Intracellular TRPA1 mediates Ca2+ release from lysosomes in dorsal root ganglion neurons.
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ABSTRACT:
SUBMITTER: Shang S
PROVIDER: S-EPMC5100290 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundDorsal root ganglia (DRG) somata from rodents have provided an excellent model system to study ion channel properties and modulation using electrophysiological investigation. As in other vertebrates, zebrafish (Danio rerio) DRG are organized segmentally and possess peripheral axons that bifurcate into each body segment. However, the electrical properties of zebrafish DRG sensory neurons, as compared with their mammalian counterparts, are relatively unexplored because a preparation suitable for electrophysiological studies has not been available.Methodology/principal findingsWe show enzymatically dissociated DRG neurons from juvenile zebrafish expressing Isl2b-promoter driven EGFP were easily identified with fluorescence microscopy and amenable to conventional whole-cell patch-clamp studies. Two kinetically distinct TTX-sensitive Na(+) currents (rapidly- and slowly-inactivating) were discovered. Rapidly-inactivating I(Na) were preferentially expressed in relatively large neurons, while slowly-inactivating I(Na) was more prevalent in smaller DRG neurons. RT-PCR analysis suggests zscn1aa/ab, zscn8aa/ab, zscn4ab and zscn5Laa are possible candidates for these I(Na) components. Voltage-gated Ca(2+) currents (I(Ca)) were primarily (87%) comprised of a high-voltage activated component arising from ω-conotoxin GVIA-sensitive Ca(V)2.2 (N-type) Ca(2+) channels. A few DRG neurons (8%) displayed a miniscule low-voltage-activated component. I(Ca) in zebrafish DRG neurons were modulated by neurotransmitters via either voltage-dependent or -independent G-protein signaling pathway with large cell-to-cell response variability.Conclusions/significanceOur present results indicate that, as in higher vertebrates, zebrafish DRG neurons are heterogeneous being composed of functionally distinct subpopulations that may correlate with different sensory modalities. These findings provide the first comparison of zebrafish and rodent DRG neuron electrical properties and thus provide a basis for future studies.
| S-EPMC3411820 | biostudies-literature
Project description:To determine whether activation and/or inhibition of α-adrenoreceptors influences substance P (SP) release from dorsal root ganglion (DRG) primary sensory neurons in vitro.DRGs were dissected from 15-day embryonic Wistar rats. DRG neurons were dissociated and cultured for 2 d and then exposed to noradrenaline (NA) alone (1×10(-4) mol/L), or along with the α1-adrenoreceptor antagonist prazosin (1×10(-6) mol/L) or the α2-adrenoreceptor antagonist yohimbine (1×10(-5) mol/L) for 4 d. Then, RT-PCR was used to determine the levels of preprotachykinin (PPT) mRNA encoding for SP and Western blot to assess the protein levels of SP. Basal and capsaicin (CAP)-evoked SP release were measured by enzyme-linked immunosorbent assay (ELISA).CAP-evoked SP release was sensitized by NA and this effect was inhibited by pre-incubation with prazosin but not with yohimbine. The levels of PPT mRNA, SP peptide, and basal SP release did not change significantly in any of the experimental conditions.NA may significantly increase CAP-evoked SP release through activation of α-adrenoreceptors, which may contribute to noradrenergic pain modulation.
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Project description:A central principle of synaptic transmission is that action potential-induced presynaptic neurotransmitter release occurs exclusively via Ca2+-dependent secretion (CDS). The discovery and mechanistic investigations of Ca2+-independent but voltage-dependent secretion (CiVDS) have demonstrated that the action potential per se is sufficient to trigger neurotransmission in the somata of primary sensory and sympathetic neurons in mammals. One key question remains, however, whether CiVDS contributes to central synaptic transmission. Here we report, in the central transmission from presynaptic (dorsal root ganglion) to postsynaptic (spinal dorsal horn) neurons in vitro, (1) excitatory postsynaptic currents (EPSCs) are mediated by glutamate transmission through both CiVDS (up to 87%) and CDS; (2) CiVDS-mediated EPSCs are independent of extracellular and intracellular Ca2+; (3) CiVDS is faster than CDS in vesicle recycling with much less short-term depression; (4) the fusion machinery of CiVDS includes Cav2.2 (voltage sensor) and SNARE (fusion pore). Together, an essential component of activity-induced EPSCs is mediated by CiVDS in a central synapse.
