Central Terminals Research Articles

Neuropeptide Y Y2 receptors in acute and chronic pain and itch

Pain and itch are regulated by a diverse array of neuropeptides and their receptors in superficial laminae of the spinal cord dorsal horn (DH). Neuropeptide Y (NPY) is normally expressed on DH neurons but not sensory neurons. By contrast, the Npy2r receptor (Y2) is expressed on the central and peripheral terminals of sensory neurons but not on DH neurons. Neurophysiological slice recordings indicate that Y2-selective agonists inhibits spinal neurotransmitter release from sensory neurons. However, behavioral pharmacology studies indicate that Y2 agonists exert minimal changes in nociception, even after injury. Additional discrepancies in the behavioral actions of the Y2-antagonist BIIE0246 – reports of either pronociception or antinociception – have now been resolved. In the normal state, spinally-directed (intrathecal) administration of BIIE0246 elicits ongoing nociception, hypersensitivity to sensory stimulation, and aversion. Conversely, in the setting of nerve injury and inflammation, intrathecal BIIE024 reduced not only mechanical and thermal hypersensitivity, but also a measure of the affective dimension of pain (conditioned place preference). When administered in chronic pain models of latent sensitization, BIIE0246 produced a profound reinstatement of pain-like behaviors. We propose that tissue or nerve injury induces a G protein switch in the action of NPY-Y2 signaling from antinociception in the naïve state to the inhibition of mechanical and heat hyperalgesia in the injured state, and then a switch back to antinociception to keep LS in a state of remission. This model clarifies the pharmacotherapeutic potential of Y2 research, pointing to the development of a new non-opioid pharmacotherapy for chronic pain using Y2 antagonists in patients who do not develop LS.

Where you are matters: Network position, designed network, and central government agency termination

Where you are matters: Network position, designed network, and central government agency termination

Neuropeptide Y Y2 Receptors in Sensory Neurons Tonically Suppress Nociception and Itch but Facilitate Postsurgical and Neuropathic Pain Hypersensitivity.

Neuropeptide Y (NPY) Y2 receptor (Y2) antagonist BIIE0246 can both inhibit and facilitate nociception. The authors hypothesized that Y2 function depends on inflammation or nerve injury status. The authors implemented a battery of behavioral tests in mice of both sexes that received (1) no injury; (2) an incision model of postoperative pain; (3) a spared nerve injury model of neuropathic pain; and (4) a latent sensitization model of chronic postsurgical pain. In addition to Y2 gene expression assays, spinal Y2 G-protein coupling was studied with guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assays. The authors report that intrathecal BIIE0246 increased mechanical and cold hypersensitivity, produced behavioral signs of spontaneous nociception and itch, and produced conditioned place aversion and preference in normal, uninjured mice. BIIE0246 did not change heat hypersensitivity or motor coordination. Conditional (sensory neuron-specific) Y2 deletion prevented BIIE0246-induced mechanical and cold hypersensitivity, nocifensive behaviors, and aversion. Both conditional deletion and pharmacologic blockade of Y2 reduced mechanical and thermal hypersensitivity after incision or nerve injury. SNI did not change the sensitivity of Y2 G-protein coupling with the Y2 agonist peptide YY (3-36) (PYY3-36), but increased the population of Y2 that effectively coupled G-proteins. Intrathecal PYY3-36 failed to reduce spared nerve injury- or incision-induced hypersensitivity in C57BL/6N mice. Incision did not change Npy2r gene expression in dorsal root ganglion. The authors conclude that Y2 at central terminals of primary afferent neurons provides tonic inhibition of mechanical and cold nociception and itch. This switches to the promotion of mechanical and thermal hyperalgesia in models of acute and chronic postsurgical and neuropathic pain, perhaps due to an increase in the population of Y2 that effectively couples to G-proteins. These results support the development of Y2 antagonists for the treatment of chronic postsurgical and neuropathic pain.

