The beta(3)-adrenergic receptor (beta(3)AR) is an essential regulator of metabolic and endocrine functions. A major cellular and clinically significant consequence of beta(3)AR activation is the substantial elevation in interleukin-6 (IL-6) levels. Although the beta(3)AR-dependent regulation of IL-6 expression is well established, the cellular pathways underlying this regulation have not been characterized. Using a novel method of homogenous reporters, we assessed the pattern of activation of 43 transcription factors in response to the specific beta(3)AR agonist CL316243 in adipocytes, cells that exhibit the highest expression of beta(3)ARs. We observed a unique and robust activation of the CRE-response element, suggesting that IL-6 transcription is regulated via the G(s)-protein/cAMP/protein kinase A (PKA) but not nuclear factor kappa B (NF-kappaB) pathway. However, pretreatment of adipocytes with pharmacologic inhibitors of PKA pathway failed to block beta(3)AR-mediated IL-6 up-regulation. Additionally, stimulation of adipocytes with the exchange protein directly activated by cAMP (Epac) agonist did not induce IL-6 expression. Instead, the beta(3)AR-mediated transcription of IL-6 required activation of both the p38 and PKC pathways. Western blot analysis further showed that transcription factors CREB and ATF-2 but not ATF-1 were activated in a p38- and PKC-dependent manner. Collectively, our results suggest that while stimulation of the beta(3)AR leads to a specific activation of CRE-dependent transcription, there are several independent cellular pathways that converge at the level of CRE-response element activation, and in the case of IL-6 this activation is mediated by p38 and PKC but not PKA pathways.

The pharmacological effect of opioids originates, at the cellular level, by their interaction with the μ-opioid receptor (mOR) resulting in the regulation of voltage-gated Ca2+ channels and inwardly rectifying K+ channels that ultimately modulate the synaptic transmission. Recently, an alternative six trans-membrane helix isoform of mOR, (6TM-mOR) has been identified, but its function and signaling are still largely unknown. Here, we present the structural and functional mechanisms of 6TM-mOR signaling activity upon binding to morphine. Our data suggest that despite the similarity of binding modes of the alternative 6TM-mOR and the dominant seven trans-membrane helix variant (7TM-mOR), the interaction with morphine generates different dynamic responses in the two receptors, thus, promoting the activation of different mOR-specific signaling pathways. We characterize a series of 6TM-mOR-specific cellular responses, and observed that they are significantly different from those for 7TM-mOR. Morphine stimulation of 6TM-mOR does not promote a cellular cAMP response, while it increases the intracellular Ca2+ concentration and reduces the cellular K+ conductance. Our findings indicate that 6TM-mOR has a unique contribution to the cellular opioid responses. Therefore, it should be considered as a relevant target for the development of novel pharmacological tools and medical protocols involving the use of opioids.

The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the integrated stress response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism.

High interindividual variability in postoperative opioid consumption is related to genetic and environmental factors. We tested the association between morphine consumption, postoperative pain, and single nucleotide polymorphisms (SNPs) within opioid receptor μ 1 (OPRM1), catechol-O-methyltransferase (COMT), uridine diphosphate glucose-glucuronosyltransferase-2B7, and estrogen receptor (ESR1) gene loci to elucidate genetic prediction of opioid consumption. We analyzed 20 SNPs in 201 unrelated Caucasian patients who underwent abdominal surgery and who were receiving postoperative patient-controlled analgesia-administered morphine. Morphine consumption and pain intensity were dependent variables; age and sex were covariates. A haplotype of 7 SNPs in OPRM1 showed significant additive effects on opioid consumption (P = .007); a linear regression model including age and 9 SNPs in ESR1, OPRM1, and COMT explained the highest proportion of variance of morphine consumption (10.7%; P = .001). The minimal model including 3 SNPs in ESR1, OPRM1, and COMT explained 5% of variance (P = .007). We found a significant interaction between rs4680 in COMT and rs4986936 in ESR1 (P = .007) on opioid consumption. SNPs rs677830 and rs540825 of OPRM1 and rs9340799 of ESR1 were nominally associated with pain Numeric Rating Scale scores. Combinations of genetic variants within OPRM1, COMT, and ESR1 better explain variability in morphine consumption than single genetic variants. Our results contribute to the development of genetic markers and statistical models for future diagnostic tools for opioid consumption/efficacy.

