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G-Protein coupled D2 autoreceptors expressed on dopamine neurons (D2Rs) inhibit transmitter release and cell firing at axonal endings and somatodendritic compartments. Mechanistic details of somatodendritic dopamine release remain unresolved, partly due to insufficient information on the subcellular distribution of D2Rs. Previous studies localizing D2Rs have been hindered by a dearth of antibodies validated for specificity in D2R knockout animals and have been limited by the small sampling areas imaged by electron microscopy. This study utilized sub-diffraction fluorescence microscopy and electron microscopy to examine D2 receptors in a superecliptic pHlourin GFP (SEP) epitope tagged D2 receptor knockin mouse. Incubating live slices with an anti-SEP antibody achieved the selective labeling of plasma membrane associated receptors for immunofluorescent imaging over a large area of the substantia nigra pars compacta (SNc). SEP-D2Rs appeared as puncta-like structures along the surface of dendrites and soma of dopamine neurons visualized by antibodies to tyrosine hydroxylase (TH). TH-associated SEP-D2Rs displayed a cell surface density of 0.66 puncta per μm(2), which corresponds to an average frequency of 1 punctum every 1.50 μm. Separate ultrastructural experiments using silver-enhanced immunogold revealed that membrane-bound particles represented 28% of total D2Rs in putative dopamine cells within the SNc. Structures immediately adjacent to dendritic membrane gold particles were unmyelinated axons or axon varicosities (40%), astrocytes (19%), other dendrites (7%), or profiles unidentified (34%) in single sections. Some apposed profiles also expressed D2Rs. Fluorescent and ultrastructural analyses also provided the first visualization of membrane-D2Rs at the axon initial segment, a compartment critical for action potential generation. The punctate appearance of anti-SEP staining indicates there is a population of D2Rs organized in discrete signaling sites along the plasma membrane, and for the first time a quantitative estimate of spatial frequency is provided.

The axon initial segment (AIS) is a specialized structure that controls neuronal excitability via action potential (AP) generation. Currently, AIS plasticity with regard to changes in length and location in response to neural activity has been extensively investigated, but how AIS diameter is regulated remains elusive. Here we report that COUP-TFI (chicken ovalbumin upstream promotor-transcription factor 1) is an essential regulator of AIS diameter in both developing and adult mouse neocortex. Either embryonic or adult ablation of COUP-TFI results in reduced AIS diameter and impaired AP generation. Although COUP-TFI ablations in sparse single neurons and in populations of neurons have similar impacts on AIS diameter and AP generation, they strengthen and weaken, respectively, the receiving spontaneous network in mutant neurons. In contrast, overexpression of COUP-TFI in sparse single neurons increases the AIS diameter and facilitates AP generation, but decreases the receiving spontaneous network. Our findings demonstrate that COUP-TFI is indispensable for both the expansion and maintenance of AIS diameter and that AIS diameter fine-tunes action potential generation and synaptic inputs in mammalian cortical neurons. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12264-021-00792-8.

The axon initial segment (AIS) is essential for maintaining neuronal polarity, modulating protein transport into the axon, and action potential generation. These functions are supported by a distinctive actin and microtubule cytoskeleton that controls axonal trafficking and maintains a high density of voltage-gated ion channels linked by scaffold proteins to the AIS cytoskeleton. However, our knowledge of the mechanisms and proteins involved in AIS cytoskeleton regulation to maintain or modulate AIS structure is limited. In this context, formins play a significant role in the modulation of actin and microtubules. We show that pharmacological inhibition of formins modifies AIS actin and microtubule characteristics in cultured hippocampal neurons, reducing F-actin density and decreasing microtubule acetylation. Moreover, formin inhibition diminishes sodium channels, ankyrinG and ßIV-spectrin AIS density, and AIS length, in cultured neurons and brain slices, accompanied by decreased neuronal excitability. We show that genetic downregulation of the mDia1 formin by interference RNAs also decreases AIS protein density and shortens AIS length. The ankyrinG decrease and AIS shortening observed in pharmacologically inhibited neurons and neuron-expressing mDia1 shRNAs were impaired by HDAC6 downregulation or EB1-GFP expression, known to increase microtubule acetylation or stability. However, actin stabilization only partially prevented AIS shortening without affecting AIS protein density loss. These results suggest that mDia1 maintain AIS composition and length contributing to the stability of AIS microtubules., Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. The work was supported by agrant from Ministerio de Ciencia y Universidades (RTI2018-095156-B-100) to JJG and INSERM funding to DD. Wei Zhang was supported by a fellowship from China Scholarship Council (No.201506300085) and Beatriz Achon by a Master fellowship from Universidad Autónoma de Madrid.

Cortical pyramidal neurons possess a persistent Na(+) current (I(NaP)), which, in contrast to the larger transient current, does not undergo rapid inactivation. Although relatively quite small, I(NaP) is active at subthreshold voltages and therefore plays an important role in neuronal input–output processing. The subcellular distribution of channels responsible for I(NaP) and the mechanisms that render them persistent are not known. Using high-speed fluorescence Na(+) imaging and whole-cell recordings in brain slices obtained from mice of either sex, we reconstructed the I(NaP) elicited by slow voltage ramps in soma and processes of cortical pyramidal neurons. We found that in all neuronal compartments, the relationship between persistent Na(+) conductance and membrane voltage has the shape of a Boltzmann function. Although the density of channels underlying I(NaP) was about twofold lower in the axon initial segment (AIS) than in the soma, the axonal channels were activated by ∼10 mV less depolarization than were somatic channels. This difference in voltage dependence explains why, at functionally critical subthreshold voltages, most I(NaP) originates in the AIS. Finally, we show that endogenous polyamines constrain I(NaP) availability in both somatodendritic and axonal compartments of nondialyzed cortical neurons. SIGNIFICANCE STATEMENT The most salient characteristic of neuronal sodium channels is fast inactivation. However, a fraction of the sodium current does not inactivate. In cortical neurons, persistent current (I(NaP)) plays a prominent role in many important functions. Its subcellular distribution and generation mechanisms are, however, elusive. Using high-speed fluorescence Na(+) imaging and electrical recordings, we reconstructed the I(NaP) in soma and processes of cortical pyramidal neurons. We found that at near-threshold voltages I(NaP) originates predominately from the axon, because of the distinctive voltage dependence of the underlying channels and not because of their high density. Finally, we show that the presence of endogenous polyamines significantly constrains I(NaP) availability in all compartments of nondialyzed cortical neurons.