Analgesic targets identified in mouse sensory neuron somata and terminal pain translatomes

The relationship between transcription and protein expression is complex. We identified polysome-associated RNA transcripts in the somata and central terminals of mouse sensory neurons in control, painful (plus nerve growth factor), and pain-free conditions (Nav1.7-null mice). The majority (98%) of translated transcripts are shared between male and female mice in both the somata and terminals. Some transcripts are highly enriched in the somata or terminals. Changes in the translatome in painful and pain-free conditions include novel and known regulators of pain pathways. Antisense knockdown of selected somatic and terminal polysome-associated transcripts that correlate with pain states diminished pain behavior. Terminal-enriched transcripts included those encoding synaptic proteins (e.g., synaptotagmin), non-coding RNAs, transcription factors (e.g., Znf431), proteins associated with transsynaptic trafficking (HoxC9), GABA-generating enzymes (Gad1 and Gad2), and neuropeptides (Penk). Thus, central terminal translation may well be a significant regulatory locus for peripheral input from sensory neurons.

Peripherally targeted analgesia via AAV-mediated sensory neuron-specific inhibition of multiple pronociceptive sodium channels.

This study reports that targeting intrinsically disordered regions of the voltage-gated sodium channel 1.7 (NaV1.7) protein facilitates discovery of sodium channel inhibitory peptide aptamers (NaViPA) for adeno-associated virus-mediated (AAV-mediated), sensory neuron-specific analgesia. A multipronged inhibition of INa1.7, INa1.6, INa1.3, and INa1.1 - but not INa1.5 and INa1.8 - was found for a prototype and named NaViPA1, which was derived from the NaV1.7 intracellular loop 1, and is conserved among the TTXs NaV subtypes. NaViPA1 expression in primary sensory neurons (PSNs) of dorsal root ganglia (DRG) produced significant inhibition of TTXs INa but not TTXr INa. DRG injection of AAV6-encoded NaViPA1 significantly attenuated evoked and spontaneous pain behaviors in both male and female rats with neuropathic pain induced by tibial nerve injury (TNI). Whole-cell current clamp of the PSNs showed that NaViPA1 expression normalized PSN excitability in TNI rats, suggesting that NaViPA1 attenuated pain by reversal of injury-induced neuronal hypersensitivity. IHC revealed efficient NaViPA1 expression restricted in PSNs and their central and peripheral terminals, indicating PSN-restricted AAV biodistribution. Inhibition of sodium channels by NaViPA1 was replicated in the human iPSC-derived sensory neurons. These results summate that NaViPA1 is a promising analgesic lead that, combined with AAV-mediated PSN-specific block of multiple TTXs NaVs, has potential as a peripheral nerve-restricted analgesic therapeutic.

Peripheral Branch Injury Induces Oxytocin Receptor Expression at the Central Axon Terminals of Primary Sensory Neurons.

Considerable evidence suggests that oxytocin, as a regulatory nonapeptide, participates in modulatory mechanisms of nociception. Nonetheless, the role of this hypothalamic hormone and its receptor in the sensory pathway has yet to be fully explored. The present study performed immunohistochemistry, enzyme-linked immunosorbent assay, and RT-qPCR analysis to assess changes in the expression of the neuronal oxytocin receptor in female rats following tight ligation of the sciatic nerve after 1, 3, and 7 days of survival. Oxytocin receptor immunoreactivity was present in both dorsal root ganglia and lumbar spinal cord segments, but not accumulated at the site of the ligation of the peripheral nerve branch. We found a time-dependent change in the expression of oxytocin receptor mRNA in L5 dorsal root ganglion neurons, as well as an increase in the level of the receptor protein in the lumbar segment of the spinal cord. A peak in the expression was observed on day 3, which downturned slightly by day 7 after the nerve ligation. These results show that OTR expression is up-regulated in response to peripheral nerve lesions. We assume that the importance of OTR is to modify spinal presynaptic inputs of the sensory neurons upon injury-induced activation, thus to be targets of the descending oxytocinergic neurons from supraspinal levels. The findings of this study support the concept that oxytocin plays a role in somatosensory transmission.

Botulinum toxin type a antinociceptive activity in trigeminal regions involves central transcytosis

