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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 in vivo to induce a strong long-term potentiation of dentate gyrus granule cells. Immunolabeling for bIV-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. [ABSTRACT FROM AUTHOR]

A large body of studies has investigated bidirectional homeostatic plasticity both in vitro and in vivo using numerous pharmacological manipulations of activity or behavioral paradigms. However, these experiments rarely explored in the same cellular system the bidirectionality of the plasticity and simultaneously on excitatory and inhibitory neurons. M-channels are voltagegated potassium channels that play a crucial role in regulating neuronal excitability and plasticity. In cultured hippocampal excitatory neurons, we previously showed that chronic exposure to the M-channel blocker XE991 leads to adaptative compensations, thereby triggering at different timescales intrinsic and synaptic homeostatic plasticity. This plastic adaptation barely occurs in hippocampal inhibitory neurons. In this study, we examined whether this homeostatic plasticity induced by M-channel inhibition was bidirectional by investigating the acute and chronic effects of the M-channel opener retigabine on hippocampal neuronal excitability. Acute retigabine exposure decreased excitability in both excitatory and inhibitory neurons. Chronic retigabine treatment triggered in excitatory neurons homeostatic adaptation of the threshold current and spontaneous firing rate at a time scale of 4– 24 h. These plastic changes were accompanied by a substantial decrease in the M-current density and by a small, though significant, proximal relocation of Kv7.3-FGF14 segment along the axon initial segment. Thus, bidirectional homeostatic changes were observed in excitatory neurons though not symmetric in kinetics and mechanisms. Contrastingly, in inhibitory neurons, the compensatory changes in intrinsic excitability barely occurred after 48 h, while no homeostatic normalization of the spontaneous firing rate was observed. Our results indicate that excitatory and inhibitory hippocampal neurons differ in their adaptation to chronic alterations in neuronal excitability induced by M-channel bidirectional modulation. [ABSTRACT FROM AUTHOR]

A large body of studies has investigated bidirectional homeostatic plasticity both in vitro and in vivo using numerous pharmacological manipulations of activity or behavioral paradigms. However, these experiments rarely explored in the same cellular system the bidirectionality of the plasticity and simultaneously on excitatory and inhibitory neurons. M-channels are voltage-gated potassium channels that play a crucial role in regulating neuronal excitability and plasticity. In cultured hippocampal excitatory neurons, we previously showed that chronic exposure to the M-channel blocker XE991 leads to adaptative compensations, thereby triggering at different timescales intrinsic and synaptic homeostatic plasticity. This plastic adaptation barely occurs in hippocampal inhibitory neurons. In this study, we examined whether this homeostatic plasticity induced by M-channel inhibition was bidirectional by investigating the acute and chronic effects of the M-channel opener retigabine on hippocampal neuronal excitability. Acute retigabine exposure decreased excitability in both excitatory and inhibitory neurons. Chronic retigabine treatment triggered in excitatory neurons homeostatic adaptation of the threshold current and spontaneous firing rate at a time scale of 4–24 h. These plastic changes were accompanied by a substantial decrease in the M-current density and by a small, though significant, proximal relocation of Kv7.3-FGF14 segment along the axon initial segment. Thus, bidirectional homeostatic changes were observed in excitatory neurons though not symmetric in kinetics and mechanisms. Contrastingly, in inhibitory neurons, the compensatory changes in intrinsic excitability barely occurred after 48 h, while no homeostatic normalization of the spontaneous firing rate was observed. Our results indicate that excitatory and inhibitory hippocampal neurons differ in their adaptation to chronic alterations in neuronal excitability induced by M-channel bidirectional modulation.

Neuronal type-specific physiologic heterogeneity can be driven by both extrinsic and intrinsic mechanisms. In retinal ganglion cells (RGCs), which carry visual information from the retina to central targets, evidence suggests intrinsic properties shaping action potential (AP) generation significantly impact the responses of RGCs to visual stimuli. Here, we explored how differences in intrinsic excitability further distinguish two RCG types with distinct presynaptic circuits, alpha ON-sustained (αON-S) cells and alpha OFFsustained (αOFF-S) cells. We found that αOFF-S RGCs are more excitable to modest depolarizing currents than αON-S RGCs but excitability plateaued earlier as depolarization increased (i.e., depolarization block). In addition to differences in depolarization block sensitivity, the two cell types also produced distinct AP shapes with increasing stimulation. αOFF-S AP width and variability increased with depolarization magnitude, which correlated with the onset of depolarization block, while αON-S AP width and variability remained stable. We then tested if differences in depolarization block observed in αON-S and αOFF-S RGCs were due to sensitivity to extracellular potassium. We found αOFF-S RGCs more sensitive to increased extracellular potassium concentration, which shifted αON-S RGC excitability to that of αOFF-S cells under baseline potassium conditions. Finally, we investigated the influence of the axon initial segment (AIS) dimensions on RGC spiking. We found that the relationship between AIS length and evoked spike rate varied not only by cell type, but also by the strength of stimulation, suggesting AIS structure alone cannot fully explain the observed differences RGC excitability. Thus, sensitivity to extracellular potassium contributes to differences in intrinsic excitability, a key factor that shapes how RGCs encode visual information. [ABSTRACT FROM AUTHOR]

Homeostatic plasticity encompasses the mechanisms by which neurons stabilize their synaptic strength and excitability in response to prolonged and destabilizing changes in their network activity. Prolonged activity blockade leads to homeostatic scaling of action potential (AP) firing rate in hippocampal neurons in part by decreased activity of N-Methyl-D-Aspartate receptors and subsequent transcriptional down-regulation of potassium channel genes including KCNQ3 which encodes Kv7.3. Neuronal Kv7 channels are mostly heterotetramers of Kv7.2 and Kv7.3 subunits and are highly enriched at the axon initial segment (AIS) where their current potently inhibits repetitive and burst firing of APs. However, whether a decrease in Kv7.3 expression occurs at the AIS during homeostatic scaling of intrinsic excitability and what signaling pathway reduces KCNQ3 transcript upon prolonged activity blockade remain unknown. Here, we report that prolonged activity blockade in cultured hippocampal neurons reduces the activity of extracellular signalregulated kinase 1/2 (ERK1/2) followed by a decrease in the activation of brain-derived neurotrophic factor (BDNF) receptor, Tropomyosin receptor kinase B (TrkB). Furthermore, both prolonged activity blockade and prolonged pharmacological inhibition of ERK1/2 decrease KCNQ3 and BDNF transcripts as well as the density of Kv7.3 and ankyrin-G at the AIS. Collectively, our findings suggest that a reduction in the ERK1/2 activity and subsequent transcriptional down-regulation may serve as a potential signaling pathway that links prolonged activity blockade to homeostatic control of BDNF-TrkB signaling and Kv7.3 density at the AIS during homeostatic scaling of AP firing rate. [ABSTRACT FROM AUTHOR]

Homeostatic plasticity encompasses the mechanisms by which neurons stabilize their synaptic strength and excitability in response to prolonged and destabilizing changes in their network activity. Prolonged activity blockade leads to homeostatic scaling of action potential (AP) firing rate in hippocampal neurons in part by decreased activity of N-Methyl-D-Aspartate receptors and subsequent transcriptional down-regulation of potassium channel genes including KCNQ3 which encodes Kv7.3. Neuronal Kv7 channels are mostly heterotetramers of Kv7.2 and Kv7.3 subunits and are highly enriched at the axon initial segment (AIS) where their current potently inhibits repetitive and burst firing of APs. However, whether a decrease in Kv7.3 expression occurs at the AIS during homeostatic scaling of intrinsic excitability and what signaling pathway reduces KCNQ3 transcript upon prolonged activity blockade remain unknown. Here, we report that prolonged activity blockade in cultured hippocampal neurons reduces the activity of extracellular signal-regulated kinase 1/2 (ERK1/2) followed by a decrease in the activation of brain-derived neurotrophic factor (BDNF) receptor, Tropomyosin receptor kinase B (TrkB). Furthermore, both prolonged activity blockade and prolonged pharmacological inhibition of ERK1/2 decrease KCNQ3 and BDNF transcripts as well as the density of Kv7.3 and ankyrin-G at the AIS. Collectively, our findings suggest that a reduction in the ERK1/2 activity and subsequent transcriptional down-regulation may serve as a potential signaling pathway that links prolonged activity blockade to homeostatic control of BDNF-TrkB signaling and Kv7.3 density at the AIS during homeostatic scaling of AP firing rate.

