Inhibition of Nonsense-Mediated Decay Induces Nociceptive Sensitization through Activation of the Integrated Stress Response.

RNA stability is meticulously controlled. Here, we sought to determine whether an essential post-transcriptional regulatory mechanism plays a role in pain. Nonsense-mediated decay (NMD) safeguards against translation of mRNAs that harbor premature termination codons and controls the stability of ∼10% of typical protein-coding mRNAs. It hinges on the activity of the conserved kinase SMG1. Both SMG1 and its target, UPF1, are expressed in murine DRG sensory neurons. SMG1 protein is present in both the DRG and sciatic nerve. Using high-throughput sequencing, we examined changes in mRNA abundance following inhibition of SMG1. We confirmed multiple NMD stability targets in sensory neurons, including ATF4. ATF4 is preferentially translated during the integrated stress response (ISR). This led us to ask whether suspension of NMD induces the ISR. Inhibition of NMD increased eIF2-α phosphorylation and reduced the abundance of the eIF2-α phosphatase constitutive repressor of eIF2-α phosphorylation. Finally, we examined the effects of SMG1 inhibition on pain-associated behaviors. Peripheral inhibition of SMG1 results in mechanical hypersensitivity in males and females that persists for several days and priming to a subthreshold dose of PGE2. Priming was fully rescued by a small-molecule inhibitor of the ISR. Collectively, our results indicate that suspension of NMD promotes pain through stimulation of the ISR.SIGNIFICANCE STATEMENT Nociceptors undergo long-lived changes in their plasticity which may contribute to chronic pain. Translational regulation has emerged as a dominant mechanism in pain. Here, we investigate the role of a major pathway of RNA surveillance called nonsense-mediated decay (NMD). Modulation of NMD is potentially beneficial for a broad array of diseases caused by frameshift or nonsense mutations. Our results suggest that inhibition of the rate-limiting step of NMD drives behaviors associated with pain through activation of the ISR. This work reveals complex interconnectivity between RNA stability and translational regulation and suggests an important consideration in harnessing the salubrious benefits of NMD disruption.

The RNA-Binding Protein HuR Is Integral to the Function of Nociceptors in Mice and Humans.

HuR is an RNA-binding protein implicated in RNA processing, stability, and translation. Previously, we examined protein synthesis in dorsal root ganglion (DRG) neurons treated with inflammatory mediators using ribosome profiling. We found that the HuR consensus binding element was enriched in transcripts with elevated translation. HuR is expressed in the soma of nociceptors and their axons. Pharmacologic inhibition of HuR with the small molecule CMLD-2 reduced the activity of mouse and human sensory neurons. Peripheral administration of CMLD-2 in the paw or genetic elimination of HuR from sensory neurons diminished behavioral responses associated with NGF- and IL-6-induced allodynia in male and female mice. Genetic disruption of HuR altered the proximity of mRNA decay factors near a key neurotrophic factor (TrkA). Collectively, the data suggest that HuR is required for local control of mRNA stability and reveals a new biological function for a broadly conserved post-transcriptional regulatory factor.SIGNIFICANCE STATEMENT Nociceptors undergo long-lived changes in excitability, which may contribute to chronic pain. Noxious cues that promote pain lead to rapid induction of protein synthesis. The underlying mechanisms that confer specificity to mRNA control in nociceptors are unclear. Here, we identify a conserved RNA-binding protein called HuR as a key regulatory factor in sensory neurons. Using a combination of genetics and pharmacology, we demonstrate that HuR is required for signaling in nociceptors. In doing so, we report an important mechanism of mRNA control in sensory neurons that ensures appropriate nociceptive responses to inflammatory mediators.

Global analyses of mRNA expression in human sensory neurons reveal eIF5A as a conserved target for inflammatory pain.

