Ganglion Neurons In Culture Research Articles

DNA origami scaffold promoting nerve guidance and regeneration.

Self-assembly of biological elements into biomimetic cargo carriers for targeting and delivery is a promising approach. However, it still holds practical challenges. We developed a functionalization approach of DNA origami (DO) nanostructures with neuronal growth factor (NGF) for manipulating neuronal systems. NGF bioactivity and its interactions with the neuronal system were demonstrated in vitro and in vivo models. The DO elements fabricated by molecular self-assembly have manipulated the surrounding environment through static spatially and temporally controlled presentation of ligands to the cell surface receptors. Our data showed effective bioactivity in differentiating PC12 cells in vitro. Furthermore, the DNA origami NGF (DON) affected the growth directionality and spatial capabilities of dorsal root ganglion neurons in culture by introducing a chemotaxis effect along a gradient of functionalized DO structures. Finally, we showed that these elements provide enhanced axonal regeneration in a rat sciatic nerve injury model in vivo. This study is a proof of principle for the functionality of DO in neuronal manipulation and regeneration. The approach proposed here, of an engineered platform formed out of programmable nanoscale elements constructed of DO, could be extended beyond the nervous system and revolutionize the fields of regenerative medicine, tissue engineering, and cell biology.

Nuclear Magnetic Resonance Treatment Accelerates the Regeneration of Dorsal Root Ganglion Neurons in vitro.

Functional recovery from peripheral nerve injuries depends on a multitude of factors. Schwann cells (SCs) are key players in the regenerative process as they develop repair-specific functions to promote axon regrowth. However, chronically denervated SCs lose their repair phenotype, which is considered as a main reason for regeneration failure. Previous studies reported a modulatory effect of low nuclear magnetic resonance therapy (NMRT) on cell proliferation and gene expression. To provide first insight into a possible effect of NMRT on cells involved in peripheral nerve regeneration, this study investigated whether NMRT is able to influence the cellular behavior of primary SC and dorsal root ganglion (DRG) neuron cultures in vitro. The effect of NMRT on rat SCs was evaluated by comparing the morphology, purity, proliferation rate, and expression levels of (repair) SC associated genes between NMRT treated and untreated SC cultures. In addition, the influence of (1) NMRT and (2) medium obtained from NMRT treated SC cultures on rat DRG neuron regeneration was examined by analyzing neurite outgrowth and the neuronal differentiation status. Our results showed that NMRT stimulated the proliferation of SCs without changing their morphology, purity, or expression of (repair) SC associated markers. Furthermore, NMRT promoted DRG neuron regeneration shown by an increased cell survival, enhanced neurite network formation, and progressed neuronal differentiation status. Furthermore, the medium of NMRT treated SC cultures was sufficient to support DRG neuron survival and neurite outgrowth. These findings demonstrate a beneficial impact of NMRT on DRG neuron survival and neurite formation, which is primarily mediated via SC stimulation. Our data suggest that NMRT could be suitable as a non-invasive auxiliary treatment option for peripheral nerve injuries and encourage future studies that investigate the effect of NMRT in a physiological context.

Prdm12 modulates pain-related behavior by remodeling gene expression in mature nociceptors.

Prdm12 is a conserved epigenetic transcriptional regulator that displays restricted expression in nociceptors of the developing peripheral nervous system. In mice, Prdm12 is required for the development of the entire nociceptive lineage. In humans, PRDM12 mutations cause congenital insensitivity to pain, likely because of the loss of nociceptors. Prdm12 expression is maintained in mature nociceptors suggesting a yet-to-be explored functional role in adults. Using Prdm12 inducible conditional knockout mouse models, we report that in adult nociceptors Prdm12 is no longer required for cell survival but continues to play a role in the transcriptional control of a network of genes, many of them encoding ion channels and receptors. We found that disruption of Prdm12 alters the excitability of dorsal root ganglion neurons in culture. Phenotypically, we observed that mice lacking Prdm12 exhibit normal responses to thermal and mechanical nociceptive stimuli but a reduced response to capsaicin and hypersensitivity to formalin-induced inflammatory pain. Together, our data indicate that Prdm12 regulates pain-related behavior in a complex way by modulating gene expression in adult nociceptors and controlling their excitability. The results encourage further studies to assess the potential of Prdm12 as a target for analgesic development.

