The striatal heterogeneous nuclear ribonucleoprotein H1 mRNA targetome associated with methamphetamine administration and behavior.

Individuals with methamphetamine use disorder (MUD) can experience significant suffering due to the harmful effects of methamphetamine (METH) on physical and mental health. Although there are no approved medications for MUD, immunotherapies, including monoclonal antibodies (mAb), could serve as treatments for this debilitating condition. Our candidate anti-METH mAb called "devextinetug" features a mouse-derived variable binding region called "7F9" and a humanized constant domain. Our earlier work has shown that devextinetug is effective in altering METH's pharmacological effects. However, it is unknown whether the effectiveness of a chimeric mAb would be altered, or possibly improved, if the variable region were humanized. Moreover, no studies have determined whether onset and offset rates of binding to METH predicts anti-METH effectiveness of mAb candidates. The goals of the present study were to compare the anti-METH effectiveness of a chimeric mAb (called "IS12") that features our parental 7F9 variable region with a panel of mAbs that have fully humanized variable regions, and to determine whether onset and/or offset rates of binding to METH are associated with anti-METH effectiveness. We humanized the variable region from ch-mAb7F9 with multiple sequences to produce 48 IgGs. The on and off rates to METH binding were determined for these IgGs and, based on their various onset/offset rats, we chose eight candidate IgGs for further testing (including our parental IS12). All eight IgGs were tested for ligand cross-reactivity and in a METH-elicited locomotor stimulation model in rats. Cross-reactivity results revealed that IS12 exhibited the greatest affinity to METH, and it also produced the largest reduction in METH-elicited locomotor stimulation compared to the other seven candidates. Furthermore, onset and offset rates of binding to METH did not appear to be associated with reducing METH's in vivo pharmacological effects. The 7F9 variable region is the most promising to treat MUD.

Locomotor stimulant and drug discrimination effects of five synthetic cathinones in rodents.

3-chloromethcathinone (3-CMC) is a synthetic cathinone that gained relevance, having been involved in a large number of seizures and poisoning reports. Despite this, literature currently lacks information on its pharmaco-toxicological effects. This study aims to investigate the acute sensorimotor and physiological effects of 3-CMC (0.1–30 mg/kg; i.p.) in male and female CD-1 mice and its effects (1 and 10 mg/kg) on Prepulse Inhibition (PPI). Furthermore, we describe a series of 3-CMC (or CMC)-related human intoxications (Italy, 2014–2025) registered by the PCC–National Toxicology Information Centre. Finally, we predicted the ADMET properties of 3-CMC compared to 2-CMC, 4-CMC, 2-MMC, and two 3-CMC metabolites. 3-CMC induced in mice locomotor stimulation in mice, relevant tachypnoea and hypothermia, sensorimotor, and PPI alterations were observed only at high doses, with minor sex differences. All intoxications were non-fatal and involved male patients showing psychomotor agitation, psychosis, aggressiveness, CNS depression, but also cardiac arrhythmias, thoracic pain, and tachypnea. N-dealkylation, N-hydroxylation, and phenyl hydroxylation were the main predicted reactions. Drug–drug interaction potential and cardiotoxicity were suggested for all compounds. This interdisciplinary study elucidates 3-CMC effects and its associated risks, opening new objectives for future studies on CMC compounds to provide critical information to clinicians and the toxicological field.

The delta opioid receptor (DOR) is expressed broadly throughout the central and peripheral nervous systems. Activation of DOR by exogenous and endogenous ligands regulates pain, motivation, emotion and memory, but the cells and neural circuits mediating these behavioral effects remain poorly characterized. Parvalbumin-expressing (PV) interneurons express high levels of DOR transcript (OPRD1) and protein (Birdsong et al 2019). Parvalbumin (PV) interneurons also play a role in pain and emotional processing, suggesting that DOR signaling on PV interneurons may modulate these behaviors. To address this question we used a conditional knockout mouse line (Floxed DOR; PV-cre) to delete DOR from PV-expressing cells. First, we validated the functional loss of DOR through whole-cell electrophysiology experiments. Next, we characterized baseline behavioral phenotypes and the convulsant, pro-locomotive and anxiolytic-like behavioral responses induced by the DOR agonist SNC80 in Floxed DOR; PV-Cre mice and their littermate controls. Interestingly, we found that the convulsant and anxiolytic effects of the DOR agonist SNC80 were diminished in Floxed DOR;PV Cre animals. However, baseline behavioral phenotypes, SNC80-induced spontaneous hyperlocomotion and respiratory stimulation were conserved. These novel findings indicate that the pro-convulsant and anxiolytic effects of DOR agonists are anatomically separable from the locomotor stimulating effects and are dependent on DOR expression on PV interneurons.