Nociceptors are a type of sensory neuron that are integral to most forms of pain. Targeted disruption of nociceptor sensitization affords unique opportunities to prevent pain. An emerging model for nociceptors are sensory neurons derived from human stem cells. Here, we subjected five groups to high-throughput sequencing: human induced pluripotent stem cells (hiPSCs) prior to differentiation, mature hiPSC-derived sensory neurons, mature co-cultures containing hiPSC-derived astrocytes and sensory neurons, mouse dorsal root ganglion (DRG) tissues, and mouse DRG cultures. Co-culture of nociceptors and astrocytes promotes expression of transcripts enriched in DRG tissues. Comparisons of the hiPSC models to tissue samples reveal that many key transcripts linked to pain are present. Markers indicative of a range of neuronal subtypes present in the DRG were detected in mature hiPSCs. Intriguingly, translation factors were maintained at consistently high expression levels across species and culture systems. As a proof of concept for the utility of this resource, we validated expression of eukaryotic initiation factor 5A (eIF5A) in DRG tissues and hiPSC samples. eIF5A is subject to a unique posttranslational hypusine modification required for its activity. Inhibition of hypusine biosynthesis prevented hyperalgesic priming by inflammatory mediators in vivo and diminished hiPSC activity in vitro. Collectively, our results illuminate the transcriptomes of hiPSC sensory neuron models. We provide a demonstration for this resource through our investigation of eIF5A. Our findings reveal hypusine as a potential target for inflammation associated pain in males.

A role for translational regulation by S6 kinase and a downstream target in inflammatory pain.

BACKGROUND AND PURPOSE: Translational controls pervade neurobiology. Nociceptors play an integral role in the detection and propagation of pain signals. Nociceptors can undergo persistent changes in their intrinsic excitability. Pharmacological disruption of nascent protein synthesis diminishes acute and chronic forms of pain-associated behaviours. However, the targets of translational controls that facilitate plasticity in nociceptors are unclear. EXPERIMENTAL APPROACH: We used ribosome profiling to probe the translational landscape in dorsal root ganglion (DRG) neurons from male Swiss-Webster mice, after treatment with nerve growth factor and IL-6. Expression dynamics of c-Fos were followed with immunoblotting and immunohistochemistry. The involvement of ribosomal protein S6 kinase 1 (S6K1), a downstream component of mTOR signalling, in the control of c-Fos levels was assessed with low MW inhibitors of S6K1 (DG2) or c-Fos (T-5224), studying their effects on nociceptor activity in vitro using multielectrode arrays (MEAs) and pain behaviour in vivo in Swiss-Webster mice using the hyperalgesic priming model. KEY RESULTS: c-Fos was expressed in sensory neurons. Inflammatory mediators that promote pain in both humans and rodents promote c-Fos translation. The mTOR effector S6K1 is essential for c-Fos biosynthesis. Inhibition of S6K1 or c-Fos with low MW compounds diminished mechanical and thermal hypersensitivity in response to inflammatory cues. Additionally, both inhibitors reduced evoked nociceptor activity. CONCLUSION AND IMPLICATIONS: Our data show a novel role of S6K1 in modulating the rapid response to inflammatory mediators, with c-Fos being one key downstream target. Targeting the S6 kinase pathway or c-Fos is an exciting new avenue for pain-modulating compounds.

Intercellular Arc Signaling Regulates Vasodilation.

Injury responses require communication between different cell types in the skin. Sensory neurons contribute to inflammation and can secrete signaling molecules that affect non-neuronal cells. Despite the pervasive role of translational regulation in nociception, the contribution of activity-dependent protein synthesis to inflammation is not well understood. To address this problem, we examined the landscape of nascent translation in murine dorsal root ganglion (DRG) neurons treated with inflammatory mediators using ribosome profiling. We identified the activity-dependent gene, Arc, as a target of translation in vitro and in vivo Inflammatory cues promote local translation of Arc in the skin. Arc-deficient male mice display exaggerated paw temperatures and vasodilation in response to an inflammatory challenge. Since Arc has recently been shown to be released from neurons in extracellular vesicles (EVs), we hypothesized that intercellular Arc signaling regulates the inflammatory response in skin. We found that the excessive thermal responses and vasodilation observed in Arc defective mice are rescued by injection of Arc-containing EVs into the skin. Our findings suggest that activity-dependent production of Arc in afferent fibers regulates neurogenic inflammation potentially through intercellular signaling.SIGNIFICANCE STATEMENT Nociceptors play prominent roles in pain and inflammation. We examined rapid changes in the landscape of nascent translation in cultured dorsal root ganglia (DRGs) treated with a combination of inflammatory mediators using ribosome profiling. We identified several hundred transcripts subject to rapid preferential translation. Among them is the immediate early gene (IEG) Arc. We provide evidence that Arc is translated in afferent fibers in the skin. Arc-deficient mice display several signs of exaggerated inflammation which is normalized on injection of Arc containing extracellular vesicles (EVs). Our work suggests that noxious cues can trigger Arc production by nociceptors which in turn constrains neurogenic inflammation in the skin.