Since the 1960s, it is held that when a neuron fires, a nerve spike passes only through the selective branches, the calculated choice is a key to learning by rewiring. It is argued by chemically estimating the membrane's ion channel density that different axonal branches get active to pass the spike-branches blink at firing at different time domains. Here, using a new time-lapse dielectric imaging, we visualize the classic branch selection process; thenceforth, hidden circuits operating at different time domains become visible. The fractal grid of coaxial probes captures wireless snapshots of material's vibration at various depths below the membrane by setting a suitable frequency. Thus far, branch selection observed emitted energy or particle but never the emitters, what they do. As each dielectric material transmits and reflects signals of different frequencies, we image live how filaments search for many branch-made circuits, choose a unique pathway 103 times faster than a single nerve spike. It reveals that neural branches and circuit visible in a microscope are not absolute, there coexist many circuits each operating in different dime domains, operating at a time.NEW & NOTEWORTHY Using dielectric resonance scanner, we show electromagnetic field connections between physically separated neurons. Electromagnetic field creates field lines that pass through gap junctions, connect Axon initial segment with the dendrites through Soma, and connect axonal or dendritic branches even if there is no synaptic junction. Consequently, many distinct loops connecting various branches form coexisting circuits. Our discovery suggests that physically appearing neural circuit is a fractional view of many simultaneously operating circuits in different time domains in a neural network.

Somatodendritic missorting of the axonal protein TAU is a hallmark of Alzheimer’s disease and related tauopathies. Cultured rodent primary neurons and iPSC-derived neurons are used for studying mechanisms of neuronal polarity, including TAU trafficking. However, these models are expensive, time-consuming and/or require the sacrification of animals. In this study, we evaluated four differentiation procedures to generate mature neuron cultures from human SH-SY5Y neuroblastoma cells, in comparison to mouse primary neurons, and tested their TAU sorting capacity. We show that SH-SY5Y-derived neurons, differentiated with sequential RA/BDNF treatment, are suitable for investigating axonal TAU sorting. These human neurons show pronounced neuronal polarity, axodendritic outgrowth, expression of the neuronal maturation markers TAU and MAP2, and, importantly, efficient axonal sorting of endogenous and transfected human wild type TAU, similar to primary neurons. We demonstrate that axonal TAU enrichment requires the presence of the C-terminal half, as a C-terminus-lacking construct (N-term-TAUHA) is not axonally enriched in both neuronal cell models. Moreover, SH-SY5Y-derived neurons do not show formation of a classical axon initial segment (AIS), indicated by the lack of Ankyrin G (ANKG) and tripartite motif-containing protein 46 (TRIM46) at the proximal axon, which suggests that successful axonal TAU sorting is independent of classical AIS formation. Taken together, our results suggest i) that SH-SY5Y-derived neurons are a valuable human neuronal cell model for studying TAU sorting, which is readily accessible at low cost and without animal need, and that ii) the mechanisms of axonal TAU targeting require the TAU C-terminal half but are independent of ANKG or TRIM46 enrichment at the proximal axon.

Alcohol intoxication at early ages is a risk factor for the development of addictive behavior. To uncover neuronal molecular correlates of acute ethanol intoxication, we used stable-isotope–labeled mice combined with quantitative mass spectrometry to screen more than 2,000 hippocampal proteins, of which 72 changed synaptic abundance up to twofold after ethanol exposure. Among those were mitochondrial proteins and proteins important for neuronal morphology, including MAP6 and ankyrin-G. Based on these candidate proteins, we found acute and lasting molecular, cellular, and behavioral changes following a single intoxication in alcohol-naïve mice. Immunofluorescence analysis revealed a shortening of axon initial segments. Longitudinal two-photon in vivo imaging showed increased synaptic dynamics and mitochondrial trafficking in axons. Knockdown of mitochondrial trafficking in dopaminergic neurons abolished conditioned alcohol preference in Drosophila flies. This study introduces mitochondrial trafficking as a process implicated in reward learning and highlights the potential of high-resolution proteomics to identify cellular mechanisms relevant for addictive behavior.

Genetic variants in TMEM106B are a major risk factor for several neurodegenerative diseases including frontotemporal degeneration, limbic‐predominant age‐related TDP‐43 encephalopathy, Parkinson's disease, late‐onset‐Alzheimer's disease and constitute a genetic determinant of differential aging. TMEM106B encodes an integral lysosomal membrane protein but its precise physiological function in the central nervous system remains enigmatic. Presently, we aimed to increase understanding of TMEM106B contribution to general brain function and aging. We analyzed an aged cohort of Tmem106b knockout‐, heterozygote and wild‐type mice in a behavioral test battery including assessments of motor function as well as, social, emotional and cognitive function. Aged Tmem106b knockout (KO) mice displayed diverse behavioral deficits including motor impairment, gait defects and reduced startle reactivity. In contrast, no prominent deficits were observed in social, emotional or cognitive behaviors. Histologically, we observed late‐onset loss of Purkinje cells followed by reactive gliosis in the cerebellum, which likely contributed to progressive decline in motor function and gait defects in particular. Reactive gliosis was not restricted to the cerebellum but observed in different areas of the brain including the brain stem and parts of the cerebral cortex. Surviving Purkinje cells showed vacuolated lysosomes in the axon initial segment, implicating TMEM106B‐dependent lysosomal trafficking defects as the underlying cause of axonal and more general neuronal dysfunction contributing to behavioral impairments. Our experiments help to elucidate how TMEM106B affects spatial neuronal homeostasis and exemplifies a critical role of TMEM106B in neuronal cells for survival., TMEM106B is a disease‐modifying gene in several neurodegenerative diseases including frontotemporal dementia. We analyzed aged Tmem106b knockout mice by a battery of behavioral tests and histology and found cerebellar dysfunction accompanying motor dysfunction at later ages.

Autism spectrum disorder can be associated with a variety of genetic findings. We report a heterozygous de novo missense variant of SCN2A, the gene coding a voltage-gated sodium ion channel enriched in the axon initial segment and nodes of Ranvier of "immature" neocortical pyramidal neurons. With further understanding of the neurodevelopmental and functional effects of this missense variant on neuronal excitability and neocortical circuitry, there may be targeted pharmacotherapeutic interventions, potentially with "disease-modifying effects."