Integrins protect sensory neurons in models of paclitaxel-induced peripheral sensory neuropathy

Chemotherapy-induced peripheral neuropathy (CIPN) is a major side effect from cancer treatment with no known method for prevention or cure in clinics. CIPN often affects unmyelinated nociceptive sensory terminals. Despite the high prevalence, molecular and cellular mechanisms that lead to CIPN are still poorly understood. Here, we used a genetically tractable Drosophila model and primary sensory neurons isolated from adult mouse to examine the mechanisms underlying CIPN and identify protective pathways. We found that chronic treatment of Drosophila larvae with paclitaxel caused degeneration and altered the branching pattern of nociceptive neurons, and reduced thermal nociceptive responses. We further found that nociceptive neuron-specific overexpression of integrins, which are known to support neuronal maintenance in several systems, conferred protection from paclitaxel-induced cellular and behavioral phenotypes. Live imaging and superresolution approaches provide evidence that paclitaxel treatment causes cellular changes that are consistent with alterations in endosome-mediated trafficking of integrins. Paclitaxel-induced changes in recycling endosomes precede morphological degeneration of nociceptive neuron arbors, which could be prevented by integrin overexpression. We used primary dorsal root ganglia (DRG) neuron cultures to test conservation of integrin-mediated protection. We show that transduction of a human integrin β-subunit 1 also prevented degeneration following paclitaxel treatment. Furthermore, endogenous levels of surface integrins were decreased in paclitaxel-treated mouse DRG neurons, suggesting that paclitaxel disrupts recycling in vertebrate sensory neurons. Altogether, our study supports conserved mechanisms of paclitaxel-induced perturbation of integrin trafficking and a therapeutic potential of restoring neuronal interactions with the extracellular environment to antagonize paclitaxel-induced toxicity in sensory neurons.

The Prenylflavonoid ENDF1 Overrules Central Nervous System Growth Inhibitors and Facilitates Regeneration of DRG Neurons.

Restoration of neuronal connectivity after lesion of the central nervous system, such as spinal cord injury, is one of the biggest challenges in modern medicine. In particular, the accumulation of axon growth inhibitory factors at the site of injury constitutes a major obstacle to structural and thus functional repair. We previously investigated a group of prenylflavonoids derived from hops for their capacity to promote neuroregeneration. We identified a molecule called ENDF1 that was very potent to enhance regrowth and branching of neurites from dorsal root ganglion neurons in culture on growth promoting substrates. In the present study, we investigated ENDF1’s capacity to promote regeneration of rat dorsal root ganglion neurons in vitro in the presence of three main components of the extracellular matrix acting as axon growth inhibitors: Semaphorin 3A, Ephrin A4 and mixed chondroitin sulfate proteoglycans. We report that ENDF1 application significantly promoted the percentages of sensory neurons able to regrow their neurites regardless of the presence of those inhibitors, and this to an extent similar to the one obtained after NGF treatment. Moreover, ENDF1 strongly enhanced the total neurite length and the complexity of neurites extending from neurons challenged with axon growth inhibitors. Although the impact of NGF and ENDF1 on the regeneration of neurons was similar, the activity of ENDF1 was not mediated by signaling through the TrkA receptor, indicating that each molecule act through different signaling pathways. In addition, ENDF1 did not decrease the phosphorylation of cofilin, a downstream effector of the regeneration-associated RhoA/ROCK signaling pathway. Hence, ENDF1 is a potent pro-neuroregenerative factors that could help in identifying new efficient targets for regenerative therapies of the nervous system.

Indirect AMP-Activated Protein Kinase Activators Prevent Incision-Induced Hyperalgesia and Block Hyperalgesic Priming, Whereas Positive Allosteric Modulators Block Only Priming in Mice.

