Dual Photoredox/Nickel Catalysis Enables Diastereoselective Synthesis of Multisubstituted γ-Lactams Using Alkyl-GeMe3 as Radical Precursors.

Herein, we report a single-step, multicomponent approach to versatile γ-lactams through dual photoredox/nickel-catalyzed dicarbofunctionalization of α,ß-unsaturated γ-butyrolactam. This reaction utilized alkyl trimethylgermanium as a radical precursor and acyl chloride as the electrophile, demonstrating remarkable functional group compatibility.

Alkyl-GeMe3 : Neutral Metalloid Radical Precursors upon Visible-Light Photocatalysis.

Single-electron transfer (SET) oxidation of ionic hypervalent complexes, in particular alkyltrifluoroborates (Alkyl-BF3 - ) and alkylbis(catecholato)silicates (Alkyl-Si(cat)2 - ), have contributed substantially to alkyl radical generation compared to alkali or alkaline earth organometallics because of their excellent activity-stability balance. Herein, another proposal is reported by using neutral metalloid compounds, Alkyl-GeMe3 , as radical precursors. Alkyl-GeMe3 shows comparable activity to that of Alkyl-BF3 - and Alkyl-Si(cat)2 - in radical addition reactions. Moreover, Alkyl-GeMe3 is the first successful group 14 tetraalkyl nucleophile in nickel-catalyzed cross-coupling. Meanwhile, the neutral nature of these organogermanes offset the limitation of ionic precursors in purification and derivatization. A preliminary mechanism study suggests that an alkyl radical is generated from a tetraalkylgermane radical cation with the assistance of a nucleophile, which may also result in the development of more non-ionic alkyl radical precursors with a metalloid center.

Alkyl Carbagermatranes Enable Practical Palladium-Catalyzed sp2-sp3 Cross-Coupling.

Pd-catalyzed cross-coupling reactions have achieved tremendous accomplishments in the past decades. However, C(sp3)-hybridized nucleophiles generally remain as challenging coupling partners due to their sluggish transmetalation compared to the C(sp2)-hybridized counterparts. While a single-electron-transfer-based strategy using C(sp3)-hybridized nucleophiles had made significant progress recently, fewer breakthroughs have been made concerning the traditional two-electron mechanism involving C(sp3)-hybridized nucleophiles. In this report, we present a series of unique alkyl carbagermatranes that were proven to be highly reactive in cross-coupling reactions with our newly developed electron-deficient phosphine ligands. Generally, secondary alkyl carbagermatranes show slightly lower, yet comparable activity to its Sn analogue. Meanwhile, primary alkyl carbagermatranes exhibit high activity, and they were also proved stable enough to be compatible with various reactions. Chiral secondary benzyl carbagermatrane gave the coupling product under base/additive-free conditions with its configuration fully inversed, suggesting that transmetalation was carried out in an "SE2(open) Inv" pathway, which is consistent with Hiyama's previous observation. Notably, the cross-coupling of primary alkyl carbagermatranes could be performed under base/additive-free conditions with excellent functional group tolerance and therefore may have potentially important applications such as stapled peptide synthesis.

The effects of intraplantar and intrathecal botulinum toxin type B on tactile allodynia in mono and polyneuropathy in the mouse.

