Depression, a prevalent mental disorder with significant global health implications, remains a complex and multifaceted challenge. Despite extensive research using traditional animal models and clinical trials, the underlying mechanisms of depression remain incompletely understood, hindering the development of effective treatments. Zebrafish (Danio rerio), with their remarkable genetic and neurobiological similarities to humans, have emerged as a powerful tool for investigating the complexities of depression. Zebrafish exhibit a rich and quantifiable behavioral repertoire, with assays such as the novel tank test, light-dark box, open field, shoaling, maze test, and tail immobilization used to assess depression-like phenotypes. Diverse modeling approaches (including genetic and pharmacological manipulations, chronic stress paradigms, and gut-brain axis studies) have been applied, enabling investigations into the physiological, genetic, pharmacological, and environmental drivers of depression. The integration of zebrafish into depression research offers unique opportunities to advance mechanistic insight and accelerate antidepressant discovery. This review summarizes recent progress, examines current challenges, and outlines future directions to maximize the translational potential of zebrafish depression models.

Alzheimer's disease (AD) is a neurodegenerative disease that effects memory and behaviour. The phytocannabinoid cannabidiol (CBD) has been found to reverse impairments of recognition as well as spatial memory deficits of AD transgenic mice but had only limited effects on disease-relevant brain pathologies. Recent evidence suggests that combining CBD with other cannabinoids including delta-9-tetrahydrocannabinol (THC) may lead to improved therapeutic outcomes. Thus, this study evaluated the chronic effects of combined treatment with 3 mg/kg THC and 20 mg/kg CBD on 14.5-month-old APP/PS1ΔE9 (APP/PS1) transgenic females and control littermates. Mice were treated with THCxCBD or vehicle (VEH) daily via intraperitoneal injections for 3 weeks before behavioural testing commenced. AD-relevant behavioural domains were analysed utilising Elevated Plus Maze (EPM), open field (OF), novel object recognition test (NORT), social interaction (SI), Y-maze (YM), and prepulse inhibition test (PPI). APP/PS1 females showed an anxiety-like phenotype and object recognition deficits that remained unchanged by cannabinoid treatment. Interestingly, some effects of THCxCBD appeared genotype-dependent with cannabinoid treatment causing an anxiogenic EPM response in APP/PS1 mice but having an anxiolytic-like effect in WT females. Moreover, THCxCBD administration disrupted the novel object preference of control females. Noteworthy, THCxCBD significantly decreased different fat depots and bodyweight of all mice across genotype. No other differences between genotypes or treatment groups were detected. In conclusion, the particular cannabinoid combination strategy utilised had no prominent therapeutic-like effect in 14.5-month-old APP/PS1 females.

Intranasal infusion of lymphocyte-activating gene-3 antibody (In-LAG3-Ab) has microglia-dependent antidepressant effects, but the underlying mechanism remains unclear. Since microglia can interact with astrocytes through purinergic signaling, we hypothesize that microglia-driven purinergic signaling may mediate the antidepressant effect of In-LAG3-Ab. The results showed that a single In-LAG3-Ab infusion in chronically stressed mice produced both an antidepressant effect and increased adenosine triphosphate (ATP) levels in the dentate gyrus, both of which were suppressed by chemogenetic inhibition of microglia in the dentate gyrus. Depletion of ATP or non-specific antagonism of purinergic receptors abolished the antidepressant effect of In-LAG3-Ab. Specific inhibition of purinergic 2Y1 receptors (P2Y1Rs), but not other purinergic receptors, in the hippocampus or conditional depletion of P2Y1Rs in astrocytes also abolished the antidepressant effect of In-LAG3-Ab. Brain-derived neurotrophic factor (BDNF) may act downstream of astrocytic P2Y1Rs to mediate the antidepressant effect of In-LAG3-Ab, as (i) chemogenetic inhibition of microglia in the dentate gyrus, specific deletion of astrocytic P2Y1Rs, and depletion of endogenous ATP abolished the reversal effect of In-LAG3-Ab on chronic stress-induced decreases in BDNF in the dentate gyrus, and (ii) infusion of BDNF-Ab into the hippocampus abolished the antidepressant effect of In-LAG3-Ab. These results suggest that astrocytic P2Y1R signaling associated with microglia stimulation may mediate the antidepressant effect of In-LAG3-Ab through BDNF.

