Preparation and Characterization of a Novel Salicin-Cyclodextrin Complex.

Bioactive compounds extracted from plants can provide wide health benefits. However, some molecules have limited applications as pharmaceuticals due to their limited solubility, poor bioavailability, and low stability when exposed to environmental factors. Their integration in formulations that can deliver them to physiological targets while preserving their biological activity can enhance their usage in improving human health. This research provides a delivery system design to enhance the solubility, stability and to mask the bitter taste of salicin. Thus, a novel salicin-ß-cyclodextrin complex was prepared and analyzed by X-ray diffraction, scanning electron microscopy, thermogravimetric analysis, FTIR, Raman and UV-Vis spectroscopy. The analytical and computational methods provided clear and distinct evidence for inclusion of salicin within the ß-cyclodextrin cavity and brought important findings for the characterization of the inclusion complex. The present study showed that salicin and ß-cyclodextrin can form inclusion complexes, both in solution and in solid state, and that the inclusion of salicin in the cavity of ß-cyclodextrin leads to the improvement of its solubility and stability. Thus, the study communicates both qualitative and quantitative knowledge about the preparation of a new salicin-ß-cyclodextrin inclusion complex suggesting its potential applications in pharmaceutical industry and medical sciences, as formulations with better compliance for the patient, with increased bioavailability, and easier control of dosage.

Increasing Electrical Resistivity of P-Type BiFeO3 Ceramics by Hydrogen Peroxide-Assisted Hydrothermal Synthesis.

Bismuth ferrite (BiFeO3, BFO) is still widely investigated both because of the great diversity of its possible applications and from the perspective of intrinsic defect engineering in the perovskite structure. Defect control in BiFeO3 semiconductors could provide a key technology for overcoming undesirable limitations, namely, a strong leakage current, which is attributed to the presence of oxygen vacancies (VO) and Bi vacancies (VBi). Our study proposes a hydrothermal method for the reduction of the concentration of VBi during the ceramic synthesis of BiFeO3.Using hydrogen peroxide (H2O2) as part of the medium, p-type BiFeO3 ceramics characterized by their low conductivity were obtained. Hydrogen peroxide acted as the electron donor in the perovskite structure, controlling VBi in the BiFeO3 semiconductor, which caused the dielectric constant and loss to decrease along with the electrical resistivity. The reduction of Bi vacancies highlighted by a FT-IR and Mott-Schottky analysis has an expected contribution to the dielectric characteristic. A decrease in the dielectric constant (with approximately 40%) and loss (3 times) and an increase of the electrical resistivity (by 3 times) was achieved by the hydrogen peroxide-assisted hydrothermal synthesized BFO ceramics, as compared with the hydrothermal synthesized BFOs.

Dermal Sensory Regenerative Peripheral Nerve Interface for Reestablishing Sensory Nerve Feedback in Peripheral Afferents in the Rat.

BACKGROUND: Without meaningful, intuitive sensory feedback, even the most advanced myoelectric devices require significant cognitive demand to control. The dermal sensory regenerative peripheral nerve interface (DS-RPNI) is a biological interface designed to establish high-fidelity sensory feedback from prosthetic limbs. METHODS: DS-RPNIs were constructed in rats by securing fascicles of residual sensory peripheral nerves into autologous dermal grafts, with the objectives of confirming regeneration of sensory afferents within DS-RPNIs and establishing the reliability of afferent neural response generation with either mechanical or electrical stimulation. RESULTS: Two months after implantation, DS-RPNIs were healthy and displayed well-vascularized dermis with organized axonal collaterals throughout and no evidence of neuroma. Electrophysiologic signals were recorded proximal from DS-RPNI's sural nerve in response to both mechanical and electrical stimuli and compared with (1) full-thickness skin, (2) deepithelialized skin, and (3) transected sural nerves without DS-RPNI. Mechanical indentation of DS-RPNIs evoked compound sensory nerve action potentials (CSNAPs) that were like those evoked during indentation of full-thickness skin. CSNAP firing rates and waveform amplitudes increased in a graded fashion with increased mechanical indentation. Electrical stimuli delivered to DS-RPNIs reliably elicited CSNAPs at low current thresholds, and CSNAPs gradually increased in amplitude with increasing stimulation current. CONCLUSIONS: These findings suggest that afferent nerve fibers successfully reinnervate DS-RPNIs, and that graded stimuli applied to DS-RPNIs produce proximal sensory afferent responses similar to those evoked from normal skin. This confirmation of graded afferent signal transduction through DS-RPNI neural interfaces validate DS-RPNI's potential role of facilitating sensation in human-machine interfacing. CLINICAL RELEVANCE STATEMENT: The DS-RPNI is a novel biotic-abiotic neural interface that allows for transduction of sensory stimuli into neural signals. It is expected to advance the restoration of natural sensation and development of sensorimotor control in prosthetics.