AMP-activated protein kinase (AMPK) is a multifunctional kinase that negatively regulates the mechanistic target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) signaling, two signaling pathways linked to pain promotion after injury, such as surgical incision. AMPK can be activated directly using positive allosteric modulators, as well as indirectly through the upregulation of upstream kinases, such as liver kinase B1 (LKB1), which is a mechanism of action of metformin. Metformin’s antihyperalgesic effects occur only in male mice, raising questions about how metformin regulates pain sensitivity. We used metformin and other structurally distinct AMPK activators narciclasine (NCLS), ZLN-024, and MK8722, to treat incision-induced mechanical hypersensitivity and hyperalgesic priming in male and female mice. Metformin was the only AMPK activator to have sex-specific effects. We also found that indirect AMPK activators metformin and NCLS were able to reduce mechanical hypersensitivity and block hyperalgesic priming, whereas direct AMPK activators ZLN-024 and MK8722 only blocked priming. Direct and indirect AMPK activators stimulated AMPK in dorsal root ganglion (DRG) neuron cultures to a similar degree; however, incision decreased phosphorylated AMPK (p-AMPK) in DRG. Because AMPK phosphorylation is required for kinase activity, we interpret our findings as evidence that indirect AMPK activators are more effective for treating pain hypersensitivity after incision because they can drive increased p-AMPK through upstream kinases like LKB1. These findings have important implications for the development of AMPK-targeting therapeutics for pain treatment.SIGNIFICANCE STATEMENTNonopioid treatments for postsurgical pain are needed. Our work focused on whether direct or indirect AMP-activated protein kinase (AMPK) activators would show greater efficacy for inhibiting incisional pain, and we also tested for potential sex differences. We conclude that indirect AMPK activators are likely to be more effective as potential therapeutics for postsurgical pain because they inhibit acute pain caused by incision and prevent the long-term neuronal plasticity that is involved in persistent postsurgical pain. Our work points to the natural product narciclasine, an indirect AMPK activator, as an excellent starting point for development of therapeutics.

Oxaliplatin treatment impairs extension of sensory neuron neurites in vitro through miR-204 overexpression

Oxaliplatin is a platinum-based drug used in the treatment of gastric cancers. Oxaliplatin treatment induces sensory neuropathy characterized by cold hypersensibility in the acute phase and sensory impairment when the neuropathy becomes chronic. In order to determine the effect of oxaliplatin on sensory neurons, we used an in vitro model in which oxaliplatin treatment reduced arborization of dorsal root ganglia neurons in a dose-dependent manner. Moreover, we characterized the role of microRNAs in oxaliplatin induced-neuropathy. In particular, we focused on microRNAs that control the expression of axon guidance molecules, and therefore, regulate neurite arborization. As a result, we highlighted the upregulation of miR-204, a microRNA that controls the expression of PlexinA2, a semaphorin receptor involved in neurite guidance. Interaction of miR-204 and Plexin A2 was confirmed by luciferase assay. In addition, overexpression of miR-204 in dorsal root ganglia neuron cultures reduced length and extension of neurites and also reduced Plexin A2 labelling without increasing apoptosis rate. On the other hand, sequestration of miR-204 by a specific microRNA sponge increases neurite length and PlexinA2 expression. Taken together, our data indicate that oxaliplatin impairs sensory neurons arborization through up-regulation of miR-204 that decreases PlexinA2 expression and neurite length.

Functions of CaBP1 and CaBP2 in the peripheral auditory system

CaBPs are a family of Ca2+ binding proteins related to calmodulin. Two CaBP family members, CaBP1 and CaBP2, are highly expressed in the cochlea. Here, we investigated the significance of CaBP1 and CaBP2 for hearing in mice lacking expression of these proteins (CaBP1 KO and CaBP2 KO) using auditory brain responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). In CaBP1 KO mice, ABR wave I was larger in amplitude, and shorter in latency and faster in decay, suggestive of enhanced synchrony of auditory nerve fibers. This interpretation was supported by the greater excitability of CaBP1 KO than WT neurons in whole-cell patch clamp recordings of spiral ganglion neurons in culture, and normal presynaptic function of CaBP1 KO IHCs. DPOAEs and ABR thresholds were normal in 4-week old CaBP1 KO mice, but elevated ABR thresholds became evident at 32 kHz at 9 weeks, and at 8 and 16 kHz by 6 months of age. In contrast, CaBP2 KO mice exhibited significant ABR threshold elevations at 4 weeks of age that became more severe in the mid-frequency range by 9 weeks. Though normal at 4 weeks, DPOAEs in CaBP2 KO mice were significantly reduced in the mid-frequency range by 9 weeks. Our results reveal requirements for CaBP1 and CaBP2 in the peripheral auditory system and highlight the diverse modes by which CaBPs influence sensory processing.