R (-)-methoxetamine exerts rapid and sustained antidepressant effects and fewer behavioral side effects relative to S (+)-methoxetamine.

The newfound antidepressant efficacy of ketamine has provided opportunities for the development of new-generation, rapid-acting, glutamate-based antidepressants. We previously identified that methoxetamine (MXE), a ketamine analog, and an N-Methyl-d-aspartate (NMDA) receptor antagonist, produced rapid and sustained antidepressant effects in mice. MXE (R, S (±)-MXE) is a racemic mixture containing equal parts of S (+)-MXE and R (-)-MXE. However, studies have yet to investigate the antidepressant effects of its enantiomers. Here, we examined the potential antidepressant properties and behavioral side effects of S- and R-MXE in mice. Both S- and R-MXE showed significant NMDA receptor affinity and appreciable inhibitory activity on serotonin transporter. Also, S- and R-MXE (10 mg kg-1) exerted antidepressant effects and increased gamma waves (electroencephalography) but were inhibited by NBQX (an AMPA receptor antagonist). Subsequently, they increased mammalian target of rapamycin phosphorylation and AMPA receptor subunits GluA1 and GluA2 protein levels in the hippocampus or prefrontal cortex. Furthermore, they increased 5HT2a and 5HT2c receptor mRNA levels in the prefrontal cortex, with their antidepressant effects inhibited by ketanserin (a 5HT2a/c receptor antagonist). Taken together, S-MXE and R-MXE elicit antidepressant effects that are probably mediated via glutamatergic and serotonergic mechanisms. Unlike S-MXE, R-MXE did not induce prepulse inhibition deficits, hyperlocomotion, conditioned place preference, and locomotor sensitization, although it acutely altered motor coordination. This suggests that R-MXE induces fewer behavioral side effects and is a safer antidepressant than S-MXE. Overall, this study provides significant implications for future research on the next generation of rapid-acting, glutamate-based antidepressant drugs.

A Highly Selective MNK Inhibitor Rescues Deficits Associated with Fragile X Syndrome in Mice.

Fragile X syndrome (FXS) is the most common inherited source of intellectual disability in humans. FXS is caused by mutations that trigger epigenetic silencing of the Fmr1 gene. Loss of Fmr1 results in increased activity of the mitogen-activated protein kinase (MAPK) pathway. An important downstream consequence is activation of the mitogen-activated protein kinase interacting protein kinase (MNK). MNK phosphorylates the mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E). Excessive phosphorylation of eIF4E has been directly implicated in the cognitive and behavioral deficits associated with FXS. Pharmacological reduction of eIF4E phosphorylation is one potential strategy for FXS treatment. We demonstrate that systemic dosing of a highly specific, orally available MNK inhibitor, eFT508, attenuates numerous deficits associated with loss of Fmr1 in mice. eFT508 resolves a range of phenotypic abnormalities associated with FXS including macroorchidism, aberrant spinogenesis, and alterations in synaptic plasticity. Key behavioral deficits related to anxiety, social interaction, obsessive and repetitive activities, and object recognition are ameliorated by eFT508. Collectively, this work establishes eFT508 as a potential means to reverse deficits associated with FXS.

Conserved Expression of Nav1.7 and Nav1.8 Contribute to the Spontaneous and Thermally Evoked Excitability in IL-6 and NGF-Sensitized Adult Dorsal Root Ganglion Neurons In Vitro.

