Intramuscular Botulinum toxin A injections induce central changes to axon initial segments and cholinergic boutons on spinal motoneurones in rats.

Intramuscular injections of botulinum toxin block pre-synaptic cholinergic release at neuromuscular junctions producing a temporary paralysis of affected motor units. There is increasing evidence, however, that the effects are not restricted to the periphery and can alter the central excitability of the motoneurones at the spinal level. This includes increases in input resistance, decreases in rheobase currents for action potentials and prolongations of the post-spike after-hyperpolarization. The aim of our experiments was to investigate possible anatomical explanations for these changes. Unilateral injections of Botulinum toxin A mixed with a tracer were made into the gastrocnemius muscle of adult rats and contralateral tracer only injections provided controls. Immunohistochemistry for Ankyrin G and the vesicular acetylcholine transporter labelled axon initial segments and cholinergic C-boutons on traced motoneurones at 2 weeks post-injection. Soma size was not affected by the toxin; however, axon initial segments were 5.1% longer and 13.6% further from the soma which could explain reductions in rheobase. Finally, there was a reduction in surface area (18.6%) and volume (12.8%) but not frequency of C-boutons on treated motoneurones potentially explaining prolongations of the after-hyperpolarization. Botulinum Toxin A therefore affects central anatomical structures controlling or modulating motoneurone excitability explaining previously observed excitability changes.

Sub-membranous actin rings in the axon initial segment are resistant to the action of latrunculin.

The axon initial segment (AIS) comprises a sub-membranous lattice containing periodic actin rings. The overall AIS structure is insensitive to actin-disrupting drugs, but the effects of actin-disrupting drugs on actin rings lack consensus. We examined the effect of latrunculin A and B on the actin cytoskeleton of neurons in culture and actin rings in the AIS. Both latrunculin A and B markedly reduced the overall amount of F-actin in treated neurons in a dose-dependent manner, but the periodicity of actin rings remained unaffected. The insensitivity of AIS actin rings to latrunculin suggests they are relatively stable.

Activation of axon initial segmental GABAA receptors inhibits action potential generation in neocortical GABAergic interneurons.

Ionotropic GABAA receptors expressing at the axon initial segment (AIS) of glutamatergic pyramidal cell (PC) in the cortex plays critical roles in regulating action potential generation. However, it remains unclear whether these receptors also express at the AIS of cortical GABAergic interneurons. In mouse prefrontal cortical slices, we performed experiments at the soma and AIS of the two most abundant GABAergic interneurons: parvalbumin (PV) and somatostatin (SST) positive neurons. Local application of GABA at the perisomatic axonal regions could evoke picrotoxin-sensitive currents with a reversal potential near the Cl- equilibrium potential. Puffing agonists to outside-out patches excised from AIS confirmed the expression of GABAA receptors. Further pharmacological experiments revealed that GABAA receptors in AIS of PV neurons contain α1 subunits, different from those containing α2/3 in AIS and α4 in axon trunk of layer-5 PCs. Cell-attached recording at the soma of PV and SST neurons revealed that the activation of AIS GABAA receptors inhibits the action potential generation induced by synaptic stimulation. Together, our results demonstrate that the AIS of PV and SST neurons express GABAA receptors with distinct subunit composition, which exert an inhibitory effect on neuronal excitability in these inhibitory interneurons.

A constitutively-active IKK-complex at the axon initial segment.

BACKGROUND: Previous studies provided evidence for an accumulation of IκB-kinase (IKK) α/ß at the axon initial segment (AIS), a neuronal compartment defined by ankyrin-G expression. Here we explored whether the presence of the IKK-complex at the AIS was associated with the activation of IKK signaling at this site. METHODS AND RESULTS: Proximity-ligation assays (PLAs) using pan-IKKα/ß, phospho-IKKα/ß-specific as well as ankyrin-G specific antibodies validated their binding to proximal epitopes in the AIS, while antibodies to other phosphorylated signaling proteins showed no preference for the AIS. Small-hairpin mediated silencing of IKKß significantly reduced anti-phospho-IKKα/ß-immunoreactivities in the AIS. ank3 gene-deficient cerebellar Purkinje cells also exhibited no phosphorylated IKKα/ß at the proximal region of their axons. Transient ankyrin-G overexpression in PC12 cells augmented NF-κB transactivation in an ankyrin-G death-domain dependent manner. Finally, small molecule inhibitors of IKK-activity, including Aspirin, inhibited the accumulation of activated IKK proteins in the AIS. CONCLUSION: Our data suggest the existence of a constitutively-active IKK signaling complex in the AIS.