Swiss-Webster and C57BL/6 mice are differentially sensitive to the stimulant effects of methamphetamine.

While alcohol use disorder can be treated with pharmacological interventions, ketosis is a recently proposed treatment option. Ketosis, defined by elevated concentrations of ketone bodies such as β-hydroxybutyrate (BHB), can be induced by a ketogenic diet or by supplements. As a supplement, both the salt and ester formulation of BHB rapidly increase blood ketone levels. Although preclinical studies have revealed that a ketogenic diet or a mix of ketone supplements reduces alcohol intake and alleviates withdrawal symptoms, the impact of BHB supplements on alcohol-related responses remains to be defined. We first assessed the efficacy of BHB in ester versus salt formulation on general locomotor activity, exogenous ketosis and alcohol-induced locomotor stimulation in male mice. We then investigated the impact of the BHB salt on alcohol intake in male and female rats. In attempts to define mechanisms influenced by the BHB salt, monoamines and their metabolites were measured in the nucleus accumbens (NAc), a brain region associated with alcohol reward. Initial results indicate that the BHB salt had a greater impact on ketone levels, glucose-ketone index and inhibition of alcohol-induced locomotor stimulation compared to the BHB ester, without altering the general locomotor activity. We further found that BHB salt dose-dependently lowered alcohol intake in rats of both sexes and that females responded to lower doses than males. Moreover, BHB salt elevated dopamine and noradrenaline and their metabolites in the NAc of male mice. Overall, this study provides insight into the role of BHB salt in modulating rodent alcohol-related behaviours.

The prevalence of fluorinated amphetamine and methamphetamine analogs on the illicit market has continued to increase in the past decade. The perceived ability of these compounds to bypass legal regulation has resulted in an increasing popularity among drug users; however, their use produces significant adverse effects, including heart failure, cerebral hemorrhage, and death. This study aimed to investigate the effects of phenyl ring fluorination on the abuse potential of 5 synthetic stimulant compounds: 2- and 3-fluoroamphetamine (FA) and 2-, 3-, and 4-fluoromethamphetamine (FMA). The open-field assay was used to observe the locomotor effects of the compounds and to evaluate effective dose ranges and time courses for psychoactive effects in male Swiss-Webster mice. Discriminative stimulus effects were evaluated using male Sprague-Dawley rats trained to discriminate methamphetamine from saline using an fixed-ratio 10 food-maintained reinforcement schedule in a 2-lever operant box. The compounds tested all resulted in time- and dose-dependent stimulation of locomotor activity. Potencies (ED50) ranged from 0.38 to 7.38 mg/kg, with rank-order potency of 2-FMA > methamphetamine > 3-FMA = 3-FA = 2-FMA > 4-FMA. Peak effects varied, with 2-FA, 3-FA, and 3-FMA showing peak effects similar to methamphetamine (5905-7758 counts), while 2-FMA and 4-FMA were weak stimulants with lower peak effects (2200-3980 counts). All fluorinated compounds elicited dose-dependent full substitution for methamphetamine with comparable potencies (ED50 values = 0.32-0.71 mg/kg). The present study indicates that these analogs may have a potential for abuse comparable to that of methamphetamine, although self-administration studies need to be conducted to confirm this, and the locomotor activity data highlight possible mechanistic differences between the positional analogs through the contrasting potencies and efficacies. SIGNIFICANCE STATEMENT: Fluorinated amphetamine analogs have appeared on the illicit market and produce significant adverse effects. The present study shows that these analogs produce methamphetamine-like locomotor stimulant and discriminative stimulus effects and so may have a potential for abuse comparable to that of methamphetamine and other psychostimulants.