BACKGROUND: Mononeuropathies (MNs: nerve ligation) and polyneuropathies (PNs: cisplatin) produce unilateral and bilateral tactile allodynia, respectively. We examined the effects of intraplantar (IPLT) and intrathecal (IT) botulinum toxin B (BoNT-B) on this allodynia. METHODS: Mice (male c57Bl/6) were prepared with an L5 nerve ligation. Others received cisplatin (IP 2.3 mg/kg/d, every other day for 6 injections). Saline and BoNT-B were administered through the IPLT or IT route. We examined mechanical allodynia (von Frey hairs) before and at intervals after BoNT. As a control, we injected IPLT BoNT-B treated with dithiothreitol to cleave heavy chain from light chain. We measured motor function using acute thermal escape and sensorimotor tests. RESULTS: MN and PN mice showed a persistent ipsilateral and bilateral allodynia, respectively. IPLT BoNT-B resulted in an ipsilateral dorsal horn reduction in the synaptic protein target of BoNT-B (vesicle-associated membrane protein) and a long-lasting (up to approximately 17 days) reversal of allodynia in PN and MN models. The predominant effect after IPLT delivery was ipsilateral to IPLT BoNT. The effects of IPLT BoNT-B in MN mice were blocked by prior reduction of BoNT-B with dithiothreitol. IT BoNT-B in mice with PN resulted in a bilateral reversal of allodynia. With these dosing parameters, hind paw placing and stepping reflexes were unaltered, and there were no changes in thermal escape latencies. After cisplatin, dorsal root ganglions displayed increases in activation transcription factor 3, which were reduced by IT, but not IPLT BoNT-B. CONCLUSIONS: BoNT-B given IPLT and IT yields a long-lasting attenuation of the allodynia in mice displaying MN and PN allodynia.

Effects of intrathecal SNC80, a delta receptor ligand, on nociceptive threshold and dorsal horn substance p release.

Delta-opioid receptors (DOR) are present in the superficial dorsal horn and are believed to regulate the release of small afferent transmitters as evidenced by the effects of spinally delivered delta-opioid preferring peptides. Here we examined the effects of intrathecal SNC80 [(+)-4-[α(R)-α-[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-3-(methoxybenzyl)-N,N-diethylbenzamide], a selective nonpeptidic DOR agonist, in three preclinical pain models, acute thermal escape, intraplantar carrageenan-tactile allodynia, and intraplantar formalin flinches, and on the evoked release of substance P (SP) from small primary afferents. Rats with chronic intrathecal catheters received intrathecal vehicle or SNC80 (100 or 200 µg). Intrathecal SNC80 did not change acute thermal latencies or carrageenan-induced thermal hyperalgesia. However, SNC80 attenuated carrageenan-induced tactile allodynia and significantly reduced both phase 1 and phase 2 formalin-induced paw flinches, as assessed by an automatic flinch counting device. These effects were abolished by naltrindole (3 mg/kg i.p.), a selective DOR antagonist, but not CTOP (10 µg i.t.), a selective MOR antagonist. Furthermore, intrathecal SNC80 (200 µg) blocked formalin-induced substance P release otherwise evoked in the ispilateral superficial dorsal horn as measured by NK1 receptor internalization. In conclusion, intrathecal SNC80 alleviated pain hypersensitivity after peripheral inflammation in a fashion paralleling its ability to block peptide transmitter release from small peptidergic afferents, which by its pharmacology appears to represent an effect mediated by a spinal DOR.

Intrathecal substance P-saporin in the dog: distribution, safety, and spinal neurokinin-1 receptor ablation.

BACKGROUND: Neurokinin-1 receptors (NK1-rs) located on superficial dorsal horn neurons are essential for integration of nociceptive input. Intrathecal injection of substance P-saporin (SP-SAP) leads to local loss of spinal NK1-r (+) neurons suggesting its potential as a therapeutic agent for chronic pain. The authors determined, in a canine model, effects of lumbar intrathecal SP-SAP. METHODS: Distribution of SP-SAP and Saporin was determined in plasma, lumbar cerebrospinal fluid, and tissue. Safety of intrathecal SP-SAP was determined in four groups (six dogs each) administered 0 (0.9% saline), 1.5, 15, or 150 µg SP-SAP through lumbar intrathecal catheters. Behavioral, physiologic, and biochemical variables were assessed. Spinal tissues were collected at 7 and approximately 90 days, or earlier if significant morbidity developed, and analyzed for NK1-r (+) neuron loss and histopathology. RESULTS: SP-SAP and Saporin were detectable in lumbar cerebrospinal fluid for up to 4 and 24 h, respectively. Animals receiving intrathecal saline, 1.5, or 15 µg of SP-SAP showed no persistent neurologic deficits. Three animals receiving 150 µg of SP-SAP developed pelvic limb paraparesis and were euthanized prematurely. Immunohistochemistry and in situ hybridization cell counts confirmed a significant reduction in NK1-r (+) in superficial dorsal horn neurons from lumbar spinal cord after intrathecal administration of 15 and 150 µg of SP-SAP. A significant loss of NK1-r neurons in the lumbar ventral horn occurred only with 150-µg SP-SAP. CONCLUSION: Intrathecal 15-µg SP-SAP reduced dorsal, but not ventral, NK1-r (+) neurons at the spinal level of delivery with minimal side effects, whereas 150-µg SP-SAP resulted in motor neuron toxicity.