In the mammalian brain, auditory brainstem nuclei are arranged topographically according to acoustic frequency responsiveness. During postnatal development, the axon initial segment (AIS) of principal neurons undergoes structural refinement depending on location along the tonotopic axis within the medial nucleus of the trapezoid body (MNTB). However, the molecular mechanisms underlying the structural refinement of the AIS along the tonotopic axis in the auditory brainstem have not been explored. We tested the hypothesis that brain-derived neurotrophic factor (BDNF) is a molecular mediator of the structural development of the MNTB in an activity-dependent manner. Using BDNF heterozygous mutant (BDNF+/–) mice, we examined the impact of global BDNF reduction on structural and functional development of MNTB neurons by assessing AIS structure and associated intrinsic neuronal properties. BDNF reduction inhibits the structural and functional differentiation of principal neurons along the tonotopic axis in the MNTB. Augmented sound input during the critical period of development has been shown to enhance the structural refinement of the AIS of MNTB neurons. However, in BDNF +/– mice, MNTB neurons did not show this activity-dependent structural modification of the AIS following repeated sound stimulation. In addition, BDNF+/– mice lacked a defined isofrequency band of neuronal activity following exposure to 16 kHz sound, suggesting degradation of tonotopy. Taken together, structural development and functional refinement of auditory brainstem neurons require physiological levels of BDNF to establish proper tonotopic gradients. [ABSTRACT FROM AUTHOR]

In the mammalian brain, auditory brainstem nuclei are arranged topographically according to acoustic frequency responsiveness. During postnatal development, the axon initial segment (AIS) of principal neurons undergoes structural refinement depending on location along the tonotopic axis within the medial nucleus of the trapezoid body (MNTB). However, the molecular mechanisms underlying the structural refinement of the AIS along the tonotopic axis in the auditory brainstem have not been explored. We tested the hypothesis that brain-derived neurotrophic factor (BDNF) is a molecular mediator of the structural development of the MNTB in an activity-dependent manner. Using BDNF heterozygous mutant (BDNF+/–) mice, we examined the impact of global BDNF reduction on structural and functional development of MNTB neurons by assessing AIS structure and associated intrinsic neuronal properties. BDNF reduction inhibits the structural and functional differentiation of principal neurons along the tonotopic axis in the MNTB. Augmented sound input during the critical period of development has been shown to enhance the structural refinement of the AIS of MNTB neurons. However, in BDNF +/– mice, MNTB neurons did not show this activity-dependent structural modification of the AIS following repeated sound stimulation. In addition, BDNF+/– mice lacked a defined isofrequency band of neuronal activity following exposure to 16 kHz sound, suggesting degradation of tonotopy. Taken together, structural development and functional refinement of auditory brainstem neurons require physiological levels of BDNF to establish proper tonotopic gradients.

Electric micro-stimulation of the nervous system is a means to restore various body functions. The stimulus amplitude necessary to generate action potentials, the lower threshold (LT), is well characterized for many neuronal populations. However, electric overstimulation above an upper threshold (UT) prevents action potential generation and therefore hinders optimal neuro-rehabilitation. Previous studies demonstrated the impact of the UT in micro-stimulation of retinal ganglion cells (RGCs). The observed phenomenon is mostly explained by (i) reversed sodium ion flow in the soma membrane, and (ii) anodal surround block that hinders spike conduction in strongly hyperpolarized regions of the axon at high stimulus intensities. However, up to now, no detailed study of the nature of these phenomena has been presented, particularly for different cell types. Here, we present computational analyses of LT and UT for layer 5 pyramidal cells (PCs) as well as alpha RGCs. Model neurons were stimulated in close vicinity to the cell body and LTs and UTs as well as the ratio UT/LT were compared. Aside from a simple point source electrode and monophasic stimuli also realistic electrode and pulse configurations were examined. The analysis showed: (i) in RGCs, the soma contributed to action potential initiation and block for small electrode distances, whereas in PCs the soma played no role in LTs or UTs. (ii) In both cell types, action potential always initiated within the axon initial segment at LT. (iii) In contrast to a complete block of spike conductance at UT that occurred in RGCs, an incomplete block of spiking appeared in PC axon collaterals. (iv) PC axon collateral arrangement influenced UTs but had small impact on LTs. (v) Population responses of RGCs change from circular regions of activation to ring-shaped patterns for increasing stimulus amplitude. A better understanding of the stimulation window that can reliably activate target neurons will benefit the future development of neuroprostheses. [ABSTRACT FROM AUTHOR]

Electric micro-stimulation of the nervous system is a means to restore various body functions. The stimulus amplitude necessary to generate action potentials, the lower threshold (LT), is well characterized for many neuronal populations. However, electric overstimulation above an upper threshold (UT) prevents action potential generation and therefore hinders optimal neuro-rehabilitation. Previous studies demonstrated the impact of the UT in micro-stimulation of retinal ganglion cells (RGCs). The observed phenomenon is mostly explained by (i) reversed sodium ion flow in the soma membrane, and (ii) anodal surround block that hinders spike conduction in strongly hyperpolarized regions of the axon at high stimulus intensities. However, up to now, no detailed study of the nature of these phenomena has been presented, particularly for different cell types. Here, we present computational analyses of LT and UT for layer 5 pyramidal cells (PCs) as well as alpha RGCs. Model neurons were stimulated in close vicinity to the cell body and LTs and UTs as well as the ratio UT/LT were compared. Aside from a simple point source electrode and monophasic stimuli also realistic electrode and pulse configurations were examined. The analysis showed: (i) in RGCs, the soma contributed to action potential initiation and block for small electrode distances, whereas in PCs the soma played no role in LTs or UTs. (ii) In both cell types, action potential always initiated within the axon initial segment at LT. (iii) In contrast to a complete block of spike conductance at UT that occurred in RGCs, an incomplete block of spiking appeared in PC axon collaterals. (iv) PC axon collateral arrangement influenced UTs but had small impact on LTs. (v) Population responses of RGCs change from circular regions of activation to ring-shaped patterns for increasing stimulus amplitude. A better understanding of the stimulation window that can reliably activate target neurons will benefit the future development of neuroprostheses.

By limiting protein exchange between the soma and the axon, the axon initial segment (AIS) enables the segregation of specific proteins and hence the differentiation of the somatodendritic compartment and the axonal compartment. Electron microscopy and super-resolution fluorescence imaging have provided important insights in the ultrastructure of the AIS. Yet, the full extent of its filtering properties is not fully delineated. In particular, it is unclear whether and how the AIS opposes the free exchange of soluble proteins. Here we describe a robust framework to combine whole-cell photobleaching and retrospective high-resolution imaging in developing neurons. With this assay, we found that cytoplasmic soluble proteins that are not excluded from the axon upon expression over tens of hours exhibit a strong mobility reduction at the AIS – i.e., are indeed compartmentalized – when monitored over tens of minutes. This form of compartmentalization is developmentally regulated, requires intact F-actin and may be correlated with the composition and ultrastructure of the submembranous spectrin cytoskeleton. Using computational modeling, we provide evidence that both neuronal morphology and the AIS regulate this compartmentalization but act on distinct time scales, with the AIS having a more pronounced effect on fast exchanges. Our results thus suggest that the rate of protein accumulation in the soma may impact to what extent and over which timescales freely moving molecules can be segregated from the axon. This in turn has important implications for our understanding of compartment-specific signaling in neurons.

Alternative splicing is a powerful mechanism for molecular and functional diversification. In neurons, alternative splicing extensively controls various developmental steps as well as the plasticity and remodeling of neuronal activity in the adult brain. The axon initial segment (AIS) is the specialized compartment of proximal axons that initiates action potential (AP). At the AIS, the ion channels and cell adhesion molecules (CAMs) required for AP initiation are densely clustered via the scaffolding and cytoskeletal proteins. Notably, recent studies have elucidated that multiple AIS proteins are controlled by extensive alternative splicing in developing and adult brains. Here, we argue the potential role of dynamic regulation of alternative splicing in the development, specification, and functions of the AIS. In particular, we propose the novel concept that alternative splicing potentially modulates the structural and functional plasticity at the AIS.

