Block Phenomena During Electric Micro-Stimulation of Pyramidal Cells and Retinal Ganglion Cells

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

Giant ankyrin-B mediates transduction of axon guidance and collateral branch pruning factor sema 3A

Variants in the high confident autism spectrum disorder (ASD) gene ANK2 target both ubiquitously expressed 220 kDa ankyrin-B and neurospecific 440 kDa ankyrin-B (AnkB440) isoforms. Previous work showed that knock-in mice expressing an ASD-linked Ank2 variant yielding a truncated AnkB440 product exhibit ectopic brain connectivity and behavioral abnormalities. Expression of this variant or loss of AnkB440 caused axonal hyperbranching in vitro, which implicated AnkB440 microtubule bundling activity in suppressing collateral branch formation. Leveraging multiple mouse models, cellular assays, and live microscopy, we show that AnkB440 also modulates axon collateral branching stochastically by reducing the number of F-actin-rich branch initiation points. Additionally, we show that AnkB440 enables growth cone (GC) collapse in response to chemorepellent factor semaphorin 3 A (Sema 3 A) by stabilizing its receptor complex L1 cell adhesion molecule/neuropilin-1. ASD-linked ANK2 variants failed to rescue Sema 3A-induced GC collapse. We propose that impaired response to repellent cues due to AnkB440 deficits leads to axonal targeting and branch pruning defects and may contribute to the pathogenicity of ANK2 variants.

Development of motor neurons and motor activity in zebrafish requires F-actin nucleation by Fmn2b

Background: Cytoskeletal remodelling plays a pivotal role in the establishment of neuronal connectivity during development and in plasticity in adults. Mutations in the cytoskeleton regulatory protein Formin-2 (Fmn2) are associated with neurodevelopmental disorders like intellectual disability, though its function in neuronal morphogenesis has not been characterised in vivo. Results: Here we develop a loss-of-function model for fmn2b, the zebrafish orthologue of Fmn2, using CRISPR/Cas9-mediated gene editing. fmn2b mutants display motor deficits starting from the earliest motor responses in the embryo. We find that fmn2b is expressed in motor neurons and its loss reduces motor neuron innervation of the axial muscles without affecting myotome integrity. The translocation of caudal primary (CaP) motor neuron outgrowth is compromised in fmn2b mutants, while rostral primary (RoP) motor neurons have missing soma or stall at the horizontal myoseptum. Strikingly, axon collateral branching of the motor neurons is severely compromised and results in reduced synaptic coverage of the myotome. Rescue experiments identify the requirement for Fmn2-mediated actin nucleation for motor neuron outgrowth and arborisation. Conclusions: The zebrafish loss-of-function model of Fmn2 reveals the specific requirement of F-actin polymerisation by Fmn2 in neuromuscular development. It also underscores the role of Fmn2 in motor neuropathies, especially as a proportion of individuals harbouring mutations in Fmn2 present with hypotonia.

Giant ankyrin-B mediates transduction of axon guidance and collateral branch pruning factor Sema 3A

Variants in the high confident autism spectrum disorder gene ANK2 target both ubiquitously expressed 220-kDa ankyrin- B and neurospecific 440-kDa ankyrin-B (AnkB440) isoforms. Previous work showed that knock-in mice expressing an ASD linked Ank2 variant yielding a truncated AnkB440 product exhibit ectopic brain connectivity and behavioral abnormalities. Expression of this variant or loss of AnkB440 caused axonal hyperbranching in vitro, which implicated AnkB440 microtubule bundling activity in suppressing collateral branch formation. Leveraging multiple mouse models, cellular assays, and live microscopy, we show that AnkB440 also modulates axon collateral branching stochastically by reducing the number of F-actin-rich branch initiation points. Additionally, we show that AnkB440 enables growth cone (GC) collapse in response to chemorepellent factor semaphorin 3A (Sema 3A) by stabilizing its receptor complex L1 cell adhesion molecule/neuropilin-1. ASD-linked ANK2 variants failed to rescue Sema 3A-induced GC collapse. We propose that impaired response to repellent cues due to AnkB440 deficits leads to axonal guidance and branch pruning defects and may contribute to the pathogenicity of ANK2 variants.

Projection Neuron Axon Collaterals in the Dorsal Horn: Placing a New Player in Spinal Cord Pain Processing

The pain experience depends on the relay of nociceptive signals from the spinal cord dorsal horn to higher brain centers. This function is ultimately achieved by the output of a small population of highly specialized neurons called projection neurons (PNs). Like output neurons in other central nervous system (CNS) regions, PNs are invested with a substantial axon collateral system that ramifies extensively within local circuits. These axon collaterals are widely distributed within and between spinal cord segments. Anatomical data on PN axon collaterals have existed since the time of Cajal, however, their function in spinal pain signaling remains unclear and is absent from current models of spinal pain processing. Despite these omissions, some insight on the potential role of PN axon collaterals can be drawn from axon collateral systems of principal or output neurons in other CNS regions, such as the hippocampus, amygdala, olfactory cortex, and ventral horn of the spinal cord. The connectivity and actions of axon collaterals in these systems have been well-defined and used to confirm crucial roles in memory, fear, olfaction, and movement control, respectively. We review this information here and propose a framework for characterizing PN axon collateral function in the dorsal horn. We highlight that experimental approaches traditionally used to delineate axon collateral function in other CNS regions are not easily applied to PNs because of their scarcity relative to spinal interneurons (INs), and the lack of cellular organization in the dorsal horn. Finally, we emphasize how the rapid development of techniques such as viral expression of optogenetic or chemogenetic probes can overcome these challenges and allow characterization of PN axon collateral function. Obtaining detailed information of this type is a necessary first step for incorporation of PN collateral system function into models of spinal sensory processing.