Missense variants in the SCN8A voltage-gated sodium channel gene are linked to early-infantile epileptic encephalopathy type 13, also known as SCN8A-related epilepsy. These patients exhibit a wide spectrum of intractable seizure types, severe developmental delay, movement disorders, and elevated risk of sudden unexpected death in epilepsy. The mechanisms by which SCN8A variants lead to epilepsy are poorly understood, although heterologous expression systems and mouse models have demonstrated altered sodium current properties. To investigate these mechanisms using a patient-specific model, we generated induced pluripotent stem cells from three patients with missense variants in SCN8A: p.R1872>L (Patient 1); p.V1592>L (Patient 2); and p.N1759>S (Patient 3). Using small molecule differentiation into excitatory neurons, induced pluripotent stem cell-derived neurons from all three patients displayed altered sodium currents. Patients 1 and 2 had elevated persistent current, while Patient 3 had increased resurgent current compared to controls. Neurons from all three patients displayed shorter axon initial segment lengths compared to controls. Further analyses focused on one of the patients with increased persistent sodium current (Patient 1) and the patient with increased resurgent current (Patient 3). Excitatory cortical neurons from both patients had prolonged action potential repolarization. Using doxycycline-inducible expression of the neuronal transcription factors neurogenin 1 and 2 to synchronize differentiation of induced excitatory cortical-like neurons, we investigated network activity and response to pharmacotherapies. Both small molecule differentiated and induced patient neurons displayed similar abnormalities in action potential repolarization. Patient induced neurons showed increased burstiness that was sensitive to phenytoin, currently a standard treatment for SCN8A-related epilepsy patients, or riluzole, an FDA-approved drug used in amyotrophic lateral sclerosis and known to block persistent and resurgent sodium currents, at pharmacologically relevant concentrations. Patch-clamp recordings showed that riluzole suppressed spontaneous firing and increased the action potential firing threshold of patient-derived neurons to more depolarized potentials. Two of the patients in this study were prescribed riluzole off-label. Patient 1 had a 50% reduction in seizure frequency. Patient 3 experienced an immediate and dramatic seizure reduction with months of seizure freedom. An additional patient with a SCN8A variant in domain IV of Nav1.6 (p.V1757>I) had a dramatic reduction in seizure frequency for several months after starting riluzole treatment, but then seizures recurred. Our results indicate that patient-specific neurons are useful for modelling SCN8A-related epilepsy and demonstrate SCN8A variant-specific mechanisms. Moreover, these findings suggest that patient-specific neuronal disease modelling offers a useful platform for discovering precision epilepsy therapies.

The structural features of dopamine (DA)-GABAergic neurons in the mouse main olfactory bulbs were examined, using both wild type and transgenic TH-GFP mice, with the combination of several methods; the immunocytochemistry, biotinylated dextran amine labeling, lucifer yellow injection in fixed slices, biocytin injection in live slice and the functional olfactory deprivation. DA-GABAergic neurons were clustered in the glomerular layer (GL) but they also scattered in other layers. DA-GABAergic juxtaglomerular neurons, were classified into 5 groups based on their structural features and named as follows: 1) Large periglomerular (LPG) cells with tuft-like glomerular dendritic branches and apparent axons extending to the distant glomeruli, which correspond to the " inhibitory juxtaglomerular association (IJGA) neurons" participating in the interglomerular association system. 2) Small periglomerular (SPG) cells including both axonic and anaxonic ones; the axonic SPG cells might correspond to the classical periglomerular cells. 3) Transglomerular cells extending dendritic processes spanning 2 or more glomeruli. 4) Incrusting cells extending their dendritic branches mainly in the periphery of the glomeruli. 5) Other various neurons not-yet classified. In the layers other than the GL various types of TH expressing neurons were scattered; some of them extended dendritic processes into the GL.

Some forms of Autism Spectrum Disorder, a neurodevelopmental syndrome characterized by impaired communication and social skills as well as repetitive behaviors, are purportedly associated with dysregulation of the excitation/inhibition balance in the cerebral cortex. Through human postmortem tissue analysis, we previously found a significant decrease in the number of a gamma-aminobutyric acid (GABA)ergic interneuron subtype, the chandelier (Ch) cell, in the prefrontal cortex of subjects with autism. Ch cells exclusively target the axon initial segment (AIS) of excitatory pyramidal (Pyr) neurons, and a single Ch cell forms synapses on hundreds of Pyr cells, indicating a possible role in maintaining electrical balance. Thus, we herein investigated this crucial link between Ch and Pyr cells in the anatomy of autism neuropathology by examining GABA receptor protein expression in the Pyr cell AIS in subjects with autism. We collected tissue from the prefrontal cortex (Brodmann Areas (BA) 9, 46, and 47) of 20 subjects with autism and 20 age- and sex-matched control subjects. Immunohistochemical staining with antibodies against the GABAA receptor subunit α2 (GABAARα2) - the subunit most prevalent in the Pyr cell AIS - revealed a significantly decreased GABAARα2 protein in the Pyr cell AIS in supragranular layers of prefrontal cortical areas BA9 and BA47 in autism. Downregulated GABAARα2 protein in the Pyr cell AIS may result from decreased GABA synthesis in the prefrontal cortex of subjects with autism, and thereby contribute to an excitation/inhibition imbalance. Our findings support the potential for GABA receptor agonists asa therapeutic tool for autism.

It has been known for a long time that inositol-trisphosphate (IP(3)) receptors are present in the axon of certain types of mammalian neurons, but their functional role has remained unexplored. Here we show that localized photolysis of IP(3) induces spatially constrained calcium rises in Purkinje cell axons. Confocal immunohistology reveals that the axon initial segment (AIS), as well as terminals onto deep cerebellar cells, express specific subtypes of Gα/q and phospholipase C (PLC) molecules, together with the upstream purinergic receptor P2Y1. By contrast, intermediate parts of the axon express another set of Gα/q and PLC molecules, indicating two spatially segregated signaling cascades linked to IP(3) generation. This prompted a search for distinct actions of IP(3) in different parts of Purkinje cell axons. In the AIS, we found that local applications of the specific P2Y1R agonist MRS2365 led to calcium elevation, and that IP(3) photolysis led to inhibition of action potential firing. In synaptic terminals on deep cerebellar nuclei neurons, we found that photolysis of both IP(3) and ATP led to GABA release. We propose that axonal IP(3) receptors can inhibit action potential firing and increase neurotransmitter release, and that these effects are likely controlled by purinergic receptors. Altogether our results suggest a rich and diverse functional role of IP(3) receptors in axons of mammalian neurons.