Perturbation of GABAergic Synapses at the Axon Initial Segment of Basolateral Amygdala Induces Trans-regional Metaplasticity at the Medial Prefrontal Cortex.

GABAergic synapses in the basolateral amygdala (BLA) play an important role in fear memory generation. We have previously reported that reduction in GABAergic synapses innervating specifically at the axon initial segment (AIS) of principal neurons of BLA, by neurofascin (NF) knockdown, impairs fear extinction. BLA is bidirectionally connected with the medial prefrontal cortex (mPFC), which is a key region involved in extinction of acquired fear memory. Here, we showed that reducing AIS GABAergic synapses within the BLA leads to impairment of synaptic plasticity in the BLA-mPFC pathway, as well as in the ventral subiculum (vSub)-mPFC pathway, which is independent of BLA involvement. The results suggest that the alteration within the BLA subsequently resulted in a form of trans-regional metaplasticity in the mPFC. In support of that notion, we observed that NF knockdown induced a severe deficit in behavioral flexibility as measured by reversal learning. Interestingly, reversal learning similar to extinction learning is an mPFC-dependent behavior. In agreement with that, measurement of the immediate-early gene, c-Fos immunoreactivity after reversal learning was reduced in the mPFC and BLA, supporting further the notion that the BLA GABAergic manipulation resulted in trans-regional metaplastic alterations within the mPFC.

Oxidative Stress Induces Disruption of the Axon Initial Segment.

The axon initial segment (AIS), the domain responsible for action potential initiation and maintenance of neuronal polarity, is targeted for disruption in a variety of central nervous system pathological insults. Previous work in our laboratory implicates oxidative stress as a potential mediator of structural AIS alterations in two separate mouse models of central nervous system inflammation, as these effects were attenuated following reactive oxygen species scavenging and NADPH oxidase-2 ablation. While these studies suggest a role for oxidative stress in modulation of the AIS, the direct effects of reactive oxygen and nitrogen species (ROS/RNS) on the stability of this domain remain unclear. Here, we demonstrate that oxidative stress, as induced through treatment with 3-morpholinosydnonimine (SIN-1), a spontaneous ROS/RNS generator, drives a reversible loss of AIS protein clustering in primary cortical neurons in vitro. Pharmacological inhibition of both voltage-dependent and intracellular calcium (Ca2+) channels suggests that this mechanism of AIS disruption involves Ca2+ entry specifically through L-type voltage-dependent Ca2+ channels and its release from IP3-gated intracellular stores. Furthermore, ROS/RNS-induced AIS disruption is dependent upon activation of calpain, a Ca2+-activated protease previously shown to drive AIS modulation. Overall, we demonstrate for the first time that oxidative stress, as induced through exogenously applied ROS/RNS, is capable of driving structural alterations in the AIS complex.

Neurons with Multiple Axons Have Functional Axon Initial Segments.

Neurons grow multiple axons after treatment with glycogen synthase kinase-3 (GSK-3) inhibitors. However, whether they are electrically active is not known. Here, we examined the role of multiple axons as electrophysiological components during neuronal firing. Combining pharmacological, immunofluorescence, and electrophysiological methods, we found that more neurons had multiple axon initial segments (AISs) after inhibition of GSK-3 activity with SB415286. The multiple AISs induced by GSK-3 inhibition were enriched with voltage-gated sodium channels. The depolarization rate of the multiple-AIS neurons was increased, but their action potential threshold and half-width were normal. By calculating derivatives of the action-potential rising phase, an extra d2 V/dt 2 peak from the extra AIS was distinguished; this indicated that the extra AIS fired ahead of the soma and increased the rate of depolarization. Our study demonstrates that the multiple axons induced by GSK-3 inhibition have AIS structures that are electrically active, and provides insight for axon and AIS studies.

Altered axon initial segment in hippocampal newborn neurons, associated with recurrence of temporal lobe epilepsy in rats.