ObjectiveBotulinum toxin type A (BoNT-A) provides lasting pain relief in patients with craniofacial pain conditions but the mechanisms of its antinociceptive activity remain unclear. Preclinical research revealed toxin axonal transport to the central afferent terminals, but it is unknown if its central effects involve transsynaptic traffic to the higher-order synapses. To answer this, we examined the contribution of central BoNT-A transcytosis to its action in experimental orofacial pain. Material and methods:Male Wistar rats, 3–4 months old, were injected with BoNT-A (7 U/kg) unilaterally into the vibrissal pad. To investigate the possible contribution of toxin's transcytosis, BoNT-A-neutralizing antiserum (5 IU) was applied intracisternally. Antinocicepive BoNT-A action was assessed by duration of nocifensive behaviors and c-Fos activation in the trigeminal nucleus caudalis (TNC) following bilateral or unilateral formalin (2.5%) application into the vibrissal pad. Additionally, cleaved synaptosomal-associated protein of 25 kDa (cl-SNAP-25) immunoreactivity was analyzed in the bilateral TNC. Results:Unilaterally injected BoNT-A reduced the nocifensive behaviors and bilateral c-Fos activation induced by formalin, which was accompanied by the toxin's enzymatic activity on both sides of the TNC. BoNT-A antinociceptive or enzymatic activities were prevented by the specific neutralizing antitoxin. BoNT-A contralateral action occurred independently from ipsilateral side nociception or contralateral trigeminal nerve-mediated axonal traffic. ConclusionHerein, we demonstrate that antinociceptive action of pericranially administered BoNT-A involves transsynaptic transport to second order synapses and contralateral trigeminal nociceptive nuclei. These results reveal more complex central toxin activity, necessary to explain its clinical effectiveness in the trigeminal region-related pain states.

TRPV1 enhances cholecystokinin signaling in primary vagal afferent neurons and mediates the central effects on spontaneous glutamate release in the NTS.

The gut peptide cholecystokinin (CCK) is released during feeding and promotes satiation by increasing excitation of vagal afferent neurons that innervate the upper gastrointestinal tract. Vagal afferent neurons express CCK1 receptors (CCK1Rs) in the periphery and at central terminals in the nucleus of the solitary tract (NTS). While the effects of CCK have been studied for decades, CCK receptor signaling and coupling to membrane ion channels are not entirely understood. Previous findings have implicated L-type voltage-gated calcium channels as well as transient receptor potential (TRP) channels in mediating the effects of CCK, but the lack of selective pharmacology has made determining the contributions of these putative mediators difficult. The nonselective ion channel transient receptor potential vanilloid subtype 1 (TRPV1) is expressed throughout vagal afferent neurons and controls many forms of signaling, including spontaneous glutamate release onto NTS neurons. Here we tested the hypothesis that CCK1Rs couple directly to TRPV1 to mediate vagal signaling using fluorescent calcium imaging and brainstem electrophysiology. We found that CCK signaling at high concentrations (low-affinity binding) was potentiated in TRPV1-containing afferents and that TRPV1 itself mediated the enhanced CCK1R signaling. While competitive antagonism of TRPV1 failed to alter CCK1R signaling, TRPV1 pore blockade or genetic deletion (TRPV1 KO) significantly reduced the CCK response in cultured vagal afferents and eliminated its ability to increase spontaneous glutamate release in the NTS. Together, these results establish that TRPV1 mediates the low-affinity effects of CCK on vagal afferent activation and control of synaptic transmission in the brainstem.NEW & NOTEWORTHY Cholecystokinin (CCK) signaling via the vagus nerve reduces food intake and produces satiation, yet the signaling cascades mediating these effects remain unknown. Here we report that the capsaicin receptor transient receptor potential vanilloid subtype 1 (TRPV1) potentiates CCK signaling in the vagus and mediates the ability of CCK to control excitatory synaptic transmission in the nucleus of the solitary tract. These results may prove useful in the future development of CCK/TRPV1-based therapeutic interventions.

Kv2 channels contribute to neuronal activity within the vagal afferent-nTS reflex arc.

Diversity in the functional expression of ion channels contributes to the unique patterns of activity generated in visceral sensory A-type myelinated neurons versus C-type unmyelinated neurons in response to their natural stimuli. In the present study, Kv2 channels were identified as underlying a previously uncharacterized delayed rectifying potassium current expressed in both A- and C-type nodose ganglion neurons. Kv2.1 and 2.2 appear confined to the soma and initial segment of these sensory neurons; however, neither was identified in their central presynaptic terminals projecting onto relay neurons in the nucleus of the solitary tract (nTS). Kv2.1 and Kv2.2 were also not detected in the peripheral axons and sensory terminals in the aortic arch. Functionally, in nodose neuron somas, Kv2 currents exhibited frequency-dependent current inactivation and contributed to action potential repolarization in C-type neurons but not A-type neurons. Within the nTS, the block of Kv2 currents does not influence afferent presynaptic calcium influx or glutamate release in response to afferent activation, supporting our immunohistochemical observations. On the other hand, Kv2 channels contribute to membrane hyperpolarization and limit action potential discharge rate in second-order neurons. Together, these data demonstrate that Kv2 channels influence neuronal discharge within the vagal afferent-nTS circuit and indicate they may play a significant role in viscerosensory reflex function.NEW & NOTEWORTHY We demonstrate the expression and function of the voltage-gated delayed rectifier potassium channel Kv2 in vagal nodose neurons. Within sensory neurons, Kv2 channels limit the width of the broader C-type but not narrow A-type action potential. Within the nucleus of the solitary tract (nTS), the location of the vagal terminal field, Kv2 does not influence glutamate release. However, Kv2 limits the action potential discharge of nTS relay neurons. These data suggest a critical role for Kv2 in the vagal-nTS reflex arc.