KEY POINTS: During maximal effort contractions, intense serotonin release via the raphe-spinal pathway spills over from the somato-dendritic compartment to activate inhibitory 5-HT1A receptors on the axon initial segment of motoneurons to reduce motoneuronal output. We investigated whether the same mechanism of central fatigue is present for low-intensity contractions, whereby weak serotonergic drive over an extended period may cause accumulation of serotonin and exacerbate central fatigue. Enhanced availability of serotonin did not directly influence motor pathways or motor performance during prolonged submaximal contraction. However, perceptions of muscle fatigue were greater, and the fatigue-induced lengthening of the silent period elicited via motor cortical stimulation was reduced with enhanced availability of serotonin. We propose that sustained low-intensity serotonergic neurotransmission influences supraspinal processes associated with fatigue, without directly influencing the output of the motor system during submaximal exercise. ABSTRACT: Enhanced availability of serotonin (5-HT) exacerbates central fatigue that occurs during maximal effort contractions. However, it is unknown if 5-HT release contributes to central fatigue during prolonged submaximal contractions. Hence, we assessed the effect that enhanced availability of 5-HT has on sustained low-intensity fatiguing contractions. Fifteen individuals (22.3 ± 2.1 years) ingested the 5-HT reuptake inhibitor paroxetine in a human, double-blinded, placebo-controlled, repeated-measures design. Participants performed a low-intensity isometric elbow flexion for 30 min (15% of maximal voluntary contraction, MVC). Throughout the protocol, brief MVCs were performed and muscle responses to transcranial magnetic stimulation (TMS) of the motor cortex, electrical stimulation of the brachial plexus, and motor point stimulation of the biceps were obtained. Ratings of perceived fatigue were also acquired. Paroxetine did not influence torque or voluntary activation during brief MVCs performed throughout the low-intensity contraction. However, paroxetine increased the perception of fatigue throughout the contraction (P = 0.005), and shortened the biceps silent period elicited via TMS during sustained submaximal contraction (P = 0.003) and brief MVCs (P = 0.011). Overall, it appears that prolonged low-intensity contractions do not cause intense 5-HT release onto motoneurons, and therefore, 5-HT does not activate inhibitory extra-synaptic 5-HT1A receptors of motoneurons to reduce their output. Although motor performance was unaffected by paroxetine, perceived fatigue was greater and intracortical inhibitory activity was reduced following the enhancement of endogenous concentrations of 5-HT during sustained submaximal contraction. Thus, 5-HT affects supraspinal processes during low-intensity contractions without directly altering motor pathways projecting to the muscle.

Summary: Genetic variations in TMEM106B, coding for a lysosomal membrane protein, affect frontotemporal lobar degeneration (FTLD) in GRN- (coding for progranulin) and C9orf72-expansion carriers and might play a role in aging. To determine the physiological function of TMEM106B, we generated TMEM106B-deficient mice. These mice develop proximal axonal swellings caused by drastically enlarged LAMP1-positive vacuoles, increased retrograde axonal transport of lysosomes, and accumulation of lipofuscin and autophagosomes. Giant vacuoles specifically accumulate at the distal end and within the axon initial segment, but not in peripheral nerves or at axon terminals, resulting in an impaired facial-nerve-dependent motor performance. These data implicate TMEM106B in mediating the axonal transport of LAMP1-positive organelles in motoneurons and axonal sorting at the initial segment. Our data provide mechanistic insight into how TMEM106B affects lysosomal proteolysis and degradative capacity in neurons. : Genetic variants in the TMEM106B gene, coding for a lysosomal transmembrane protein, are linked to various neurodegenerative diseases. The function of TMEM106B remains enigmatic. Lüningschrör et al. analyze Tmem106b-knockout mice and find drastically enlarged LAMP1-positive vacuoles in proximal axons of selected motoneuron nuclei. Vacuolization is caused by impaired axonal transport. Keywords: TMEM106B, frontotemporal lobar degeneration, lysosome, retrograde, axon, axon initial segment, motoneurons, FTLD

In excitable membranes, the clustering of voltage-gated sodium channels (VGSC) serves to enhance excitability at critical sites. The two most profoundly studied sites of channel clustering are the axon initial segment, where action potentials are generated and the node of Ranvier, where action potentials propagate along myelinated axons. The clustering of VGSC is found, however, in other highly excitable sites such as axonal terminals, postsynaptic membranes of dendrites and muscle fibers, and pre-myelinated axons. In this review, different examples of axonal as well as non-axonal clustering of VGSC are discussed and the underlying mechanisms are compared. Whether the clustering of channels is intrinsically or extrinsically induced, it depends on the submembranous actin-based cytoskeleton that organizes these highly specialized membrane microdomains through specific adaptor proteins.

Prickle2 has been identified in genetic studies of subjects with autism spectrum disorder (ASD) and epilepsy, but the pathological mechanism of Prickle2 remains to be fully understood. Proteomic analysis of Prickle2 with mass spectrometry revealed twenty-eight Prickle2 interactors, including immunoglobulin superfamily member 9b (Igsf9b), in the brain. Here, because Igsf9 family proteins are associated with psychiatric diseases and seizures, we studied the physiological interaction between Prickle2 and Igsf9b. Prickle2 colocalized with Igsf9b in cultured hippocampal neurons. Knockdown of Prickle2 affected the subcellular localization of Igsf9b. Interestingly, Igsf9b localized along axonal processes in a pattern opposite to the ASD-related molecule ANK3/AnkG. AnkG is a major component of the axon initial segment (AIS), where a variety of ASD and epilepsy susceptibility proteins accumulate. Igsf9b-knockdown neurons displayed altered AnkG localization. Prickle2 depletion caused defects in AnkG and voltage-gated Na+ channel localization, resulting in altered network activity. These results support the idea that Prickle2 regulates AnkG distribution by controlling the proper localization of Igsf9b. The novel function of Prickle2 in AIS cytoarchitecture provides new insights into the shared pathology of ASD and epilepsy.Key words: Prickle2, Igsf9b, axon initial segment, neuronal excitability, ASD.