Methamphetamine addiction remains a major public health concern in the United States that has paralleled the opioid epidemic. Psychostimulant use disorders have a heritable genetic component that remains unexplained. Methamphetamine targets membrane and vesicular transporters to increase synaptic dopamine, norepinephrine, and serotonin. We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene underlying methamphetamine behavioral sensitivity. Hnrnph1 encodes the RNA-binding protein hnRNP H1 that is ubiquitously expressed in neurons throughout the brain. Gene-edited mice with a heterozygous frameshift deletion in Hnrnph1’s first coding exon of showed reduced methamphetamine-induced dopamine release and behaviors. To inform the mechanism linking hnRNP H with methamphetamine neurobehavioral effects, we surveyed the mRNA targetome of hnRNP H via cross-linking immunoprecipitation coupled with RNA-sequencing in striatal tissue at baseline and at 30 min post-methamphetamine in wild-type male and female C57BL/6J mice. Methamphetamine induced changes in RNA-binding targets of hnRNP H in mice, including differential binding to 3’UTR targets and multiple enriched mRNAs involved in synaptic plasticity. Targetome, transcriptome, and spliceome analyses triangulated on Cacna2d2 as a suggestive target, with differences in hnRNP H binding, gene expression and splicing following methamphetamine treatment (2 mg/kg, i.p.). Furthermore, pre-treatment with pregabalin, an inhibitor of α2δ2 and α2δ1 voltage-gated calcium channel subunits, attenuated methamphetamine-induced locomotor activity in male and female mice, supporting a role for Cacna2d1/d2 in methamphetamine locomotor stimulant sensitivity. Our study identifies a dynamic hnRNP H RNA targetome that can rapidly and adaptively respond to methamphetamine to regulate gene expression and likely synaptic plasticity and behavior.

An inhibitory GLP-1 circuit in the lateral septum modulates reward processing and alcohol intake in rodents.

Ketamine, a fast-acting antidepressant, is a racemic mixture, composed of equal amounts of R- and S-ketamine. Preclinical studies are comparing them to better understand their role in therapeutic and undesirable effects. An important research gap is that studies do not use long clinically relevant protocols to compare the desired and undesired effects of ketamine enantiomers in modeled and control animals. In our preclinical study, we explored the behavioral effects of R- and S-ketamine at 10mg/kg in clinically relevant treatment protocol using Wistar-Kyoto rats as a depression model and Wistar rats as a control. Undesirable psychomotor effects were evaluated with locomotor stimulation and sensitization, ataxia, and stereotypy. Persistent effects associated with therapeutic outcomes were evaluated by measuring working memory, anxiety, and behavioral despair. We found that S-ketamine has stronger acute psychomotor effects compared to R-ketamine and that Wistar-Kyoto rats are more sensitive to these effects compared to Wistar rats. After repeated treatment, sensitization to locomotor stimulating effects, and tolerance to ataxic effects of S-ketamine develops. We found no persistent changes due to ketamine treatments. Taken together, our results may indicate that depressed patients would be more prone to negative side effects of ketamine, compared to healthy controls. However, after repeated treatment, tolerance to side effects may develop and make the treatment more tolerable. Future preclinical and clinical studies are needed to address neurobiological mechanisms and clinical relevance of higher sensitivity to the psychomotor effects of ketamine, and the development of tolerance to psychomotor effects of ketamine in depressed individuals.