Elaboration of New Materials Using Hydrothermal Methods.

"Elaboration of New Materials Using Hydrothermal Methods" is a new and open Special Issue of Materials, which aims to publish original research and review papers on that present state-of-the-art advances in the research on the hydrothermal synthesis of new materials [...].

Experimental and Computational Studies on Bio-Inspired Flavylium Salts as Sensitizers for Dye-Sensitized Solar Cells.

Six new bio-inspired flavylium salts were synthesized and investigated by a combined computational and experimental study for dye-sensitized solar cell applications. The compounds were characterized by FT-IR, UV-Vis, NMR spectroscopy, and LC-MS spectrometry techniques. The pH-dependent photochromic properties of the flavylium dyes were investigated through a UV-Vis spectroscopy study and revealed that they follow the same network of chemical reactions as anthocyanins upon pH changes. The structural and electronic properties of the dyes were investigated using density functional theory (DFT) and time-dependent density functional theory (TD-DFT). Geometry optimization calculation revealed that all dyes, regardless of the specie, flavylium cations or quinoidal bases, present a planar geometry. The photovoltaic performances of the dyes, in both flavylium and quinoidal base forms, were evaluated by the HOMO and LUMO energies and by calculating the light-harvesting efficiencies, the free energy change of electron injection, and the free energy change regeneration. The MO analysis showed that all dyes can inject electrons into the conduction band of the TiO2 upon excitation and that the redox couple can regenerate the oxidized dyes. The results obtained for the free energy change of electron injection suggest that the quinoidal bases should inject electrons into the semiconductor more efficiently than the flavylium cations. The values for the free energy change regeneration showed that the redox electrolyte can easily regenerate all dyes. Dipole moment analysis was also performed. DSSCs based on the dyes, in both flavylium and quinoidal base forms, were assembled, and their photovoltaic performances were evaluated by measuring the open-circuit voltage, the short circuit current density, the fill factor, and the energy conversion efficiency. Results obtained by both experimental and computational studies showed that the overall performances of the DSSCs with the quinoidal forms were better than those obtained with the flavylium cations dyes.

Novel Chloro-Substituted Salicylanilide Derivatives and Their ß-Cyclodextrin Complexes: Synthesis, Characterization, and Antibacterial Activity.

The goal of this research was to design novel chloro-substituted salicylanilide derivatives and their ß-cyclodextrin complexes in order to obtain efficient antibacterial compounds and to demonstrate the beneficial role of complexation on the efficiency of these compounds. Thus, salicylanilide derivatives, esters, and hydrazides were obtained by microwave-assisted synthesis and their structure proven based on FTIR and NMR spectra. In order to improve water solubility, chemical and physical stability, and drug distribution through biological membranes, the inclusion complexes of the ethyl esters in ß-cyclodextrin were also obtained using kneading. Inclusion-complex characterization was accomplished by modern analytical methods, X-ray diffraction, SEM, TGA, FTIR, and UV-vis spectroscopy. The newly synthesized compounds were tested against some Gram-positive and Gram-negative bacteria. Antimicrobial tests revealed good activity on Gram-positive bacteria and no inhibition against Gram-negative strains. The MIC and MBC values for compounds derived from N-(2-chlorophenyl)-2-hydroxybenzamide were 0.125-1.0 mg/mL. N-(4-chlorophenyl)-2-hydroxybenzamide derivatives were found to be less active. The inclusion complexes generally behaved similarly to the guest compounds, and antibacterial activity was not been altered by complexation.