Palladium-Catalyzed Cross-Coupling of Tertiary Cyclopropyl Carbagermatranes with Acyl Chlorides.

A simple method for the synthesis of cyclopropane compounds via cross-coupling reaction between tertiary cyclopropyl carbagermatranes and acyl chlorides was reported. Derivatives of acryloyl chloride and aliphatic acyl chloride also performed to be suitable substrates. This process can be used to introduce a wide range of functionalized cyclopropane groups and acyl groups directly.

A Broad Range Triboelectric Stiffness Sensor for Variable Inclusions Recognition.

With the development of artificial intelligence, stiffness sensors are extensively utilized in various fields, and their integration with robots for automated palpation has gained significant attention. This study presents a broad range self-powered stiffness sensor based on the triboelectric nanogenerator (Stiff-TENG) for variable inclusions in soft objects detection. The Stiff-TENG employs a stacked structure comprising an indium tin oxide film, an elastic sponge, a fluorinated ethylene propylene film with a conductive ink electrode, and two acrylic pieces with a shielding layer. Through the decoupling method, the Stiff-TENG achieves stiffness detection of objects within 1.0 s. The output performance and characteristics of the TENG for different stiffness objects under 4 mm displacement are analyzed. The Stiff-TENG is successfully used to detect the heterogeneous stiffness structures, enabling effective recognition of variable inclusions in soft object, reaching a recognition accuracy of 99.7%. Furthermore, its adaptability makes it well-suited for the detection of pathological conditions within the human body, as pathological tissues often exhibit changes in the stiffness of internal organs. This research highlights the innovative applications of TENG and thereby showcases its immense potential in healthcare applications such as palpation which assesses pathological conditions based on organ stiffness.

Spinal phosphinositide 3-kinase-Akt-mammalian target of rapamycin signaling cascades in inflammation-induced hyperalgesia.

Phosphinositide 3-kinase (PI3K), Akt, and their downstream kinase, mammalian target of rapamycin (mTOR), are implicated in neural plasticity. The functional linkages of this signaling cascade in spinal dorsal horn and their role in inflammatory hyperalgesia have not been elucidated. In the present work, we identified the following characteristics of this cascade. (1) Local inflammation led to increase in rat dorsal horn phosphorylation (activation) of Akt (pAkt) and mTOR (pmTOR), as assessed by Western blotting and immunocytochemistry. (2) Increased pAkt and pmTOR were prominent in neurons in laminae I, III, and IV, whereas pmTOR and its downstream targets (pS6, p4EBP) were also observed in glial cells. (3) Intrathecal treatment with inhibitors to PI3K or Akt attenuated Formalin-induced second-phase flinching behavior, as well as carrageenan-induced thermal hyperalgesia and tactile allodynia. (4) Intrathecal rapamycin (an mTORC1 inhibitor) displayed anti-hyperalgesic effect in both inflammatory pain models. Importantly, intrathecal wortmannin at anti-hyperalgesic doses reversed the evoked increase not only in Akt but also in mTORC1 signaling (pS6/p4EBP). (5) pAkt and pmTOR are expressed in neurokinin 1 receptor-positive neurons in laminae I-III after peripheral inflammation. Intrathecal injection of Substance P activated this cascade (increased phosphorylation) and resulted in hyperalgesia, both of which effects were blocked by intrathecal wortmannin and rapamycin. Together, these findings reveal that afferent inputs trigged by peripheral inflammation initiate spinal activation of PI3K-Akt-mTOR signaling pathway, a component of which participates in neuronal circuits of facilitated pain processing.

