Remote nongenetic optical modulation of neuronal activity using fuzzy graphene.

The ability to modulate cellular electrophysiology is fundamental to the investigation of development, function, and disease. Currently, there is a need for remote, nongenetic, light-induced control of cellular activity in two-dimensional (2D) and three-dimensional (3D) platforms. Here, we report a breakthrough hybrid nanomaterial for remote, nongenetic, photothermal stimulation of 2D and 3D neural cellular systems. We combine one-dimensional (1D) nanowires (NWs) and 2D graphene flakes grown out-of-plane for highly controlled photothermal stimulation at subcellular precision without the need for genetic modification, with laser energies lower than a hundred nanojoules, one to two orders of magnitude lower than Au-, C-, and Si-based nanomaterials. Photothermal stimulation using NW-templated 3D fuzzy graphene (NT-3DFG) is flexible due to its broadband absorption and does not generate cellular stress. Therefore, it serves as a powerful toolset for studies of cell signaling within and between tissues and can enable therapeutic interventions.

Sustained stimulation of ß2- and ß3-adrenergic receptors leads to persistent functional pain and neuroinflammation.

Functional pain syndromes, such as fibromyalgia and temporomandibular disorder, are associated with enhanced catecholamine tone and decreased levels of catechol-O-methyltransferase (COMT; an enzyme that metabolizes catecholamines). Consistent with clinical syndromes, our lab has shown that sustained 14-day delivery of the COMT inhibitor OR486 in rodents results in pain at multiple body sites and pain-related volitional behaviors. The onset of COMT-dependent functional pain is mediated by peripheral ß2- and ß3-adrenergic receptors (ß2- and ß3ARs) through the release of the pro-inflammatory cytokines tumor necrosis factor α (TNFα), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6). Here, we first sought to investigate the role of ß2- and ß3ARs and downstream mediators in the maintenance of persistent functional pain. We then aimed to characterize the resulting persistent inflammation in neural tissues (neuroinflammation), characterized by activated glial cells and phosphorylation of the mitogen-activated protein kinases (MAPKs) p38 and extracellular signal-regulated kinase (ERK). Separate groups of rats were implanted with subcutaneous osmotic mini-pumps to deliver OR486 (15 mg/kg/day) or vehicle for 14 days. The ß2AR antagonist ICI118551 and ß3AR antagonist SR59230A were co-administrated subcutaneously with OR486 or vehicle either on day 0 or day 7. The TNFα inhibitor Etanercept, the p38 inhibitor SB203580, or the ERK inhibitor U0126 were delivered intrathecally following OR486 cessation on day 14. Behavioral responses, pro-inflammatory cytokine levels, glial cell activation, and MAPK phosphorylation were measured over the course of 35 days. Our results demonstrate that systemic delivery of OR486 leads to mechanical hypersensitivity that persists for at least 3 weeks after OR486 cessation. Corresponding increases in spinal TNFα, IL-1ß, and IL-6 levels, microglia and astrocyte activation, and neuronal p38 and ERK phosphorylation were observed on days 14-35. Persistent functional pain was alleviated by systemic delivery of ICI118551 and SR59230A beginning on day 0, but not day 7, and by spinal delivery of Etanercept or SB203580 beginning on day 14. These results suggest that peripheral ß2- and ß3ARs drive persistent COMT-dependent functional pain via increased activation of immune cells and production of pro-inflammatory cytokines, which promote neuroinflammation and nociceptor activation. Thus, therapies that resolve neuroinflammation may prove useful in the management of functional pain syndromes.

Nuclear factor-kappa B regulates pain and COMT expression in a rodent model of inflammation.

Nuclear factor-kappa B (NF-κB) is a ubiquitously expressed protein complex regulating the transcription of genes involved in inflammation and pain. Increased NF-κB activity in immune and nervous system cells is linked to several chronic pain conditions in humans as well as inflammation and nerve injury-evoked pain in animals. A recent in vitro study further demonstrates that increased NF-κB activity in astrocytes decreases transcription of catechol-o-methyltransferase (COMT), an enzyme that inactivates catecholamines that cause pain. The purpose of the present study was to examine the relationship between systemic and astrocytic NF-κB activity, pain, and COMT expression in an animal model of inflammation. Results demonstrated that administration of the inflammatory stimulant complete Freund's adjuvant (CFA) led to increased pain and decreased COMT protein expression in an NF-κB-dependent manner. Specifically, we found that rats and mice receiving intraplantar CFA exhibited increased behavioral responses to mechanical and thermal heat stimuli. CFA-evoked pain was blocked in rats receiving a pre-emptive systemic dose of the NF-κB inhibitor MG132 and exacerbated in IKKca mice with constitutive NF-κB activity in astrocytes. Furthermore, we observed NF-κB-linked reductions in COMT expression in midbrain at 6h and 1d following CFA in rats and at 1h and 1d in forebrain and midbrain following CFA in IKKca mice. Collectively, these results demonstrate that systemic and astrocytic NF-κB activity drive inflammatory pain and regulate the expression of COMT in forebrain and midbrain structures.