The axon initial segment (AIS) is the site of action potential (AP) initiation in most neurons and is thus a critical site in the regulation of neuronal excitability. Normal function within the discrete AIS compartment requires intricate molecular machinery to ensure the proper concentration and organization of voltage-gated and ligand-gated ion channels; in humans, dysfunction at the AIS due to channel mutations is commonly associated with epileptic disorders. In this review, we will examine the molecular mechanisms underlying the formation of the only synapses found at the AIS: synapses containing γ-aminobutyric type A receptors (GABAARs). GABAARs are heteropentamers assembled from 19 possible subunits and are the primary mediators of fast synaptic inhibition in the brain. Although the total GABAAR population is incredibly heterogeneous, only one specific GABAAR subtype—the α2-containing receptor—is enriched at the AIS. These AIS synapses are innervated by GABAergic chandelier cells, and this inhibitory signaling is thought to contribute to the tight control of AP firing. Here, we will summarize the progress made in understanding the regulation of GABAAR synapse formation, concentrating on post-translational modifications of subunits and on interactions with intracellular proteins. We will then discuss subtype-specific synapse formation, with a focus on synapses found at the AIS, and how these synapses influence neuronal excitation.

Sound input critically influences the development and maintenance of neuronal circuits in the mammalian brain throughout life. We investigate the structural and functional plasticity of auditory neurons in response to various auditory experiences during development, adulthood, and aging. Using electrophysiology, computer simulation, and immunohistochemistry, we study the structural plasticity of the axon initial segment (AIS) in the medial nucleus of the trapezoid body (MNTB) from the auditory brainstem of the mice (either sex), in different ages and auditory environments. The structure and spatial location of the AIS of MNTB neurons depend on their functional topographic location along the tonotopic axis, aligning high- to low-frequency sound-responding neurons (HF or LF neurons). HF neurons dramatically undergo structural remodeling of the AIS throughout life. The AIS progressively shortens during development, is stabilized in adulthood, and becomes longer in aging. Sound inputs are critically associated with setting and maintaining AIS plasticity and tonotopy at various ages. Sound stimulation increases the excitability of auditory neurons. Computer simulation shows that modification of the AIS length, location, and diameter can affect firing properties of MNTB neurons in the developing brainstem. The adaptive capability of axonal structure in response to various auditory experiences at different ages suggests that sound input is important for the development and maintenance of the structural and functional properties of the auditory brain throughout life.

By limiting protein exchange between the soma and the axon, the axon initial segment (AIS) enables the segregation of specific proteins and hence the differentiation of the somatodendritic compartment and the axonal compartment. Electron microscopy and super-resolution fluorescence imaging have provided important insights in the ultrastructure of the AIS. Yet, the full extent of its filtering properties is not fully delineated. In particular, it is unclear whether and how the AIS opposes the free exchange of soluble proteins. Here we describe a robust framework to combine whole-cell photobleaching and retrospective high-resolution imaging in developing neurons. With this assay, we found that cytoplasmic soluble proteins that are not excluded from the axon upon expression over tens of hours exhibit a strong mobility reduction at the AIS – i.e., are indeed compartmentalized – when monitored over tens of minutes. This form of compartmentalization is developmentally regulated, requires intact F-actin and may be correlated with the composition and ultrastructure of the submembranous spectrin cytoskeleton. Using computational modeling, we provide evidence that both neuronal morphology and the AIS regulate this compartmentalization but act on distinct time scales, with the AIS having a more pronounced effect on fast exchanges. Our results thus suggest that the rate of protein accumulation in the soma may impact to what extent and over which timescales freely moving molecules can be segregated from the axon. This in turn has important implications for our understanding of compartment-specific signaling in neurons. [ABSTRACT FROM AUTHOR]

Alternative splicing is a powerful mechanism for molecular and functional diversification. In neurons, alternative splicing extensively controls various developmental steps as well as the plasticity and remodeling of neuronal activity in the adult brain. The axon initial segment (AIS) is the specialized compartment of proximal axons that initiates action potential (AP). At the AIS, the ion channels and cell adhesion molecules (CAMs) required for AP initiation are densely clustered via the scaffolding and cytoskeletal proteins. Notably, recent studies have elucidated that multiple AIS proteins are controlled by extensive alternative splicing in developing and adult brains. Here, we argue the potential role of dynamic regulation of alternative splicing in the development, specification, and functions of the AIS. In particular, we propose the novel concept that alternative splicing potentially modulates the structural and functional plasticity at the AIS. [ABSTRACT FROM AUTHOR]

The axon initial segment (AIS) is the site of action potential (AP) initiation in most neurons and is thus a critical site in the regulation of neuronal excitability. Normal function within the discrete AIS compartment requires intricate molecular machinery to ensure the proper concentration and organization of voltage-gated and ligand-gated ion channels; in humans, dysfunction at the AIS due to channel mutations is commonly associated with epileptic disorders. In this review, we will examine the molecular mechanisms underlying the formation of the only synapses found at the AIS: synapses containing γ-aminobutyric type A receptors (GABAARs). GABAARs are heteropentamers assembled from 19 possible subunits and are the primary mediators of fast synaptic inhibition in the brain. Although the total GABAAR population is incredibly heterogeneous, only one specific GABAAR subtype—the α2-containing receptor—is enriched at the AIS. These AIS synapses are innervated by GABAergic chandelier cells, and this inhibitory signaling is thought to contribute to the tight control of AP firing. Here, we will summarize the progress made in understanding the regulation of GABAAR synapse formation, concentrating on post-translational modifications of subunits and on interactions with intracellular proteins. We will then discuss subtype-specific synapse formation, with a focus on synapses found at the AIS, and how these synapses influence neuronal excitation. [ABSTRACT FROM AUTHOR]

Sound input critically influences the development and maintenance of neuronal circuits in the mammalian brain throughout life. We investigate the structural and functional plasticity of auditory neurons in response to various auditory experiences during development, adulthood, and aging. Using electrophysiology, computer simulation, and immunohistochemistry, we study the structural plasticity of the axon initial segment (AIS) in the medial nucleus of the trapezoid body (MNTB) from the auditory brainstem of the mice (either sex), in different ages and auditory environments. The structure and spatial location of the AIS of MNTB neurons depend on their functional topographic location along the tonotopic axis, aligning high- to low-frequency sound-responding neurons (HF or LF neurons). HF neurons dramatically undergo structural remodeling of the AIS throughout life. The AIS progressively shortens during development, is stabilized in adulthood, and becomes longer in aging. Sound inputs are critically associated with setting and maintaining AIS plasticity and tonotopy at various ages. Sound stimulation increases the excitability of auditory neurons. Computer simulation shows that modification of the AIS length, location, and diameter can affect firing properties of MNTB neurons in the developing brainstem. The adaptive capability of axonal structure in response to various auditory experiences at different ages suggests that sound input is important for the development and maintenance of the structural and functional properties of the auditory brain throughout life. [ABSTRACT FROM AUTHOR]

The anatomical properties of the axon initial segment (AIS) are tailored in certain types of CNS neurons to help optimize different aspects of neuronal function. Here, we questioned whether the AISs of retinal ganglion cells (RGC) were similarly customized, and if so, whether they supported specific RGC functions. To explore this, we measured the AIS properties in alpha sustained RGCs (α S RGCs) of mouse; α S RGCs sizes vary systematically along the nasal temporal axis of the retina, making these cells an attractive population with which to study potential correlations between AIS properties and cell size. Measurements of AIS length as well as distance from the soma revealed that both were scaled to cell size, i.e., cells with large dendritic fields had long AISs that were relatively far from the soma. Within the AIS, the percentage of Nav1.6 voltage-gated sodium channels remained highly consistent, regardless of cell size or other AIS properties. Although ON RGCs were slightly larger than OFF cells at any given location of the retina, the level of scaling and relative distribution of voltage-gated sodium channels were highly similar. Computational modeling revealed that AIS scaling influenced spiking thresholds, spike rate as well as the kinetics of individual action potentials, Interestingly, the effect of individual features of the AIS varied for different neuronal functions, e.g., AIS length had a larger effect on the efficacy by which the AIS initiated spike triggered the somatic spike than it did on repetitive spiking. The polarity of the effect varied for different properties, i.e., increases to soma size increased spike threshold while increases to AIS length decreased threshold. Thus, variations in the relative level of scaling for individual components could fine tune threshold or other neuronal functions. Light responses were highly consistent across the full range of cell sizes suggesting that scaling may post-synaptically shape response stability, e.g., in addition to several well-known pre-synaptic contributors.