Graphene – Coated Microelectrode Arrays

Graphene – Coated Microelectrode Arrays

Nanoparticle Delivery of Fidgetin siRNA as a Microtubule-based Therapy to Augment Nerve Regeneration

Microtubule-stabilizing drugs have gained popularity for treating injured adult axons, the rationale being that increased stabilization of microtubules will prevent the axon from retracting and fortify it to grow through inhibitory molecules associated with nerve injury. We have posited that a better approach would be not to stabilize the microtubules, but to increase labile microtubule mass to levels more conducive to axonal growth. Recent work on fetal neurons suggests this can be accomplished using RNA interference to reduce the levels of fidgetin, a microtubule-severing protein. Methods to introduce RNA interference into adult neurons, in vitro or in vivo, have been problematic and not translatable to human patients. Here we show that a novel nanoparticle approach, previously shown to deliver siRNA into tissues and organs, enables siRNA to gain entry into adult rat dorsal root ganglion neurons in culture. Knockdown of fidgetin is partial with this approach, but sufficient to increase the labile microtubule mass of the axon, thereby increasing axonal growth. The increase in axonal growth occurs on both a favorable substrate and a growth-inhibitory molecule associated with scar formation in injured spinal cord. The nanoparticles are readily translatable to in vivo studies on animals and ultimately to clinical applications.

Identification of interleukin-1 beta as a key mediator in the upregulation of Cav3.2-USP5 interactions in the pain pathway.

We recently reported that nerve injury or peripheral inflammation triggers an upregulation of the deubiquitinase, USP5 in mouse dorsal root ganglion and spinal dorsal horn. This leads to dysregulated ubiquitination of Cav3.2 T-type calcium channels, thus increasing Cav3.2 channel plasma membrane expression and nociceptive signaling in the primary afferent pain pathway. This phenomenon could be recapitulated by noninvasive, optogenetic activation of transient receptor potential vanilloid-1–expressing nociceptors, indicating that neuronal activity is a key player in this process. Given the relevance of the pro-inflammatory cytokine interleukin-1 beta in many forms of pathological pain, we hypothesized that interleukin-1 beta may be a critical cofactor required to drive upregulation of interactions between USP5 and Cav3.2 channels. Here, we report that gene expression, as well as protein levels for interleukin-1 beta and the endogenous interleukin-1 receptor-I antagonist, IL-1Ra are unaltered following conditioning stimulation of optogenetically targeted cutaneous nociceptors, indicating that neuronal activity is not a driver of interleukin-1 beta signaling. In contrast, co-immunoprecipitation experiments revealed that intrathecal administration of interleukin-1 beta in wild-type mice led to an increase in the interaction between USP5 and Cav3.2 in the spinal dorsal horn. Moreover, disruption of the interaction between USP5 and Cav3.2 with TAT peptides suppressed acute nocifensive responses produced by interleukin-1 beta, which was similar to that achieved by elimination of T-type channel activity with the channel blockers, mibefradil, or TTA-A2. Finally, this upregulation could be maintained in dorsal root ganglion neuron cultures exposed overnight to interleukin-1 beta, while the copresence of interleukin-1 receptor antagonist or the dampening of neuronal cell activity with tetrodotoxin attenuated this response. Altogether, our findings identify interleukin-1 beta as an upstream trigger for the upregulation of interactions between USP5 and Cav3.2 channels in the pain pathway, presumably by triggering increased firing activity in afferent fibers.

Ovariectomy-Induced Mitochondrial Oxidative Stress, Apoptosis, and Calcium Ion Influx Through TRPA1, TRPM2, and TRPV1 Are Prevented by 17β-Estradiol, Tamoxifen, and Raloxifene in the Hippocampus and Dorsal Root Ganglion of Rats.