Cav1.2 L-type Ca²âº channels mediate cocaine-induced GluA1 trafficking in the nucleus accumbens, a long-term adaptation dependent on ventral tegmental area Ca(v)1.3 channels.

AMPA receptor (AMPAR) plasticity at glutamatergic synapses in the mesoaccumbal dopaminergic pathway has been implicated in persistent cocaine-induced behavioral responses; however, the precise mechanism underlying these changes remains unknown. Utilizing cocaine psychomotor sensitization, we have examined phosphorylation of GluA1 at key residues serine 845 (S845) and S831, as well as GluA1 cell surface levels in the nucleus accumbens (NAc) of cocaine-preexposed mice and the role of brain-specific Ca(v)1.2 and Ca(v)1.3 L-type Ca²âº channels (LTCCs), therein. We found higher basal levels of S845 phospho-GluA1 (P-GluA1) and cell surface GluA1 in the NAc following protracted withdrawal from cocaine exposure, changes that occur independently of LTCCs. In contrast, we found that a cocaine challenge that elicits expression of the cocaine-sensitized response increases S831 P-GluA1 that further increases surface GluA1 beyond the higher basal levels. Intra-NAc pharmacological manipulations indicate that the Ca(v)1.2-activated CaM kinase II (CaMKII) mediates cocaine-induced increase in S831 P-GluA1 and that both Ca(v)1.2-activated CaMKII and extracellular signal-regulated kinase 2 (ERK2) mediate the increase in GluA1 cell surface levels specific to the sensitized response. Experiments using adenoassociated viral vectors expressing Ca(v)1.3 and ERK2 siRNA further indicate that recruitment of the Ca(v)1.2 pathway in the NAc is dependent on ventral tegmental area Ca(v)1.3 LTCCs and ERK2. Together, these results identify candidate pathways that mediate cocaine-induced AMPAR plasticity in the NAc and provide a mechanism linking LTCCs and GluA1 plasticity to cocaine-induced persistent behavioral changes.

Aptasensor based on triplex switch for SERS detection of cytochrome c.

We report a sensitive approach for SERS detection of cytochrome c using target binding-induced conformational changes of signal transduction probe (STP). STP labeled with a SERS-active molecule, carboxy-X-rhodamine (ROX), is immobilized on the substrate where the formation of a rigid triplex switching structure with aptamers does not allow SERS amplification to take place. The target binding event leads to an enhancement in SERS intensity of ROX adsorbed on the gold surface. Meanwhile, we found that an appropriate STP surface density could shield the SERS signal produced by protein adsorption which would foul the sensing surface. In addition, cytochrome c aptamers used were not the original sequence but reorganized in the nonspecific binding site to adapt to our design. This method provides a low detection limit of 2 nM (10 fmol within 5 µL sample solution), and shows good selectivity toward cytochrome c compared to interfering proteins such as hemoglobin and immunoglobulin G. The general strategy of the method can also be extended to aptamer or DNA based sensors.

Beyond the Pore Size Limitation of a Nanoporous Graphene Monolayer Membrane for Water Desalination Assisted by an External Electric Field.

One efficient strategy for addressing the global water shortage is advanced membrane separation, which depends on the precise pore size being close to the hydrated ion size and other surface properties like charge and polarity. However, it is very difficult to fabricate uniform pores with diameters of <1 nm on monolayer membranes. By applying an electric field (bias voltage) perpendicular to the direction of the pressure difference, herein we demonstrate for the first time that a monolayer nanoporous graphene membrane with pores much larger than hydrated ions exhibits high salt rejection and allows a high rate of water transport. This theoretical proposal goes beyond the pore size limitation and shows promise for the design of high-performance reverse osmosis membranes.

Self-Powered Electrodeposition System for Sub-10-nm Silver Nanoparticles with High-Efficiency Antibacterial Activity.