A key component allowing a neuron to function properly within its dynamic environment is the axon initial segment (AIS), the site of action potential generation. In visual cortex, AIS of pyramidal neurons undergo periods of activity-dependent structural plasticity during development. However, it remains unknown how AIS morphology is organized during development for downstream cells in the visual pathway (retinal ganglion cells; RGCs) and whether AIS retain the ability to dynamically adjust to changes in network state. Here, we investigated the maturation of AIS in RGCs during mouse retinal development, and tested putative activity-dependent mechanisms by applying visual deprivation with a focus on the AIS-specific cisternal organelle (CO), a presumed Ca2+-store. Whole-mount retinae from wildtype and Thy1-GFP transgenic mice were processed for multi-channel immunofluorescence using antibodies against AIS scaffolding proteins ankyrin-G, βIV-spectrin and the CO marker synaptopodin (synpo). Confocal microscopy in combination with morphometrical analysis of AIS length and position as well as synpo cluster size was performed. Data indicated that a subset of RGC AIS contains synpo clusters and that these show significant dynamic regulation in size during development as well as after visual deprivation. Using super resolution microscopy, we addressed the subcellular localization of synpo in RGC axons. Similar to cortical neurons, RGCs show a periodic distribution of AIS scaffolding proteins. A previously reported scaffold-deficient nanodomain correlating with synpo localization is not evident in all RGC AIS. In summary, our work demonstrates a dynamic regulation of both the AIS and synpo in RGCs during retinal development and after visual deprivation, providing first evidence that the AIS and CO in RGCs can undergo structural plasticity in response to changes in network activity.

Morphological and functional polarization of neurons depends on the generation and maintenance of the axon initial segment (AIS). This axonal domain maintains axonal properties but is also the place where the action potential (AP) is generated. All these functions require the AIS, a complex structure that is not fully understood. An integrated structure of voltage-gated ion channels, specific cytoskeleton architecture, as well as, scaffold proteins contributes to these functions. Among them, ankyrinG plays a crucial role to maintain ion channels and membrane proteins. However, it is still elusive how the AIS performs its complex structural and functional regulation. Recent studies reveal that AIS is dynamically regulated in molecular composition, length and location in response to neuronal activity. Some mechanisms acting on AIS plasticity have been uncovered recently, including Ca2+, calpain or calmodulin-mediated modulation, as well as post-translational modifications of cytoskeleton proteins and actin-associated proteins. Neurons are able to respond to different kind of physiological and pathological stimuli from development to maturity by adapting their AIS composition, position and length. This raises the question of which are the neuronal receptors that contribute to the modulation of AIS plasticity. Previous studies have shown that purinergic receptor P2X7 activation is detrimental to AIS maintenance. During initial axonal elongation, P2X7 is coordinated with P2Y1, another purinergic receptor that is essential for proper axon elongation. In this study, we focus on the role of P2Y1 receptor on AIS development and maintenance. Our results show that P2Y1 receptor activity and expression are necessary during AIS initial development, while has no role once AIS maturity is achieved. P2Y1 inhibition or suppression results in a decrease in ankyrinG, βIV-spectrin and voltage-gated sodium channels accumulation that can be rescued by actin stabilization or the modulation of actin-binding proteins at the AIS. Moreover, P2X7 or calpain inhibition also rescues ankyrinG decrease. Hence, a dynamic balance of P2Y1 and P2X7 receptors expression and function during AIS assembly and maturation may represent a fine regulatory mechanism in response to physiological or pathological extracellular purines concentration.

Non-invasive electrical stimulation can be used to study and control neural activity in the brain or to alleviate somatosensory dysfunctions. One intriguing prospect is to precisely stimulate individual targeted neurons. Here, we investigated single-neuron current and voltage stimulation in vitro using high-density microelectrode arrays featuring 26,400 bidirectional electrodes at a pitch of 17.5 μm and an electrode area of 5 × 9 μm2. We determined optimal waveforms, amplitudes and durations for both stimulation modes. Owing to the high spatial resolution of our arrays and the close proximity of the electrodes to the respective neurons, we were able to stimulate the axon initial segments (AIS) with charges of less than 2 pC. This resulted in minimal artifact production and reliable readout of stimulation efficiency directly at the soma of the stimulated cell. Stimulation signals as low as 70 mV or 100 nA, with pulse durations as short as 18 μs, yielded measurable action potential initiation and propagation. We found that the required stimulation signal amplitudes decreased with cell growth and development and that stimulation efficiency did not improve at higher electric fields generated by simultaneous multi-electrode stimulation.

The axon initial segment (AIS) is a specialized region within the proximal portion of the axon that initiates action potentials thanks in large part to an enrichment of sodium channels. The scaffolding protein ankyrinG (AnkG) is essential for the recruitment of sodium channels as well as several other intracellular and extracellular proteins to the AIS. In the present study, we explore the role of the cell adhesion molecule (CAM) neurofascin-186 (NF-186) in arranging the individual molecular components of the AIS in cultured rat hippocampal neurons. Using a CRISPR depletion strategy to ablate NF expression, we found that the loss of NF selectively perturbed AnkG accumulation and its relative proximal distribution within the AIS. We found that the overexpression of sodium channels could restore AnkG accumulation, but not its altered distribution within the AIS without NF present. We go on to show that although the loss of NF altered AnkG distribution, sodium channel function within the AIS remained normal. Taken together, these results demonstrate that the regulation of AnkG and sodium channel accumulation within the AIS can occur independently of one another, potentially mediated by other binding partners such as NF.

The anatomical properties of the axon initial segment (AIS) are tailored in certain types of CNS neurons to help optimize different aspects of neuronal function. Here, we questioned whether the AISs of retinal ganglion cells (RGC) were similarly customized, and if so, whether they supported specific RGC functions. To explore this, we measured the AIS properties in alpha sustained RGCs (α S RGCs) of mouse; α S RGCs sizes vary systematically along the nasal temporal axis of the retina, making these cells an attractive population with which to study potential correlations between AIS properties and cell size. Measurements of AIS length as well as distance from the soma revealed that both were scaled to cell size, i.e., cells with large dendritic fields had long AISs that were relatively far from the soma. Within the AIS, the percentage of Na v 1.6 voltage-gated sodium channels remained highly consistent, regardless of cell size or other AIS properties. Although ON RGCs were slightly larger than OFF cells at any given location of the retina, the level of scaling and relative distribution of voltage-gated sodium channels were highly similar. Computational modeling revealed that AIS scaling influenced spiking thresholds, spike rate as well as the kinetics of individual action potentials, Interestingly, the effect of individual features of the AIS varied for different neuronal functions, e.g., AIS length had a larger effect on the efficacy by which the AIS initiated spike triggered the somatic spike than it did on repetitive spiking. The polarity of the effect varied for different properties, i.e., increases to soma size increased spike threshold while increases to AIS length decreased threshold. Thus, variations in the relative level of scaling for individual components could fine tune threshold or other neuronal functions. Light responses were highly consistent across the full range of cell sizes suggesting that scaling may post-synaptically shape response stability, e.g., in addition to several well-known pre-synaptic contributors. [ABSTRACT FROM AUTHOR]

A key component allowing a neuron to function properly within its dynamic environment is the axon initial segment (AIS), the site of action potential generation. In visual cortex, AIS of pyramidal neurons undergo periods of activity-dependent structural plasticity during development. However, it remains unknown how AIS morphology is organized during development for downstream cells in the visual pathway (retinal ganglion cells; RGCs) and whether AIS retain the ability to dynamically adjust to changes in network state. Here, we investigated the maturation of AIS in RGCs during mouse retinal development, and tested putative activity-dependent mechanisms by applying visual deprivation with a focus on the AIS-specific cisternal organelle (CO), a presumed Ca2+-store. Whole-mount retinae from wildtype and Thy1-GFP transgenic mice were processed for multi-channel immunofluorescence using antibodies against AIS scaffolding proteins ankyrin-G, βIV-spectrin and the CO marker synaptopodin (synpo). Confocal microscopy in combination with morphometrical analysis of AIS length and position as well as synpo cluster size was performed. Data indicated that a subset of RGC AIS contains synpo clusters and that these show significant dynamic regulation in size during development as well as after visual deprivation. Using super resolution microscopy, we addressed the subcellular localization of synpo in RGC axons. Similar to cortical neurons, RGCs show a periodic distribution of AIS scaffolding proteins. A previously reported scaffold-deficient nanodomain correlating with synpo localization is not evident in all RGC AIS. In summary, our work demonstrates a dynamic regulation of both the AIS and synpo in RGCs during retinal development and after visual deprivation, providing first evidence that the AIS and CO in RGCs can undergo structural plasticity in response to changes in network activity. [ABSTRACT FROM AUTHOR]