Relative 17β-estradiol (E2) deprivation and excessive production of mitochondrial oxygen free radicals (OFRs) with a high amount of Ca2+ influx TRPA1, TRPM2, and TRPV1 activity is one of the main causes of neurodegenerative disease in postmenopausal women. In addition to the roles of tamoxifen (TMX) and raloxifene (RLX) in cancer and bone loss treatments, regulator roles in Ca2+ influx and mitochondrial oxidative stress in neurons have not been reported. The aim of this study was to evaluate whether TMX and RLX interactions with TRPA1, TRPM2, and TRPV1 in primary hippocampal (HPC) and dorsal root ganglion (DRG) neuron cultures of ovariectomized (OVX) rats. Forty female rats were divided into five groups: a control group, an OVX group, an OVX+E2 group, an OVX+TMX group, and an OVX+RLX group. The OVX+E2, OVX+TMX, and OVX+RLX groups received E2, TMX, and RLX, respectively, for 14days after the ovariectomy. E2, ovariectomy-induced TRPA1, TRPM2, and TRPV1 current densities, as well as accumulation of cytosolic free Ca2+ in the neurons, were returned to the control levels by E2, TMX, and RLX treatments. In addition, E2, TMX, and RLX via modulation of TRPM2 and TRPV1 activity reduced ovariectomy-induced mitochondrial membrane depolarization, apoptosis, and cytosolic OFR production. TRPM2, TRPV1, PARP, and caspase-3 and caspase-9 expressions were also decreased in the neurons by the E2, TMX, and RLX treatments. In conclusion, we first reported the molecular effects of E2, TMX, and RLX on TRPA1, TRPM2, and TRPV1 channel activation in the OVX rats. In addition, we observed neuroprotective effects of E2, RLX, and TMX on oxidative and apoptotic injuries of the hippocampus and peripheral pain sensory neurons (DRGs) in the OVX rats. Graphical Abstract Possible molecular pathways of involvement of DEX in cerebral ischemia-induced apoptosis, oxidative stress, and calcium accumulation through TRPA1,TRPM2 and TRPV1 in the hippocampus and DRG neurons of rats. The N domain of the TRPM2 contains ADP-ribose (ADPR) pyrophosphate enzyme, which is separately activated by ADPR and oxidative stress, although the channel is reversibly inhibited by N-(p-amylcinnamoyl) anthranilic acid (ACA). The TRPV1 is also activated by mitochondrial oxidative stress and capsaicin, and it is blocked by capsazepine (CPZ). TRPA1 is also activated by oxidative stress it is inhibited by AP18. Increased cytosolic Ca2+ concentration through TRPA1,TRPM2 and TRPV1 in ovariectomized (OVX) rats may lead to neuronal toxicity, reactive oxygen species (ROS) processes, and eventual cell death. 17β-Estradiol (E2), tamoxifen (TMX), and raloxifene (RLX) reduced oxidative stress, apoptosis (including caspase-3 and caspase-9), mitochondrial membrane depolarization, and Ca2+ influx through the inhibition of TRPA1,TRPM2 and TRPV1 activation.

Cyclic Nucleotide Control of Microtubule Dynamics for Axon Guidance.