Alcohol is well known for impairing motor coordination and increasing subjective intoxication. Previous research has found that these effects are exacerbated in women, but such observations were limited to times when blood alcohol concentrations (BACs) were at or near peak. Interestingly, alcohol-induced impairment of motor coordination and subjective intoxication both demonstrate acute tolerance, meaning they recover faster than the decline of BAC as alcohol is eliminated. Consideration of acute tolerance to both measures in tandem is particularly important because if recovery from subjective intoxication outpaces recovery from objective motor impairment, a drinker may develop a false sense of freedom from the impairing effects of alcohol. Such a misjudgment can lead the drinker to engage in risky behavior as BAC declines. The present study examined whether sex differences were present in the acute tolerance to motor impairment and subjective intoxication. Twenty-five women and 25 men participated in a placebo-controlled study of their acute tolerance to motor impairment and subjective intoxication following a moderate dose of alcohol, 0.60 g/kg for women and 0.64 g/kg for men. Repeated assessments of motor coordination with a grooved pegboard and subjective intoxication with a visual analog scale were conducted seven times as BAC declined. While all participants demonstrated acute tolerance to both motor impairment and subjective intoxication, women exhibited significantly faster recovery from subjective intoxication than men. Consequently, women may be more likely than men to engage in risky behavior on the descending limb, such as alcohol-impaired driving.

Clinical and preclinical literature has linked prenatal nicotine exposure to several cognitive effects, including memory and attentional deficits. However, many preclinical studies focus on effects during adulthood and do not use clinically relevant nicotine delivery system.

Bipolar disorder is a chronic psychiatric condition marked by alternating episodes of mania and depression. Current pharmacological treatments show limited efficacy and are often associated with incomplete remission. Creatine, a compound involved in energy buffering, mitochondrial function, and neuromodulation, has demonstrated neuroprotective and potential mood-stabilizing properties. However, the underlying mechanisms remain poorly understood. This study evaluated the effects of acute oral creatine administration (300 mg/kg) on behavioral and molecular changes in a validated rat model of mania induced by intracerebroventricular ouabain (10 M), a Na/K-ATPase inhibitor. Behavioral alterations were assessed using the open field test, and hippocampal samples were analyzed for enzyme activity and key signaling proteins. Creatine significantly attenuated ouabain-induced hyperactivity and increased latency to symptom onset. At the molecular level, creatine preserved Na/K-ATPase activity and reduced phosphorylation of its α-subunit at a regulatory site. Additionally, creatine enhanced activation of the PI3K/AKT/mTOR/p70S6K pathway and prevented the ouabain-induced activation of GSK-3β, a kinase involved in mood regulation. Notably, rapamycin, an mTOR inhibitor, reversed the behavioral effects of creatine, suggesting a mechanistic role for this pathway. These findings demonstrate that acute creatine administration confers both behavioral and neurochemical protection in a mania model and supports its potential as an adjunctive therapeutic strategy for individuals with bipolar disorder.

Opioid use during pregnancy has increased drastically in the last two decades. Pregnant women who use opioids are often prescribed Medications for Opioid Use Disorder (MOUDs), including buprenorphine (BUP), to mitigate negative effects on the fetus. However, BUP exposure during pregnancy may still negatively impact maternal care behavior and offspring neurodevelopment. In the current study, we used a translational rodent model to investigate the effects of continued morphine or BUP use from preconception (7 days prior to mating) to the early postpartum period, as well as the transition from morphine to BUP during early pregnancy (gestational day (GD) 5), on both maternal care behaviors and acute offspring neurodevelopmental outcomes. Our results reveal that exposure to BUP beginning before pregnancy or on GD5 resulted in decreased nesting quality, maternal motivation, and pup-directed care behaviors as compared to controls. For the offspring, BUP-exposure resulted in increased pup mortality, more neonatal opioid withdrawal syndrome-like (NOWS) symptoms, altered norepinephrine levels in the brain, and decreased offspring weight, body length, and presence of milk bands compared to vehicle pups. Importantly, maternal care behavior was significantly correlated with offspring mortality, physical maturation, and NOWS-like scores, suggesting that at least some of the adverse effects were driven by impairments in maternal care. Morphine-exposed dams and pups showed overall fewer impairments compared to BUP-exposed dams and pups. This highlights that more research is needed to further understand the unique impact of BUP on the maternal brain and subsequent infant outcomes to mitigate potential adverse effects in pregnant women with MOUD prescriptions.