Morphological and functional polarization of neurons depends on the generation and maintenance of the axon initial segment (AIS). This axonal domain maintains axonal properties but is also the place where the action potential (AP) is generated. All these functions require the AIS, a complex structure that is not fully understood. An integrated structure of voltage-gated ion channels, specific cytoskeleton architecture, as well as, scaffold proteins contributes to these functions. Among them, ankyrinG plays a crucial role to maintain ion channels and membrane proteins. However, it is still elusive how the AIS performs its complex structural and functional regulation. Recent studies reveal that AIS is dynamically regulated in molecular composition, length and location in response to neuronal activity. Some mechanisms acting on AIS plasticity have been uncovered recently, including Ca2+, calpain or calmodulin-mediated modulation, as well as post-translational modifications of cytoskeleton proteins and actin-associated proteins. Neurons are able to respond to different kind of physiological and pathological stimuli from development to maturity by adapting their AIS composition, position and length. This raises the question of which are the neuronal receptors that contribute to the modulation of AIS plasticity. Previous studies have shown that purinergic receptor P2X7 activation is detrimental to AIS maintenance. During initial axonal elongation, P2X7 is coordinated with P2Y1, another purinergic receptor that is essential for proper axon elongation. In this study, we focus on the role of P2Y1 receptor on AIS development and maintenance. Our results show that P2Y1 receptor activity and expression are necessary during AIS initial development, while has no role once AIS maturity is achieved. P2Y1 inhibition or suppression results in a decrease in ankyrinG, βIV-spectrin and voltage-gated sodium channels accumulation that can be rescued by actin stabilization or the modulation of actin-binding proteins at the AIS. Moreover, P2X7 or calpain inhibition also rescues ankyrinG decrease. Hence, a dynamic balance of P2Y1 and P2X7 receptors expression and function during AIS assembly and maturation may represent a fine regulatory mechanism in response to physiological or pathological extracellular purines concentration. [ABSTRACT FROM AUTHOR]

Non-invasive electrical stimulation can be used to study and control neural activity in the brain or to alleviate somatosensory dysfunctions. One intriguing prospect is to precisely stimulate individual targeted neurons. Here, we investigated single-neuron current and voltage stimulation in vitro using high-density microelectrode arrays featuring 26,400 bidirectional electrodes at a pitch of 17.5 μm and an electrode area of 5 × 9 μm2. We determined optimal waveforms, amplitudes and durations for both stimulation modes. Owing to the high spatial resolution of our arrays and the close proximity of the electrodes to the respective neurons, we were able to stimulate the axon initial segments (AIS) with charges of less than 2 pC. This resulted in minimal artifact production and reliable readout of stimulation efficiency directly at the soma of the stimulated cell. Stimulation signals as low as 70 mV or 100 nA, with pulse durations as short as 18 μs, yielded measurable action potential initiation and propagation. We found that the required stimulation signal amplitudes decreased with cell growth and development and that stimulation efficiency did not improve at higher electric fields generated by simultaneous multi-electrode stimulation. [ABSTRACT FROM AUTHOR]

The axon initial segment (AIS) is a specialized region within the proximal portion of the axon that initiates action potentials thanks in large part to an enrichment of sodium channels. The scaffolding protein ankyrinG (AnkG) is essential for the recruitment of sodium channels as well as several other intracellular and extracellular proteins to the AIS. In the present study, we explore the role of the cell adhesion molecule (CAM) neurofascin-186 (NF-186) in arranging the individual molecular components of the AIS in cultured rat hippocampal neurons. Using a CRISPR depletion strategy to ablate NF expression, we found that the loss of NF selectively perturbed AnkG accumulation and its relative proximal distribution within the AIS. We found that the overexpression of sodium channels could restore AnkG accumulation, but not its altered distribution within the AIS without NF present. We go on to show that although the loss of NF altered AnkG distribution, sodium channel function within the AIS remained normal. Taken together, these results demonstrate that the regulation of AnkG and sodium channel accumulation within the AIS can occur independently of one another, potentially mediated by other binding partners such as NF. [ABSTRACT FROM AUTHOR]

Cognitive and mood impairments are common central nervous system complications of type 2 diabetes, although the neuronal mechanism(s) remains elusive. Previous studies focused mainly on neuronal inputs such as altered synaptic plasticity. Axon initial segment (AIS) is a specialized functional domain within neurons that regulates neuronal outputs. Structural changes of AIS have been implicated as a key pathophysiological event in various psychiatric and neurological disorders. Here we evaluated the structural integrity of the AIS in brains of db/db mice, an established animal model of type 2 diabetes associated with cognitive and mood impairments. We assessed the AIS before (5 weeks of age) and after (10 weeks) the development of type 2 diabetes, and after daily exercise treatment of diabetic condition. We found that the development of type 2 diabetes is associated with significant AIS shortening in both medial prefrontal cortex and hippocampus, as evident by immunostaining of the AIS structural protein βIV spectrin. AIS shortening occurs in the absence of altered neuronal and AIS protein levels. We found no change in nodes of Ranvier, another neuronal functional domain sharing a molecular organization similar to the AIS. This is the first study to identify AIS alteration in type 2 diabetes condition. Since AIS shortening is known to lower neuronal excitability, our results may provide a new avenue for understanding and treating cognitive and mood impairments in type 2 diabetes.

The neurodevelopmental disorder Angelman syndrome (AS) is characterized by intellectual disability, motor dysfunction, distinct behavioral aspects, and epilepsy. AS is caused by a loss of the maternally expressed UBE3A gene, and many of the symptoms are recapitulated in a Ube3a mouse model of this syndrome. At the cellular level, changes in the axon initial segment (AIS) have been reported, and changes in vesicle cycling have indicated the presence of presynaptic deficits. Here we studied the role of UBE3A in the auditory system by recording synaptic transmission at the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) through in vivo whole cell and juxtacellular recordings. We show that MNTB principal neurons in Ube3a mice exhibit a hyperpolarized resting membrane potential, an increased action potential (AP) amplitude and a decreased AP half width. Moreover, both the pre- and postsynaptic AP in the calyx of Held synapse of Ube3a mice showed significantly faster recovery from spike depression. An increase in AIS length was observed in the principal MNTB neurons of Ube3a mice, providing a possible substrate for these gain-of-function changes. Apart from the effect on APs, we also observed that EPSPs showed decreased short-term synaptic depression (STD) during long sound stimulations in AS mice, and faster recovery from STD following these tones, which is suggestive of a presynaptic gain-of-function. Our findings thus provide in vivo evidence that UBE3A plays a critical role in controlling synaptic transmission and excitability at excitatory synapses.

The microdomain that orchestrates action potential initiation in neurons is the axon initial segment (AIS). It has long been considered to be a rather homogeneous domain at the very proximal axon hillock with relatively stable length, particularly in cortical pyramidal cells. However, studies in other brain regions paint a different picture. In hippocampal CA1, up to 50% of axons emerge from basal dendrites. Further, in about 30% of thick-tufted layer V pyramidal neurons in rat somatosensory cortex, axons have a dendritic origin. Consequently, the AIS is separated from the soma. Recent in vitro and in vivo studies have shown that cellular excitability is a function of AIS length/position and somatodendritic morphology, undermining a potentially significant impact of AIS heterogeneity for neuronal function. We therefore investigated neocortical axon morphology and AIS composition, hypothesizing that the initial observation of seemingly homogeneous AIS is inadequate and needs to take into account neuronal cell types. Here, we biolistically transfected cortical neurons in organotypic cultures to visualize the entire neuron and classify cell types in combination with immunolabeling against AIS markers. Using confocal microscopy and morphometric analysis, we investigated axon origin, AIS position, length, diameter as well as distance to the soma. We find a substantial AIS heterogeneity in visual cortical neurons, classified into three groups: (I) axons with somatic origin with proximal AIS at the axon hillock; (II) axons with somatic origin with distal AIS, with a discernible gap between the AIS and the soma; and (III) axons with dendritic origin (axon-carrying dendrite cell, AcD cell) and an AIS either starting directly at the axon origin or more distal to that point. Pyramidal cells have significantly longer AIS than interneurons. Interneurons with vertical columnar axonal projections have significantly more distal AIS locations than all other cells with their prevailing phenotype as an AcD cell. In contrast, neurons with perisomatic terminations display most often an axon originating from the soma. Our data contribute to the emerging understanding that AIS morphology is highly variable, and potentially a function of the cell type.