Development of P301S tau seeded organotypic hippocampal slice cultures to study potential therapeutics.

Intracellular tau inclusions are a pathological hallmark of Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration and other sporadic neurodegenerative tauopathies. Recent in vitro and in vivo studies have shown that tau aggregates may spread to neighbouring cells and functionally connected brain regions, where they can seed further tau aggregation. This process is referred to as tau propagation. Here we describe an ex vivo system using organotypic hippocampal slice cultures (OHCs) which recapitulates aspects of this phenomenon. OHCs are explants of hippocampal tissue which may be maintained in culture for months. They maintain their synaptic connections and multicellular 3D architecture whilst also permitting direct control of the environment and direct access for various analysis types. We inoculated OHCs prepared from P301S mouse pups with brain homogenate from terminally ill P301S mice and then examined the slices for viability and the production and localization of insoluble phosphorylated tau. We show that following seeding, phosphorylated insoluble tau accumulate in a time and concentration dependent manner within OHCs. Furthermore, we show the ability of the conformation dependent anti-tau antibody, MC1, to compromise tau accrual in OHCs, thus showcasing the potential of this therapeutic approach and the utility of OHCs as an ex vivo model system for assessing such therapeutics.

Regenerative peripheral nerve interface free muscle graft mass and function.

BACKGROUND: Regenerative peripheral nerve interfaces (RPNIs) transduce neural signals to provide high-fidelity control of neuroprosthetic devices. Traditionally, rat RPNIs are constructed with ~150 mg of free skeletal muscle grafts. It is unknown whether larger free muscle grafts allow RPNIs to transduce greater signal. METHODS: RPNIs were constructed by securing skeletal muscle grafts of various masses (150, 300, 600, or 1200 mg) to the divided peroneal nerve. In the control group, the peroneal nerve was transected without repair. Endpoint assessments were conducted 3 mo postoperatively. RESULTS: Compound muscle action potentials (CMAPs), maximum tetanic isometric force, and specific muscle force were significantly higher for both the 150 and 300 mg RPNI groups compared to the 600 and 1200 mg RPNIs. Larger RPNI muscle groups contained central areas lacking regenerated muscle fibers. CONCLUSIONS: Electrical signaling and tissue viability are optimal in smaller as opposed to larger RPNI constructs in a rat model.

Sumatriptan improves the locomotor activity and neuropathic pain by modulating neuroinflammation in rat model of spinal cord injury.

OBJECTIVES: To investigate the therapeutic effects of sumatriptan in a rat model of spinal cord injury (SCI) and possible anti-inflammatory and analgesic mechanisms underlying this effect. METHODS: Using an aneurysm mini-clip model of contusive SCI, T9-10 laminectomies were performed for 60 male rats. Animals were divided into six experimental groups (n = 10 per group) as follows: a minocycline administered positive control group, a saline-vehicle negative control group, a sham-operated group, and three experimental groups which received separate doses of sumatriptan (0.1, 0.3 and 1 mg/kg). Behavioural assessments were used to evaluate locomotor activity and neuropathic pain for 28 days. At the end of the study, spinal cord tissues were collected from sacrificed animals for histopathological analysis. Levels of calcitonin gene-related peptide (CGRP) and two pro-inflammatory cytokines (tumor necrosis factor [TNF]-α and interleukin [IL]-1ß) were assessed by the enzyme-linked immunosorbent assay (ELISA). RESULTS: Sumatriptan significantly (P < 0.001) improved the locomotor activity in SCI group. Sumatriptan was also more effective than the positive control, i.e. minocycline (0.3 mg/kg). Additionally, sumatriptan and minocycline similarly attenuated the mechanical and thermal allodynia in SCI (P < 0.001). TNF-α, IL-1ß and CGRP levels in sumatriptan- and minocycline-treated groups significantly (P < 0.001) decreased compared to controls. Histopathological analysis also revealed a markedly improvement in hemorrhage followed by inflammatory cell invasion, neuronal vacuolation, and cyst formation in both sumatriptan- and minocycline-treated groups compared to control animals. CONCLUSIONS: Sumatriptan improves functional recovery from SCI through its anti-inflammatory effects and reducing pro-inflammatory and pain mediators.