Anxiety-like complex behavioral and psychological constructs are difficult to evaluate in rodents. Many studies have investigated which techniques are appropriate for measuring anxiety and related physiological parameters. Here, we used network meta-analysis to compare the current methods of assessing anxiety. We performed a comprehensive review and network meta-analysis by searching PubMed, EMBASE, the Cochrane Library, SCOPUS, and Web of Science for studies involving rodents with anxiety-related behaviors undergoing behavioral tests with certain keywords: The common parameters that emerged were total distance traveled, fecal boli count, and rearing behavior. In the 46 studies reviewed, the open-field test (OFT) and elevated plus maze (EPM) test appeared most often (in 45 and 43 studies, respectively), while the light-dark box (LDB) and elevated zero maze tests appeared less frequently (in two studies and one study, respectively Subsequently, the tests were ranked based on their likelihood of being the most effective measure for each outcome. For total distance traveled, the OFT showed a significant disadvantage over the EPM and LDB. For fecal boli, there was a significant difference between the LDB and OFT. There were no variations between tests in terms of rearing. Our findings reinforce the importance of considering each behavioral test's unique characteristics when selecting appropriate measures for anxiety-like behaviors. Researchers should exercise caution when interpreting single-measure outcomes and adopt a holistic approach that integrates multiple test results to achieve reliable and relevant conclusions. Network meta-analysis is a powerful tool for identifying the highlights, complexities, and inconsistencies of anxiety-related behaviors in rodents in preclinical anxiety models.

Cocaine use disorder (CUD) remains a major public health challenge with no effective pharmacological treatments to date. Although extensive preclinical research using animal models has advanced our understanding, these findings have yet to translate into clinical success. This review highlights key independent variables that should be incorporated to improve the validity and translational relevance of animal studies. The discussion is organized around three primary domains: the agent; the host; and the environment. Most studies in this review focus on cocaine as the agent and host factors such as species and sex are emphasized as important independent variables influencing outcomes. The central theme of this review is to emphasize, from a preclinical model perspective, the critical role of environmental context, including operant conditioning parameters like schedules of reinforcement and maintaining events, as well as social housing conditions, which profoundly impact cocaine-maintained behavior and the effectiveness of interventions for cocaine self-administration.

Synthetic cathinones are synthesized as legal alternatives to methamphetamine, cocaine and MDMA, and can produce serious health risks. The DEA identified five synthetic cathinones of interest: N-ethylpentedrone, N-ethylheptedrone, N-butylhexedrone, 4-methylpentedrone, and 4-methyl-α-ethylaminopentiophenone (4-MEAP). These compounds were tested to determine their locomotor stimulant and psychostimulant-like discriminative stimulus effects. Locomotor activity was tested in male Swiss-Webster mice to identify behaviorally active dose ranges and time courses. Discriminative stimulus effects of the five synthetic cathinones were tested in male Sprague-Dawley rats trained to discriminate cocaine (10 mg/kg, 10-min pretreatment) or methamphetamine (1 mg/kg, 10-min pretreatment) from saline vehicle. Four of the test compounds produced locomotor stimulant effects comparable in efficacy to methamphetamine and cocaine, with rank order of potency: 4-MEAP > N-ethylpentedrone > N-ethylheptedrone >4-methyl-pentedrone. N-butylhexedrone depressed locomotor activity. N-Ethylpentedrone and N-ethylheptedrone substituted for the discriminative stimulus effects produced by methamphetamine and cocaine. 4-MEAP fully substituted for methamphetamine. 4-Methylpentedrone fully substituted for cocaine, but only partially substituted for methamphetamine, while N-butylhexedrone partially substituted for cocaine in DD and failed to substitute for methamphetamine. Three of the compounds, N-ethylpentedrone, N-ethylheptedrone, and 4-MEAP produced robust psychostimulant-like effects, which suggests they will have abuse liability similar to cocaine or methamphetamine. 4-Methylpentedrone was a slow onset locomotor stimulant, and substituted for cocaine and only partially substituted for methamphetamine, which may indicate a weaker abuse liability compared to methamphetamine or cocaine. N-butylhexedrone produced weak psychostimulant-like effects, which suggests it may have limited use as a street drug on its own.