Significance The Mauthner cell is the largest neuron known in the vertebrate brain and, in fish, mediates rapid escape behavior. Ablating this neuron has repeatedly failed to eliminate rapid escapes, a survival role of these escapes has not been supported experimentally, and it is unknown which advantage the enormous size and complexity of the cell conveys. By taking care to ensure ablations remove not only the soma but also the giant axon, we find that rapid escapes are lost forever and that this loss directly affects survival in predator–prey assays. The Mauthner cell thus is an example in which a survival-critical function depends on an individual neuron whose axon appears to have unusual capacities to remain functional after severe injury., The giant Mauthner (M) cell is the largest neuron known in the vertebrate brain. It has enabled major breakthroughs in neuroscience but its ultimate function remains surprisingly unclear: An actual survival value of M cell-mediated escapes has never been supported experimentally and ablating the cell repeatedly failed to eliminate all rapid escapes, suggesting that escapes can equally well be driven by smaller neurons. Here we applied techniques to simultaneously measure escape performance and the state of the giant M axon over an extended period following ablation of its soma. We discovered that the axon survives remarkably long and remains still fully capable of driving rapid escape behavior. By unilaterally removing one of the two M axons and comparing escapes in the same individual that could or could not recruit an M axon, we show that the giant M axon is essential for rapid escapes and that its loss means that rapid escapes are also lost forever. This allowed us to directly test the survival value of the M cell-mediated escapes and to show that the absence of this giant neuron directly affects survival in encounters with a natural predator. These findings not only offer a surprising solution to an old puzzle but demonstrate that even complex brains can trust vital functions to individual neurons. Our findings suggest that mechanisms must have evolved in parallel with the unique significance of these neurons to keep their axons alive and connected.

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.

The axon initial segment (AIS), the site of action potential initiation in neurons, is a critical determinant of neuronal excitability. Growing evidence indicates that appropriate recruitment of the AIS macrocomplex is essential for synchronized firing. However, disruption of the AIS structure is linked to the etiology of multiple disorders, including autism spectrum disorder (ASD), a condition characterized by deficits in social communication, stereotyped behaviors, and very limited interests. To date, a complete understanding of the molecular components that underlie the AIS in ASD has remained elusive. In this research, we examined the AIS structure in a BTBR T+Itpr3tf/J mouse model (BTBR), a valid model that exhibits behavioral, electrical, and molecular features of autism, and compared this to the C57BL/6J wild-type control mouse. Using Western blot studies and high-resolution confocal microscopy in the prefrontal frontal cortex (PFC), our data indicate disrupted expression of different isoforms of the voltage-gated sodium channels (NaV) at the AIS, whereas other components of AIS such as ankyrin-G and fibroblast growth factor 14 (FGF14) and contactin-associated protein 1 (Caspr) in BTBR were comparable to those in wild-type control mice. A Western blot assay showed that BTBR mice exhibited a marked increase in different sodium channel isoforms in the PFC compared to wild-type mice. Our results provide potential evidence for previously undescribed mechanisms that may play a role in the pathogenesis of autistic-like phenotypes in BTBR mice.

Nonpyramidal GABAergic interneurons in the basolateral nuclear complex (BNC) of the amygdala are critical for the regulation of emotion. Remarkably, there have been no Golgi studies of these neurons in nonhuman primates. Therefore, in the present study we investigated the morphology of nonpyramidal neurons (NPNs) in the BNC of the baboon and monkey using the Golgi technique. NPNs were scattered throughout all nuclei of the BNC and had aspiny or spine-sparse dendrites. NPNs were morphologically heterogeneous and could be divided into small, medium, large, and giant types based on the size of their somata. NPNs could be further divided on the basis of their somatodendritic morphology into four types: multipolar, bitufted, bipolar, and irregular. NPN axons, when stained, formed dense local arborizations that overlapped their dendritic fields to varying extents. These axons always exhibited varying numbers of varicosities representing axon terminals. Three specialized NPN subtypes were recognized because of their unique anatomical features: axo-axonic cells, neurogliaform cells, and giant cells. The axons of axo-axonic cells formed “axonal cartridges,” with clustered varicosities that contacted the axon initial segments of pyramidal neurons (PNs). Neurogliaform cells had small somata and numerous short dendrites that formed a dense dendritic arborization; they also exhibited a very dense axonal arborization that overlapped the dendritic field. Giant cells had very large irregular somata and long, thick dendrites; their distal dendrites often branched extensively and had long appendages. In general, the NPNs of the baboon and monkey BNC, including the specialized subtypes, were similar to those of rodents.

The extent of functional maturation and integration of nonproliferative neuronal precursors, becoming neurons in the adult murine piriform cortex, is largely unexplored. We thus questioned whether precursors eventually become equivalent to neighboring principal neurons or whether they represent a novel functional network element. Adult brain neuronal precursors and immature neurons (complex cells) were labeled in transgenic mice (DCX-DsRed and DCX-CreERT2 /flox-EGFP), and their cell fate was characterized with patch clamp experiments and morphometric analysis of axon initial segments. Young (DCX+) complex cells in the piriform cortex of 2- to 4-month-old mice received sparse synaptic input and fired action potentials at low maximal frequency, resembling neonatal principal neurons. Following maturation, the synaptic input detected on older (DCX−) complex cells was larger, but predominantly GABAergic, despite evidence of glutamatergic synaptic contacts. Furthermore, the rheobase current of old complex cells was larger and the maximal firing frequency was lower than those measured in neighboring age-matched principal neurons. The striking differences between principal neurons and complex cells suggest that the latter are a novel type of neuron and new coding element in the adult brain rather than simple addition or replacement for preexisting network components.