Pharmacokinetic and pharmacodynamic actions of clozapine-N-oxide, clozapine, and compound 21 in DREADD-based chemogenetics in mice.

Muscarinic Designer Receptors Exclusively Activated by Designer Drugs (DREADD) gated by clozapine-N-oxide (CNO) allow selective G-protein cascade activation in genetically specified cell-types in vivo. Here we compare the pharmacokinetics, off-target effects and efficacy of CNO, clozapine (CLZ) and compound 21 (Cmpd-21) at the inhibitory DREADD human Gi-coupled M4 muscarinic receptor (hM4Di). The half maximal effective concentration (EC50) of CLZ was substantially lower (0.42 nM) than CNO (8.1 nM); Cmpd-21 was intermediate (2.95 nM). CNO was back-converted to CLZ in mice, and CLZ accumulated in brain tissue. However, CNO itself also entered the brain, and free cerebrospinal fluid (CSF) levels were within the range to activate hM4Di directly, while free (CSF) CLZ levels remained below the detection limit. Furthermore, directly injected CLZ was strongly converted to its pharmacologically active metabolite, norclozapine. Cmpd-21 showed a superior brain penetration and long-lasting presence. Although we identified a wide range of CNO and Cmpd-21 off-targets, there was hardly any nonspecific behavioural effects among the parameters assessed by the 5-choice-serial-reaction-time task. Our results suggest that CNO (3-5 mg/kg) and Cmpd-21 (0.4-1 mg/kg) are suitable DREADD agonists, effective at latest 15 min after intraperitoneal application, but both require between-subject controls for unspecific effects.

Regenerative peripheral nerve interfaces for real-time, proportional control of a Neuroprosthetic hand.

INTRODUCTION: Regenerative peripheral nerve interfaces (RPNIs) are biological constructs which amplify neural signals and have shown long-term stability in rat models. Real-time control of a neuroprosthesis in rat models has not yet been demonstrated. The purpose of this study was to: a) design and validate a system for translating electromyography (EMG) signals from an RPNI in a rat model into real-time control of a neuroprosthetic hand, and; b) use the system to demonstrate RPNI proportional neuroprosthesis control. METHODS: Animals were randomly assigned to three experimental groups: (1) Control; (2) Denervated, and; (3) RPNI. In the RPNI group, the extensor digitorum longus (EDL) muscle was dissected free, denervated, transferred to the lateral thigh and neurotized with the residual end of the transected common peroneal nerve. Rats received tactile stimuli to the hind-limb via monofilaments, and electrodes were used to record EMG. Signals were filtered, rectified and integrated using a moving sample window. Processed EMG signals (iEMG) from RPNIs were validated against Control and Denervated group outputs. RESULTS: Voluntary reflexive rat movements produced signaling that activated the prosthesis in both the Control and RPNI groups, but produced no activation in the Denervated group. Signal-to-Noise ratio between hind-limb movement and resting iEMG was 3.55 for Controls and 3.81 for RPNIs. Both Control and RPNI groups exhibited a logarithmic iEMG increase with increased monofilament pressure, allowing graded prosthetic hand speed control (R2 = 0.758 and R2 = 0.802, respectively). CONCLUSION: EMG signals were successfully acquired from RPNIs and translated into real-time neuroprosthetic control. Signal contamination from muscles adjacent to the RPNI was minimal. RPNI constructs provided reliable proportional prosthetic hand control.

Synthesis and Pharmacological Characterization of C4ß-Amide-Substituted 2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylates. Identification of (1 S,2 S,4 S,5 R,6 S)-2-Amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (LY2794193), a Highly Potent and Selective mGlu3 Receptor Agonist.