Sensory neurons respond to noxious stimuli by relaying information from the periphery to the central nervous system via action potentials driven by voltage-gated sodium channels, specifically Nav1.7 and Nav1.8. These channels play a key role in the manifestation of inflammatory pain. The ability to screen compounds that modulate voltage-gated sodium channels using cell-based assays assumes that key channels present in vivo is maintained in vitro. Prior electrophysiological work in vitro utilized acutely dissociated tissues, however, maintaining this preparation for long periods is difficult. A potential alternative involves multi-electrode arrays which permit long-term measurements of neural spike activity and are well suited for assessing persistent sensitization consistent with chronic pain. Here, we demonstrate that the addition of two inflammatory mediators associated with chronic inflammatory pain, nerve growth factor (NGF) and interleukin-6 (IL-6), to adult DRG neurons increases their firing rates on multi-electrode arrays in vitro. Nav1.7 and Nav1.8 proteins are readily detected in cultured neurons and contribute to evoked activity. The blockade of both Nav1.7 and Nav1.8, has a profound impact on thermally evoked firing after treatment with IL-6 and NGF. This work underscores the utility of multi-electrode arrays for pharmacological studies of sensory neurons and may facilitate the discovery and mechanistic analyses of anti-nociceptive compounds.

RNA control in pain: Blame it on the messenger.

mRNA function is meticulously controlled. We provide an overview of the integral role that posttranscriptional controls play in the perception of painful stimuli by sensory neurons. These specialized cells, termed nociceptors, precisely regulate mRNA polarity, translation, and stability. A growing body of evidence has revealed that targeted disruption of mRNAs and RNA-binding proteins robustly diminishes pain-associated behaviors. We propose that the use of multiple independent regulatory paradigms facilitates robust temporal and spatial precision of protein expression in response to a range of pain-promoting stimuli. This article is categorized under: RNA in Disease and Development > RNA in Disease Translation > Translation Regulation RNA Turnover and Surveillance > Regulation of RNA Stability.

5-Methoxy-α-methyltryptamine (5-MeO-AMT), a tryptamine derivative, induces head-twitch responses in mice through the activation of serotonin receptor 2a in the prefrontal cortex.

5-Methoxy-α-methyltryptamine (5-MeO-AMT) is a tryptamine derivative that is used recreationally because of its reported hallucinogenic and mood elevating effects. Studies suggest that the psychopharmacological effects of tryptamines involve serotonin receptor 2a (5-HTR2a) activation in the brain. The head-twitch response (HTR) is widely used as a behavioral correlate for assessing 5-HTR2a agonist activity of a drug. Thus, we investigated whether 5-MeO-AMT induces HTR in mice and explored its mechanism of action. 5-MeO-AMT (0.3, 1, 3, 10 mg/kg) was administered once a day for 7 days, and the HTR was measured after 1 day (acute) and 7 days (repeated) of administration. Another cohort of mice was treated with 5-HTR2a antagonist ketanserin (KS) before 5-MeO-AMT administration. We measured 5-HTR2a and 5-HTR2c mRNA levels in the prefrontal cortex of the mice treated acutely or repeatedly with 5-MeO-AMT. We performed western blotting to determine the effects of the drug on the expression of G protein (Gq/11), protein kinase C gamma (PKC-γ), and extracellular signal-regulated kinases 1/2 (ERK1/2), in addition to PKC-γ and ERK1/2 phosphorylation. Additionally, we evaluated potential rewarding and reinforcing effects of 5-MeO-AMT using locomotor sensitization, conditioned place preference (CPP), and self-administration (SA) paradigms. Acute 5-MeO-AMT administration elicited the HTR, while repeated administration resulted in tolerance. KS blocked the 5-MeO-AMT-induced HTR. 5-MeO-AMT increased 5-HTR2a mRNA levels and induced PKC-γ phosphorylation in the prefrontal cortex. 5-MeO-AMT did not induce locomotor sensitization, CPP, or SA. This study shows that 5-MeO-AMT induces HTR through 5-HTR2a activation in the prefrontal cortex, and may have low potential for abuse.