Cognitive and mood impairments are common central nervous system complications of type 2 diabetes, although the neuronal mechanism(s) remains elusive. Previous studies focused mainly on neuronal inputs such as altered synaptic plasticity. Axon initial segment (AIS) is a specialized functional domain within neurons that regulates neuronal outputs. Structural changes of AIS have been implicated as a key pathophysiological event in various psychiatric and neurological disorders. Here we evaluated the structural integrity of the AIS in brains of db/db mice, an established animal model of type 2 diabetes associated with cognitive and mood impairments. We assessed the AIS before (5 weeks of age) and after (10 weeks) the development of type 2 diabetes, and after daily exercise treatment of diabetic condition. We found that the development of type 2 diabetes is associated with significant AIS shortening in both medial prefrontal cortex and hippocampus, as evident by immunostaining of the AIS structural protein bIV spectrin. AIS shortening occurs in the absence of altered neuronal and AIS protein levels. We found no change in nodes of Ranvier, another neuronal functional domain sharing a molecular organization similar to the AIS. This is the first study to identify AIS alteration in type 2 diabetes condition. Since AIS shortening is known to lower neuronal excitability, our results may provide a new avenue for understanding and treating cognitive and mood impairments in type 2 diabetes. [ABSTRACT FROM AUTHOR]

The neurodevelopmental disorder Angelman syndrome (AS) is characterized by intellectual disability, motor dysfunction, distinct behavioral aspects, and epilepsy. AS is caused by a loss of the maternally expressed UBE3A gene, and many of the symptoms are recapitulated in a Ube3a mouse model of this syndrome. At the cellular level, changes in the axon initial segment (AIS) have been reported, and changes in vesicle cycling have indicated the presence of presynaptic deficits. Here we studied the role of UBE3A in the auditory system by recording synaptic transmission at the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) through in vivo whole cell and juxtacellular recordings. We show that MNTB principal neurons in Ube3a mice exhibit a hyperpolarized resting membrane potential, an increased action potential (AP) amplitude and a decreased AP half width. Moreover, both the pre- and postsynaptic AP in the calyx of Held synapse of Ube3a mice showed significantly faster recovery from spike depression. An increase in AIS length was observed in the principal MNTB neurons of Ube3a mice, providing a possible substrate for these gain-of-function changes. Apart from the effect on APs, we also observed that EPSPs showed decreased shortterm synaptic depression (STD) during long sound stimulations in AS mice, and faster recovery from STD following these tones, which is suggestive of a presynaptic gain-of-function. Our findings thus provide in vivo evidence that UBE3A plays a critical role in controlling synaptic transmission and excitability at excitatory synapses. [ABSTRACT FROM AUTHOR]

The microdomain that orchestrates action potential initiation in neurons is the axon initial segment (AIS). It has long been considered to be a rather homogeneous domain at the very proximal axon hillock with relatively stable length, particularly in cortical pyramidal cells. However, studies in other brain regions paint a different picture. In hippocampal CA1, up to 50% of axons emerge from basal dendrites. Further, in about 30% of thick-tufted layer V pyramidal neurons in rat somatosensory cortex, axons have a dendritic origin. Consequently, the AIS is separated from the soma. Recent in vitro and in vivo studies have shown that cellular excitability is a function of AIS length/position and somatodendritic morphology, undermining a potentially significant impact of AIS heterogeneity for neuronal function. We therefore investigated neocortical axon morphology and AIS composition, hypothesizing that the initial observation of seemingly homogeneous AIS is inadequate and needs to take into account neuronal cell types. Here, we biolistically transfected cortical neurons in organotypic cultures to visualize the entire neuron and classify cell types in combination with immunolabeling against AIS markers. Using confocal microscopy and morphometric analysis, we investigated axon origin, AIS position, length, diameter as well as distance to the soma. We find a substantial AIS heterogeneity in visual cortical neurons, classified into three groups: (I) axons with somatic origin with proximal AIS at the axon hillock; (II) axons with somatic origin with distal AIS, with a discernible gap between the AIS and the soma; and (III) axons with dendritic origin (axon-carrying dendrite cell, AcD cell) and an AIS either starting directly at the axon origin or more distal to that point. Pyramidal cells have significantly longer AIS than interneurons. Interneurons with vertical columnar axonal projections have significantly more distal AIS locations than all other cells with their prevailing phenotype as an AcD cell. In contrast, neurons with perisomatic terminations display most often an axon originating from the soma. Our data contribute to the emerging understanding that AIS morphology is highly variable, and potentially a function of the cell type. [ABSTRACT FROM AUTHOR]

The selective transport of different cargoes into axons and dendrites underlies the polarized organization of the neuron. Although it has become clear that the combined activity of different motors determines the destination and selectivity of transport, little is known about the mechanistic details of motor cooperation. For example, the exact role of myosin-V in opposing microtubule-based axon entries has remained unclear. Here we use two orthogonal chemically-induced heterodimerization systems to independently recruit different motors to cargoes. We find that recruiting myosin-V to kinesin-propelled cargoes at approximately equal numbers is sufficient to stall motility. Kinesin-driven cargoes entering the axon were arrested in the axon initial segment (AIS) upon myosin-V recruitment and accumulated in distinct actin-rich hotspots. Importantly, unlike proposed previously, myosin-V did not return these cargoes to the cell body, suggesting that additional mechanism are required to establish cargo retrieval from the AIS.

The axon initial segment (AIS) is the site of action potential (AP) initiation, thus a crucial regulator of neuronal activity. In excitatory pyramidal neurons, the high density of voltage-gated sodium channels (NaV1.6) at the distal AIS regulates AP initiation. A surrogate AIS marker, ankyrin-G (ankG) is a structural protein regulating neuronal functional via clustering voltage-gated ion channels. In neuronal circuits, changes in presynaptic input can alter postsynaptic output via AIS structural-functional plasticity. Recently, we showed experimental mild traumatic brain injury (mTBI) evokes neocortical circuit disruption via diffuse axonal injury (DAI) of excitatory and inhibitory neuronal systems. A key finding was that mTBI-induced neocortical electrophysiological changes involved non-DAI/ intact excitatory pyramidal neurons consistent with AIS-specific alterations. In the current study we employed Thy1-yellow fluorescent protein (YFP)-H mice to test if mTBI induces AIS structural and/or functional plasticity within intact pyramidal neurons 2 days after mTBI. We used confocal microscopy to assess intact YFP+ pyramidal neurons in layer 5 of primary somatosensory barrel field (S1BF), whose axons were continuous from the soma of origin to the subcortical white matter (SCWM). YFP+ axonal traces were superimposed on ankG and NaV1.6 immunofluorescent profiles to determine AIS position and length. We found that while mTBI had no effect on ankG start position, the length significantly decreased from the distal end, consistent with the site of AP initiation at the AIS. However, NaV1.6 structure did not change after mTBI, suggesting uncoupling from ankG. Parallel quantitative analysis of presynaptic inhibitory terminals along the postsynaptic perisomatic domain of these same intact YFP+ excitatory pyramidal neurons revealed a significant decrease in GABAergic bouton density. Also within this non-DAI population, patch-clamp recordings of intact YFP+ pyramidal neurons showed AP acceleration decreased 2 days post-mTBI, consistent with AIS functional plasticity. Simulations of realistic pyramidal neuron computational models using experimentally determined AIS lengths showed a subtle decrease is NaV1.6 density is sufficient to attenuate AP acceleration. Collectively, these findings highlight the complexity of mTBI-induced neocortical circuit disruption, involving changes in extrinsic/presynaptic inhibitory perisomatic input interfaced with intrinsic/postsynaptic intact excitatory neuron AIS output.

Neurons are highly specialized cells of the nervous system that receive, process and transmit electrical signals critical for normal brain function. Here, we review the intricate organization of axonal membrane domains that facilitate rapid action potential conduction underlying communication between complex neuronal circuits. Two critical excitable domains of vertebrate axons are the axon initial segment (AIS) and the nodes of Ranvier, which are characterized by the high concentrations of voltage-gated ion channels, cell adhesion molecules and specialized cytoskeletal networks. The AIS is located at the proximal region of the axon and serves as the site of action potential initiation, while nodes of Ranvier, gaps between adjacent myelin sheaths, allow rapid propagation of the action potential through saltatory conduction. The AIS and nodes of Ranvier are assembled by ankyrins, spectrins and their associated binding partners through the clustering of membrane proteins and connection to the underlying cytoskeleton network. Although the AIS and nodes of Ranvier share similar protein composition, their mechanisms of assembly are strikingly different. Here we will cover the mechanisms of formation and maintenance of these axonal excitable membrane domains, specifically highlighting the similarities and differences between them. We will also discuss recent advances in super resolution fluorescence imaging which have elucidated the arrangement of the submembranous axonal cytoskeleton revealing a surprising structural organization necessary to maintain axonal organization and function. Finally, human mutations in axonal domain components have been associated with a growing number of neurological disorders including severe cognitive dysfunction, epilepsy, autism, neurodegenerative diseases and psychiatric disorders. Overall, this review highlights the assembly, maintenance and function of axonal excitable domains, particularly the AIS and nodes of Ranvier, and how abnormalities in these processes may contribute to disease.