Depression is a chronic mental disorder characterized by alternations in emotions, thoughts, physical condition, and behavior. Using the natural product database Compound Combination-Oriented Natural Product Database with Unified Terminology (COCONUT) and the bioinformatics tool CODA (Context-Oriented Directed Associations), we screened and identified 1,4-naphthoquinone (1,4-NQ) as a promising candidate for depression treatment. Oral administration of 1,4-NQ attenuated the depressive-like behaviors in the open field test (OFT), elevated plus maze test (EPM) and forced swim test (FST) in a chronic restraint stress (CRS)-induced depressive-like mouse model. Real-time PCR analysis demonstrated that 1,4-NQ increased the mRNA levels of 5-HT1A and BDNF in the hippocampus of mouse brains. The expression level of glucocorticoid receptor (GR) in the hippocampus was increased by 1,4-NQ treatment in both CRS- and corticosterone-induced depression mouse models. We confirmed that 1,4-NQ has anti-neuroinflammatory efficacy by suppressing the levels of IL-6, TNF-α and IL-1β in LPS-stimulated BV2 microglial cell line. The western blot and real-time PCR analysis demonstrated that 1,4-NQ increased the level of GR in both the U-138 MG glial cell line and the SH-SY5Y neuronal cell line. In conclusion, 1,4-NQ is supposed to have anti-depressive efficacy by alleviating depressive-like behaviors through modulation of neuroinflammatory mediators and GR expression in the nervous system.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline and behavioral impairments. Thiethylperazine, a dopamine receptor antagonist with antiemetic and antidopaminergic properties, has been proposed as a potential therapeutic agent for AD. However, its impact on cognitive function in AD remains unclear.

The initial neurobehavioral adaptations following the first drug exposure may underlie the transition from recreational to compulsive use in vulnerable individuals. Compelling evidence indicates that early life adversity (ELA) is a significant risk factor for drug dependence. To better understand the relationship between ELA and initial drug experiences, we investigated whether chronic unpredictable stress (CUS) during adolescence modifies the affective (ultrasonic vocalizations, USVs), psychomotor (rearing and locomotion), and neural responses to a single dose of amphetamine in rats. CUS alone led to open-field hyperactivity and reduced flat USVs. CUS significantly blunted amphetamine-induced hyperactivity -suggesting a cross-tolerance effect- while it augmented amphetamine-induced appetitive 50-kHz calls, indicating a cross-sensitization effect. These results might suggest that CUS increases the rewarding and reduces the anxiogenic properties of initial amphetamine experience. At the neural level, amphetamine increased the expression of corticotropin-releasing factor (Crf)-related genes and the 2B subunit of the N-methyl-d-aspartate glutamate receptor (Nr2b) in a region-dependent manner. CUS upregulated the expression of brain-derived neurotrophic factor (Bdnf) in the medial prefrontal cortex (mPFC) and actin-related protein 2 (Arp2) in the nucleus accumbens (NAc). A cross-tolerance effect was observed for Bdnf, tropomyosin receptor kinase B (TrkB), and Cofilin-1 in the mPFC. Conversely, the expression of Rho GTPase-activating protein 32 (P250gap), cAMP-response element binding protein (Creb), and DNA methyltransferase 3A (Dnmt3a) was cross-sensitized in the NAc. The coexistence of cross-sensitization and cross-tolerance neurobehavioral effects between CUS and amphetamine supports the idea that ELA can simultaneously blunt and heighten different brain substrates, collectively increasing the risk of drug dependence.

In addition to cognitive decline, 90 % of dementia patients experience behavioral and psychological symptoms of dementia, for which no safe and effective pharmacotherapy currently exists. Our study aimed to determine the therapeutic potential of the new dual 5-HT/SERT-acting ligand MM394 in the context of pro-cognitive and neuroprotective properties, as well as its effects on behavioral symptoms and mood disorders in patients diagnosed with Alzheimer's disease. We performed in vitro experiments to examine the neuroprotective and antioxidant properties of MM394, as well as in vivo studies on male rats to evaluate its pharmacokinetics and potential in behavioral modulation of memory and mood. Following this, we attempted to determine the molecular mechanism of action of the dual 5-HT/SERT targeting compound in ex vivo experiments using rats' hippocampus and prefrontal cortex. The results of our study in the HT-22 cell line support the potential of MM394 to target excitotoxicity and oxidative stress, which play a key role in neurodegeneration. Furthermore, the compound exhibits antidepressant-like activity in the forced swim test and counteracts the memory impairments caused by MK-801 in the novel object recognition assessment in rats. Ex vivo findings demonstrate that MM394 modulates mTOR and ERK1/2 phosphorylation in the studied brain areas and increases the level of BDNF in the hippocampus when co-administered with MK-801 in the novel object recognition test. As a result of these findings, MM394 may be useful as a therapeutic for BPSD, which should be further explored.