Graded distribution of intracellular second messengers, such as Ca(2+) and cyclic nucleotides, mediates directional cell migration, including axon navigational responses to extracellular guidance cues, in the developing nervous system. Elevated concentrations of cAMP or cGMP on one side of the neuronal growth cone induce its attractive or repulsive turning, respectively. Although effector processes downstream of Ca(2+) have been extensively studied, very little is known about the mechanisms that enable cyclic nucleotides to steer migrating cells. Here, we show that asymmetric cyclic nucleotide signaling across the growth cone mediates axon guidance via modulating microtubule dynamics and membrane organelle transport. In embryonic chick dorsal root ganglion neurons in culture, contact of an extending microtubule with the growth cone leading edge induces localized membrane protrusion at the site of microtubule contact. Such a contact-induced protrusion requires exocytosis of vesicle-associated membrane protein 7 (VAMP7)-positive vesicles that have been transported centrifugally along the microtubule. We found that the two cyclic nucleotides counteractively regulate the frequency of microtubule contacts and targeted delivery of VAMP7 vesicles: cAMP stimulates and cGMP inhibits these events, thereby steering the growth cone in the opposite directions. By contrast, Ca(2+) signals elicit no detectable change in either microtubule contacts or VAMP7 vesicle delivery during Ca(2+)-induced growth cone turning. Our findings clearly demonstrate growth cone steering machinery downstream of cyclic nucleotide signaling and highlight a crucial role of dynamic microtubules in leading-edge protrusion for cell chemotaxis. Developing neurons can extend long axons toward their postsynaptic targets. The tip of each axon, called the growth cone, recognizes extracellular guidance cues and navigates the axon along the correct path. Here we show that asymmetric cyclic nucleotide signaling across the growth cone mediates axon guidance through localized regulation of microtubule dynamics and resulting recruitment of specific populations of membrane vesicles to the growth cone's leading edge. Remarkably, cAMP stimulates microtubule growth and membrane protrusion, whereas cGMP promotes microtubule retraction and membrane senescence, explaining the opposite directional polarities of growth cone turning induced by these cyclic nucleotides. This study reveals a novel microtubule-based mechanism through which cyclic nucleotides polarize the growth cone steering machinery for bidirectional axon guidance.

Anti-glycan antibodies halt axon regeneration in a model of Guillain Barrè Syndrome axonal neuropathy by inducing microtubule disorganization via RhoA–ROCK-dependent inactivation of CRMP-2

Several reports have linked the presence of high titers of anti-Gg Abs with delayed recovery/poor prognosis in GBS. In most cases, failure to recover is associated with halted/deficient axon regeneration. Previous work identified that monoclonal and patient-derived anti-Gg Abs can act as inhibitory factors in an animal model of axon regeneration. Further studies using primary dorsal root ganglion neuron (DRGn) cultures demonstrated that anti-Gg Abs can inhibit neurite outgrowth by targeting gangliosides via activation of the small GTPase RhoA and its associated kinase (ROCK), a signaling pathway common to other established inhibitors of axon regeneration. We aimed to study the molecular basis of the inhibitory effect of anti-Gg abs on neurite outgrowth by dissecting the molecular dynamics of growth cones (GC) cytoskeleton in relation to the spatial–temporal analysis of RhoA activity. We now report that axon growth inhibition in DRGn induced by a well characterized mAb targeting gangliosides GD1a/GT1b involves: i) an early RhoA/ROCK-independent collapse of lamellipodia; ii) a RhoA/ROCK-dependent shrinking of filopodia; and iii) alteration of GC microtubule organization/and presumably dynamics via RhoA/ROCK-dependent phosphorylation of CRMP-2 at threonine 555. Our results also show that mAb 1B7 inhibits peripheral axon regeneration in an animal model via phosphorylation/inactivation of CRMP-2 at threonine 555. Overall, our data may help to explain the molecular mechanisms underlying impaired nerve repair in GBS. Future work should define RhoA-independent pathway/s and effectors regulating actin cytoskeleton, thus providing an opportunity for the design of a successful therapy to guarantee an efficient target reinnervation.

Structural and Functional Interactions between Transient Receptor Potential Vanilloid Subfamily 1 and Botulinum Neurotoxin Serotype A.

BackgroundBotulinum neurotoxins are produced by Clostridium botulinum bacteria. There are eight serologically distinct botulinum neurotoxin isoforms (serotypes A–H). Currently, botulinum neurotoxin serotype A (BoNT⁄A) is commonly used for the treatment of many disorders, such as hyperactive musculoskeletal disorders, dystonia, and pain. However, the effectiveness of BoNT⁄A for pain alleviation and the mechanisms that mediate the analgesic effects of BoNT⁄A remain unclear. To define the antinociceptive mechanisms by which BoNT/A functions, the interactions between BoNT⁄A and the transient receptor potential vanilloid subfamily 1 (TRPV1) were investigated using immunofluorescence, co-immunoprecipitation, and western blot analysis in primary mouse embryonic dorsal root ganglion neuronal cultures.Results1) Three-week-old cultured dorsal root ganglion neurons highly expressed transient TRPV1, synaptic vesicle 2A (SV2A) and synaptosomal-associated protein 25 (SNAP-25). SV2A and SNAP-25 are the binding receptor and target protein, respectively, of BoNT⁄A. 2) TRPV1 colocalized with both BoNT⁄A and cleaved SNAP-25 when BoNT⁄A was added to dorsal root ganglia neuronal cultures. 3) After 24 hours of BoNT⁄A treatment (1 nmol⁄l), both TRPV1 and BoNT⁄A positive bands were detected in western blots of immunoprecipitated pellets. 4) Blocking TRPV1 with a specific antibody decreased the cleavage of SNAP-25 by BoNT⁄A.ConclusionBoNT/A interacts with TRPV1 both structurally and functionally in cultured mouse embryonic dorsal root ganglion neurons. These results suggest that an alternative mechanism is used by BoNT⁄A to mediate pain relief.