The circadian gene, Per2, influences methamphetamine sensitization and reward through the dopaminergic system in the striatum of mice.

Drug addiction is a chronic and relapsing brain disorder, influenced by complex interactions between endogenous and exogenous factors. Per2, a circadian gene, plays a role in drug addiction. Previous studies using Per2-knockout mice have shown a role for Per2 in cocaine, morphine and alcohol addiction. In the present study, we investigated the role of Per2 in methamphetamine (METH) addiction using Per2-overexpression and knockout mice. We observed locomotor sensitization responses to METH administration, and rewarding effects using a conditioned place preference test. In addition, we measured expression levels of dopamine and dopamine-related genes (monoamine oxidase A, DA receptor 1, DA receptor 2, DA active transporter, tyrosine hydroxylase and cAMP response element-binding protein 1) in the striatum of the mice after repeated METH treatments, using qRT-PCR. Per2-overexpressed mice showed decreased locomotor sensitization and rewarding effects of METH compared to the wildtype mice, whereas the opposite was observed in Per2 knockout mice. Both types of transgenic mice showed altered expression levels of dopamine-related genes after repeated METH administration. Specifically, we observed lower dopamine levels in Per2-overexpressed mice and higher levels in Per2-knockout mice. Taken together, Per2 expression levels may influence the addictive effects of METH through the dopaminergic system in the striatum of mice.

Methoxetamine: A foe or friend?

Methoxetamine (MXE) is an N-methyl-D-aspartate (NMDA) receptor antagonist that is chemically and pharmacologically similar to other dissociative substances, such as ketamine and phencyclidine. There are reports on the misuse of MXE, which sometimes resulted in adverse consequences and death. Studies have also shown that MXE has abuse liability and stimulates dopamine neurotransmission in the mesolimbic reward pathway in the brain. These findings have contributed to the negative impression on MXE. However, recent preclinical studies have identified the antidepressant properties of MXE, which are attributed to its ability to affect the glutamatergic and serotonergic systems. MXE is also reported to have analgesic effects. These findings show some of the "redeeming qualities" of MXE and indicate its possible therapeutic uses. In this paper, we have reviewed the findings that provide insights into the adverse and potential therapeutic effects of MXE. We compiled studies on the toxicity, psychotomimetic effects, and abuse liability of MXE, as well as its promising antidepressant and analgesic properties. We also have discussed the mechanism of action that might mediate the somewhat paradoxical effects observed. Importantly, this review provides valuable information on MXE for future research and will enable a better understanding of its psychopharmacological properties and the mechanisms responsible for its unique effects.

The Atxn7-overexpressing mice showed hyperactivity and impulsivity which were ameliorated by atomoxetine treatment: A possible animal model of the hyperactive-impulsive phenotype of ADHD.

Attention-deficit/hyperactivity disorder (ADHD) is a heterogeneous neurodevelopmental disorder characterized by varying levels of hyperactivity, inattention, and impulsivity. Patients with ADHD are often classified as (1) predominantly hyperactive-impulsive, (2) predominantly inattentive, and (3) combined type. There is a growing interest in developing specific animal models that would recapitulate specific clinical forms of ADHD, with the goal of developing specific therapeutic strategies. In our previous study, we have identified Ataxin-7 (Atxn7) as a hyperactivity-associated gene. Here, we generated Atxn7 overexpressing (Atxn7 OE) mice to investigate whether the increased Atxn7 expression in the brain correlates with ADHD-like behaviors. Quantitative real-time polymerase chain reaction and immunofluorescence confirmed overexpression of the Atxn7 gene and protein in the prefrontal cortex (PFC) and striatum (STR) of the Atxn7 OE mice. The Atxn7 OE mice displayed hyperactivity and impulsivity, but not inattention. Interestingly, treatment with the ADHD drug, atomoxetine (3 mg/kg, intraperitoneal), attenuated ADHD-like behaviors and reduced Atxn7 gene expression in the PFC and STR of these mice. These findings suggest that Atxn7 plays a role in the pathophysiology of ADHD, and that the Atxn7 OE mice can be used as an animal model of the hyperactive-impulsive phenotype of this disorder. Although confirmatory studies are warranted, the present study provides valuable information regarding the potential genetic underpinnings of ADHD.