Multiple therapeutic opportunities have been suggested for compounds capable of selective activation of metabotropic glutamate 3 (mGlu3) receptors, but small molecule tools are lacking. As part of our ongoing efforts to identify potent, selective, and systemically bioavailable agonists for mGlu2 and mGlu3 receptor subtypes, a series of C4ß-N-linked variants of (1 S,2 S,5 R,6 S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 1 (LY354740) were prepared and evaluated for both mGlu2 and mGlu3 receptor binding affinity and functional cellular responses. From this investigation we identified (1 S,2 S,4 S,5 R,6 S)-2-amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 8p (LY2794193), a molecule that demonstrates remarkable mGlu3 receptor selectivity. Crystallization of 8p with the amino terminal domain of hmGlu3 revealed critical binding interactions for this ligand with residues adjacent to the glutamate binding site, while pharmacokinetic assessment of 8p combined with its effect in an mGlu2 receptor-dependent behavioral model provides estimates for doses of this compound that would be expected to selectively engage and activate central mGlu3 receptors in vivo.

Current State of the Surgical Treatment of Terminal Neuromas.

Painful terminal neuromas resulting from nerve injury following amputation are common. However, there is currently no universally accepted gold standard of treatment for this condition. A comprehensive literature review is presented on the treatment of terminal neuromas. Four categories of terminal neuroma surgical procedures are assessed: epineurial closure; nerve transposition with implantation; neurorrhaphy, and alternate target reinnervation. Significant patient and case studies are highlighted in each section, focusing on surgical technique and patient outcome metrics. Studies presented consisted of a PubMed search for "terminal neuromas," without year limitation. The current available research supports the use of implantation into muscle for the surgical treatment of terminal neuromas. However, this technique has several fundamental flaws that limit its utility, as it does not address the underlying physiology behind neuroma formation. Regenerative peripheral nerve interfaces and targeted muscle reinnervation are 2 techniques that seem to offer the most promise in preventing and treating terminal neuroma formation. Both techniques are also capable of generating control signals which can be used for both motor and sensory prosthetic control. Such technology has the potential to lead to the future restoration of lost limb function in amputees. Further clinical research employing larger patient groups with high-quality control groups and reproducible outcome measures is needed to determine the most effective and beneficial surgical treatment for terminal neuromas. Primary focus should be placed on investigating techniques that most closely approximate the theoretically ideal neuroma treatment, including targeted muscle reinnervation and regenerative peripheral nerve interfaces.

A high-throughput model for investigating neuronal function and synaptic transmission in cultured neuronal networks.

Loss of synapses or alteration of synaptic activity is associated with cognitive impairment observed in a number of psychiatric and neurological disorders, such as schizophrenia and Alzheimer's disease. Therefore successful development of in vitro methods that can investigate synaptic function in a high-throughput format could be highly impactful for neuroscience drug discovery. We present here the development, characterisation and validation of a novel high-throughput in vitro model for assessing neuronal function and synaptic transmission in primary rodent neurons. The novelty of our approach resides in the combination of the electrical field stimulation (EFS) with data acquisition in spatially separated areas of an interconnected neuronal network. We integrated our methodology with state of the art drug discovery instrumentation (FLIPR Tetra) and used selective tool compounds to perform a systematic pharmacological validation of the model. We investigated pharmacological modulators targeting pre- and post-synaptic receptors (AMPA, NMDA, GABA-A, mGluR2/3 receptors and Nav, Cav voltage-gated ion channels) and demonstrated the ability of our model to discriminate and measure synaptic transmission in cultured neuronal networks. Application of the model described here as an unbiased phenotypic screening approach will help with our long term goals of discovering novel therapeutic strategies for treating neurological disorders.

Characterisation of Nav1.7 functional expression in rat dorsal root ganglia neurons by using an electrical field stimulation assay.