Single injection sensitization studies model the early transition to compulsive drug-seeking behavior, independent of other biological processes (e.g., drug tolerance). This paper examined a) the temporal persistence of (Experiment 1), and b) the role of catecholaminergic-mediated post-conditioning memory processes (Experiments 2a and 2b), in conditioned hyperactivity and behavioral sensitization after a single methamphetamine injection in male, Swiss Webster mice. Mice received either a single injection (intraperitoneal, i.p.) of physiological saline (vehicle) or methamphetamine (2.0 mg/kg) prior to a 30-minute locomotor activity session (Conditioning). Tests for conditioned hyperactivity (CR Test) and behavioral sensitization (Methamphetamine Challenge Test) occurred after a delay of 2 and 3 days (Immediate), 6 and 7 days (Short), 14 and 15 days (Moderate), or 27 and 28 days (Long), respectively. To assess the role of catecholaminergic activity, specifically dopamine D2 receptors (Experiments 2a) or β-adrenergic receptors (Experiment 2b) on memory consolidation, and its subsequent effect of conditioned hyperactivity and behavioral sensitization, the dopamine D2 antagonist, haloperidol (40 μg/kg), or the non-selective β-adrenergic (β1/β2) antagonist, propranolol (16 and 32 mg/kg), was administered immediately or 2.5 h after methamphetamine conditioning. Conditioned hyperactivity and behavioral sensitization were detected at all time points (Experiment 1). Furthermore, post-conditioning administration of haloperidol or propranolol failed to alter conditioned hyperactivity or behavioral sensitization (Experiment 2a and 2b). Collectively, these results suggest that the 1) conditioned and pharmacological responses persisted unchanged for equal durations, and 2) targeting the catecholaminergic system during memory consolidation did not disrupt induction of either conditioning hyperactivity or sensitization.

The nicotine analog 6-methyl nicotine (6-MN) has recently appeared in non-tobacco nicotine delivery products, including oral pouches and e-cigarette liquids, in an apparent ploy to evade regulation of nicotine by the United States Food and Drug Administration or other public health agencies. Unfortunately, only minimal scientific information on the effects of 6-MN is available.

Norepinephrine (NE) signaling is disrupted in stress disorders, with insufficient NE signaling implicated in major depressive disorder and hyperactive NE signaling associated with post-traumatic stress disorder, suggesting that adequate mood regulation requires optimal NE levels. The basolateral amygdala (BLA) is a hub for stress processing and receives dense noradrenergic innervation from the locus coeruleus (LC), the primary noradrenergic nucleus in the brain. The relationship between LC activity and cognitive/behavioral function during fear conditioning has been described as an inverted U, in which moderate LC activity, and subsequent NE release, is required for adaptive coping to threats, while hyperactive LC-NE signaling drives maladaptive behavioral responses. We used fiber photometry to measure NE signaling in the mouse BLA during acute behavioral responses to escapable and inescapable stressors, and then used an optogenetic approach to stimulate the noradrenergic terminals in the BLA at different frequencies to evaluate effects on coping behavior and cFos expression in the LC-BLA circuit. We found that low-frequency stimulation of the circuit inhibited both passive coping and BLA neuronal activity, while high-frequency stimulation had the opposite effect; the behavioral effects were not mediated by sex, but the cFos effects were specific to males. This study represents an expansion of the inverted U framework to encompass LC-BLA signaling driving acute behavioral responses to stress.