Inhibitory neurons in mammalian cortex exhibit diverse physiological, morphological, molecular, and connectivity signatures. While considerable work has measured the average connectivity of several interneuron classes, there remains a fundamental lack of understanding of the connectivity distribution of distinct inhibitory cell types with synaptic resolution, how it relates to properties of target cells, and how it affects function. Here, we used large-scale electron microscopy and functional imaging to address these questions for chandelier cells in layer 2/3 of the mouse visual cortex. With dense reconstructions from electron microscopy, we mapped the complete chandelier input onto 153 pyramidal neurons. We found that synapse number is highly variable across the population and is correlated with several structural features of the target neuron. This variability in the number of axo-axonic ChC synapses is higher than the variability seen in perisomatic inhibition. Biophysical simulations show that the observed pattern of axo-axonic inhibition is particularly effective in controlling excitatory output when excitation and inhibition are co-active. Finally, we measured chandelier cell activity in awake animals using a cell-type-specific calcium imaging approach and saw highly correlated activity across chandelier cells. In the same experiments, in vivo chandelier population activity correlated with pupil dilation, a proxy for arousal. Together, these results suggest that chandelier cells provide a circuit-wide signal whose strength is adjusted relative to the properties of target neurons.

Knowledge on cortical development is based mainly on rodent besides primates and carnivores, all being altricial. Here, we analyzed a precocial animal, the pig, looking at dorsoparietal cortex from E45 to P90. At E45, most ionized calcium-binding adapter molecule 1-positive (Iba1+) cells had a macrophage-like morphology and resided in meninges and choroid plexus. Only a few cells were scattered in ventricular and subventricular zone (VZ and SVZ). At E60/E70, all laminar compartments displayed microglia cells at a low-to-moderate density, being highest in VZ and SVZ followed by intermediate zone/white matter (IZ/WM). Cortical plate and marginal zone displayed only a few Iba1+ cells. Cells were intensely labeled, but still had poorly arborized somata and many resembled ameboid, macrophage-like microglia. Concurrent with a massive increase in cortical volume, microglia cell density increased until E85, and further until E100/E110 (birth at E114) to densities that resemble those seen postnatally. A fraction of microglia co-labeled with Ki67 suggesting proliferation in all laminar compartments. Cell-to-cell distance decreased substantially during this time, and the fraction of microglia to all nuclei and to neurons increases in the laminar compartments. Eventually, of all cortical DAPI+ nuclei 7-12% were Iba1+ microglia. From E70 onwards, more and more cells with ramified processes were present in MZ down to IZ/WM, showing for instance a close association with NeuN+, NPY+ and GAD65/67+ somata and axon initial segments. These results suggested that development of microglia cell density and morphology proceeds rapidly from mid-gestation onwards reaching near-adult status already before birth. This article is protected by copyright. All rights reserved.

Η εγκεφαλική υποαιμάτωση εμφανίζεται στη φυσιολογική γήρανση και προτείνεται ότι προκαλεί διαταραχές στη λευκή ουσία οι οποίες παρατηρούνται στους γερασμένους εγκεφάλους. Επιπλέον, η εγκεφαλική υποαιμάτωση και οι αλλοιώσεις της λευκής ουσίας είναι πρώιμα συμβάντα στην εξέλιξη της νόσου του Αλτσχάιμερ (AD). Η λευκή ουσία αποτελείται κυρίως από μυελινωμένους άξονες που έχουν μία ιδιαίτερη πρωτεϊνική αρχιτεκτονική καθώς διαχωρίζονται σε συγκεκριμένες περιοχές οι οποίες είναι το αρχικό τμήμα του άξονα (AIS), ο κόμβος του Ranvier, ο paranode, ο juxtaparanode, και ο internode. Αυτές οι διαφορετικές περιοχές είναι απαραίτητες για την έναρξη και τη διάδοση του δυναμικού δράσης και κατά συνέπεια για την αποτελεσματική λειτουργία του εγκεφάλου. Ένα εύρος δεδομένων συνηγορούν ότι οι διαφορετικές περιοχές εντός των εμμύελων αξόνων είναι ευάλωτες σε περιπτώσεις τραυματισμού ή ασθένειας. Για αυτό υποθέτουμε ότι ως απόκριση στη φυσιολογική γήρανση και στην εγκεφαλική υποαιμάτωση αυτές οι περιοχές θα αλλοιώνονται οδηγώντας σε γνωστική εξασθένηση και ότι αυτές οι επιδράσεις θα επιδεινώνονται σε ένα διαγονιδιακό μοντέλο ποντικού (σειρά APPSw, Ind, J9) που αναπτύσσει παθολογία β-αμυλοειδoύς (Αβ) εξαρτώμενη από την ηλικία. Η πρώτη μελέτη στοχεύει στη διερεύνηση της επίδρασης της φυσιολογικής γήρανσης και της εναπόθεσης του Αβ στους μυελινωμένους άξονες καθώς και στη μνήμη και τη μάθηση. Για αυτό, οι επιδράσεις της φυσιολογικής γήρανσης στην ακεραιότητα του AIS, των κόμβων του Ranvier, της μυελίνης, των αξόνων, των συνάψεων και της χωρικής μνήμης εργασίας εξετάστηκαν σε νεαρα και ηλικιωμένα wild-type και TgAPPSw,Ind ποντίκια. Παρατηρήθηκε σημαντική μείωση του μήκους των κόμβων του Ranvier στα ηλικιωμένα wild-type και TgAPPSw,Ind ποντίκια. Επιπλέον, το μήκος του AIS μειώνεται σημαντικά στα ηλικιωμένα wild-type ζώα, ενώ τα νεαρά TgAPPSw,Ind έχουν σημαντικά μικρότερο AIS από τα νεαρά wild-type ποντίκια. Αυτές οι επιδράσεις δεν επηρεάζονται από την παρουσία του Αβ. Η ακεραιότητα της μυελίνης επηρεάζεται από την ηλικία, αλλά αυτό είναι πιο εμφανές στα wild-type ζώα, ενώ η ακεραιότητα των νευραξόνων είναι ανέπαφη. Επιπλέον, υπάρχει μια σχετιζόμενη με την ηλικία μείωση των προσυναπτικών buttons μόνο στα TgAPPSw,Ind ποντίκια. Σε αντίθεση με την αρχική υπόθεση, η μνήμη εργασία δεν μεταβάλλεται με την ηλικία ούτε επηρεάζεται από την αύξηση των επιπέδων Αβ. Η δεύτερη μελέτη στοχεύει να εξετάσει τις επιδράσεις της εγκεφαλικής υποαιμάτωσης σε συνδυασμό με την παθολογία του Αβ και τη γήρανση στις γνωστικές λειτουργίες και στη δομή των μυελινωμένων αξόνων. Για αυτό, διερευνήθηκαν τα αποτελέσματα της χειρουργικά επαγόμενης εγκεφαλικής υποαιμάτωσης στην ακεραιότητα των κόμβων του Ranvier, των paranodes, της μυελίνης, των αξόνων και στη χωρική μνήμη εργασίας σε νεαρά και ηλικιωμένα wild-type και TgAPPSw,Ind ποντίκια. Παρατηρείται μείωση του μήκους των κόμβων του Ranvier ως απόκριση στην υποαιμάτωση σε νεαρά και ηλικιωμένα ζώα. Αυτή η επίδραση φαίνεται να επιδεινώνεται στα νεαρά TgAPPSw,Ind ζώα. Επιπλέον, η διαταραχή της περιοχής του κόμβου φαίνεται να συμβαίνει χωρίς μεγάλες αλλαγές στη μυελίνη ή στην ακεραιότητα του νευράξονα. Αποδεικνύεται επίσης ότι ως απόκριση στην υποαιμάτωση, η χωρική μνήμη εργασίας είναι ελαττωματική τόσο στα νεαρά όσο και στα ηλικιωμένα ζώα και των δύο γονότυπων. Αυτό η βλάβη επιδεινώνεται στα νεαρά TgAPPSw,Ind ζώα. Οι παρατηρούμενες αλλαγές στη δομή του κόμβου του Ranvier συσχετίζονται με την μείωση της μνήμης εργασίας, υποδεικνύοντας τις λειτουργικές επιπτώσεις στις αλλαγές της δομής του κόμβου του Ranvier. Αυτά τα δεδομένα υπογραμμίζουν ότι οι δομές εντός των μυελινωμένων αξόνων είναι ευάλωτες στη γήρανση και στην εγκεφαλική υποαιμάτωση. Επομένως, η ανάπτυξη στρατηγικών που ελαχιστοποιούν τον τραυματισμό ή οδηγούν στην αποκατάσταση αυτών των περιοχών είναι απαραίτητη μαζί με θεραπευτικές προσεγγίσεις κατά των αγγειακών προσβολών που προκαλούν υποαιμάτωση και οδηγούν σε αλλοίωση της λευκής ουσίας και γνωστική έκπτωση. Στο μέλλον, θα ήταν ενδιαφέρον να διερευνηθεί πώς οι αλλαγές στο AIS και τους κόμβους του Ranvier επηρεάζουν τη νευρωνική διεγερσιμότητα.