BackgroundMethoxphenidine (MXP) is a new psychoactive substance that belongs to the group of arylethylamines. Even though MXP has a different pharmacophore than ketamine, both of these substances belong to the dissociatives. To date, MXP has been associated with several intoxications and despite it being banned in most countries it is still available over the internet.1Aims & ObjectivesEven though MXP started to be abused approximately a decade ago, there is still a lack of data about it. Considering the knowledge gap on MXP toxicological data, its mostly unknown metabolism and overall the significant potential of dissociative anaesthetics for depression treatment, we aimed to thoroughly evaluate this substance.MethodThe MXP, nor-MXP and MXP-d3 were in-house synthesized and characterized using an 400 MHz NMR spectrometer (JEOL, Japan) and HRMS (LTQ Orbitrap Velos, ThermoFisher, USA), which was also used for the untargeted screening. The targeted screening was performed using the LC system Dionex Ultimate 3000 (Thermo-Fisher, USA) and the chromatographic separation was performed using Poroshell 120, Phenyl Hexyl (2.1 × 100 mm, 2.7 μ m) column (Agilent, USA). The behavioural experiments were performed on Wistar rats acquired from VELAZ (Prague, CZ). The open field test (OFT) was done in a sound-proof and evenly lit chamber with low levels of light intensity and pre-pulse inhibition (PPI) took place in startle chambers (SR-LAB, San Diego Instruments, USA). The rats were decapitated after 0.5, 1, 2, 4, 8 and 24 hours.ResultsThe MXP (60 mg/kg s.c.) was administered to the Wistar rats, the urine was collected and analysed. Using the untargeted screening we proposed structures of several metabolites and we synthesized the nor-MXP metabolite and deuterium labelled analogue for the upcoming pharmacokinetics. The MXP was administered to the Wistar rats (10, 20 and 40 mg/kg s.c.) for behavioural experiments in which we studied locomotor effects in OFT and sensorimotor gating in PPI. The obtained serum and brain were analysed using targeted LC/MS screening and the concentrations were used for calculation of the pharmacokinetics.Discussion & ConclusionThe maximal effect of the MXP was observed around 30 minutes after administration and the maximal effect prevailed another 0.5 hour. These data were consistent with the pharmacokinetic data, in which the concentration of MXP and its nor-MXP metabolite rapidly increased with a maximum of around 1 hour. The pharmacokinetic profile MXP and the nor-MXP suggest a slower metabolism and excretion of the MXP compared to the ketamine. Lower doses (10 and 20 mg/kg) induced significant locomotor stimulation suggesting MXP has a stimulating effect, which is typical for other dissociative anaesthetics, on the other hand, the higher dose increased the time spent in the centre of the arena, which suggests rather sedating effects. Even though MXP is a substance with a high potential for abuse, further research is needed to provide deep insight into its antidepressant effects.AcknowledgementThis study was funded by the Ministry of Interior of the Czech Republic (New psychoactive substances: forensic-toxicology research center, VK01010212).ReferencesJurá sek, B. et al. (2023) 'A structural spectroscopic study of dissociative anaesthetic methoxphenidine’, New Journal of Chemistry, 47(9), pp. 4543–4551. doi: 10.1039/D2NJ06126K.

Identification of N,N-dimethylpentylone (DMP) in counterfeit "Ecstasy" and "Molly" tablets poses risk to public health due to its adverse effects. Little information is available regarding the pharmacological activity or relevant blood or tissue concentrations of DMP, and even less is known about other structurally related beta-keto methylenedioxyamphetamine analogs on recreational drug markets, such as N-propyl butylone. Here, a novel toxicological assay utilizing liquid chromatography-tandem quadrupole mass spectrometry was developed and validated for the quantitation of DMP and five related synthetic cathinones [eutylone, pentylone, N-ethyl pentylone (NEP), N-propyl butylone, and N-cyclohexyl butylone], with chromatographic resolution from isomeric variants and quantitation performed by standard addition. A forensic series of 125 cases is presented for DMP and related analogs, along with pharmacological activity assessments using monoamine transporter and mouse behavioral assays. The blood concentration range for DMP in postmortem forensic cases was 3.3-4600 ng/mL (mean: 320 ± 570 ng/mL, median: 150 ng/mL), whereas pentylone, the primary N-desmethyl metabolite of DMP, was identified in 98% of cases with a concentration range 1.3-710 ng/mL (mean ± SD: 105 ± 120 ng/mL, median: 71 ng/mL). N-Propyl butylone, a newly identified synthetic cathinone, was quantitated in seven cases (mean ± SD: 82 ± 75 ng/mL, median: 50 ng/mL, range: 1.7-200 ng/mL). DMP displayed potent uptake inhibition at the dopamine transporter [half maximal inhibitory concentration (IC50) of 49 nM], with 100-fold weaker potency at the serotonin transporter (IC50 = 4990 nM). DMP was a locomotor stimulant in mice [medium effective dose (ED50) of 3.5 mg/kg] exhibiting potency relatively similar to eutylone, NEP, and pentylone. Our results show that DMP is a psychomotor stimulant associated with adverse clinical outcomes leading to death. Forensic laboratories must continue to update testing methods to capture emerging drugs, with specific emphasis on resolution and identification of isomeric species. Following the scheduling of DMP in early 2024, there could be an anticipated market shift toward a new unregulated synthetic stimulant to replace DMP.

