The axon initial segment (AIS) constitutes not only the site of action potential initiation, but also a hub for activity-dependent modulation of output generation. Recent studies shedding light on AIS function used predominantly post-hoc approaches since no robust murine in vivo live reporters exist. Here, we introduce a reporter line in which the AIS is intrinsically labeled by an ankyrin-G-GFP fusion protein activated by Cre recombinase, tagging the native Ank3 gene. Using confocal, superresolution, and two-photon microscopy as well as whole-cell patch-clamp recordings in vitro, ex vivo, and in vivo, we confirm that the subcellular scaffold of the AIS and electrophysiological parameters of labeled cells remain unchanged. We further uncover rapid AIS remodeling following increased network activity in this model system, as well as highly reproducible in vivo labeling of AIS over weeks. This novel reporter line allows longitudinal studies of AIS modulation and plasticity in vivo in real-time and thus provides a unique approach to study subcellular plasticity in a broad range of applications.

Changes in mitochondrial distribution are a feature of numerous age-related neurodegenerative diseases. In Drosophila, reducing the activity of Cdk5 causes a neurodegenerative phenotype and is known to affect several mitochondrial properties. Therefore, we investigated whether alterations of mitochondrial distribution are involved in Cdk5-associated neurodegeneration. We find that reducing Cdk5 activity does not alter the balance of mitochondrial localization to the somatodendritic versus axonal neuronal compartments of the mushroom body, the learning and memory center of the Drosophila brain. We do, however, observe changes in mitochondrial distribution at the axon initial segment (AIS), a neuronal compartment located in the proximal axon involved in neuronal polarization and action potential initiation. Specifically, we observe that mitochondria are partially excluded from the AIS in wild-type neurons, but that this exclusion is lost upon reduction of Cdk5 activity, concomitant with the shrinkage of the AIS domain that is known to occur in this condition. This mitochondrial redistribution into the AIS is not likely due to the shortening of the AIS domain itself but rather due to altered Cdk5 activity. Furthermore, mitochondrial redistribution into the AIS is unlikely to be an early driver of neurodegeneration in the context of reduced Cdk5 activity.

Parvalbumin-expressing (PV+) inhibitory interneurons drive gamma oscillations (30-80 Hz), which underlie higher cognitive functions. In this review, we discuss two groups/aspects of fundamental properties of PV+ interneurons. In the first group (dubbed Before Axon), we list properties representing optimal synaptic integration in PV+ interneurons designed to support fast oscillations. For example: [i] Information can neither enter nor leave the neocortex without the engagement of fast PV+ -mediated inhibition; [ii] Voltage responses in PV+ interneuron dendrites integrate linearly to reduce impact of the fluctuations in the afferent drive; and [iii] Reversed somatodendritic Rm gradient accelerates the time courses of synaptic potentials arriving at the soma. In the second group (dubbed After Axon), we list morphological and biophysical properties responsible for (a) short synaptic delays, and (b) efficient postsynaptic outcomes. For example: [i] Fast-spiking ability that allows PV+ interneurons to outpace other cortical neurons (pyramidal neurons). [ii] Myelinated axon (which is only found in the PV+ subclass of interneurons) to secure fast-spiking at the initial axon segment; and [iii] Inhibitory autapses - autoinhibition, which assures brief biphasic voltage transients and supports postinhibitory rebounds. Recent advent of scientific tools, such as viral strategies to target PV cells and the ability to monitor PV cells via in vivo imaging during behavior, will aid in defining the role of PV cells in the CNS. Given the link between PV+ interneurons and cognition, in the future, it would be useful to carry out physiological recordings in the PV+ cell type selectively and characterize if and how psychiatric and neurological diseases affect initiation and propagation of electrical signals in this cortical sub-circuit. Voltage imaging may allow fast recordings of electrical signals from many PV+ interneurons simultaneously.