| S-SCDT-EMBOR-2021-54507V1 | biostudies-other
Project description:Autonomous activation of calcium-calmodulin kinase (CaMKII) has been proposed as a molecular mechanism for decoding Ca(2+) spike frequencies resulting from action potential firing, but this has not been investigated in intact neurons. This was studied in mouse DRG neurons in culture using confocal measurements of [Ca(2+)](i) and biochemical measurements of CaMKII autophosphorylation and autonomous activity. Using electrical stimulation at different frequencies, we find that CaMKII autonomous activity reached near maximal levels after approximately 45 impulses, regardless of firing frequency (1-10 Hz), and autonomous activity declined with prolonged stimulation. Frequency-dependent activation of CaMKII was limited to spike frequencies in the range of 0.1-1 Hz, despite marked increases in [Ca(2+)](i) at higher frequencies (1-30 Hz). The high levels of autonomous activity measured before stimulation and the relatively long duration of Ca(2+) spikes induced by action potentials ( approximately 300 msec) are consistent with the lower frequency range of action potential decoding by CaMKII. The high autonomous activity under basal conditions was associated with extracellular [Ca(2+)], independently from changes in [Ca(2+)](i), and unrelated to synaptic or spontaneous impulse activity. CaMKII autonomous activity in response to brief bursts of action potentials correlated better with the frequency of Ca(2+) transients than with the concentration of [Ca(2+)](i). In conclusion, CaMKII may decode frequency-modulated responses between 0.1 and 1 Hz in these neurons, but other mechanisms may be required to decode higher frequencies. Alternatively, CaMKII may mediate high-frequency responses in subcellular microdomains in which the enzyme is maintained at a low level of autonomous activity or the Ca(2+) transients have faster kinetics.
| S-EPMC6763116 | biostudies-literature
Project description:Recently, studies have focused on the antihyperalgesic activity of the A3 adenosine receptor (A3AR) in several chronic pain models, but the cellular and molecular basis of this effect is still unknown. Here, we investigated the expression and functional effects of A3AR on the excitability of small- to medium-sized, capsaicin-sensitive, dorsal root ganglion (DRG) neurons isolated from 3- to 4-week-old rats. Real-time quantitative polymerase chain reaction experiments and immunofluorescence analysis revealed A3AR expression in DRG neurons. Patch-clamp experiments demonstrated that 2 distinct A3AR agonists, Cl-IB-MECA and the highly selective MRS5980, inhibited Ca-activated K (KCa) currents evoked by a voltage-ramp protocol. This effect was dependent on a reduction in Ca influx via N-type voltage-dependent Ca channels, as Cl-IB-MECA-induced inhibition was sensitive to the N-type blocker PD173212 but not to the L-type blocker, lacidipine. The endogenous agonist adenosine also reduced N-type Ca currents, and its effect was inhibited by 56% in the presence of A3AR antagonist MRS1523, demonstrating that the majority of adenosine's effect is mediated by this receptor subtype. Current-clamp recordings demonstrated that neuronal firing of rat DRG neurons was also significantly reduced by A3AR activation in a MRS1523-sensitive but PD173212-insensitive manner. Intracellular Ca measurements confirmed the inhibitory role of A3AR on DRG neuronal firing. We conclude that pain-relieving effects observed on A3AR activation could be mediated through N-type Ca channel block and action potential inhibition as independent mechanisms in isolated rat DRG neurons. These findings support A3AR-based therapy as a viable approach to alleviate pain in different pathologies.
| S-EPMC6669900 | biostudies-literature
Project description:Available evidence indicates voltage-gated Na+ channels (VGSCs) in peripheral sensory neurons are essential for the pain and hypersensitivity associated with tissue injury. However, our understanding of the biophysical and pharmacological properties of the channels in sensory neurons is largely based on the study of heterologous systems or rodent tissue, despite evidence that both expression systems and species differences influence these properties. Therefore, we sought to determine the extent to which the biophysical and pharmacological properties of VGSCs were comparable in rat and human sensory neurons. Whole cell patch clamp techniques were used to study Na+ currents in acutely dissociated neurons from human and rat. Our results indicate that while the two major current types, generally referred to as tetrodotoxin (TTX)-sensitive and TTX-resistant were qualitatively similar in neurons from rats and humans, there were several differences that have important implications for drug development as well as our understanding of pain mechanisms.