Comparative morphology of serotonin-immunoreactive neurons innervating the central complex in the brain of dicondylian insects.

Serotonin (5-hydroxytryptamine) acts as a widespread neuromodulator in the nervous system of vertebrates and invertebrates. In insects, it promotes feeding, enhances olfactory sensitivity, modulates aggressive behavior, and, in the central complex of Drosophila, serves a role in sleep homeostasis. In addition to a role in sleep-wake regulation, the central complex has a prominent role in spatial orientation, goal-directed locomotion, and navigation vector memory. To further understand the role of serotonergic signaling in this brain area, we analyzed the distribution and identity of serotonin-immunoreactive neurons across a wide range of insect species. While one bilateral pair of tangential neurons innervating the central body was present in all species studied, a second type was labeled in all neopterans but not in dragonflies and firebrats. Both cell types show conserved major fiber trajectories but taxon-specific differences in dendritic targets outside the central body and axonal terminals in the central body, noduli, and lateral accessory lobes. In addition, numerous tangential neurons of the protocerebral bridge were labeled in all studied polyneopteran species except for Phasmatodea, but not in Holometabola. Lepidoptera and Diptera showed additional labeling of two bilateral pairs of neurons of a third type. The presence of serotonin in systems of columnar neurons apparently evolved independently in dragonflies and desert locusts. The data suggest distinct evolutionary changes in the composition of serotonin-immunolabeled neurons of the central complex and provides a promising basis for a phylogenetic study in a wider range of arthropod species.

MGluR5 from Primary Sensory Neurons Promotes Opioid-Induced Hyperalgesia and Tolerance by Interacting with and Potentiating Synaptic NMDA Receptors.

Aberrant activation of presynaptic NMDARs in the spinal dorsal horn is integral to opioid-induced hyperalgesia and analgesic tolerance. However, the signaling mechanisms responsible for opioid-induced NMDAR hyperactivity remain poorly identified. Here, we show that repeated treatment with morphine or fentanyl reduced monomeric mGluR5 protein levels in the dorsal root ganglion (DRG) but increased levels of mGluR5 monomers and homodimers in the spinal cord in mice and rats of both sexes. Coimmunoprecipitation analysis revealed that monomeric and dimeric mGluR5 in the spinal cord, but not monomeric mGluR5 in the DRG, directly interacted with GluN1. By contrast, mGluR5 did not interact with μ-opioid receptors in the DRG or spinal cord. Repeated morphine treatment markedly increased the mGluR5-GluN1 interaction and protein levels of mGluR5 and GluN1 in spinal synaptosomes. The mGluR5 antagonist MPEP reversed morphine treatment-augmented mGluR5-GluN1 interactions, GluN1 synaptic expression, and dorsal root-evoked monosynaptic EPSCs of dorsal horn neurons. Furthermore, CRISPR-Cas9-induced conditional mGluR5 knockdown in DRG neurons normalized mGluR5 levels in spinal synaptosomes and NMDAR-mediated EPSCs of dorsal horn neurons increased by morphine treatment. Correspondingly, intrathecal injection of MPEP or conditional mGluR5 knockdown in DRG neurons not only potentiated the acute analgesic effect of morphine but also attenuated morphine treatment-induced hyperalgesia and tolerance. Together, our findings suggest that opioid treatment promotes mGluR5 trafficking from primary sensory neurons to the spinal dorsal horn. Through dimerization and direct interaction with NMDARs, presynaptic mGluR5 potentiates and/or stabilizes NMDAR synaptic expression and activity at primary afferent central terminals, thereby maintaining opioid-induced hyperalgesia and tolerance.SIGNIFICANCE STATEMENT Opioids are essential analgesics for managing severe pain caused by cancer, surgery, and tissue injury. However, these drugs paradoxically induce pain hypersensitivity and tolerance, which can cause rapid dose escalation and even overdose mortality. This study demonstrates, for the first time, that opioids promote trafficking of mGluR5, a G protein-coupled glutamate receptor, from peripheral sensory neurons to the spinal cord; there, mGluR5 proteins dimerize and physically interact with NMDARs to augment their synaptic expression and activity. Through dynamic interactions, the two distinct glutamate receptors mutually amplify and sustain nociceptive input from peripheral sensory neurons to the spinal cord. Thus, inhibiting mGluR5 activity or disrupting mGluR5-NMDAR interactions could reduce opioid-induced hyperalgesia and tolerance and potentiate opioid analgesic efficacy.