Aims: Iodine is critical for synthesis of thyroid hormones (TH). And iodine deficiency (ID) is one of the most significant reasons of intellectual disability and motor memory impairment, although the potential mechanisms are still under investigation. Presently, mild ID and marginal ID are largely ignored problems for women of child bearing age. Mild ID is a subtle form of TH deficiency, which shows low levels of free thyroxine (FT4) and relatively normal free triiodothyronine (FT3) or thyroid stimulation hormone (TSH). And marginal ID is a milder form of ID with decreased total T4 (TT4) but relatively normal FT3, FT4, and TSH. Therefore, we investigated the effects of maternal different degrees of ID on the development of pinceau in cerebellar purkinje cells (PCs) and studied the expression of pinceau related protein, which is crucial for the development and maturation of pinceau.Methods and Results: Three developmental iodine deficient rat models were created by feeding dam rats with an iodine-deficient diet and deionized water supplemented with potassiumiodide. Our study showed that different degrees of ID inhibited cerebellar pinceau synapse development and maturation on postnatal day (PN) 14 and PN21. What's more, mild and severe ID reduced the expression of AnkG, β4-spectrin, neurofascin186 and NrCAM on PN7, PN14, and PN21. However, marginal ID rarely altered expression of these proteins in the offspring.Conclusion: These results suggested that maternal mild and severe ID impaired the development and maturation of cerebellar pinceau, which may be attributed to the decrease of AnkG, β4-spectrin, neurofascin 186, and NrCAM. And the alteration of development and maturation in cerebellar pinceau in the offspring were also observed following maternal marginal ID, which is slighter than that of mild ID.

Neurons are highly polarized cells with apparent functional and morphological differences between dendrites and axon. A critical determinant for the molecular and functional identity of axonal and dendritic segments is the restricted expression of voltage-gated ion channels (VGCs). Several studies show an uneven distribution of ion channels and their differential regulation within dendrites and axons, which is a prerequisite for an appropriate integration of synaptic inputs and the generation of adequate action potential (AP) firing patterns. This review article will focus on the signaling pathways leading to segmented expression of voltage-gated potassium and sodium ion channels at the neuronal plasma membrane and the regulatory mechanisms ensuring segregated functions. We will also discuss the relevance of proper ion channel targeting for neuronal physiology and how alterations in polarized distribution contribute to neuronal pathology.

The axon initial segment (AIS) is a specialised neuronal compartment involved in the maintenance of axo-dendritic polarity and in the generation of action potentials. It is also a site of significant structural plasticity – manipulations of neuronal activity in vitro and in vivo can produce changes in AIS position and/or size that are associated with alterations in intrinsic excitability. However, to date all activity-dependent AIS changes have been observed in experiments carried out on fixed samples, offering only a snapshot, population-wide view of this form of plasticity. To extend these findings by following morphological changes at the AIS of individual neurons requires reliable means of labelling the structure in live preparations. Here we assessed five different immunofluorescence-based and genetically-encoded tools for live-labelling the AIS of dentate granule cells in dissociated hippocampal cultures. We found that an antibody targeting the extracellular domain of neurofascin provided accurate live label of AIS structure at baseline, but could not follow rapid activity-dependent changes in AIS length. Three different fusion constructs of GFP with full-length AIS proteins also proved unsuitable: while neurofascin-186-GFP and NaVβ4-GFP did not localise to the AIS in our experimental conditions, overexpressing 270kDa-AnkyrinG-GFP produced abnormally elongated AISs in mature neurons. In contrast, a genetically-encoded construct consisting of a voltage-gated sodium channel intracellular domain fused to yellow fluorescent protein (YFP-NaVII-III) fulfilled all of our criteria for successful live AIS label: this construct specifically localised to the AIS, accurately revealed plastic changes at the structure within hours, and, crucially, did not alter normal cell firing properties. We therefore recommend this probe for future studies of live AIS plasticity in vitro and in vivo.

The axon initial segment (AIS) is positioned between the axonal and somato-dendritic compartments and plays a pivotal role in triggering action potentials and determining neuronal output. It is now widely accepted that structural properties of the AIS, such as length and/or location relative to the soma, change in an activity-dependent manner. This structural plasticity of the AIS is known to be crucial for homeostatic control of neuronal excitability. However, it is obvious that the impact of the AIS on neuronal excitability is critically dependent on the biophysical properties of the AIS, which are primarily determined by the composition and characteristics of ion channels in this domain. Moreover, these properties can be altered via phosphorylation and/or redistribution of the channels. Recently, studies in auditory neurons showed that alterations in the composition of voltage-gated K+ channels at the AIS coincide with elongation of the AIS, thereby enhancing the neuronal excitability, suggesting that the interaction between structural and functional plasticities of the AIS is important in the control of neuronal excitability. In this review, we will summarize the current knowledge regarding structural and functional alterations of the AIS and discuss how they interact and contribute to regulating the neuronal output.

The selective transport of different cargoes into axons and dendrites underlies the polarized organization of the neuron. Although it has become clear that the combined activity of different motors determines the destination and selectivity of transport, little is known about the mechanistic details of motor cooperation. For example, the exact role of myosin-V in opposing microtubule-based axon entries has remained unclear. Here we use two orthogonal chemically-induced heterodimerization systems to independently recruit different motors to cargoes. We find that recruiting myosin-V to kinesin-propelled cargoes at approximately equal numbers is sufficient to stall motility. Kinesin-driven cargoes entering the axon were arrested in the axon initial segment (AIS) upon myosin-V recruitment and accumulated in distinct actin-rich hotspots. Importantly, unlike proposed previously, myosin-V did not return these cargoes to the cell body, suggesting that additional mechanism are required to establish cargo retrieval from the AIS. [ABSTRACT FROM AUTHOR]

The axon initial segment (AIS) is the site of action potential (AP) initiation, thus a crucial regulator of neuronal activity. In excitatory pyramidal neurons, the high density of voltage-gated sodium channels (NaV1.6) at the distal AIS regulates AP initiation. A surrogate AIS marker, ankyrin-G (ankG) is a structural protein regulating neuronal functional via clustering voltage-gated ion channels. In neuronal circuits, changes in presynaptic input can alter postsynaptic output via AIS structural-functional plasticity. Recently, we showed experimental mild traumatic brain injury (mTBI) evokes neocortical circuit disruption via diffuse axonal injury (DAI) of excitatory and inhibitory neuronal systems. A key finding was that mTBI-induced neocortical electrophysiological changes involved non-DAI/ intact excitatory pyramidal neurons consistent with AIS-specific alterations. In the current study we employed Thy1-yellow fluorescent protein (YFP)-H mice to test if mTBI induces AIS structural and/or functional plasticity within intact pyramidal neurons 2 days after mTBI. We used confocal microscopy to assess intact YFP+ pyramidal neurons in layer 5 of primary somatosensory barrel field (S1BF), whose axons were continuous from the soma of origin to the subcortical white matter (SCWM). YFP+ axonal traces were superimposed on ankG and NaV1.6 immunofluorescent profiles to determine AIS position and length. We found that while mTBI had no effect on ankG start position, the length significantly decreased from the distal end, consistent with the site of AP initiation at the AIS. However, NaV1.6 structure did not change after mTBI, suggesting uncoupling from ankG. Parallel quantitative analysis of presynaptic inhibitory terminals along the postsynaptic perisomatic domain of these same intact YFP+ excitatory pyramidal neurons revealed a significant decrease in GABAergic bouton density. Also within this non-DAI population, patch-clamp recordings of intact YFP+ pyramidal neurons showed AP acceleration decreased 2 days post-mTBI, consistent with AIS functional plasticity. Simulations of realistic pyramidal neuron computational models using experimentally determined AIS lengths showed a subtle decrease is NaV1.6 density is sufficient to attenuate AP acceleration. Collectively, these findings highlight the complexity of mTBI-induced neocortical circuit disruption, involving changes in extrinsic/presynaptic inhibitory perisomatic input interfaced with intrinsic/postsynaptic intact excitatory neuron AIS output. [ABSTRACT FROM AUTHOR]