Hippocampal neurogenesis in temporal lobe epilepsy (TLE) may result in alteration of the excitability of neurons, which contributes to spontaneous recurrent seizures. Axon initial segment (AIS) structural and functional plasticity is important in the control of neuronal excitability. It remains to be elucidated whether the plasticity of AIS occurs in hippocampal newly­generated neurons that are involved in recurrent seizures following pilocarpine­induced status epilepticus (SE). The present study first established a pilocarpine­induced TLE rat model to assess the features of newborn neurons and AIS plasticity alterations using double immunofluorescence staining of Ankyrin G and doublecortin (DCX). AIS plasticity alterations include length and distance from soma in the hippocampal newly­generated neurons post­SE. The results of the present study demonstrated that pilocarpine­induced epileptic rats exhibited aberrant hippocampal neurogenesis and longer DCX­labeled cell dendrites in the dentate gyrus. Pilocarpine­induced epileptic rats demonstrated shortened lengths of AIS and an increased distance from the soma in hippocampal newborn neurons. Mibefradil, a T/L­type calcium blocker, reversed the alterations in length and position of AIS in hippocampal newborn neurons post­SE, accompanied by decreased long­term seizure activity without increased aberrant neurogenesis. These findings indicate that the plasticity of AIS in hippocampal neurogenesis may have profound consequences in epilepsy, at least in animals.

Acute neuroinflammation induces AIS structural plasticity in a NOX2-dependent manner.

BACKGROUND: Chronic microglia-mediated inflammation and oxidative stress are well-characterized underlying factors in neurodegenerative disease, whereby reactive inflammatory microglia enhance ROS production and impact neuronal integrity. Recently, it has been shown that during chronic inflammation, neuronal integrity is compromised through targeted disruption of the axon initial segment (AIS), the axonal domain critical for action potential initiation. AIS disruption was associated with contact by reactive inflammatory microglia which wrap around the AIS, increasing association with disease progression. While it is clear that chronic microglial inflammation and enhanced ROS production impact neuronal integrity, little is known about how acute microglial inflammation influences AIS stability. Here, we demonstrate that acute neuroinflammation induces AIS structural plasticity in a ROS-mediated and calpain-dependent manner. METHODS: C57BL/6J and NOX2-/- mice were given a single injection of lipopolysaccharide (LPS; 5 mg/kg) or vehicle (0.9% saline, 10 mL/kg) and analyzed at 6 h-2 weeks post-injection. Anti-inflammatory Didox (250 mg/kg) or vehicle (0.9% saline, 10 mL/kg) was administered beginning 24 h post-LPS injection and continued for 5 days; animals were analyzed 1 week post-injection. Microglial inflammation was assessed using immunohistochemistry (IHC) and RT-qPCR, and AIS integrity was quantitatively analyzed using ankyrinG immunolabeling. Data were statistically compared by one-way or two-way ANOVA where mean differences were significant as assessed using Tukey's post hoc analysis. RESULTS: LPS-induced neuroinflammation, characterized by enhanced microglial inflammation and increased expression of ROS-producing enzymes, altered AIS protein clustering. Importantly, inflammation-induced AIS changes were reversed following resolution of microglial inflammation. Modulation of the inflammatory response using anti-inflammatory Didox, even after significant AIS disruption occurred, increased the rate of AIS recovery. qPCR and IHC analysis revealed that expression of microglial NOX2, a ROS-producing enzyme, was significantly increased correlating with AIS disruption. Furthermore, ablation of NOX2 prevented inflammation-induced AIS plasticity, suggesting that ROS drive AIS structural plasticity. CONCLUSIONS: In the presence of acute microglial inflammation, the AIS undergoes an adaptive change that is capable of spontaneous recovery. Moreover, recovery can be therapeutically accelerated. Together, these findings underscore the dynamic capabilities of this domain in the presence of a pathological insult and provide evidence that the AIS is a viable therapeutic target.

Myosin II activity is required for structural plasticity at the axon initial segment.