Recently, silver nanoparticles (AgNPs) have been widely applied in sterilization due to their excellent antibacterial properties. However, AgNPs require rigorous storage conditions because their antibacterial performances are significantly affected by environmental conditions. Instant fabrication provides a remedy for this drawback. In this study, we propose a self-powered electrodeposition system to synthesize sub-10-nm AgNPs, consisting of a triboelectric nanogenerator (TENG) as the self-powered source, a capacitor for storing electrical energy from the TENG, and an electrochemical component for electrodeposition. The self-powered system with larger capacitance and discharging voltage tends to deliver smaller AgNPs due to the nucleation mechanism dominated by current density. Furthermore, antibacterial tests reveal that compared to direct current (DC) electrodeposition, the TENG-based electrodeposition can synthesize finer-sized AgNPs (<10 nm) with overwhelming antibacterial effect against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) (with 100% efficiency at 2 h). This work provides a new strategy for the self-powered, instant, and controllable electrodeposition of nanoparticles.

A brief comparison of the pathophysiology of inflammatory versus neuropathic pain.

PURPOSE OF REVIEW: The causes of inflammatory pain and neuropathic pain are fundamentally different. There are, however, common mechanisms underlying the generation of each pain state. We will discuss some specific elements observed in both tissue and nerve injury pain states and consider the hypothesis that these two states actually demonstrate a convergence over time. RECENT FINDINGS: The increased pain sensation following tissue and nerve injury results from several mechanisms, including altered ion channel expression in dorsal root ganglion neurons, enhanced dorsal horn glutamate release from primary afferents, enhanced glutamate receptor function in second-order neurons, disinhibition in the dorsal horn and glia cell activation. The role of specific subtypes of receptors, ion channels and glutamate transporters is revealed at peripheral and central sites. Importantly over time, a number of changes, in the dorsal root ganglion and in dorsal horn observed after tissue injury resemble changes observed after nerve injury. SUMMARY: Recognition of mechanisms common to both inflammatory pain and neuropathic pain might shed light on the understanding of the transition from acute pain to persistent pain.

Exercise Thermal Sensation: Physiological Response to Dynamic-Static Steps at Moderate Exercise.

The study of exercise thermal sensation is more difficult than that of static thermal sensation in the human body. This work's main purpose was to examine specific changes in human physiological parameters and subjective perceptions during the exercise process, especially around dynamic-static steps, and to assess exercise thermal sensation. Experiments were conducted in a climate chamber. A total of 16 subjects participated in two activities of different intensities on a treadmill, namely at 4.5 km/h and 6 km/h. The experimental procedure was set to static-dynamic-static. Skin temperature (Tsk), oral temperature (Tor), heart rate (HR), heart rate variability (HRV) parameters, and electrodermal activity (EDA) were measured at fixed time points, and thermal sensation values, thermal comfort values, and sweat feeling index were collected. The results showed complex changes in physiological indicators around the dynamic-static steps. Some important physio-logical indicators can be used as valid parameters for exercise thermal sensation models, such as Tsk, Tor, and EDA. This study highlighted that prediction models using average change and rate of change of measurements were better than using the original measurements. Our findings suggest that the exercise thermal sensation prediction models should be constructed according to the dynamic-static state and that psychological factors cannot be ignored.

A portable triboelectric spirometer for wireless pulmonary function monitoring.