Neurons are highly specialized cells of the nervous system that receive, process and transmit electrical signals critical for normal brain function. Here, we review the intricate organization of axonal membrane domains that facilitate rapid action potential conduction underlying communication between complex neuronal circuits. Two critical excitable domains of vertebrate axons are the axon initial segment (AIS) and the nodes of Ranvier, which are characterized by the high concentrations of voltage-gated ion channels, cell adhesion molecules and specialized cytoskeletal networks. The AIS is located at the proximal region of the axon and serves as the site of action potential initiation, while nodes of Ranvier, gaps between adjacent myelin sheaths, allow rapid propagation of the action potential through saltatory conduction. The AIS and nodes of Ranvier are assembled by ankyrins, spectrins and their associated binding partners through the clustering of membrane proteins and connection to the underlying cytoskeleton network. Although the AIS and nodes of Ranvier share similar protein composition, their mechanisms of assembly are strikingly different. Here we will cover the mechanisms of formation and maintenance of these axonal excitable membrane domains, specifically highlighting the similarities and differences between them. We will also discuss recent advances in super resolution fluorescence imaging which have elucidated the arrangement of the submembranous axonal cytoskeleton revealing a surprising structural organization necessary to maintain axonal organization and function. Finally, human mutations in axonal domain components have been associated with a growing number of neurological disorders including severe cognitive dysfunction, epilepsy, autism, neurodegenerative diseases and psychiatric disorders. Overall, this review highlights the assembly, maintenance and function of axonal excitable domains, particularly the AIS and nodes of Ranvier, and how abnormalities in these processes may contribute to disease. [ABSTRACT FROM AUTHOR]

Neurons are highly polarized cells with apparent functional and morphological differences between dendrites and axon. A critical determinant for the molecular and functional identity of axonal and dendritic segments is the restricted expression of voltage-gated ion channels (VGCs). Several studies show an uneven distribution of ion channels and their differential regulation within dendrites and axons, which is a prerequisite for an appropriate integration of synaptic inputs and the generation of adequate action potential (AP) firing patterns. This review article will focus on the signaling pathways leading to segmented expression of voltage-gated potassium and sodium ion channels at the neuronal plasma membrane and the regulatory mechanisms ensuring segregated functions. We will also discuss the relevance of proper ion channel targeting for neuronal physiology and how alterations in polarized distribution contribute to neuronal pathology. [ABSTRACT FROM AUTHOR]

The axon initial segment (AIS) is a highly specialized neuronal compartment that plays a key role in neuronal development and excitability. It concentrates multiple membrane proteins such as ion channels and cell adhesion molecules (CAMs) that are recruited to the AIS by the scaffold protein ankyrin G (ankG). The crucial function of ankG in the anchoring of AIS membrane components is well established, but a reciprocal role of membrane partners in ankG targeting and stabilization remained elusive. In rat cultured hippocampal neurons and cortical organotypic slices, we found that shRNA-mediated knockdown of ankG membrane partners (voltage-gated sodium channels (Nav) or neurofascin-186) led to a decrease of ankG concentration and perturbed the AIS formation and maintenance. These effects were rescued by expressing a recombinant AIS-targeted Nav or by a minimal construct containing the ankyrin-binding domain of Nav1.2 and a membrane anchor (mABD). Moreover, overexpressing mABD in mature neurons led to ankG mislocalization. Altogether, these results demonstrate that a tight and precocious association of ankG with its membrane partners is a key step for the establishment and maintenance of the AIS. [ABSTRACT FROM AUTHOR]

Neuronal polarization underlies the ability of neurons to integrate and transmit information. This process begins early in development with axon outgrowth, followed by dendritic growth and subsequent maturation. In between these two steps, the axon initial segment (AIS), a subcellular domain crucial for generating action potentials (APs) and maintaining the morphological and functional polarization, starts to develop. However, the cellular/molecular mechanisms and receptors involved in AIS initial development and maturation are mostly unknown. In this study, we have focused on the role of the type-1 cannabinoid receptor (CB1R), a highly abundant G-protein coupled receptor (GPCR) in the nervous system largely involved in different phases of neuronal development and differentiation. Although CB1R activity modulation has been related to changes in axons or dendrites, its possible role as a modulator of AIS development has not been yet explored. Here we analyzed the potential role of CB1R on neuronal morphology and AIS development using pharmacological and RNA interference approaches in cultured hippocampal neurons. CB1R inhibition, at a very early developmental stage, has no effect on axonal growth, yet CB1R activation can promote it. By contrast, subsequent dendritic growth is impaired by CB1R inhibition, which also reduces ankyrinG density at the AIS. Moreover, our data show a significant correlation between early dendritic growth and ankyrinG density. However, CB1R inhibition in later developmental stages after dendrites are formed only reduces ankyrinG accumulation at the AIS. In conclusion, our data suggest that neuronal CB1R basal activity plays a role in initial development of dendrites and indirectly in AIS proteins accumulation. Based on the lack of CB1R expression at the AIS, we hypothesize that CB1R mediated modulation of dendritic arbor size during early development indirectly determines the accumulation of ankyrinG and AIS development. Further studies will be necessary to determine which CB1R-dependent mechanisms can coordinate these two domains, and what may be the impact of these early developmental changes once neurons mature and are embedded in a functional brain network. [ABSTRACT FROM AUTHOR]

Axon initial segments (AIS) and nodes of Ranvier are highly specialized axonal membrane domains enriched in Na+ channels. These Na+ channel clusters play essential roles in action potential initiation and propagation. AIS and nodal Na+ channel complexes are linked to the actin cytoskeleton through βIV spectrin. However, neuronal βIV spectrin exists as two main splice variants: a longer βIVΣ1 variant with canonical N-terminal actin and αII spectrin-binding domains, and a shorter βIVΣ6 variant lacking these domains. Here, we show that the predominant neuronal βIV spectrin splice variant detected in the developing brain switches from βIVΣ1 to βIVΣ6, and that this switch is correlated with expression changes in ankyrinG (ankG) splice variants. We show that βIVΣ1 is the predominant splice variant at nascent and developing AIS and nodes of Ranvier, but with increasing age and in adults βIVΣ6 becomes the main splice variant. Remarkably, super-resolution microscopy revealed that the spacing of spectrin tetramers between actin rings remains unchanged, but that shorter spectrin tetramers may also be present. Thus, during development βIV spectrin may undergo a switch in the splice variants found at AIS and nodes of Ranvier. [ABSTRACT FROM AUTHOR]

The axon initial segment (AIS) is a specialized neuronal compartment involved in the maintenance of axo-dendritic polarity and in the generation of action potentials. It is also a site of significant structural plasticity-manipulations of neuronal activity in vitro and in vivo can produce changes in AIS position and/or size that are associated with alterations in intrinsic excitability. However, to date all activity-dependent AIS changes have been observed in experiments carried out on fixed samples, offering only a snapshot, population-wide view of this form of plasticity. To extend these findings by following morphological changes at the AIS of individual neurons requires reliable means of labeling the structure in live preparations. Here, we assessed five different immunofluorescence-based and genetically-encoded tools for live-labeling the AIS of dentate granule cells (DGCs) in dissociated hippocampal cultures. We found that an antibody targeting the extracellular domain of neurofascin provided accurate live label of AIS structure at baseline, but could not follow rapid activity-dependent changes in AIS length. Three different fusion constructs of GFP with full-length AIS proteins also proved unsuitable: while neurofascin-186-GFP and NaVβ4-GFP did not localize to the AIS in our experimental conditions, overexpressing 270kDa-AnkyrinG-GFP produced abnormally elongated AISs in mature neurons. In contrast, a genetically-encoded construct consisting of a voltage-gated sodium channel intracellular domain fused to yellow fluorescent protein (YFP-NaVII-III) fulfilled all of our criteria for successful live AIS label: this construct specifically localized to the AIS, accurately revealed plastic changes at the structure within hours, and, crucially, did not alter normal cell firing properties. We therefore recommend this probe for future studies of live AIS plasticity in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Neurons are highly polarized cells exhibiting axonal and somatodendritic domains with distinct complements of cytoplasmic organelles. Although some organelles are widely distributed throughout the neuronal cytoplasm, others are segregated to either the axonal or somatodendritic domains. Recent findings show that organelle segregation is largely established at a pre-axonal-exclusion zone (PAEZ) within the axon hillock. Polarized sorting of cytoplasmic organelles at the PAEZ is proposed to depend mainly on their selective association with different microtubule motors and, in turn, with distinct microtubule arrays. Somatodendritic organelles that escape sorting at the PAEZ can be subsequently retrieved at the axon initial segment (AIS) by a microtubule- and/or actin-based mechanism. Dynamic sorting along the PAEZ-AIS continuum can thus explain the polarized distribution of cytoplasmic organelles between the axonal and somatodendritic domains.