In neurons, axons possess a molecularly defined and highly organised proximal region - the axon initial segment (AIS) - that is a key regulator of both electrical excitability and cellular polarity. Despite existing as a large, dense structure with specialised cytoskeletal architecture, the AIS is surprisingly plastic, with sustained alterations in neuronal activity bringing about significant alterations to its position, length or molecular composition. However, although the upstream activity-dependent signalling pathways that lead to such plasticity have begun to be elucidated, the downstream mechanisms that produce structural changes at the AIS are completely unknown. Here, we use dissociated cultures of rat hippocampus to show that two forms of AIS plasticity in dentate granule cells - long-term relocation, and more rapid shortening - are completely blocked by treatment with blebbistatin, a potent and selective myosin II ATPase inhibitor. These data establish a link between myosin II and AIS function, and suggest that myosin II's primary role at the structure may be to effect activity-dependent morphological alterations.

RACK1 is necessary for the formation of point contacts and regulates axon growth.

Receptor for activated C kinase 1 (RACK1) is a multifunctional ribosomal scaffolding protein that can interact with multiple signaling molecules concurrently through its seven WD40 repeats. We recently found that RACK1 is localized to mammalian growth cones, prompting an investigation into its role during neural development. Here, we show for the first time that RACK1 localizes to point contacts within mouse cortical growth cones. Point contacts are adhesion sites that link the actin network within growth cones to the extracellular matrix, and are necessary for appropriate axon guidance. Our experiments show that RACK1 is necessary for point contact formation. Brain-derived neurotrophic factor (BDNF) stimulates an increase in point contact density, which was eliminated by RACK1 shRNA or overexpression of a nonphosphorylatable mutant form of RACK1. We also found that axonal growth requires both RACK1 expression and phosphorylation. We have previously shown that the local translation of ß-actin mRNA within growth cones is necessary for appropriate axon guidance and is dependent on RACK1. Thus, we examined the location of members of the local translation complex relative to point contacts. Indeed, both ß-actin mRNA and RACK1 colocalize with point contacts, and this colocalization increases following BDNF stimulation. This implies the novel finding that local translation is regulated at point contacts. Taken together, these data suggest that point contacts are a targeted site of local translation within growth cones, and RACK1 is a critical member of the point contact complex and necessary for appropriate neural development. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1038-1056, 2017.

The impact of early environmental interventions on structural plasticity of the axon initial segment in neocortex.

Plasticity of the axon initial segment (AIS) is a newly discovered type of structural plasticity that regulates cell excitability. AIS plasticity has been reported to happen during normal development of neocortex and also in a few pathological conditions involving disruption of the inhibition/excitation balance. Here we report on the impact of early environmental interventions on structural plasticity of AIS in the mouse neocortex. C57BL/6 mice were raised in standard or enriched environment (EE) from birth up to the time of experiments and were injected with saline or MK-801 [N-Methyl-D-Aspartate (NMDA) receptor antagonist, 1 mg/kg] on postnatal days (P) 6-10. We used Ankyrin G immunoreactivity to mark the AIS of cortical neurons in two sub-regions of frontal cortex (frontal association area, FrA and secondary motor cortex, M2) and in the secondary visual cortex (V2). In 1-month-old mice, the mean AIS length differed between three areas, with the shortest AISs being observed in V2. Postnatal MK-801 or EE led to shortening of AIS only in the frontal areas. However, exposure to EE restored AIS shortening induced by MK-801. Chronic postnatal MK-801 results in structural plasticity of AIS exclusive to the frontal cortex. EE may modify underlying neuronal mechanisms resulting in restoration of AIS length.

Rats with Malformations of Cortical Development Exhibit Decreased Length of AIS and Hypersensitivity to Pilocarpine-Induced Status Epilepticus.

Malformations of cortical development (MCD) are critical brain development disorders associated with varied abnormalities in both anatomic structures and neural functioning. It is also a very common etiology to the epilepsy, in which the alteration on excitability of cortical neurons is hypothesized as one of important causes to the epileptic seizures. Due to the key role in regulating neuron firing properties, the plasticity of axon initial segment (AIS) was investigated in present study to further determine the relation between MCD and epilepsy. Our results showed a prolonged decrease in the length of AIS occurred in MCD animal models. Besides, the AIS was also found greatly shortened in MCD models during the acute, but not chronic phase of status epileptics compared with intact controls. Our findings of identification of AIS plasticity in MCD animal models and its hypersensitivity to status epilepsy are significant in furthering our understanding of the pathophysiological mechanisms involved in this disorder.