Coronavirus disease 2019 (COVID-19) as a severe acute respiratory syndrome infection has spread rapidly across the world since its emergence in 2019 and drastically altered our way of life. Patients who have recovered from COVID-19 may still face persisting respiratory damage from the virus, necessitating long-term supervision after discharge to closely assess pulmonary function during rehabilitation. Therefore, developing portable spirometers for pulmonary function tests is of great significance for convenient home-based monitoring during recovery. Here, we propose a wireless, portable pulmonary function monitor for rehabilitation care after COVID-19. It is composed of a breath-to-electrical (BTE) sensor, a signal processing circuit, and a Bluetooth communication unit. The BTE sensor, with a compact size and light weight of 2.5 cm3 and 1.8 g respectively, is capable of converting respiratory biomechanical motions into considerable electrical signals. The output signal stability is greater than 93% under 35%-81% humidity, which allows for ideal expiration airflow sensing. Through a wireless communication circuit system, the signals can be received by a mobile terminal and processed into important physiological parameters, such as forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC). The FEV1/FVC ratio is then calculated to further evaluate pulmonary function of testers. Through these measurement methods, the acquired pulmonary function parameters are shown to exhibit high accuracy (>97%) in comparison to a commercial spirometer. The practical design of the self-powered flow spirometer presents a low-cost and convenient method for pulmonary function monitoring during rehabilitation from COVID-19.

Activation of the neuronal extracellular signal-regulated kinase 2 in the spinal cord dorsal horn is required for complete Freund's adjuvant-induced pain hypersensitivity.

Extracellular signal-regulated kinase 1 (ERK1) and ERK2 signaling in the spinal cord dorsal horn (SCDH) has been implicated in injury-induced pain hypersensitivity. Available ERK pathway inhibitors cannot distinguish between ERK1 and ERK2, nor can they differentially target the expression of neuronal or glial ERK1/2. We selectively inhibited the expression of ERK2 in neurons of the adult mouse SCDH by use of an ERK2 small interfering RNA (siRNA) delivered by a neurotropic adenoassociated viral vector. In situ hybridization revealed a siRNA vector-induced decrease in ERK2 mRNA in the ipsilateral SCDH. Immunohistochemistry showed a decreased neuronal phospho-ERK1/2 (pERK1/2), and Western blot analysis revealed that both ERK2 expression and phosphorylation were reduced by the siRNA vector. In contrast, basal ERK1 expression was not affected, although pERK1 was slightly increased. The siRNA vector-induced knockdown of ERK2 expression in the SCDH did not alter the baseline mechanical or thermal paw withdrawal thresholds. Hindpaw intraplantar injection of complete Freund's adjuvant (CFA) produced peripheral inflammation, mechanical allodynia, and thermal hyperalgesia in vector control animals that persisted for at least 96 h. It also caused an increase in SCDH ERK1 and ERK2 levels at 96 h and pERK1 and pERK2 levels at 1 and 96 h. The ERK2 siRNA vector prevented changes in ERK1, ERK2, and pERK2. In addition, the siRNA vector protected the animals from developing mechanical allodynia and thermal hyperalgesia throughout the 96 h after CFA. These findings indicate that ERK2 in the SCDH neurons is critical for the development of inflammatory pain hypersensitivity.

Inhibition of spinal 15-LOX-1 attenuates TLR4-dependent, nonsteroidal anti-inflammatory drug-unresponsive hyperalgesia in male rats.

Although nonsteroidal anti-inflammatory drugs are the first line of therapeutics for the treatment of mild to moderate somatic pain, they are not generally considered to be effective for neuropathic pain. In the current study, direct activation of spinal Toll-like 4 receptors (TLR4) by the intrathecal (IT) administration of KDO2 lipid A (KLA), the active component of lipopolysaccharide, elicits a robust tactile allodynia that is unresponsive to cyclooxygenase inhibition, despite elevated expression of cyclooxygenase metabolites in the spinal cord. Intrathecal KLA increases 12-lipoxygenase-mediated hepoxilin production in the lumbar spinal cord, concurrent with expression of the tactile allodynia. The TLR4-induced hepoxilin production was also observed in primary spinal microglia, but not in astrocytes, and was accompanied by increased microglial expression of the 12/15-lipoxygenase enzyme 15-LOX-1. Intrathecal KLA-induced tactile allodynia was completely prevented by spinal pretreatment with the 12/15-lipoxygenase inhibitor CDC or a selective antibody targeting rat 15-LOX-1. Similarly, pretreatment with the selective inhibitors ML127 or ML351 both reduced activity of the rat homolog of 15-LOX-1 heterologously expressed in HEK-293T cells and completely abrogated nonsteroidal anti-inflammatory drug-unresponsive allodynia in vivo after IT KLA. Finally, spinal 12/15-lipoxygenase inhibition by nordihydroguaiaretic acid (NDGA) both prevents phase II formalin flinching and reverses formalin-induced persistent tactile allodynia. Taken together, these findings suggest that spinal TLR4-mediated hyperpathic states are mediated at least in part through activation of microglial 15-LOX-1.