Analysing Wilson’s and Goldberger’s Central Terminals: Theoretical Redesign of a Novel Central Terminal for Precordial Leads

This study delves into the foundational aspects of electrocardiographic (ECG) lead systems, specifically focusing on Einthoven’s Triangle and Wilson’s Central Terminal. We introduce the innovative concept of a Precordial Central Terminal, a strategic arrangement of thoracic electrodes that establishes two Einthoven’s triangles within the horizontal plane, in accordance with physical-mathematical principles. Furthermore, we meticulously scrutinize Goldberger’s contributions to the field of ECG, particularly augmented leads, which shed light on the development of our theory. Additionally, we propose the implementation of a novel standardized electrocardiograph, characterized by its use of exclusively “precordial” electrodes, yet capable of providing a comprehensive three-dimensional perspective of cardiac electrical activity. By amalgamating theoretical underpinnings with empirical experimentation, our study lays the groundwork for future investigations, including the experimental validation of our innovative Precordial Terminal. This validation holds the promise of yielding substantial advancements in the realm of clinical electrocardiography.

The phospho-regulated amphiphysin/endophilin interaction is required for synaptic vesicle endocytosis.

The multidomain adaptor protein amphiphysin-1 (Amph1) is an important coordinator of clathrin-mediated endocytosis in non-neuronal cells and synaptic vesicle (SV) endocytosis at central nerve terminals. Amph1 contains a lipid-binding N-BAR (Bin/Amphiphysin/Rvs) domain, central proline-rich (PRD) and clathrin/AP2 (CLAP) domains, and a C-terminal SH3 domain. Amph1 interacts with both lipids and proteins, with all of these interactions required for SV endocytosis, with the exception of the Amph1 PRD. The Amph1 PRD associates with the endocytosis protein endophilin A1, however, the role of this interaction in SV endocytosis has not been investigated. In this study, we set out to determine whether the Amph1 PRD and its interaction with endophilin A1 was essential for efficient SV endocytosis at typical small central synapses. To achieve this, domain-specific interactions of Amph1 were validated using in vitro GST pull-down assays, with the role of these interactions in SV endocytosis determined in molecular replacement experiments in primary neuronal culture. Using this approach, we confirmed important roles for CLAP and SH3 domain interactions of Amph1 in the control of SV endocytosis. Importantly, we identified the interaction site for endophilin A1 within the Amph1 PRD and exploited specific binding mutants to reveal a key role for this interaction in SV endocytosis. Finally, we determined that the formation of the Amph1-endophilin A1 complex is dependent on the phosphorylation status of Amph1-S293 within the PRD and that the phosphorylation status of this residue is essential for efficient SV regeneration. This work, therefore, reveals a key role for the dephosphorylation-dependent Amph1-endophilin A1 interaction in efficient SV endocytosis.

Collagen mimetic peptide repair of the corneal nerve bed in a mouse model of dry eye disease.