The ability to perceive noxious stimuli is critical for an animal's survival in the face of environmental danger, and thus pain perception is likely to be under stringent evolutionary pressure. Using a neuronal-specific RNAi knock-down strategy in adult Drosophila, we recently completed a genome-wide functional annotation of heat nociception that allowed us to identify α2δ3 as a novel pain gene. Here we report construction of an evolutionary-conserved, system-level, global molecular pain network map. Our systems map is markedly enriched for multiple genes associated with human pain and predicts a plethora of novel candidate pain pathways. One central node of this pain network is phospholipid signaling, which has been implicated before in pain processing. To further investigate the role of phospholipid signaling in mammalian heat pain perception, we analysed the phenotype of PIP5Kα and PI3Kγ mutant mice. Intriguingly, both of these mice exhibit pronounced hypersensitivity to noxious heat and capsaicin-induced pain, which directly mapped through PI3Kγ kinase-dead knock-in mice to PI3Kγ lipid kinase activity. Using single primary sensory neuron recording, PI3Kγ function was mechanistically linked to a negative regulation of TRPV1 channel transduction. Our data provide a systems map for heat nociception and reinforces the extraordinary conservation of molecular mechanisms of nociception across different species.

Carbonic anhydrase-8 (CA8) is an intracellular protein that functions as an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1) critical to intracellular Ca++ release, synaptic functions and neuronal excitability. We showed previously that murine nociception and analgesic responses are regulated by the expression of this gene in dorsal root ganglion (DRG) associated with a cis-eQTL. In this report, we identify an exon-level cis-eQTL (rs6471859) that regulates human DRG CA8 alternative splicing, producing a truncated 1,697bp transcript (e.g., CA8-204). Our functional genomic studies show the "G" allele at rs6471859 produces a cryptic 3'UTR splice site regulating expression of CA8-204. We developed constructs to study the expression and function of the naturally occurring CA8-204G transcript (G allele at rs6471859), CA8-204C (C allele at rs6471859 reversion mutation) and CA8-201 (full length transcript). CA8-204G transcript expression occurred predominantly in non-neuronal cells (HEK293), while CA8-204C expression was restricted to neuronal derived cells (NBL) in vitro. CA8-204G produced a stable truncated transcript in HEK293 cells that was barely detectable in NBL cells. We also show CA8-204 produces a stable peptide that inhibits pITPR1 and Ca++ release in HEK293 cells. These results imply homozygous G/G individuals at rs6471859, which are common in the general population, produce exclusively CA8-204G that is barely detectable in neuronal cells. CA8 null mutations that greatly impact neuronal functions are associated with severe forms of spinal cerebellar ataxia, and our data suggest G/G homozygotes should display a similar phenotype. To address this question, we show in vivo using AAV8-FLAG-CA8-204G and AAV8-V5-CA8-201 gene transfer delivered via intra-neural sciatic nerve injection (SN), that these viral constructs are able to transduce DRG cells and produce similar analgesic and anti-hyperalgesic responses to inflammatory pain. Immunohistochemistry (IHC) examinations of DRG tissues further show CA8-204G peptide is expressed in advillin expressing neuronal cells, but to a lesser extent compared to glial cells. These findings explain why G/G homozygotes that exclusively produce this truncated functional peptide in DRG evade a severe phenotype. These genomic studies significantly advance the literature regarding structure-function studies on CA8-ITPR1 critical to calcium signaling pathways, synaptic functioning, neuronal excitability and analgesic responses.