Neural activity selects myosin IIB and VI with a specific time window in distinct dynamin isoform-mediated synaptic vesicle reuse pathways.

Presynaptic nerve terminals must maintain stable neurotransmissions via synaptic vesicle (SV) resupply despite encountering wide fluctuations in the number and frequency of incoming action potentials (APs). However, the molecular mechanism linking variation in neural activity to SV resupply is unknown. Myosins II and VI are actin-based cytoskeletal motors that drive dendritic actin dynamics and membrane transport, respectively, at brain synapses. Here we combined genetic knockdown or molecular dysfunction and direct physiological measurement of fast synaptic transmission from paired rat superior cervical ganglion neurons in culture to show that myosins IIB and VI work individually in SV reuse pathways, having distinct dependency and time constants with physiological AP frequency. Myosin VI resupplied the readily releasable pool (RRP) with slow kinetics independently of firing rates but acted quickly within 50 ms after AP. Under high-frequency AP firing, myosin IIB resupplied the RRP with fast kinetics in a slower time window of 200 ms. Knockdown of both myosin and dynamin isoforms by mixed siRNA microinjection revealed that myosin IIB-mediated SV resupply follows amphiphysin/dynamin-1-mediated endocytosis, while myosin VI-mediated SV resupply follows dynamin-3-mediated endocytosis. Collectively, our findings show how distinct myosin isoforms work as vesicle motors in appropriate SV reuse pathways associated with specific firing patterns.

Spinorphin inhibits membrane depolarization- and capsaicin-induced intracellular calcium signals in rat primary nociceptive dorsal root ganglion neurons in culture

Objective: Spinorphin is a potential endogenous antinociceptive agent although the mechanism(s) of its analgesic effect remain unknown. We conducted this study to investigate, by considering intracellular calcium concentrations as a key signal for nociceptive transmission, the effects of spinorphin on cytoplasmic Ca2+ ([Ca2+]i) transients, evoked by high-K+ (30 mM) depolariasation or capsaicin, and to determine whether there were any differences in the effects of spinorphin among subpopulation of cultured rat dorsal root ganglion (DRG) neurons. Methods: DRG neurons were cultured on glass coverslips following enzymatic digestion and mechanical agitation, and loaded with the calcium sensitive dye fura-2 AM (1 µM). Intracellular calcium responses in individual DRG neurons were quantified using standard fura-2 based ratiometric calcium imaging technique. All data were analyzed by using unpaired t test, p < 0.05 defining statistical significance. Results: Here we found that spinorphin inhibited cytoplasmic Ca2+ ([Ca2+]i) transients, evoked by depolarization and capsaicin selectively in medium and small cultured rat DRG neurons. Spinorphin (10–300 µM) inhibited the Ca2+ signals in concentration dependant manner in small- and medium diameter DRG neurons. Capsaicin produced [Ca2+]i responses only in small- and medium-sized DRG neurons, and pre-treatment with spinorphin significantly attenuated these [Ca2+]i responses. Conclusion: Results from this study indicates that spinorphin significantly inhibits [Ca2+]i signaling, which are key for the modulation of cell membrane excitability and neurotransmitter release, preferably in nociceptive subtypes of this primary sensory neurons suggesting that peripheral site is involved in the pain modulating effect of this endogenous agent.

Highly efficient method for gene delivery into mouse dorsal root ganglia neurons.