The intraepithelial sub-basal nerve plexus of the cornea is characterized by a central swirl of nerve processes that terminate between the apical cells of the epithelium. This plexus is a critical component of maintaining homeostatic function of the ocular surface. The cornea contains a high concentration of collagen, which is susceptible to damage in conditions such as neuropathic pain, neurotrophic keratitis, and dry eye disease. Here we tested whether topical application of a collagen mimetic peptide (CMP) is efficacious in repairing the corneal sub-basal nerve plexus in a mouse model of ocular surface desiccation. We induced corneal tear film reduction, epithelial damage, and nerve bed degradation through a combination of environmental and pharmaceutical (atropine) desiccation. Mice were subjected to desiccating air flow and bilateral topical application of 1% atropine solution (4× daily) for 2 weeks. During the latter half of this exposure, mice received topical vehicle [phosphate buffered saline (PBS)] or CMP [200 μm (Pro-Pro-Gly)7, 10 μl] once daily, 2 h prior to the first atropine treatment for that day. After euthanasia, cornea were labeled with antibodies against βIII tubulin to visualize and quantify changes to the nerve bed. For mice receiving vehicle only, the two-week desiccation regimen reduced neuronal coverage of the central sub-basal plexus and epithelial terminals compared to naïve, with some corneas demonstrating complete degeneration of nerve beds. Accordingly, both sub-basal and epithelial βIII tubulin-labeled processes demonstrated increased fragmentation, indicative of nerve disassembly. Treatment with CMP significantly reduced nerve fragmentation, expanded both sub-basal and epithelial neuronal coverage compared to vehicle controls, and improved corneal epithelium integrity, tear film production, and corneal sensitivity. Together, these results indicate that topical CMP significantly counters neurodegeneration characteristic of corneal surface desiccation. Repairing underlying collagen in conditions that damage the ocular surface could represent a novel therapeutic avenue in treating a broad spectrum of diseases or injury.

West Nile Fever in the Russian Federation in 2022, the Incidence Forecast for 2023

The review presents an analysis of West Nile fever (WNF) epidemiological situation in the Russian Federation in 2022 and the summarized results of monitoring over the pathogen. The following features of the WNF epidemic process in Russia are outlined: the decrease (by 4 times) in the incidence rates compared to the long-term average values against an extremely extensive nature of manifestations (16 constituent entities in 6 federal districts), including the detection of local cases in 5 new territories (Tver, Tambov, Vladimir Regions, Khanty-Mansi Autonomous District, and Karachay-Cherkess Republic), predominant registration of incidence in the Central Federal District (51 %), early start and termination of the epidemic season. There is a decrease in the proportion of neuroinvasive forms in the structure of incidence, prevalence of females and the age group of “60 years and over”, as well as record rate values of people infected at the place of residence in cities, which is associated with the peculiarities of case detection, namely, screening examinations of febrile patients in hospitals by efforts of the Reference Center, according to the results of which 60 % of all registered patients with WNF in Russia were diagnosed. Monitoring studies of the Reference Center in the territory of the Central, Southern and North-Caucasian Federal Districts confirmed the intensive circulation of the pathogen. The low level of officially registered incidence is caused by insufficiently effective identification of patients. Molecular genetic studies have shown that West Nile virus lineage 2 circulated in the European part of Russia, both in epizootic and epidemic cycles. Phylogenetic analysis has revealed that 4 West Nile virus isolates found in Astrakhan, Volgograd Regions, the Republic of Kalmykia and Stavropol Territory in 2022 belong to a new genovariant of the genotype 2, first identified in Russia in 2021. Possible complication of the epidemiological situation in all federal districts of the center and south of the European part of Russia is predicted.

Epilepsy-Related CDKL5 Deficiency Slows Synaptic Vesicle Endocytosis in Central Nerve Terminals.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a severe early-onset epileptic encephalopathy resulting mainly from de novo mutations in the X-linked CDKL5 gene. To determine whether loss of presynaptic CDKL5 function contributes to CDD, we examined synaptic vesicle (SV) recycling in primary hippocampal neurons generated from Cdkl5 knockout rat males. Using a genetically encoded reporter, we revealed that CDKL5 is selectively required for efficient SV endocytosis. We showed that CDKL5 kinase activity is both necessary and sufficient for optimal SV endocytosis, since kinase-inactive mutations failed to correct endocytosis in Cdkl5 knockout neurons, whereas the isolated CDKL5 kinase domain fully restored SV endocytosis kinetics. Finally, we demonstrated that CDKL5-mediated phosphorylation of amphiphysin 1, a putative presynaptic target, is not required for CDKL5-dependent control of SV endocytosis. Overall, our findings reveal a key presynaptic role for CDKL5 kinase activity and enhance our insight into how its dysfunction may culminate in CDD.SIGNIFICANCE STATEMENT Loss of cyclin-dependent kinase like 5 (CDKL5) function is a leading cause of monogenic childhood epileptic encephalopathy. However, information regarding its biological role is scarce. In this study, we reveal a selective presynaptic role for CDKL5 in synaptic vesicle endocytosis and that its protein kinase activity is both necessary and sufficient for this role. The isolated protein kinase domain is sufficient to correct this loss of function, which may facilitate future gene therapy strategies if presynaptic dysfunction is proven to be central to the disorder. It also reveals that a CDKL5-specific substrate is located at the presynapse, the phosphorylation of which is required for optimal SV endocytosis.