Acetylcholine modulates the activity of the direct and indirect pathways within the striatum through interaction with muscarinic M4 and M1 receptors. M4 receptors are uniquely positioned to regulate plasticity within the direct pathway and play a substantial role in reward and addiction-related behaviors. However, the role of M4 receptors on cholinergic neurons has been less explored. This study aims to fill this gap by addressing the role of M4 receptors on cholinergic neurons in these behaviors. To investigate the significance of M4-dependent inhibitory signaling in cholinergic neurons we created mutant mice that lack M4 receptors on cholinergic neurons. Cholinergic neuron-specific depletion was confirmed using in situ hybridization. We aimed to untangle the possible contribution of M4 autoreceptors to the effects of the global M4 knockout by examining aspects of basal locomotion and dose-dependent reactivity to the psychostimulant and rewarding properties of cocaine, haloperidol-induced catalepsy, and examined both the anti-cataleptic and locomotion-inducing effects of the non-selective anticholinergic drug scopolamine. Basal phenotype assessment revealed no developmental deficits in knockout mice. Cocaine stimulated locomotion in both genotypes, with no differences observed at lower doses. However, at the highest cocaine dose tested, male knockout mice displayed significantly less activity compared to wild type littermates (p = 0.0084). Behavioral sensitization to cocaine was similar between knockout and wild type mice. Conditioned place preference tests indicated no differences in the rewarding effects of cocaine between genotypes. In food-reinforced operant tasks knockout and wild type mice successfully acquired the tasks with comparable performance results. M4 receptor depletion did not affect haloperidol-induced catalepsy and scopolamine reversal of catalepsy but attenuated scopolamine-induced locomotion in females (p = 0.04). Our results show that M4 receptor depletion attenuated the locomotor response to high doses of cocaine in males and scopolamine in females, suggesting sex-specific regulation of cholinergic activity. Depletion of M4 receptors on cholinergic neurons does not significantly impact basal behavior or cocaine-induced hyperactivity but may modulate the response to high doses of cocaine in male mice and the response to scopolamine in female mice. Overall, our findings suggest that M4-dependent autoregulation plays a minor but delicate role in modulating specific behavioral responses to pharmacological challenges, possibly in a sex-dependent manner.

Conditioned place preference with low dose mixtures of α-pyrrolidinopentiophenone (α-PVP) and 3,4-methylenedioxypyrovalerone (MDPV) in male and female Sprague-Dawley rats

The underlying neurobiology of alcohol use disorder (AUD) is complex and needs further unraveling, with one of the key mechanisms being the gut-brain peptide ghrelin and its receptor (GHSR). However, additional substrates of the ghrelin pathway, such as liver-expressed antimicrobial peptide 2 (LEAP2), an endogenous GHSR inverse agonist, may contribute to this neurobiological framework. While LEAP2 modulates feeding and reward through central mechanisms, its effects on alcohol responses are unknown. The aim of the present study was therefore to identify the impact of central LEAP2 on the ability of alcohol to activate the mesolimbic dopamine system and to define its ability to control alcohol intake. These experiments revealed that central LEAP2 (i.e. into the third ventricle) prevented the ability of alcohol to cause locomotor stimulation in male mice, suppressed the memory of alcohol reward and attenuated the dopamine release in the nucleus accumbens caused by alcohol. Moreover, central LEAP2 reduced alcohol consumption in both male and female rats exposed to alcohol for 6 weeks before treatment. However, the serum levels of LEAP2 were similar between high- and low- alcohol-consuming (male) rats. Furthermore, central LEAP2 lowered the food intake in the alcohol-consuming male rats and reduced the body weight in the females. Collectively, the present study revealed that central LEAP2 mitigates alcohol-related responses in rodents, contributing to our understanding of the ghrelin pathway’s role in alcohol effects.