We are studying the mechanisms of H-reflex operant conditioning, a simple form of learning. Modelling studies in the literature and our previous data suggested that changes in the axon initial segment (AIS) might contribute. To explore this, we used blinded quantitative histological and immunohistochemical methods to study in adult rats the impact of H-reflex conditioning on the AIS of the spinal motoneuron that produces the reflex. Successful, but not unsuccessful, H-reflex up-conditioning was associated with greater AIS length and distance from soma; greater length correlated with greater H-reflex increase. Modelling studies in the literature suggest that these increases may increase motoneuron excitability, supporting the hypothesis that they may contribute to H-reflex increase. Up-conditioning did not affect AIS ankyrin G (AnkG) immunoreactivity (IR), p-p38 protein kinase IR, or GABAergic terminals. Successful, but not unsuccessful, H-reflex down-conditioning was associated with more GABAergic terminals on the AIS, weaker AnkG-IR, and stronger p-p38-IR. More GABAergic terminals and weaker AnkG-IR correlated with greater H-reflex decrease. These changes might potentially contribute to the positive shift in motoneuron firing threshold underlying H-reflex decrease; they are consistent with modelling suggesting that sodium channel change may be responsible. H-reflex down-conditioning did not affect AIS dimensions. This evidence that AIS plasticity is associated with and might contribute to H-reflex conditioning adds to evidence that motor learning involves both spinal and brain plasticity, and both neuronal and synaptic plasticity. AIS properties of spinal motoneurons are likely to reflect the combined influence of all the motor skills that share these motoneurons. KEY POINTS: Neuronal action potentials normally begin in the axon initial segment (AIS). AIS plasticity affects neuronal excitability in development and disease. Whether it does so in learning is unknown. Operant conditioning of a spinal reflex, a simple learning model, changes the rat spinal motoneuron AIS. Successful, but not unsuccessful, H-reflex up-conditioning is associated with greater AIS length and distance from soma. Successful, but not unsuccessful, down-conditioning is associated with more AIS GABAergic terminals, less ankyrin G, and more p-p38 protein kinase. The associations between AIS plasticity and successful H-reflex conditioning are consistent with those between AIS plasticity and functional changes in development and disease, and with those predicted by modelling studies in the literature. Motor learning changes neurons and synapses in spinal cord and brain. Because spinal motoneurons are the final common pathway for behaviour, their AIS properties probably reflect the combined impact of all the behaviours that use these motoneurons.

In neurons, the microtubule (MT) cytoskeleton forms the basis for long-distance protein transport from the cell body into and out of dendrites and axons. To maintain neuronal polarity, the axon initial segment (AIS) serves as a physical barrier, separating the axon from the somatodendritic compartment and acting as a filter for axonal cargo. Selective trafficking is further instructed by axonal enrichment of MT post-translational modifications, which affect MT dynamics and the activity of motor proteins. Here, we compared two knockout mouse lines lacking the respective enzymes for MT tyrosination and detyrosination, and found that both knockouts led to a shortening of the AIS. Neurons from both lines also showed an increased immobile fraction of endolysosomes present in the axon, whereas mobile organelles displayed shortened run distances in the retrograde direction. Overall, our results highlight the importance of maintaining the balance of tyrosinated and detyrosinated MTs for proper AIS length and axonal transport processes.

In a variety of neurons, action potentials (APs) initiate at the proximal axon, within a region called the axon initial segment (AIS), which has a high density of voltage-gated sodium channels (NaVs) on its membrane. In pyramidal neurons, the proximal AIS has been reported to exhibit a higher proportion of NaVs with gating properties that are \"right-shifted\" to more depolarized voltages, compared to the distal AIS. Further, recent experiments have revealed that as neurons develop, the spatial distribution of NaV subtypes along the AIS can change substantially, suggesting that neurons tune their excitability by modifying said distribution. When neurons are stimulated axonally, computational modelling has shown that this spatial separation of gating properties in the AIS enhances the backpropagation of APs into the dendrites. In contrast, in the more natural scenario of somatic stimulation, our simulations show that the same distribution can impede backpropagation, suggesting that the choice of orthodromic versus antidromic stimulation can bias or even invert experimental findings regarding the role of NaV subtypes in the AIS. We implemented a range of hypothetical NaV distributions in the AIS of three multicompartmental pyramidal cell models and investigated the precise kinetic mechanisms underlying such effects, as the spatial distribution of NaV subtypes is varied. With axonal stimulation, proximal NaV availability dominates, such that concentrating right-shifted NaVs in the proximal AIS promotes backpropagation. However, with somatic stimulation, the models are insensitive to availability kinetics. Instead, the higher activation threshold of right-shifted NaVs in the AIS impedes backpropagation. Therefore, recently observed developmental changes to the spatial separation and relative proportions of NaV1.2 and NaV1.6 in the AIS differentially impact activation and availability. The observed effects on backpropagation, and potentially learning via its putative role in synaptic plasticity (e.g. through spike-timing-dependent plasticity), are opposite for orthodromic versus antidromic stimulation, which should inform hypotheses about the impact of the developmentally regulated subcellular localization of these NaV subtypes.

