial morphology and synaptic pruning was mimicked by treatment of acute hippocampal slices of normally reared rats with vasopressin. ELS rats exhibited increased freezing behaviour during auditory fear memory testing, which was more pronounced in female subjects and corresponded with increased Fos expression in dorsal and ventral dentate granule cells. Thus, microglial synaptic pruning in dentate AIS of hippocampus is influenced by ELS, with demonstrable sex bias regarding its anatomical characteristics and subsequent fear-induced defensive behaviours.


Assuntos
Giro Denteado/fisiologia , Medo/psicologia , Microglia/fisiologia , Plasticidade Neuronal/fisiologia , Estresse Psicológico , Envelhecimento/psicologia , Animais , Animais Recém-Nascidos , Segmento Inicial do Axônio/fisiologia , Espinhas Dendríticas/fisiologia , Giro Denteado/citologia , Feminino , Masculino , Privação Materna , Microglia/citologia , Gravidez , Ratos , Ratos Wistar , Caracteres Sexuais , Estresse Psicológico/fisiopatologia , Estresse Psicológico/psicologia
20.
STAR Protoc ; 2(1): 100336, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33644771

RESUMO

This protocol allows repeated whole-cell patch-clamp recordings from individual rodent CA1 hippocampal neurons, followed by immunohistological labeling of the axon initial segment. This overcomes the need to maintain whole-cell recordings over the timescales required for homeostatic modification to cellular excitability, allowing for correlative analysis of the structure and function of neurons. Moreover, this protocol allows for paired analysis of physiological properties assessed before and after pharmacological treatment, thus providing increased statistical power, despite the relatively low-throughput nature of the recordings. For complete details on the use and execution of this protocol, please refer to Booker et al. (2020a).


Assuntos
Segmento Inicial do Axônio/metabolismo , Região CA1 Hipocampal/metabolismo , Células Piramidais/metabolismo , Animais , Região CA1 Hipocampal/citologia , Masculino , Camundongos , Técnicas de Patch-Clamp , Células Piramidais/citologia , Ratos , Ratos Long-Evans
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