| S-EPMC5433841 | biostudies-literature
Project description:Somatosensory neurons with cell bodies in the dorsal root ganglia (DRG) project to the skin, muscles, bones, and viscera to detect touch and temperature as well as to mediate proprioception and many types of interoception. In addition, the somatosensory system conveys the clinically relevant noxious sensations of pain and itch. Here, we used single nuclear transcriptomics to characterize transcriptomic classes of human DRG neurons that detect these diverse types of stimuli. Notably, multiple types of human DRG neurons have transcriptomic features that resemble their mouse counterparts although expression of genes considered important for sensory function often differed between species. More unexpectedly, we identified several transcriptomic classes with no clear equivalent in the other species. This dataset should serve as a valuable resource for the community, for example as means of focusing translational efforts on molecules with conserved expression across species.
| S-EPMC8626086 | biostudies-literature
Project description:The dorsal root ganglia (DRG) and trigeminal ganglia (TG) are clusters of cell bodies of highly specialized sensory neurons which are responsible for relaying information about our environment to the central nervous system. Despite previous efforts to characterize sensory neurons at the molecular level, it is still unknown whether those present in DRG and TG have distinct expression profiles and therefore a unique molecular fingerprint. To address this question, we isolated lumbar DRG and TG neurons using fluorescence-activated cell sorting from Advillin-GFP transgenic mice and performed RNA sequencing. Our transcriptome analyses showed that, despite being overwhelmingly similar, a number of genes are differentially expressed in DRG and TG neurons. Importantly, we identified 24 genes which were uniquely expressed in either ganglia, including an arginine vasopressin receptor and several homeobox genes, giving each population a distinct molecular fingerprint. We compared our findings with published studies to reveal that many genes previously reported to be present in neurons are in fact likely to originate from other cell types in the ganglia. Additionally, our neuron-specific results aligned well with a dataset examining whole human TG and DRG. We propose that the data can both improve our understanding of primary afferent biology and help contribute to the development of drug treatments and gene therapies which seek targets with unique or restricted expression patterns.
| S-EPMC5623188 | biostudies-literature
Project description:AbstractVoltage-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.
| S-EPMC8882208 | biostudies-literature
Project description:Gastric hypersensitivity contributes to abdominal pain in patients with functional dyspepsia. Recent studies showed that hormones induced by stress are correlated with visceral hypersensitivity. However, the precise mechanisms underlying gastric hypersensitivity remain largely unknown. The aim of the present study was designed to investigate the roles of corticosterone (CORT) on excitability of dorsal root ganglion (DRG) neurons innervating the stomach.DRG neurons innervating the stomach were labeled by DiI injection into the stomach wall. Patch clamp recordings were employed to examine neural excitability and voltage-gated sodium channel currents. Electromyograph technique was used to determine the responses of neck muscles to gastric distension.Incubation of acutely isolated DRG neurons with CORT significantly depolarized action potential threshold and enhanced the number of action potentials induced by current stimulation of the neuron. Under voltage-clamp mode, incubation of CORT enhanced voltage-gated sodium current density of the recorded neurons. Pre-incubation of GF109203X, an inhibitor of protein kinase C, blocked the CORT-induced hyperexcitability and potentiation of sodium currents. However, pre-incubation of H-89, an inhibitor of protein kinase A, did not alter the sodium current density. More importantly, intraperitoneal injection of CORT produced gastric hypersensitivity of healthy rats, which was blocked by pre-administration of GF109203X but not H-89.Our data strongly suggest that CORT rapidly enhanced neuronal excitability and sodium channel functions, which is most likely mediated by protein kinase C but not protein kinase A signaling pathway in DRG neurons innervating the stomach, thus underlying the gastric hypersensitivity induced by CORT injection.
| S-EPMC5503297 | biostudies-other