Chronic pain is a prevalent disease with increasing clinical challenges. Genome-wide association studies in chronic pain patients have identified hundreds of common pathogenic variants, yet they only explained a portion of individual variance of chronic pain. With the advances in next-generation sequencing technologies, it is now feasible to conduct rarer variants studies in large-scale databases. Here, we performed gene-based rare variant analyses in 200,000 human subjects in the UK biobank whole-exome sequencing database for investigating 9 different chronic pain states and validated our findings in 3 other large-scale databases. Our analyses identified the SLC13A1 gene coding for sodium/sulfate symporter associated with chronic back pain and multisite pain at the genome-wide level and with chronic headache, knee, and neck and shoulder pain at the nominal level. Seven loss-of-function rare variants were identified within the gene locus potentially contributing to the development of chronic pain, with 2 of them individually associated with back pain and multisite pain. These 2 rare variants were then tested for replication in 3 other biobanks, and the strongest evidence was found for rs28364172 as an individual contributor. Transcriptional analyses of Slc13a1 in rodents showed substantial regulation of its expression in the dorsal root ganglia and the sciatic nerve in neuropathic pain assays. Our results stress the importance of the SLC13A1 gene in sulfate homeostasis in the nervous system and its critical role in preventing pain states, thus suggesting new therapeutic approaches for treating chronic pain in a personalized manner, especially in people with mutations in the SLC13A1 gene.

Calcium dysregulation is causally linked with various forms of neuropathology including seizure disorders, multiple sclerosis, Huntington's disease, Alzheimer's, spinal cerebellar ataxia (SCA) and chronic pain. Carbonic anhydrase-8 (Car8) is an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1), which regulates intracellular calcium release fundamental to critical cellular functions including neuronal excitability, neurite outgrowth, neurotransmitter release, mitochondrial energy production and cell fate. In this report we test the hypothesis that Car8 regulation of ITPR1 and cytoplasmic free calcium release is critical to nociception and pain behaviors. We show Car8 null mutant mice (MT) exhibit mechanical allodynia and thermal hyperalgesia. Dorsal root ganglia (DRG) from MT also demonstrate increased steady-state ITPR1 phosphorylation (pITPR1) and cytoplasmic free calcium release. Overexpression of Car8 wildtype protein in MT nociceptors complements Car8 deficiency, down regulates pITPR1 and abolishes thermal and mechanical hypersensitivity. We also show that Car8 nociceptor overexpression alleviates chronic inflammatory pain. Finally, inflammation results in downregulation of DRG Car8 that is associated with increased pITPR1 expression relative to ITPR1, suggesting a possible mechanism of acute hypersensitivity. Our findings indicate Car8 regulates the ITPR1-cytosolic free calcium pathway that is critical to nociception, inflammatory pain and possibly other neuropathological states. Car8 and ITPR1 represent new therapeutic targets for chronic pain.

Worldwide, acute, and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knockdown in Drosophila, we report a global screen for an innate behavior and identify hundreds of genes implicated in heat nociception, including the α2δ family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (α2δ3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, α2δ3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in α2δ3 mutant mice revealed impaired transmission of thermal pain-evoked signals from the thalamus to higher-order pain centers. Intriguingly, in α2δ3 mutant mice, thermal pain and tactile stimulation triggered strong cross-activation, or synesthesia, of brain regions involved in vision, olfaction, and hearing.

Catechol-O-methyltransferase (COMT) is a major enzyme controlling catecholamine levels that plays a central role in cognition, affective mood and pain perception. There are three common COMT haplotypes in the human population reported to have functional effects, divergent in two synonymous and one nonsynonymous position. We demonstrate that one of the haplotypes, carrying the non-synonymous variation known to code for a less stable protein, exhibits increased protein expression in vitro. This increased protein expression, which would compensate for lower protein stability, is solely produced by a synonymous variation (C(166)T) situated within the haplotype and located in the 5' region of the RNA transcript. Based on mRNA secondary structure predictions, we suggest that structural destabilization near the start codon caused by the T allele could be related to the observed increase in COMT expression. Our folding simulations of the tertiary mRNA structures demonstrate that destabilization by the T allele lowers the folding transition barrier, thus decreasing the probability of occupying its native state. These data suggest a novel structural mechanism whereby functional synonymous variations near the translation initiation codon affect the translation efficiency via entropy-driven changes in mRNA dynamics and present another example of stable compensatory genetic variations in the human population.