RNA-binding proteins as targets for pain therapeutics.

RNA-protein interactions permeate biology. Transcription, translation, processing, and mRNA decay all hinge on widespread use of regulatory information decoded by RNA-binding proteins. The final committed step of protein synthesis, translation, is intimately linked to nociceptor excitability. Understanding the factors that control translation is essential as nociceptor plasticity is a hallmark of persistent pain. Here, we review the growing body of evidence for widespread involvement of RNA-binding proteins in pain. Many of the relevant factors have been implicated in post-transcriptional and translational mechanisms of mRNA control. We propose that recent advances in the development of RNA-based therapeutics provide a potential means to exploit our current understanding of liaisons between RNAs and proteins for therapeutic purposes.

Overexpression of the Thyroid Hormone-Responsive (THRSP) Gene in the Striatum Leads to the Development of Inattentive-like Phenotype in Mice.

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder that affects 8-12% of children globally. Factor analyses have divided ADHD symptoms into two domains: inattention and a combination of hyperactivity and impulsivity. The identification of domain-specific genetic risk variants may help uncover potential genetic mechanisms underlying ADHD. We have previously identified that thyroid hormone-responsive (THRSP) gene expression is upregulated in spontaneously hypertensive rats (SHR/NCrl) and Wistar-Kyoto (WKY/NCrl) rats which exhibited inattention behavior. Thus, we established a line of THRSP overexpressing (OE) mice and assessed their behavior through an array of behavioral tests. The gene and protein overexpression of THRSP in the striatum (STR) was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The THRSP OE mice exhibited inattention in the novel-object recognition and Y-maze test, but not hyperactivity in the open-field test and impulsivity in the cliff-avoidance and delay-discounting task. We have also found that expression of dopamine-related genes (dopamine transporter, tyrosine hydroxylase, and dopamine D1 and D2 receptors) in the STR increased. Treatment with methylphenidate (5 mg/kg), the most commonly used medication for ADHD, improved attention and normalized expression levels of dopamine-related genes in THRSP OE mice. Our findings suggest that THRSP plays a role in the inattention phenotype of ADHD and that the THRSP OE mice may be used as an animal model to elucidate the genetic mechanisms of the disorder.

Exploring the Validity of Proposed Transgenic Animal Models of Attention-Deficit Hyperactivity Disorder (ADHD).

Attention-deficit/hyperactivity disorder (ADHD) is a common, behavioral, and heterogeneous neurodevelopmental condition characterized by hyperactivity, impulsivity, and inattention. Symptoms of this disorder are managed by treatment with methylphenidate, amphetamine, and/or atomoxetine. The cause of ADHD is unknown, but substantial evidence indicates that this disorder has a significant genetic component. Transgenic animals have become an essential tool in uncovering the genetic factors underlying ADHD. Although they cannot accurately reflect the human condition, they can provide insights into the disorder that cannot be obtained from human studies due to various limitations. An ideal animal model of ADHD must have face (similarity in symptoms), predictive (similarity in response to treatment or medications), and construct (similarity in etiology or underlying pathophysiological mechanism) validity. As the exact etiology of ADHD remains unclear, the construct validity of animal models of ADHD would always be limited. The proposed transgenic animal models of ADHD have substantially increased and diversified over the years. In this paper, we compiled and explored the validity of proposed transgenic animal models of ADHD. Each of the reviewed transgenic animal models has strengths and limitations. Some fulfill most of the validity criteria of an animal model of ADHD and have been extensively used, while there are others that require further validation. Nevertheless, these transgenic animal models of ADHD have provided and will continue to provide valuable insights into the genetic underpinnings of this complex disorder.

Neurobiological Functions of the Period Circadian Clock 2 Gene, Per2.