Photothermal Excitation of Neurons Using MXene: Cellular Stress and Phototoxicity Evaluation.

Understanding the communication of individual neurons necessitates precise control of neural activity. Photothermal modulation is a remote and non-genetic technique to control neural activity with high spatiotemporal resolution. The local heat release by photothermally active nanomaterial will change the membrane properties of the interfaced neurons during light illumination. Recently, it is demonstrated that the two-dimensional Ti3 C2 Tx MXene is an outstanding candidate to photothermally excite neurons with low incident energy. However, the safety of using Ti3 C2 Tx for neural modulation is unknown. Here, the biosafety of Ti3 C2 Tx -based photothermal modulation is thoroughly investigated, including assessments of plasma membrane integrity, mitochondrial stress, and oxidative stress. It is demonstrated that culturing neurons on 25 µg cm-2 Ti3 C2 Tx films and illuminating them with laser pulses (635 nm) with different incident energies (2-10 µJ per pulse) and different pulse frequencies (1 pulse, 1 Hz, and 10 Hz) neither damage the cell membrane, induce cellular stress, nor generate oxidative stress. The threshold energy to cause damage (i.e., 14 µJ per pulse) exceeded the incident energy for neural excitation (<10 µJ per pulse). This multi-assay safety evaluation provides crucial insights for guiding the establishment of light conditions and protocols in the clinical translation of photothermal modulation.

Voltage-gated calcium currents in human dorsal root ganglion neurons.

ABSTRACT: Voltage-gated calcium channels in sensory neurons underlie processes ranging from neurotransmitter release to gene expression and remain a therapeutic target for the treatment of pain. Yet virtually all we know about voltage-gated calcium channels has been obtained through the study of rodent sensory neurons and heterologously expressed channels. To address this, high voltage-activated (HVA) Ca2+ currents in dissociated human and rat dorsal root ganglion neurons were characterized with whole-cell patch clamp techniques. The HVA currents from both species shared basic biophysical and pharmacological properties. However, HVA currents in human neurons differed from those in the rat in at least 3 potentially important ways: (1) Ca2+ current density was significantly smaller, (2) the proportion of nifedipine-sensitive currents was far greater, and (3) a subpopulation of human neurons displayed relatively large constitutive current inhibition. These results highlight the need to for the study of native proteins in their native environment before initiating costly clinical trials.

Ti3C2Tx MXene Flakes for Optical Control of Neuronal Electrical Activity.

Understanding cellular electrical communications in both health and disease necessitates precise subcellular electrophysiological modulation. Nanomaterial-assisted photothermal stimulation was demonstrated to modulate cellular activity with high spatiotemporal resolution. Ideal candidates for such an application are expected to have high absorbance at the near-infrared window, high photothermal conversion efficiency, and straightforward scale-up of production to allow future translation. Here, we demonstrate two-dimensional Ti3C2Tx (MXene) as an outstanding candidate for remote, nongenetic, optical modulation of neuronal electrical activity with high spatiotemporal resolution. Ti3C2Tx's photothermal response measured at the single-flake level resulted in local temperature rises of 2.31 ± 0.03 and 3.30 ± 0.02 K for 635 and 808 nm laser pulses (1 ms, 10 mW), respectively. Dorsal root ganglion (DRG) neurons incubated with Ti3C2Tx film (25 µg/cm2) or Ti3C2Tx flake dispersion (100 µg/mL) for 6 days did not show a detectable influence on cellular viability, indicating that Ti3C2Tx is noncytotoxic. DRG neurons were photothermally stimulated using Ti3C2Tx films and flakes with as low as tens of microjoules per pulse incident energy (635 nm, 2 µJ for film, 18 µJ for flake) with subcellular targeting resolution. Ti3C2Tx's straightforward and large-scale synthesis allows translation of the reported photothermal stimulation approach in multiple scales, thus presenting a powerful tool for modulating electrophysiology from single-cell to additive manufacturing of engineered tissues.

GCaMP as an indirect measure of electrical activity in rat trigeminal ganglion neurons.