The development of gene transfection technologies has greatly advanced our understanding of life sciences. While use of viral vectors has clear efficacy, it requires specific expertise and biological containment conditions. Electroporation has become an effective and commonly used method for introducing DNA into neurons and in intact brain tissue. The present study describes the use of the Neon® electroporation system to transfect genes into dorsal root ganglia neurons isolated from embryonic mouse Day 13.5–16. This cell type has been particularly recalcitrant and refractory to physical or chemical methods for introduction of DNA. By optimizing the culture condition and parameters including voltage and duration for this specific electroporation system, high efficiency (60–80%) and low toxicity (>60% survival) were achieved with robust differentiation in response to Nerve growth factor (NGF). Moreover, 3–50 times fewer cells are needed (6 × 104) compared with other traditional electroporation methods. This approach underlines the efficacy of this type of electroporation, particularly when only limited amount of cells can be obtained, and is expected to greatly facilitate the study of gene function in dorsal root ganglia neuron cultures.

Piezo2 is the major transducer of mechanical forces for touch sensation in mice.

The sense of touch provides critical information about our physical environment by transforming mechanical energy into electrical signals. It is postulated that mechanically activated cation channels initiate touch sensation, but the identity of these molecules in mammals has been elusive. Piezo2 is a rapidly adapting, mechanically activated ion channel expressed in a subset of sensory neurons of the dorsal root ganglion and in cutaneous mechanoreceptors known as Merkel-cell-neurite complexes. It has been demonstrated that Merkel cells have a role in vertebrate mechanosensation using Piezo2, particularly in shaping the type of current sent by the innervating sensory neuron; however, major aspects of touch sensation remain intact without Merkel cell activity. Here we show that mice lacking Piezo2 in both adult sensory neurons and Merkel cells exhibit a profound loss of touch sensation. We precisely localize Piezo2 to the peripheral endings of a broad range of low-threshold mechanoreceptors that innervate both hairy and glabrous skin. Most rapidly adapting, mechanically activated currents in dorsal root ganglion neuronal cultures are absent in Piezo2 conditional knockout mice, and ex vivo skin nerve preparation studies show that the mechanosensitivity of low-threshold mechanoreceptors strongly depends on Piezo2. This cellular phenotype correlates with an unprecedented behavioural phenotype: an almost complete deficit in light-touch sensation in multiple behavioural assays, without affecting other somatosensory functions. Our results highlight that a single ion channel that displays rapidly adapting, mechanically activated currents in vitro is responsible for the mechanosensitivity of most low-threshold mechanoreceptor subtypes involved in innocuous touch sensation. Notably, we find that touch and pain sensation are separable, suggesting that as-yet-unknown mechanically activated ion channel(s) must account for noxious (painful) mechanosensation.

A family of excitatory peptide toxins from venomous crassispirine snails: Using Constellation Pharmacology to assess bioactivity

The toxinology of the crassispirine snails, a major group of venomous marine gastropods within the superfamily Conoidea, is largely unknown. Here we define the first venom peptide superfamily, the P-like crassipeptides, and show that the organization of their gene sequences is similar to conotoxin precursors. We provide evidence that one peptide family within the P-like crassipeptide superfamily includes potassium-channel (K-channel) blockers, the κP-crassipeptides. Three of these peptides were chemically synthesized (cce9a, cce9b and iqi9a). Using conventional electrophysiology, cce9b was shown to be an antagonist of both a human Kv1.1 channel isoform (Shaker subfamily of voltage-gated K channels) and a Drosophila K-channel isoform. We assessed the bioactivity of these peptides in native mammalian dorsal root ganglion neurons in culture. We demonstrate that two of these crassipeptides, cce9a and cce9b, elicited an excitatory phenotype in a subset of small-diameter capsaicin-sensitive mouse DRG neurons that were also affected by κJ-conotoxin PlXIVA (pl14a), a blocker of Kv1.6 channels. Given the vast complexity of heteromeric K-channel isoforms, this study demonstrates that the crassispirine venoms are a potentially rich source for discovering novel peptides that can help to identify and characterize the diversity of K-channel subtypes expressed in native neurons and other cell types.