Opioid-environment interaction: Contrasting effects of morphine administered in a novel versus familiar environment on acute and repeated morphine induced behavioral effects and on acute morphine ERK activation in reward associated brain areas

Under conditions of repeated exposure to ethanol, a sensitized locomotor stimulant response develops in some strains of mice. It has been hypothesized that the sensitized response is a consequence of tolerance development to the sedative/incoordinating effects of ethanol. Conversely, ethanol-induced sensitization and tolerance may be independent effects of repeated ethanol exposure. A published study in C57BL/6J by DBA/2J recombinant inbred strains concluded that the two phenomena are not genetically related and thus perhaps mechanistically distinct. To extend evaluation beyond the genetic variance found in C57BL/6J and DBA/2J mice and examine phenotypic associations, we simultaneously measured ethanol-induced sensitization and tolerance in a genetically diverse panel of 15 standard inbred mouse strains and a genetically heterogeneous stock that was produced by the intercrossing of eight inbred mouse strains. Changes in activity counts and ataxia ratio across repeated ethanol treatments indexed sensitization and tolerance, respectively. Photocell beam breaks provided the measure of activity, and foot slip errors corrected for activity in a grid test provided a measure of coordination. The results were strain and individual dependent. The genetic correlation between magnitude of sensitization and tolerance was not significant in the panel of inbred strains, but when individual data were correlated, without regard to strain, there was a significant correlation. This relationship was also significant in the genetically heterogeneous population of mice. However, magnitude of tolerance explained only 10% of the variance in sensitization among individuals of the inbred strain population, whereas it explained 44% of the variance among individuals of the eight-strain cross. When repeated exposures to ethanol were disassociated from the test apparatus, this relationship in the eight-strain cross disappeared. Furthermore, days to peak sensitization and tolerance across days did not perfectly mirror each other. Overall, our data do not support shared genetic mechanisms in sensitization and tolerance development but suggest a partial relationship among individuals that could be related to drug–environment associations.

Sex has a strong influence on the prevalence and course of brain conditions, including autism spectrum disorders. The mechanistic basis for these sex differences remains poorly understood, due in part to historical bias in biomedical research favoring analysis of male subjects, and the exclusion of female subjects. For example, studies of male mice carrying autism-associated mutations in neuroligin-3 are over-represented in the literature, including our own prior work showing diminished responses to chronic morphine exposure in male neuroligin-3 knockout mice. We therefore studied how constitutive and conditional genetic knockout of neuroligin-3 affects morphine sensitivity of female mice, using locomotor activity as a proxy for differences in opioid sensitivity that may be related to the pathophysiology and treatment of autism spectrum disorders. In contrast to male mice, female neuroligin-3 knockout mice showed normal psychomotor sensitization after chronic morphine exposure. However, in the absence of neuroligin-3 expression, both female and male mice show a similar change in the topography of locomotor stimulation produced by morphine. Conditional genetic deletion of neuroligin-3 from dopamine neurons increased the locomotor response of female mice to high doses of morphine, contrasting with the decrease in psychomotor sensitization caused by the same manipulation in male mice. Together, our data reveal that knockout of neuroligin-3 has both common and distinct effects on morphine sensitivity in female and male mice. These results also support the notion that female sex can confer resilience against the impact of autism-associated gene variants.

The mechanisms contributing to alcohol use disorder (AUD) are complex and the orexigenic peptide ghrelin, which enhances alcohol reward, is implied as a crucial modulator. The major proportion of circulating ghrelin is however the non-octanoylated form of ghrelin, des-acyl ghrelin (DAG), whose role in reward processes is unknown. As recent studies show that DAG decreases food intake, we hypothesize that DAG attenuates alcohol-related responses in animal models. Acute and repeated DAG treatment dose-dependently decreased alcohol drinking in male and female rats. In these alcohol-consuming male rats, repeated DAG treatment causes higher levels of dopamine metabolites in the ventral tegmental area, an area central to reward processing. The role of DAG in reward processing is further supported as DAG prevents alcohol-induced locomotor stimulation, reward in the conditioned place preference paradigm, and dopamine release in the nucleus accumbens in male rodents. On the contrary, DAG does not alter the memory of alcohol reward or affect neurotransmission in the hippocampus, an area central to memory. Further, circulating DAG levels are positively correlated with alcohol drinking in female but not male rats. Studies were conducted in attempts to identify tentative targets of DAG, which currently are unknown. Data from these recombinant cell system revealed that DAG does not bind to either of the monoamine transporters, 5HT2A, CB1, or µ-opioid receptors. Collectively, our data show that DAG attenuates alcohol-related responses in rodents, an effect opposite to that of ghrelin, and contributes towards a deeper insight into behaviors regulated by the ghrelinergic signaling pathway.