While debate continues over whether somatosensory information is transmitted via labeled line, population coding, frequency coding, or some combination therein, researchers have begun to address this question at the level of the primary afferent by using optical approaches that enable the assessment of neural activity in hundreds to even thousands of neurons simultaneously. However, with limited availability of tools to optically assess electrical activity in large populations of neurons, researchers have turned to genetically encoded Ca2+ indicators (GECIs) including GCaMP to enable the detection of increases in cytosolic Ca2+ concentrations as a correlate for neuronal activity. One of the most widely used GECIs is GCaMP6, which is available in three different versions tuned for sensitivity (GCaMP6s), speed (GCaMP6f), or a balance of the two (GCaMP6m). In order to determine if these issues were unique to GCaMP6 itself, or if they were inherent to more than one generation of GCaMP, we also characterized jGCaMP7. In the present study, we sought to determine the utility of the three GCaMP6 isoforms to detect changes in activity in primary afferents at frequencies ranging from 0.1-30 Hz. Given the heterogeneity of sensory neurons, we also compared the performance of each GCaMP6 isoform in subpopulations of neurons defined by properties used to identify putative nociceptive afferents: cell body size, isolectin B4 (IB4) binding, and capsaicin sensitivity. Finally, we compared results generated with GCaMP6 with that generated from neurons expressing the next generation of GCaMP, jGCaMP7s and jGCaMP7f. A viral approach, with AAV9-CAG-GCaMP6s/m/f, was used to drive GECI expression in acutely dissociated rat trigeminal ganglion (TG) neurons, and neural activity was driven by electrical field stimulation. Infection efficiency with the AAV serotype was high >95 %, and the impact of GCaMP6 expression in TG neurons over the period of study (<10 days) on the regulation of intracellular Ca2+, as assessed with fura-2, was minimal. Having confirmed that the field stimulation evoked Ca2+ transients were dependent on Ca2+ influx secondary to the activation of action potentials and voltage-gated Ca2+ channels, we also confirmed that the signal-to-noise ratio for each of the isoforms was excellent, enabling detection of a single spike in>90% of neurons. However, the utility of the GCaMP6 isoforms to enable an assessment of the firing frequency let alone changes in firing frequency of each neuron was relatively limited and isoform specific: GCaMP6s and 6m had the lowest resolution, enabling detection of spikes at 3 Hz in 15% and 32% of neurons respectively, but it was possible to resolve discrete single spikes up to 10 Hz in 36% of GCaMP6f neurons. Unfortunately, using other parameters of the Ca2+ transient, such as magnitude of the transient or the rate of rise, did not improve the range over which these indicators could be used to assess changes in spike number or firing frequency. Furthermore, in the presence of ongoing neural activity, it was even more difficult to detect a change in firing frequency. The frequency response relationship for the increase in Ca2+ was highly heterogeneous among sensory neurons and was influenced by both the GCaMP6 isoform used to assess it, the timing between the delivery of stimulation trains (inter-burst interval), and afferent subpopulation. Notably, the same deficiencies were observed with jGCaMP7s and 7f in resolving the degree of activity as were present for the GCaMP6 isoforms. Together, these data suggest that while both GCaMP6 and jGCaMP7 are potentially useful tools in sensory neurons to determine the presence or absence of neural activity, the ability to discriminate changes in firing frequency ≥ 3 Hz is extremely limited. As a result, GECIs should probably not be used in sensory neurons to assess changes in activity within or between subpopulations of neurons.

Distribution of functional opioid receptors in human dorsal root ganglion neurons.

Preclinical evidence has highlighted the importance of the µ-opioid peptide (MOP) receptor on primary afferents for both the analgesic actions of MOP receptor agonists, as well as the development of tolerance, if not opioid-induced hyperalgesia. There is also growing interest in targeting other opioid peptide receptor subtypes (δ-opioid peptide [DOP], κ-opioid peptide [KOP], and nociceptin/orphanin-FQ opioid peptide [NOP]) on primary afferents, as alternatives to MOP receptors, which may not be associated with as many deleterious side effects. Nevertheless, results from several recent studies of human sensory neurons indicate that although there are many similarities between rodent and human sensory neurons, there may also be important differences. Thus, the purpose of this study was to assess the distribution of opioid receptor subtypes among human sensory neurons. A combination of pharmacology, patch-clamp electrophysiology, Ca imaging, and single-cell semiquantitative polymerase chain reaction was used. Our results suggest that functional MOP-like receptors are present in approximately 50% of human dorsal root ganglion neurons. δ-opioid peptide-like receptors were detected in a subpopulation largely overlapping that with MOP-like receptors. Furthermore, KOP-like and NOP-like receptors are detected in a large proportion (44% and 40%, respectively) of human dorsal root ganglion neurons with KOP receptors also overlapping with MOP receptors at a high rate (83%). Our data confirm that all 4 opioid receptor subtypes are present and functional in human sensory neurons, where the overlap of DOP, KOP, and NOP receptors with MOP receptors suggests that activation of these other opioid receptor subtypes may also have analgesic efficacy.

Nicotine Evoked Currents in Human Primary Sensory Neurons.