The firing activity of ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) dopaminergic (DA) neurons is an important factor in shaping DA release and its role in motivated behavior. Dendrites in DA neurons are the main postsynaptic compartment and, along with cell body and axon initial segment, contribute to action potential generation and firing pattern. In this study, the organization of the dendritic domain in individual VTA and SNc DA neurons of adult male mice, and their relationship to in vivo spontaneous firing, are described. In comparison with dorsal VTA DA neurons, ventrally located VTA neurons (as measured by cell body location) possess a shorter total dendritic length and simpler dendritic architecture, and exhibit the most irregular in vivo firing patterns among DA neurons. In contrast, for DA neurons in the SNc, the higher irregularity of firing was related to a smaller dendritic domain, as measured by convex hull volumes. However, firing properties were also related to the specific regional distribution of the dendritic tree. Thus, VTA DA neurons with a larger extension of their dendritic tree within the parabrachial pigmented (PBP) nucleus fired more regularly compared with those with relatively more dendrites extending outside the PBP. For DA neurons in the SNc, enhanced firing irregularity was associated with a smaller proportion of dendrites penetrating the substantia nigra pars reticulata. These results suggest that differences in dendritic morphology contribute to the in vivo firing properties of individual DA neurons, and that the existence of region-specific synaptic connectivity rules that shape firing diversity.

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

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

Retinal prostheses are a promising therapeutic intervention for patients afflicted by outer retinal degenerative diseases such as retinitis pigmentosa and age‐related macular degeneration. Although significant advances in the development of retinal implants have been made, the quality of vision elicited by these devices remains largely suboptimal. The variability in the responses produced by retinal devices is most likely due to the differences between the natural cell‐type‐specific signaling that occur in the healthy retina versus the nonspecific activation of multiple cell types arising from artificial stimulation. To replicate these natural signaling patterns, stimulation strategies must be capable of preferentially activating specific retinal ganglion cell (RGC) types. To design more selective stimulation strategies, a better understanding of the morphological factors that underlie the sensitivity to prosthetic stimulation must be developed. Herein, the role that different anatomical components play in driving the direct activation of RGCs by extracellular stimulation is focused on. Briefly, 1) the variability in morphological properties of α‐RGCs is characterized, 2) the influence of morphology on the direct activation of RGCs by electric stimulation is detailed, and 3) some of the potential biophysical mechanisms that could explain differences in activation thresholds and electrically evoked responses between RGC types are described.

The position of the axon initial segment (AIS) is thought to play a critical role in neuronal excitability. In particular, empirical studies have found correlations between a distal shift in AIS position and a reduction of excitability. Yet, theoretical work has suggested that the neuron should become more excitable as the distance between soma and AIS is increased, because of increased electrical isolation. Specifically, resistive coupling theory predicts that the action potential (AP) threshold decreases with the logarithm of the axial resistance (Ra) between the middle of the AIS and the soma. However, no direct experimental evidence has been provided so far to support this theoretical prediction. We therefore examined how changes in Ra at the axon hillock impact the voltage threshold (Vth) of the somatic AP in L5 pyramidal neurons. Increasing Ra by mechanically pinching the axon between the soma and the AIS was found to lower the spike threshold by ~6 mV. Conversely, decreasing Ra by replacing a weakly mobile ion (gluconate) by a highly mobile ion (chloride) elevated the spike threshold. All Ra-dependent changes in spike threshold could be reproduced in a Hodgkin-Huxley compartmental model. We conclude that in L5 pyramidal neurons, excitability increases with axial resistance, and therefore with a distal shift of the AIS.