Background The Nav1.7 subtype of voltage-gated sodium channels is specifically expressed in sensory and sympathetic ganglia neurons where it plays an important role in the generation and transmission of information related to pain sensation. Human loss or gain-of-function mutations in the gene encoding Nav1.7 channels (SCN9A) are associated with either absence of pain, as reported for congenital insensitivity to pain, or with exacerbation of pain, as reported for primary erythromelalgia and paroxysmal extreme pain disorder. Based on this important human genetic evidence, numerous drug discovery efforts are ongoing in search for Nav1.7 blockers as a novel therapeutic strategy to treat pain conditions. Results We are reporting here a novel approach to study Nav1.7 function in cultured rat sensory neurons. We used live cell imaging combined with electrical field stimulation to evoke and record action potential-driven calcium transients in the neurons. We have shown that the tarantula venom peptide Protoxin-II, a known Nav1.7 subtype selective blocker, inhibited electrical field stimulation-evoked calcium responses in dorsal root ganglia neurons with an IC50 of 72 nM, while it had no activity in embryonic hippocampal neurons. The results obtained in the live cell imaging assay were supported by patch-clamp studies as well as by quantitative PCR and Western blotting experiments that confirmed the presence of Nav1.7 mRNA and protein in dorsal root ganglia but not in embryonic hippocampal neurons. Conclusions The findings presented here point to a selective effect of Protoxin-II in sensory neurons and helped to validate a new method for investigating and comparing Nav1.7 pharmacology in sensory versus central nervous system neurons. This will help in the characterisation of the selectivity of novel Nav1.7 modulators using native ion channels and will provide the basis for the development of higher throughput models for enabling pain-relevant phenotypic screening.

Adjacent regenerative peripheral nerve interfaces produce phase-antagonist signals during voluntary walking in rats.

BACKGROUND: Regenerative Peripheral Nerve Interfaces (RPNIs) are neurotized muscle grafts intended to produce electromyographic signals suitable for motorized prosthesis control. Two RPNIs producing independent agonist/antagonist signals are required for each control axis; however, it is unknown whether signals from adjacent RPNIs are independent. The purpose of this work was to determine signaling characteristics from two adjacent RPNIs, the first neurotized by a foot dorsi-flexor nerve and the second neurotized by a foot plantar-flexor nerve in a rodent model. METHODS: Two Control group rats had electrodes implanted onto the soleus (tibial nerve) and extensor digitorum longus (peroneal nerve) muscles in the left hind limb. Two Dual-RPNI group rats had two separate muscles grafted to the left thigh and each implanted with electrodes: the extensor digitorum longus was neurotized with a transected fascicle from the tibial nerve, and the tibialis anterior was implanted with a transected peroneal nerve. Four months post-surgery, rats walked on a treadmill, were videographed, and electromyographic signals were recorded. Amplitude and periodicity of all signals relative to gait period were quantified. To facilitate comparisons across groups, electromyographic signals were expressed as a percent of total stepping cycle activity for each stance and swing gait phase. Independence between peroneal and tibial nerve activations were assessed by statistical comparisons between groups during stance and swing. RESULTS: Electromyographic activity for Control and Dual-RPNI rats displayed alternating activation patterns coinciding with stance and swing. Significant signal amplitude differences between the peroneal and tibial nerves were found in both the Control and Dual-RPNI groups. Non-inferiority tests performed on Dual-RPNI group signal confidence intervals showed that activation was equivalent to the Control group in all but the peroneal RPNI construct during stance. The similar electromyographic activity obtained for Control and RPNI suggests the latter constructs activate independently during both stance and swing, and contain minimal crosstalk. CONCLUSIONS: In-vivo myoelectric RPNI activity encodes neural activation patterns associated with gait. Adjacent RPNIs neurotized with agonist/antagonist nerves display activity amplitudes similar to Control during voluntary walking. The distinct and expected activation patterns indicate the RPNI may provide independent signaling in humans, suitable for motorized prosthesis control.

The role of Cx36 and Cx43 in 4-aminopyridine-induced rhythmic activity in the spinal nociceptive dorsal horn: an electrophysiological study in vitro.