Sensory neuron nicotinic acetylcholine receptors (nAChRs) contribute to pain associated with tissue injury. However, there are marked differences between rats and mice with respect to both the properties and distribution of nAChR currents in sensory neurons. Because both species are used to understand pain signaling in humans, we sought to determine whether the currents present in either species was reflective of those present in human sensory neurons. Neurons from the L4/L5 dorsal root ganglia were obtained from adult male and female organ donors. Nicotine evoked currents were detected in 40 of 47 neurons (85%). In contrast with the naïve mouse, in which almost all nAChR currents are transient, or the rat, in which both mouse-like transient and more slowly activating and inactivating currents are detected, all the currents in human DRG neurons were slow, but slower than those in the rat. Currents were blocked by the nAChR antagonists mecamylamine (30 µmol/L), but not by the TRPA1 selective antagonist HC-030031 (10 µmol/L). Single cell polymerase chain reaction analysis of nicotinic receptor subunit expression in human DRG neurons are consistent with functional data indicating that receptor expression is detected 85 ± 2.1% of neurons assessed (n = 48, from 4 donors). The most prevalent coexpression pattern was α3/ß2 (95 ± 4% of neurons with subunits), but α7 subunits were detected in 70 ± 3.4% of neurons. These results suggest that there are not only species differences in the sensory neuron distribution of nAChR currents between rodent and human, but that the subunit composition of the channel underlying human nAChR currents may be different from those in the mouse or rat. PERSPECTIVE: The properties and distribution of nicotine evoked currents in human sensory neurons were markedly different from those previously observed in mice and rats. These observations add additional support to the suggestion that human sensory neurons may be an essential screening tool for those considering moving novel therapeutics targeting primary afferents into clinical trials.

Circulating Omentin-1 and Chronic Painful Temporomandibular Disorders.

AIMS: To investigate the relationship between omentin-1 levels and painful temporomandibular disorders (TMD). METHODS: In a case-control design, chronic painful TMD cases (n = 90) and TMD-free controls (n = 54) were selected from participants in the multisite OPPERA study (Orofacial Pain: Prospective Evaluation and Risk Assessment). Painful TMD case status was determined by examination using established Research Diagnostic Criteria for TMD (RDC/TMD). Levels of omentin-1 in stored blood plasma samples were measured by using an enzyme linked immunosorbent assay. Binary logistic regression was used to calculate the odds ratios (ORs) and 95% confidence limits (CLs) for the association between omentin-1 and painful TMD. Models were adjusted for study site, age, sex, and body mass index. RESULTS: The unadjusted association between omentin-1 and chronic painful TMD was statistically nonsignificant (P = .072). Following adjustment for covariates, odds of TMD pain decreased 36% per standard deviation increase in circulating omentin-1 (adjusted OR = 0.64; 95% CL: 0.43, 0.96; P = .031). CONCLUSION: Circulating levels of omentin-1 were significantly lower in painful TMD cases than controls, suggesting that TMD pain is mediated by inflammatory pathways.

ß2- and ß3-adrenergic receptors drive COMT-dependent pain by increasing production of nitric oxide and cytokines.

Decreased activity of catechol-O-methyltransferase (COMT), an enzyme that metabolizes catecholamines, contributes to pain in humans and animals. Previously, we demonstrated that development of COMT-dependent pain is mediated by both ß2- and ß3-adrenergic receptors (ß2ARs and ß3ARs). Here we investigated molecules downstream of ß2- and ß3ARs driving pain in animals with decreased COMT activity. Based on evidence linking their role in pain and synthesis downstream of ß2- and ß3AR stimulation, we hypothesized that nitric oxide (NO) and proinflammatory cytokines drive COMT-dependent pain. To test this, we measured plasma NO derivatives and cytokines in rats receiving the COMT inhibitor OR486 in the presence or absence of the ß2AR antagonist ICI118,551+ß3AR antagonist SR59320A. We also assessed whether the NO synthase inhibitor L-N(G)-nitroarginine methyl ester (L-NAME) and cytokine-neutralizing antibodies block the development of COMT-dependent pain. Results showed that animals receiving OR486 exhibited higher levels of NO derivatives, tumor necrosis factor α (TNFα), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and chemokine (C-C motif) ligand 2 (CCL2) in a ß2- and ß3AR-dependent manner. Additionally, inhibition of NO synthases and neutralization of the innate immunity cytokines TNFα, IL-1ß, and IL-6 blocked the development of COMT-dependent pain. Finally, we found that NO influences TNFα, IL-1ß, IL-6, and CCL2 levels, whereas TNFα and IL-6 influence NO levels. Altogether, these results demonstrate that ß2- and ß3ARs contribute to COMT-dependent pain, at least partly, by increasing NO and cytokines. Furthermore, they identify ß2- and ß3ARs, NO, and proinflammatory cytokines as potential therapeutic targets for pain patients with abnormalities in COMT physiology.