Genetic variants in SCN2A, encoding the NaV1.2 voltage-gated sodium channel, are associated with a range of neurodevelopmental disorders with overlapping phenotypes. Some variants fit into a framework wherein gain-of-function missense variants that increase neuronal excitability lead to infantile epileptic encephalopathy, while loss-of-function variants that reduce neuronal excitability lead to developmental delay and/or autism spectrum disorder with or without co- morbid seizures. One unique case less easily classified using this binary paradigm is the de novo missense variant SCN2A p.K1422E, associated with infant-onset developmental delay, infantile spasms, and features of autism spectrum disorder. Prior structure-function studies demonstrated that K1422E substitution alters ion selectivity of NaV1.2, conferring Ca2+ permeability, lowering overall conductance, and conferring resistance to tetrodotoxin (TTX). Based on heterologous expression of K1422E, we developed a compartmental neuron model that predicted mixed effects on channel function and neuronal activity. We also generated Scn2aK1422E mice and characterized effects on neurons and neurological/neurobehavioral phenotypes. Dissociated neurons from heterozygous Scn2aK1422E/+ mice exhibited a novel TTX-resistant current with a reversal potential consistent with mixed ion permeation. Cortical slice recordings from Scn2aK1442E/+ tissue demonstrated impaired action potential initiation and larger Ca2+ transients at the axon initial segment during the rising phase of the action potential, suggesting mixed effects on channel function. Scn2aK1422E/+ mice exhibited rare spontaneous seizures, interictal EEG abnormalities, altered response to induced seizures, reduced anxiety-like behavior and alterations in olfactory-guided social behavior. Overall, Scn2aK1422E/+ mice present with phenotypes similar yet distinct from Scn2a knockout models, consistent with mixed effects of K1422E on NaV1.2 channel function.Significance StatementThe early-onset epilepsy variant SCN2A-p.K1422E displays unique biophysical properties in vitro. To model the impact of this rare variant, we generated Scn2aK1422E mice. Neurons from heterozygous Scn2aK1422E/+ mice showed functional deficits similar to the loss-of-function effects observed in the Scn2a haploinsufficiency model, as well as gain-of-function effects specific to the K1422E variant. There is also some overlap in neurobehavioral phenotypes between Scn2aK1422E/+ and Scn2a haploinsufficient mice. However, Scn2aK1422E/+ mice exhibited unique epilepsy-related phenotypes, including epileptiform events and seizures. Scn2aK1422E/+ mice serve as a useful platform to investigate phenotypic complexity of SCN2A-associated disorders.

In neurons, the axon and axon initial segment (AIS) are critical structures for action potential initiation and propagation. Their formation and function rely on tight compartmentalisation, a process where specific proteins are trafficked to and retained at distinct subcellular locations. One mechanism which regulates protein trafficking and association with lipid membranes is the modification of protein cysteine residues with the 16-carbon palmitic acid, known as S-acylation or palmitoylation. Palmitoylation, akin to phosphorylation, is reversible, with palmitate cycling being mediated by substrate-specific enzymes. Palmitoylation is well-known to be highly prevalent among neuronal proteins and is well studied in the context of the synapse. Comparatively, how palmitoylation regulates trafficking and clustering of axonal and AIS proteins remains less understood. This review provides an overview of the current understanding of the biochemical regulation of palmitoylation, its involvement in various neurological diseases, and the most up-to-date perspective on axonal palmitoylation. Through a palmitoylation analysis of the AIS proteome, we also report that an overwhelming proportion of AIS proteins are likely palmitoylated. Overall, our review and analysis confirm a central role for palmitoylation in the formation and function of the axon and AIS and provide a resource for further exploration of palmitoylation-dependent protein targeting to and function at the AIS.

Mutations in the ER-network forming GTPase atlastin3 (ATL3) can cause axon degeneration of sensory neurons by not fully understood mechanisms. We here show that the hereditary sensory and autonomous neuropathy (HSAN)-causing ATL3 Y192C or P338R are excluded from distal axons by a barrier at the axon initial segment (AIS). This barrier is selective for mutated ATL3, but not wildtype ATL3 or unrelated ER-membrane proteins. Actin-depolymerization partially restores the transport of ATL3 Y192C into distal axons. The results point to the existence of a selective diffusion barrier in the ER membrane at the AIS, analogous to the AIS-based barriers for plasma membrane and cytosolic proteins. Functionally, the absence of ATL3 at the distal axon reduces axonal autophagy and the ER network deformation in the soma causes a reduction in axonal lysosomes. Both could contribute to axonal degeneration and eventually to HSAN.

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

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

Locomotion generates adventitious sounds which enable detection and localization of predators and prey. Such sounds contain brisk changes or transients in amplitude. We investigated the hypothesis that ill-understood temporal specializations in binaural circuits subserve lateralization of such sound transients, based on different time of arrival at the ears (interaural time differences, ITDs). We find that Lateral Superior Olive (LSO) neurons show exquisite ITD-sensitivity, reflecting extreme precision and reliability of excitatory and inhibitory postsynaptic potentials, in contrast to Medial Superior Olive neurons, traditionally viewed as the ultimate ITD-detectors. In vivo, inhibition blocks LSO excitation over an extremely short window, which, in vitro, required synaptically evoked inhibition. Light and electron microscopy revealed inhibitory synapses on the axon initial segment as the structural basis of this observation. These results reveal a neural vetoing mechanism with extreme temporal and spatial precision and establish the LSO as the primary nucleus for binaural processing of sound transients. ispartof: ELIFE vol:10 ispartof: location:England status: published

Autism spectrum condition or ‘autism’ is associated with numerous monogenic and polygenic genetic risk factors including the polygenic 16p11.2 microdeletion. A central question is what neural cells are affected. To systematically investigate we analysed single cell transcriptomes from gestational week (GW) 8-26 human foetal prefrontal cortex and identified a subset of interneurons (INs) first appearing at GW23 with enriched expression of a disproportionately large fraction of risk factor transcripts. This suggests the hypothesis that these INs are disproportionately vulnerable to mutations causing autism. We investigated this in a rat model of the 16p11.2 microdeletion. We found no change in the numbers or position of either excitatory or inhibitory neurons in the somatosensory cortex or CA1 of 16p11.2+/- rats but found that CA1 Sst INs were hyperexcitable with an enlarged axon initial segment, which was not the case for CA1 pyramidal cells. This study prompts deeper investigation of IN development as a convergent target for autism genetic risk factors.