Botulinum toxin B in the sensory afferent: transmitter release, spinal activation, and pain behavior.

We addressed the hypothesis that intraplantar botulinum toxin B (rimabotulinumtoxin B: BoNT-B) has an early local effect upon peripheral afferent terminal releasing function and, over time, will be transported to the central terminals of the primary afferent. Once in the terminals it will cleave synaptic protein, block spinal afferent transmitter release, and thereby prevent spinal nociceptive excitation and behavior. In mice, C57Bl/6 males, intraplantar BoNT-B (1 U) given unilaterally into the hind paw had no effect upon survival or motor function, but ipsilaterally decreased: (1) intraplantar formalin-evoked flinching; (2) intraplantar capsaicin-evoked plasma extravasation in the hind paw measured by Evans blue in the paw; (3) intraplantar formalin-evoked dorsal horn substance P (SP) release (neurokinin 1 [NK1] receptor internalization); (4) intraplantar formalin-evoked dorsal horn neuronal activation (c-fos); (5) ipsilateral dorsal root ganglion (DRG) vesicle-associated membrane protein (VAMP); (6) ipsilateral SP release otherwise evoked bilaterally by intrathecal capsaicin; (7) ipsilateral activation of c-fos otherwise evoked bilaterally by intrathecal SP. These results indicate that BoNT-B, after unilateral intraplantar delivery, is taken up by the peripheral terminal, is locally active (blocking plasma extravasation), is transported to the ipsilateral DRG to cleave VAMP, and is acting presynaptically to block release from the spinal peptidergic terminal. The observations following intrathecal SP offer evidence for a possible transsynaptic effect of intraplantar BoNT. These results provide robust evidence that peripheral BoNT-B can alter peripheral and central terminal release from a nociceptor and attenuate downstream nociceptive processing via a presynaptic effect, with further evidence suggesting a possible postsynaptic effect.

Intrathecal P/Q- and R-type calcium channel blockade of spinal substance P release and c-Fos expression.

Intrathecal (IT) studies have shown that several voltage sensitive calcium channels (VSCCs), such as the L-, N- and T-type may play roles in nociception and that of these only the N-type regulates primary afferent substance P (SP) release. However, the actions of other VSCCs at the spinal level are not well known. We investigated the roles of spinal P/Q- and R-type VSCCs, by IT administration of R-type (SNX-482) and P/Q-type (ω-agatoxin IVA) VSCC blockers on intraplantar formalin-evoked flinching, SP release from primary afferents and c-Fos expression in spinal dorsal horn. Intraplantar injection of formalin (2.5%, 50 µL) produced an intense, characteristic biphasic paw flinching response. In rats with IT catheters, IT SNX-482 (0.5 µg) reduced formalin-evoked paw flinching in both phase 1 and 2 compared with vehicle. Intraplantar formalin caused robust neurokinin 1 receptor (NK1r) internalization (indicating SP release) and c-Fos expression in the ipsilateral dorsal horn, which were blocked by IT SNX-482. IT ω-agatoxin IVA (0.03, 0.125 and 0.5 µg) did not reduce formalin-evoked paw flinching or c-Fos expression at any doses, with higher doses resulting in motor dysfunction. Thus, we demonstrated that blockade of spinal R-type, but not P/Q type VSCCs attenuated formalin-induced pain behavior, NK1r internalization and c-Fos expression in the superficial dorsal horn. This study supports a role for Cav2.3 in presynaptic neurotransmitter release from peptidergic nociceptive afferents and pain behaviors.