The toxin AaH-II, from the scorpion Androctonus australis Hector venom, is a 64 amino acid peptide that targets voltage-gated Na+ channels (VGNCs) and slows their inactivation. While at macroscopic cellular level AaH-II prolongs the action potential (AP), a functional analysis of the effect of the toxin in the axon initial segment (AIS), where VGNCs are highly expressed, was never performed so far. Here, we report an original analysis of the effect of AaH-II on the AP generation in the AIS of neocortical layer-5 pyramidal neurons from mouse brain slices. After determining that AaH-II does not discriminate between Nav1.2 and Nav1.6, i.e. between the two VGNC isoforms expressed in this neuron, we established that 7 nM was the smallest toxin concentration producing a minimal detectable deformation of the somatic AP after local delivery of the toxin. Using membrane potential imaging, we found that, at this minimal concentration, AaH-II substantially widened the AP in the AIS. Using ultrafast Na+ imaging, we found that local application of 7 nM AaH-II caused a large increase in the slower component of the Na+ influx in the AIS. Finally, using ultrafast Ca2+ imaging, we observed that 7 nM AaH-II produces a spurious slow Ca2+ influx via Ca2+-permeable VGNCs. Molecules targeting VGNCs, including peptides, are proposed as potential therapeutic tools. Thus, the present analysis in the AIS can be considered a general proof-of-principle on how high-resolution imaging techniques can disclose drug effects that cannot be observed when tested at the macroscopic level.

Due to its proximity to the axon initial segment (AIS), the paranode of the first myelin segment can influence the threshold for action potentials and how a neuron participates in a neuronal circuit. Using serial section electron microscopy, we examined its three-dimensional (3D) organization in the ventral horn of the mouse spinal cord. The myelin loops of postnatal day 18 mice resemble those at the node of Ranvier. However, in 3-month-old mice, 13 of 22 para-AIS showed 4 types of alteration: (A) A cytoplasmic foot process, with ultrastructural characteristics of an astrocyte, was interposed between the axolemma and the myelin loops. (B) A thin extension of the inner tongue was present between the foot process and axolemma. (C) The foot process was absent. The inner tongue extension was a broad lamella from which a thin extension reached beyond the loops and spiraled around axon. (D) One set of loops was adjacent to the axon, and another was further back and underlain by compact myelin. We suggest that (A)-(C) are steps in a progression toward (D). In this progression, a glial process displaces the original loops, the inner tongue reactivates and extends beneath the foot process, then wraps around the axon to form a new set of loops. This is the first study of the 3D organization of myelin at the AIS and provides evidence for glia-mediated age-dependent remodeling at this critical region.

Dysfunction in sodium channels and their ankyrin scaffolding partners have both been implicated in neurodevelopmental disorders, including autism spectrum disorder (ASD). In particular, the genes SCN2A, which encodes the sodium channel NaV1.2, and ANK2, which encodes ankyrin-B, have strong ASD association. Recent studies indicate that ASD-associated haploinsufficiency in Scn2a impairs dendritic excitability and synaptic function in neocortical pyramidal cells, but how NaV1.2 is anchored within dendritic regions is unknown. Here, we show that ankyrin-B is essential for scaffolding NaV1.2 to the dendritic membrane of mouse neocortical neurons and that haploinsufficiency of Ank2 phenocopies intrinsic dendritic excitability and synaptic deficits observed in Scn2a+/- conditions. These results establish a direct, convergent link between two major ASD risk genes and reinforce an emerging framework suggesting that neocortical pyramidal cell dendritic dysfunction can contribute to neurodevelopmental disorder pathophysiology.

Static magnetic stimulation (SMS) is a form of non-invasive brain stimulation that alters neural activity and induces neural plasticity that outlasts the period of stimulation. This can modify corticospinal excitability or motor behaviours, suggesting that SMS may alter the intrinsic excitability of neurons. In mammalian neurons, the axon initial segment (AIS) is the site of action potential initiation and undergoes structural plasticity (changes in length and position from the soma) as a homeostatic mechanism to counteract chronic changes in neuronal activity. We investigated whether the chronic application of SMS (6 and 48 h, 0.5 T) induces structural AIS plasticity in postnatally derived primary cortical neurons. Following 6 h of SMS, we observed a shortening in mean AIS length compared to control, that persisted 24 h post stimulation. In contrast, 48 h of SMS induced an immediate distal shift that persisted 24 h post-stimulation. Pharmacological blockade of voltage gated L/T-type calcium channels during stimulation did not prevent SMS-induced AIS structural plasticity. Our findings provide the foundation to expand the use of chronic SMS as a non-invasive method to promote AIS plasticity.