Most organisms have adapted to a circadian rhythm that follows a roughly 24-hour cycle, which is modulated by both internal (clock-related genes) and external (environment) factors. In such organisms, the central nervous system (CNS) is influenced by the circadian rhythm of individual cells. Furthermore, the period circadian clock 2 (Per2) gene is an important component of the circadian clock, which modulates the circadian rhythm. Per2 is mainly expressed in the suprachiasmatic nucleus (SCN) of the hypothalamus as well as other brain areas, including the midbrain and forebrain. This indicates that Per2 may affect various neurobiological activities such as sleeping, depression, and addiction. In this review, we focus on the neurobiological functions of Per2, which could help to better understand its roles in the CNS.

A new synthetic drug 5-(2-aminopropyl)indole (5-IT) induces rewarding effects and increases dopamine D1 receptor and dopamine transporter mRNA levels.

In recent years, there has been a marked increase in the use of recreational synthetic psychoactive substances, which is a cause of concern among healthcare providers and legal authorities. In particular, there have been reports on the misuse of 5-(2-aminopropyl)indole (5-API; 5-IT), a new synthetic drug, and of fatal and non-fatal intoxication. Despite these reports, little is known about its psychopharmacological effects and abuse potential. Here, we investigated the abuse potential of 5-IT by evaluating its rewarding and reinforcing effects through conditioned place preference (CPP) (1, 10, and 30 mg/kg, i.p.) in mice and self-administration test (0.1, 0.3, 1, and 3 mg/kg/inf., i.v.) in rats. We also examined whether 5-IT (1, 3, and 10 mg/kg, i.p.) induces locomotor sensitization in mice following a 7-day treatment and drug challenge. Then, we explored the effects of 5-IT (10 mg/kg, i.p.) on dopamine-related genes in the striatum, prefrontal cortex (PFC), and substantia nigra pars compacta (SNc)/ventral tegmental (VTA) of mice by quantitative real-time polymerase chain reaction. 5-IT produced CPP in mice but was not reliably self-administered by rats. 5-IT also induced locomotor sensitization following repeated administration and drug challenge. Moreover, 5-IT increased mRNA levels of dopamine D1 receptor in the striatum and PFC and dopamine transporter in the SNc/VTA of mice. These results indicate that 5-IT has psychostimulant and rewarding properties, which may be attributed to its ability to affect the dopaminergic system in the brain. These findings suggest that 5-IT poses a substantial risk for abuse and addiction in humans.

Methylphenidate and Atomoxetine-Responsive Prefrontal Cortical Genetic Overlaps in "Impulsive" SHR/NCrl and Wistar Rats.

Impulsivity, the predisposition to act prematurely without foresight, is associated with a number of neuropsychiatric disorders, including attention-deficit/hyperactivity disorder (ADHD). Identifying genetic underpinnings of impulsive behavior may help decipher the complex etiology and neurobiological factors of disorders marked by impulsivity. To identify potential genetic factors of impulsivity, we examined common differentially expressed genes (DEGs) in the prefrontal cortex (PFC) of adolescent SHR/NCrl and Wistar rats, which showed marked decrease in preference for the large but delayed reward, compared with WKY/NCrl rats, in the delay discounting task. Of these DEGs, we examined drug-responsive transcripts whose mRNA levels were altered following treatment (in SHR/NCrl and Wistar rats) with drugs that alleviate impulsivity, namely, the ADHD medications methylphenidate and atomoxetine. Prefrontal cortical genetic overlaps between SHR/NCrl and Wistar rats in comparison with WKY/NCrl included genes associated with transcription (e.g., Btg2, Fos, Nr4a2), synaptic plasticity (e.g., Arc, Homer2), and neuron apoptosis (Grik2, Nmnat1). Treatment with methylphenidate and/or atomoxetine increased choice of the large, delayed reward in SHR/NCrl and Wistar rats and changed, in varying degrees, mRNA levels of Nr4a2, Btg2, and Homer2, genes with previously described roles in neuropsychiatric disorders characterized by impulsivity. While further studies are required, we dissected potential genetic factors that may influence impulsivity by identifying genetic overlaps in the PFC of "impulsive" SHR/NCrl and Wistar rats. Notably, these are also drug-responsive transcripts which may be studied further as biomarkers to predict response to ADHD drugs, and as potential targets for the development of treatments to improve impulsivity.