Simulation optimization for parcel hub scheduling problem in closed-loop sortation system with shortcuts

With the growth of e-commerce, the quantity of delivery orders and parcels is steadily increasing. Reliable and fast parcel delivery services are of utmost importance to customers. To achieve this target, the central parcel consolidation terminals (CPCT) known as parcel sorting hubs are dedicated to improving the operational efficiency especially in the optimal scheduling of inbound trucks. This study aims to determine the optimal schedule and assignment of inbound trucks to the inbound docks, known as the parcel hub scheduling problem with shortcuts (PHSPwS), for minimizing the expected makespan and expected waiting time of inbound trucks in the closed-loop automated sorting conveyor (ASC) system with shortcuts of the parcel sorting hubs. We first construct a mathematical formulation to present the PHSPwS problem based on the traffic control model. However, due to the congestion behavior on the conveyors and stochastic and dynamic nature of the ASC system, the expected objective functions are not analytically available from the mathematical model. Then, a simulation optimization (SO) approach combined with an adaptive genetic-based discrete particle swarm optimization (AGDPSO) algorithm is proposed to tackle the PHSPwS problem in a stochastic and dynamic ASC environment. The closed-loop ASC simulation model is first established to describe the detail operation of the closed-loop ASC sortation system with shortcut. Based on performance evaluation of this simulation model, a novelty algorithm AGDPSO, which hybridized genetic algorithm and particle swarm optimization algorithm with self-tuning parameters is developed to search and find the optimal inbound truck schedule in the large-scale solution space. In particular, the time-varying acceleration coefficients embedded in the AGDPSO are adopted to amplify the effectiveness of the global and local best solutions to obtain a better balance between exploration and exploitation of the searching space. The experimental results reveal that AGDPSO outperforms other algorithms and provides a better and more stable solution. Moreover, the impact of the strategic and operational factors of the closed-loop ASC system for the expected makespan and truck queueing time are also investigated, while the automatic truck unloading mode is confirmed to significantly improve the system efficiency.

Primary Afferent Depolarization and the Gate Control Theory of Pain: A Tutorial Simulation.

The gate control theory of pain postulates that the sensation of pain can be reduced or blocked by closing a "gate" at the earliest synaptic level in the spinal cord, where nociceptive (pain) afferents excite the ascending interneurons that transmit the signal to the brain. Furthermore, the gate can be induced to close by stimulating touch afferents with receptive fields in the same general area as the trauma that is generating the pain (the "rub it to make it better" effect). A considerable volume of research has substantiated the theory and shown that a key mechanism mediating the gate is pre-synaptic inhibition, and that this inhibition is generated by depolarizing IPSPs in the nociceptor central terminals (primary afferent depolarization; PAD). Both pre-synaptic inhibition and depolarizing IPSPs are topics that students often regard as matters of secondary importance (if they are aware of them at all), and yet they are crucial to a matter of primary importance to us all - pain control. This report describes some simple computer simulations that illustrate pre-synaptic inhibition and explore the importance of the depolarizing aspect of the IPSPs. These concepts are then built into a model of the gate control of pain itself. Finally, the simulations show how a small change in chloride homeostasis can generate the dorsal root reflex, in which nociceptor afferents generate antidromic spikes which may increase neurogenic inflammation and actually exacerbate pain. The hope is that the simulations will increase awareness and understanding of a topic that is important in both basic neuroscience and medical neurology.

HDAC2 in Primary Sensory Neurons Constitutively Restrains Chronic Pain by Repressing α2δ-1 Expression and Associated NMDA Receptor Activity.