This paper describes methods used in the project "Orofacial Pain Prospective Evaluation and Risk Assessment" (OPPERA) and evaluates sociodemographic characteristics associated with temporomandibular disorders (TMD) in the OPPERA case-control study. Representativeness was investigated by comparing sociodemographic profiles of OPPERA participants with population census profiles of counties near study sites and by comparing age and gender associations with TMD in OPPERA and the 2007 to 2009 US National Health Interview Survey. Volunteers aged 18 to 44 years were recruited at 4 US study sites: 3,263 people without TMD were enrolled into the prospective cohort study; 1,633 of them were selected as controls for the baseline case-control study. Cases were 185 volunteers with examiner-classified TMD. Distributions of some demographic characteristics among OPPERA participants differed from census profiles, although there was less difference in socioeconomic profiles. Odds of TMD was associated with greater age in this 18 to 44 year range; females had 3 times the odds of TMD as males; and relative to non-Hispanic-Whites, other racial groups had one-fifth the odds of TMD. Age and gender associations with chronic TMD were strikingly similar to associations observed in the US population. Assessments of representativeness in this demographically diverse group of community volunteers suggest that OPPERA case-control findings have good internal validity.

Opioids that stimulate the μ-opioid receptor (MOR1) are the most frequently prescribed and effective analgesics. Here we present a structural model of MOR1. Molecular dynamics simulations show a ligand-dependent increase in the conformational flexibility of the third intracellular loop that couples with the G protein complex. These simulations likewise identified residues that form frequent contacts with ligands. We validated the binding residues using site-directed mutagenesis coupled with radioligand binding and functional assays. The model was used to blindly screen a library of ∼1.2 million compounds. From the 34 compounds predicted to be strong binders, the top three candidates were examined using biochemical assays. One compound showed high efficacy and potency. Post hoc testing revealed this compound to be nalmefene, a potent clinically used antagonist, thus further validating the model. In summary, the MOR1 model provides a tool for elucidating the structural mechanism of ligand-initiated cell signaling and for screening novel analgesics.

Catechol-O-methyltransferase (COMT) is an enzyme that plays a key role in the modulation of catechol-dependent functions such as cognition, cardiovascular function, and pain processing. Three common haplotypes of the human COMT gene, divergent in two synonymous and one nonsynonymous (val(158)met) position, designated as low (LPS), average (APS), and high pain sensitive (HPS), are associated with experimental pain sensitivity and risk of developing chronic musculoskeletal pain conditions. APS and HPS haplotypes produce significant functional effects, coding for 3- and 20-fold reductions in COMT enzymatic activity, respectively. In the present study, we investigated whether additional minor single nucleotide polymorphisms (SNPs), accruing in 1 to 5% of the population, situated in the COMT transcript region contribute to haplotype-dependent enzymatic activity. Computer analysis of COMT ESTs showed that one synonymous minor SNP (rs769224) is linked to the APS haplotype and three minor SNPs (two synonymous: rs6267, rs740602 and one nonsynonymous: rs8192488) are linked to the HPS haplotype. Results from in silico and in vitro experiments revealed that inclusion of allelic variants of these minor SNPs in APS or HPS haplotypes did not modify COMT function at the level of mRNA folding, RNA transcription, protein translation, or enzymatic activity. These data suggest that neutral variants are carried with APS and HPS haplotypes, while the high activity LPS haplotype displays less linked variation. Thus, both minor synonymous and nonsynonymous SNPs in the coding region are markers of functional APS and HPS haplotypes rather than independent contributors to COMT activity.