Antagonistic activities of Fmn2 and ADF regulate axonal F-actin patch dynamics and the initiation of collateral branching

ABSTRACTInterstitial collateral branching of axons is a critical component in the development of functional neural circuits. Axon collateral branches are established through a series of cellular processes initiated by the development of a specialized, focal F-actin network in axons. The formation, maintenance and remodelling of this F-actin patch is critical for the initiation of axonal protrusions that are subsequently consolidated to form a collateral branch. However, the mechanisms regulating F-actin patch dynamics are poorly understood.Fmn2 is a formin family member implicated in multiple neurodevelopmental disorders. We find that Fmn2 regulates the initiation of axon collateral protrusions. Fmn2 localises to the protrusion-initiating axonal F-actin patches and regulates the lifetime and size of these F-actin networks. The F-actin nucleation activity of Fmn2 is necessary for F-actin patch stability but not for initiating patch formation. We show that Fmn2 insulates the F-actin patches from disassembly by the actin-depolymerizing factor, ADF, and promotes long-lived, larger patches that are competent to initiate axonal protrusions.The regulation of axonal branching can contribute to the neurodevelopmental pathologies associated with Fmn2 and the dynamic antagonism between Fmn2 and ADF may represent a general mechanism of formin-dependent protection of Arp2/3-initiated F-actin networks from disassembly.

Giant ankyrin-B suppresses stochastic collateral axon branching through direct interaction with microtubules

Giant ankyrin-B (gAnkB) is a 440-kD neurospecific ankyrin-B isoform and a high-confidence target for autism mutations. gAnkB suppresses axon branching through coordination of cortical microtubules, and autism-related mutation of gAnkB results in ectopic neuronal connectivity. We identified a bipartite motif from gAnkB, which bundles and avidly binds to microtubules in vitro. This motif is composed of a module of 15 tandem repeats followed by a short, conserved fragment also found in giant ankyrin-G (BG-box). Combination of these two parts synergistically increases microtubule-binding avidity. Transfection of astrocytes (which lack gAnkB) with WT gAnkB resulted in prominent bundling of microtubules, which did not occur with mutant gAnkB with impaired microtubule-binding activity. Similarly, rescue of gAnkB-deficient neurons with WT gAnkB suppressed axonal branching and invasion of EB3-tagged microtubules into filopodia, which did not occur with the same mutant gAnkB. Together, these findings demonstrate that gAnkB suppresses axon collateral branching and prevents microtubule invasion of nascent axon branches through direct interaction with microtubules.

Branch-restricted localization of phosphatase Prl-1 specifies axonal synaptogenesis domains

Central nervous system (CNS) circuit development requires subcellular control of synapse formation and patterning of synapse abundance. We identified the Drosophila membrane-anchored phosphatase of regenerating liver (Prl-1) as an axon-intrinsic factor that promotes synapse formation in a spatially restricted fashion. The loss of Prl-1 in mechanosensory neurons reduced the number of CNS presynapses localized on a single axon collateral and organized as a terminal arbor. Flies lacking all Prl-1 protein had locomotor defects. The overexpression of Prl-1 induced ectopic synapses. In mechanosensory neurons, Prl-1 modulates the insulin receptor (InR) signaling pathway within a single contralateral axon compartment, thereby affecting the number of synapses. The axon branch–specific localization and function of Prl-1 depend on untranslated regions of the prl-1 messenger RNA (mRNA). Therefore, compartmentalized restriction of Prl-1 serves as a specificity factor for the subcellular control of axonal synaptogenesis.

Photoactivation of olfactory sensory neurons does not affect action potential conduction in individual trigeminal sensory axons innervating the rodent nasal cavity

Olfactory and trigeminal chemosensory systems reside in parallel within the mammalian nose. Psychophysical studies in people indicate that these two systems interact at a perceptual level. Trigeminal sensations of pungency mask odour perception, while olfactory stimuli can influence trigeminal signal processing tasks such as odour localization. While imaging studies indicate overlap in limbic and cortical somatosensory areas activated by nasal trigeminal and olfactory stimuli, there is also potential cross-talk at the level of the olfactory epithelium, the olfactory bulb and trigeminal brainstem. Here we focused on potential interactions between olfactory and trigeminal signaling in the nasal cavity. We first used a forced choice paradigm to ascertain whether trigeminal and olfactory stimuli could influence behavior in mice. Mice avoided water sources associated with volatile TRPV1 and TRPA1 irritants, however, this aversion was mitigated when combined with a pure odorant (rose fragrance, phenylethyl alcohol, PEA). To determine whether olfactory-trigeminal interactions within the nose could potentially account for this behavioral effect we recorded from single trigeminal sensory axons innervating the nasal epithelium using an isolated in vitro preparation. To circumvent non-specific effects of chemical stimuli, optical stimulation was used to excite olfactory sensory neurons in a mouse line expressing channel-rhodopsin under the olfactory marker protein. During photoactivation of olfactory sensory neurons there was no modulation of action potential conduction in individual trigeminal axons. Similarly, no evidence was found for trigeminal axon collateral branching that might serve as a conduit for cross-talk between the olfactory epithelium and olfactory dura mater. Using direct assessment of trigeminal signals emanating from the mouse nasal cavity we see no evidence for paracrine nor axon reflex mediated cross-talk between olfactory and trigeminal sensory systems in the nasal cavity.