α2δ-1 (encoded by the Cacna2d1 gene) is a newly discovered NMDA receptor-interacting protein and is the therapeutic target of gabapentinoids (e.g., gabapentin and pregabalin) frequently used for treating patients with neuropathic pain. Nerve injury causes sustained α2δ-1 upregulation in the dorsal root ganglion (DRG), which promotes NMDA receptor synaptic trafficking and activation in the spinal dorsal horn, a hallmark of chronic neuropathic pain. However, little is known about how nerve injury initiates and maintains the high expression level of α2δ-1 to sustain chronic pain. Here, we show that nerve injury caused histone hyperacetylation and diminished enrichment of histone deacetylase-2 (HDAC2), but not HDAC3, at the Cacna2d1 promoter in the DRG. Strikingly, Hdac2 knockdown or conditional knockout in DRG neurons in male and female mice consistently induced long-lasting mechanical pain hypersensitivity, which was readily reversed by blocking NMDA receptors, inhibiting α2δ-1 with gabapentin or disrupting the α2δ-1-NMDA receptor interaction at the spinal cord level. Hdac2 deletion in DRG neurons increased histone acetylation levels at the Cacna2d1 promoter, upregulated α2δ-1 in the DRG, and potentiated α2δ-1-dependent NMDA receptor activity at primary afferent central terminals in the spinal dorsal horn. Correspondingly, Hdac2 knockdown-induced pain hypersensitivity was blunted in Cacna2d1 knockout mice. Thus, our findings reveal that HDAC2 functions as a pivotal transcriptional repressor of neuropathic pain via constitutively suppressing α2δ-1 expression and ensuing presynaptic NMDA receptor activity in the spinal cord. HDAC2 enrichment levels at the Cacna2d1 promoter in DRG neurons constitute a unique epigenetic mechanism that governs acute-to-chronic pain transition.SIGNIFICANCE STATEMENT Excess α2δ-1 proteins produced after nerve injury directly interact with glutamate NMDA receptors to potentiate synaptic NMDA receptor activity in the spinal cord, a prominent mechanism of nerve pain. Because α2δ-1 upregulation after nerve injury is long lasting, gabapentinoids relieve pain symptoms only temporarily. Our study demonstrates for the first time the unexpected role of intrinsic HDAC2 activity at the α2δ-1 gene promoter in limiting α2δ-1 gene transcription, NMDA receptor-dependent synaptic plasticity, and chronic pain development after nerve injury. These findings challenge the prevailing view about the role of general HDAC activity in promoting chronic pain. Restoring the repressive HDAC2 function and/or reducing histone acetylation at the α2δ-1 gene promoter in primary sensory neurons could lead to long-lasting relief of nerve pain.

TRPV1: A Common Denominator Mediating Antinociceptive and Antiemetic Effects of Cannabinoids.

The most common medicinal claims for cannabis are relief from chronic pain, stimulation of appetite, and as an antiemetic. However, the mechanisms by which cannabis reduces pain and prevents nausea and vomiting are not fully understood. Among more than 450 constituents in cannabis, the most abundant cannabinoids are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Cannabinoids either directly or indirectly modulate ion channel function. Transient receptor potential vanilloid 1 (TRPV1) is an ion channel responsible for mediating several modalities of pain, and it is expressed in both the peripheral and the central pain pathways. Activation of TRPV1 in sensory neurons mediates nociception in the ascending pain pathway, while activation of TRPV1 in the central descending pain pathway, which involves the rostral ventral medulla (RVM) and the periaqueductal gray (PAG), mediates antinociception. TRPV1 channels are thought to be implicated in neuropathic/spontaneous pain perception in the setting of impaired descending antinociceptive control. Activation of TRPV1 also can cause the release of calcitonin gene-related peptide (CGRP) and other neuropeptides/neurotransmitters from the peripheral and central nerve terminals, including the vagal nerve terminal innervating the gut that forms central synapses at the nucleus tractus solitarius (NTS). One of the adverse effects of chronic cannabis use is the paradoxical cannabis-induced hyperemesis syndrome (HES), which is becoming more common, perhaps due to the wider availability of cannabis-containing products and the chronic use of products containing higher levels of cannabinoids. Although, the mechanism of HES is unknown, the effective treatment options include hot-water hydrotherapy and the topical application of capsaicin, both activate TRPV1 channels and may involve the vagal-NTS and area postrema (AP) nausea and vomiting pathway. In this review, we will delineate the activation of TRPV1 by cannabinoids and their role in the antinociceptive/nociceptive and antiemetic/emetic effects involving the peripheral, spinal, and supraspinal structures.