Opioid and α2-adrenoceptor (AR) agonists are analgesic when administered in the spinal cord and show a clinically beneficial synergistic interaction when co-administered. However, α2-AR antagonists can also inhibit opioid antinociception, suggesting a complex interaction between the two systems. The α2A-AR subtype is necessary for spinal adrenergic analgesia and synergy with opioids for most agonist combinations. Therefore, we investigated whether spinal opioid antinociception and opioid-adrenergic synergy were under allosteric control of the α2A-AR. Drugs were administered intrathecally in wild type (WT) and α2A-knock-out (KO) mice and antinociception was measured using the hot water tail immersion or substance P behavioral assays. The α2A-AR agonist clonidine was less effective in α2A-KO mice in both assays. The absence of the α2A-AR resulted in 10-70-fold increases in the antinociceptive potency of the opioid agonists morphine and DeltII. In contrast, neither morphine nor DeltII synergized with clonidine in α2A-KO mice, indicating that the α2AAR has both positive and negative modulatory effects on opioid antinociception. Depletion of descending adrenergic terminals with 6-OHDA resulted in a significant decrease in morphine efficacy in WT but not in α2A-KO mice, suggesting that endogenous norepinephrine acts through the α2A-AR to facilitate morphine antinociception. Based on these findings, we propose a model whereby ligand-occupied versus ligand-free α2A-AR produce distinct patterns of modulation of opioid receptor activation. In this model, agonist-occupied α2A-ARs potentiate opioid analgesia, while non-occupied α2A-ARs inhibit opioid analgesia. Exploiting such interactions between the two receptors could lead to the development of better pharmacological treatments for pain management.

Carbonic anhydrase-8 (Car8; murine gene symbol) is an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1), which regulates neuronal intracellular calcium release. We previously reported that wild-type Car8 overexpression corrects the baseline allodynia and hyperalgesia associated with calcium dysregulation in the waddle (wdl) mouse due to a 19 bp deletion in exon 8 of the Car8 gene. In this report, we provide preliminary evidence that overexpression of the human wild-type ortholog of Car8 (CA8WT), but not the reported CA8 S100P loss-of-function mutation (CA8MT), inhibits nerve growth factor (NGF)-induced phosphorylation of ITPR1, TrkA (NGF high-affinity receptor), and ITPR1-mediated cytoplasmic free calcium release in vitro. In addition, we show that gene transfer using AAV8-V5-CA8WT viral particles via sciatic nerve injection demonstrates retrograde transport to dorsal root ganglia (DRG) producing prolonged V5-CA8WT expression, pITPR1 and pTrkA inhibition, and profound analgesia and anti-hyperalgesia in male C57BL/6J mice. AAV8-V5-CA8WT-mediated overexpression prevented and treated allodynia and hyperalgesia associated with chronic neuropathic pain produced by the spinal nerve ligation (SNL) model. These AAV8-V5-CA8 data provide a proof-of-concept for precision medicine through targeted gene therapy of NGF-responsive somatosensory neurons as a long-acting local analgesic able to prevent and treat chronic neuropathic pain through regulating TrkA signaling, ITPR1 activation, and intracellular free calcium release by ITPR1.

Parasitic helminths cause significant damage as they migrate through host tissues to complete their life cycle. While chronic helminth infections are characterized by a well-described Type 2 immune response, the early, tissue-invasive stages are not well understood. Here we investigate the immune pathways activated during the early stages of Heligmosomoides polygyrus bakeri (Hpb), a natural parasitic roundworm of mice. In contrast to the Type 2 immune response present at later stages of infection, a robust Type 1 immune signature including IFNg production was dominant at the time of parasite invasion and granuloma formation. This early response was associated with an accumulation of activated Natural Killer (NK) cells, with no increase of other innate lymphoid cell populations. Parabiosis and confocal microscopy studies indicated that NK cells were recruited from circulation to the small intestine, where they surrounded parasitic larvae. NK cell recruitment required IFNγ receptor signaling, but was independent of CXCR3 expression. The depletion of tissue-infiltrating NK cells altered neither worm burden nor parasite fitness, but increased vascular injury, suggesting a role for NK cells in mediating tissue protection. Together, these data identify an unexpected role for NK cells in promoting disease tolerance during the invasive stage of an enteric helminth infection.