Epilepsy is a chronic neurological condition characterized by recurrent seizures that affects millions of people worldwide. Recent studies have reported altered expression of early growth response 1 (Egr1), an activity-inducible immediate early gene, and histone deacetylase 2 (HDAC2), a regulator of gene transcription through the removal of acetyl groups from histone tails, in the hippocampus of epilepsy patients and animal models. Here, we investigated the roles of EGR1 and HDAC2 in pentylenetetrazol (PTZ)-induced seizures in kindled mice. Chronic PTZ treatment increased EGR1 and decreased HDAC2 expression in the hippocampus. Furthermore, EGR1 bound to the Hdac2 promoter and repressed its activity, but it had no effect when the predicted EGR1 binding site was deleted. Subsequently, we investigated the role of the HDAC2 in chemically induced kindling in mice with Hdac2 deletion. Hdac2 deletion suppressed PTZ-induced kindled seizures in mice compared with control mice. The upregulation of brain-derived neurotrophic factor (BDNF), a neurotrophin crucial for brain development, was observed in the hippocampi of control mice chronically treated with PTZ, but this increase was absent in mice with Hdac2 deletion. Additionally, continuous microinjection of recombinant BDNF protein into the ventricle accelerated kindling in mice with Hdac2 deletion, suggesting that HDAC2 contributes to the development of kindled seizures by regulating BDNF expression. In summary, HDAC2, which is negatively regulated by EGR1, induces BDNF expression in the hippocampus of PTZ-treated mice, resulting in the development of kindled seizures. These findings indicate that the EGR1-HDAC2-BDNF molecular pathway may serve as a therapeutic target in epilepsy.

Major depressive disorder (MDD) causes great physical and mental suffering to patients while also imposing a tremendous economic burden on the global economy. Psychedelics, also known as serotonergic hallucinogens, are potent psychoactive compounds known for their ability to alter mood, perception, and a range of cognitive functions. Increasing evidence suggests that some psychedelics positively facilitate individual social functions, with rapid and sustained improvement in symptoms associated with MDD. Consequently, the application of psychedelics in the treatment of MDD has garnered considerable attention from researchers in recent years. This review examines recent advancements in evaluating the behavioral and physiological effects of psychedelics in both preclinical animal models and clinical trials focused on MDD. Additionally, we summarize and discuss the cellular, brain region, and circuit-level mechanisms, as well as potential intracellular signaling pathways, that may contribute to the antidepressant effects of psychedelics. Based on current evidence, we conclude that psychedelics hold significant promise as therapeutic agents for MDD.

Melanin-concentrating hormone (MCH) system within the nucleus accumbens (Acb) has been shown to regulate feeding behavior; however, the interaction between MCH and opioid-driven hedonic eating remains unclear. This study investigated the effects of intra-Acb administration of the MCHR1 antagonist SNAP-94847 on opioid-driven sucrose pellet intake in male and female rats. Subjects received MCHR1 antagonist (N-(3-{1-[4-(3,4-difluoro-phenoxy)-benzyl]-piperdin-4-yl}-4-methyl-phenyl)-isobutyramide (SNAP-94847; 0 μg, 1.5 μg, and 15 μg/0.5 μl/side) immediately prior to intra-Acb administration of the μ-opioid receptor agonist, D-Ala2, NMe-Phe4, Glyol5-enkephalin (DAMGO; 0 μg and 0.025 μg/0.5 μl/side) before completing free-feeding tests. Results showed that DAMGO significantly increased sucrose pellet intake in both sexes, while SNAP-94847 alone had no effect, nor did it block the DAMGO-induced increase in either males or females. This finding suggests that MCHR1 within the Acb does not play a significant role in low-effort palatability-driven motivation for food consumption, contrasting with its role in effort-based motivation tasks.

Although three dual orexin receptor antagonists (DORAs), suvorexant, lemborexant, and daridorexant, are currently available and widely used to treat insomnia, the differences in their elimination half-lives are not sufficient. As a result, clinicians have limited ability to tailor therapy to individual sleep complaints. The emergence of vornorexant, with a notably short half-life comparable to that of zolpidem, may substantially expand the clinical utility of DORAs. This broader spectrum of pharmacokinetic profiles enables more individualized treatment strategies that align with patients' specific sleep complaints. This approach, in turn, potentially reshapes the therapeutic paradigm of insomnia management. However, several challenges remain to be addressed in order to fully realize the clinical potential of DORAs. This review identifies four key challenges requiring resolution to advance their optimal use in clinical practice.