Connexin (Cx) proteins and gap junctions support the formation of neuronal and glial syncytia that are linked to different forms of rhythmic firing and oscillatory activity in the CNS. In this study, quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to profile developmental expression of two specific Cx proteins, namely glial Cx43 and neuronal Cx36, in postnatal lumbar spinal cord aged 4, 7, and 14 days. Extracellular electrophysiology was used to determine the contribution of Cx36 and Cx43 to a previously described form of 4-aminopyridine (4-AP)-induced 4-12 Hz rhythmic activity within substantia gelatinosa (SG) of rat neonatal dorsal horn (DH) in vitro. The involvement of Cx36 and Cx43 was probed pharmacologically using quinine, a specific uncoupler of Cx36 and the mimetic peptide blocker Gap 26 which targets Cx43. After establishment of 4-12 Hz rhythmic activity by 4-AP (25 µmol/L), coapplication of quinine (250 µmol/L) reduced 4-AP-induced 4-12 Hz rhythmic activity (P < 0.05). Preincubation of spinal cord slices with Gap 26 (100 µmol/L), compromised the level of 4-AP-induced 4-12 Hz rhythmic activity in comparison with control slices preincubated in ACSF alone (P < 0.05). Conversely, the nonselective gap junction "opener" trimethylamine (TMA) enhanced 4-12 Hz rhythmic behavior (P < 0.05), further supporting a role for Cx proteins and gap junctions. These data have defined a physiological role for Cx36 and Cx43 in rhythmic firing in SG, a key nociceptive processing area of DH. The significance of these data in the context of pain and Cx proteins as a future analgesic drug target requires further study.

An Integrated Approach for Screening and Identification of Positive Allosteric Modulators of N-Methyl-D-Aspartate Receptors.

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that play an important role in synaptic plasticity and learning and memory formation. Malfunctioning of NMDARs, in particular the reduction in NMDAR activity, is thought to be implicated in major neurological disorders. NMDAR positive allosteric modulators (PAMs) represent potential therapeutic interventions for restoring normal NMDAR function. We report a novel screening approach for identification and characterization of NMDAR-PAMs. The approach combines high-throughput fluorescence imaging with automated electrophysiological recording of glutamate-evoked responses in HEK-293 cells expressing NR1/NR2A NMDAR subunits. Initial high-throughput screening (HTS) of a chemical library containing >810,000 compounds using a calcium flux assay in 1536-well plate format identified a total of 864 NMDAR-PAMs. Concentration response determination in both calcium flux and automated electrophysiological assays found several novel chemical series with EC50 values between 0.49 and 10 µM. A small subset (six series) was selected and analyzed for pharmacological properties, subtype selectivity, mode of action, and activity at native NMDARs. Our approach demonstrates the successful application of HTS functional assays that led to identification of NMDAR-PAMs providing the foundation for further medicinal chemistry work that may lead to novel therapies for treatment of cognitive impairment associated with Alzheimer's disease and schizophrenia.

In vivo characterization of regenerative peripheral nerve interface function.

OBJECTIVE: Regenerative peripheral nerve interfaces (RPNIs) are neurotized free autologous muscle grafts equipped with electrodes to record myoelectric signals for prosthesis control. Viability of rat RPNI constructs have been demonstrated using evoked responses. In vivo RPNI characterization is the next critical step for assessment as a control modality for prosthetic devices. APPROACH: Two RPNIs were created in each of two rats by grafting portions of free muscle to the ends of divided peripheral nerves (peroneal in the left and tibial in the right hind limb) and placing bipolar electrodes on the graft surface. After four months, we examined in vivo electromyographic signal activity and compared these signals to muscular electromyographic signals recorded from autologous muscles in two rats serving as controls. An additional group of two rats in which the autologous muscles were denervated served to quantify cross-talk in the electrode recordings. Recordings were made while rats walked on a treadmill and a motion capture system tracked the hind limbs. Amplitude and periodicity of signals relative to gait were quantified, correlation between electromyographic and motion recording were assessed, and a decoder was trained to predict joint motion. MAIN RESULTS: Raw RPNI signals were active during walking, with amplitudes of 1 mVPP, and quiet during standing, with amplitudes less than 0.1 mVPP. RPNI signals were periodic and entrained with gait. A decoder predicted bilateral ankle motion with greater than 80% reliability. Control group signal activity agreed with literature. Denervated group signals remained quiescent throughout all evaluations. SIGNIFICANCE: In vivo myoelectric RPNI activity encodes neural activation patterns associated with gait. Signal contamination from muscles adjacent to the RPNI is minimal, as demonstrated by the low amplitude signals obtained from the Denervated group. The periodicity and entrainment to gait of RPNI recordings suggests the transduced signals were generated via central nervous system control.