Background: Mild traumatic brain injury (mTBI) often results in post-concussion symptoms, chronic pain, and sleepiness. Genetic factors are thought to play an important role in poor prognosis. Aims: The aims of this study are to (1) document the prevalence of pain and post-concussion symptoms in mTBI patients in acute and chronic phases (2) determine whether candidate genes predispose to post-concussive symptoms and pain. Methods: Posttraumatic symptoms, evaluated using the Rivermead Post-Concussion Symptoms Questionnaire, and pain were assessed in 94 mTBI patients in the acute phase as well as in 22 healthy controls. Assessment was repeated in 36 patients after one year who agreed to participate in the follow-up visit. Gene polymorphisms and expression were assessed in mTBI patients and healthy controls. Results: In the acute phase, mTBI patients with pain (69%) presented more psychological symptoms and sleepiness and were less able to return to work than those without pain. At one year, 19% of mTBI patients had persistent pain and psychological distress. Two haplotypes (H2 and H3) in the brain-derived neurotrophic factor (BDNF) gene were shown to be respectively deleterious and protective against post-concussion symptoms and pain in both acute and chronic phases. Protective haplotype H3 was associated with a decreased expression of the anti-sense of BDNF (BDNF-AS). Deleterious haplotype H2 predicted the development of chronic pain at one year, whereas H3 was protective. Conclusions: This pilot study suggests a protective mechanism of a multilocus effect in BDNF, through BDNF-AS, against post-concussion symptoms and pain in the acute phase and possibly chronic pain at one year post-mTBI. The role of antisense RNA should be validated in larger cohorts.

Activation of microglia in the spinal cord following peripheral nerve injury is critical for the development of long-lasting pain hypersensitivity. However, it remains unclear whether distinct microglia subpopulations or states contribute to different stages of pain development and maintenance. Using single-cell RNA-sequencing, we show that peripheral nerve injury induces the generation of a male-specific inflammatory microglia subtype, and demonstrate increased proliferation of microglia in male as compared to female mice. We also show time- and sex-specific transcriptional changes in different microglial subpopulations following peripheral nerve injury. Apolipoprotein E (Apoe) is the top upregulated gene in spinal cord microglia at chronic time points after peripheral nerve injury in mice. Furthermore, polymorphisms in the APOE gene in humans are associated with chronic pain. Single-cell RNA sequencing analysis of human spinal cord microglia reveals a subpopulation with a disease-related transcriptional signature. Our data provide a detailed analysis of transcriptional states of mouse and human spinal cord microglia, and identify a link between ApoE and chronic pain in humans.

A functional allele of the mouse catechol-O-methyltransferase (Comt) gene is defined by the insertion of a B2 short interspersed repeat element in its 3'-untranslated region (UTR). This allele has been associated with a number of phenotypes, such as pain and anxiety. In comparison with mice carrying the ancestral allele (Comt+), Comt B2i mice show higher Comt mRNA and enzymatic activity levels. Here, we investigated the molecular genetic mechanisms underlying this allelic specific regulation of Comt expression. Insertion of the B2 element introduces an early polyadenylation signal generating a shorter Comt transcript, in addition to the longer ancestral mRNA. Comparative analysis and in silico prediction of Comt mRNA potential targets within the transcript 3' to the B2 element was performed and allowed choosing microRNA (miRNA) candidates for experimental screening: mmu-miR-3470a, mmu-miR-3470b, and mmu-miR-667. Cell transfection with each miRNA downregulated the expression of the ancestral transcript and COMT enzymatic activity. Our in vivo experiments showed that mmu-miR-667-3p is strongly correlated with decreasing amounts of Comt mRNA in the brain, and lentiviral injections of mmu-miR-3470a, mmu-miR-3470b, and mmu-miR-667 increase hypersensitivity in the mouse formalin model, consistent with reduced COMT activity. In summary, our data demonstrate that the Comt+ transcript contains regulatory miRNA signals in its 3'-untranslated region leading to mRNA degradation; these signals, however, are absent in the shorter transcript, resulting in higher mRNA expression and activity levels.

Opioids are the most widely used analgesics for the treatment of clinical pain. They produce their therapeutic effects by binding to mu-opioid receptors (MORs), which are 7 transmembrane domain (7TM) G-protein-coupled receptors (GPCRs), and inhibiting cellular activity. However, the analgesic efficacy of opioids is compromised by side-effects such as analgesic tolerance, dependence and opioid-induced hyperalgesia (OIH). In contrast to opioid analgesia these side effects are associated with cellular excitation. Several hypotheses have been advanced to explain these phenomena, yet the molecular mechanisms underlying tolerance and OIH remain poorly understood.