Electronic cigarette liquids (e-liquids) often contain flavors and solvents that may influence nicotine addiction. In this study, we characterized the dose-response relationship of commercial unflavored nicotine e-liquids and investigated the impact of vanilla-flavored e-liquids on nicotine vapor self-administration (VSA) and withdrawal in rats. Male adolescent Sprague Dawley rats self-administered aerosols generated from commercial e-liquids containing 0, 3, 6, or 12 mg/ml nicotine in a propylene glycol (PG) and glycerol (G) vehicle. The vehicle (0 mg/ml nicotine) supported robust VSA, indicating the reinforcing effects of PG/G vapor. 3 mg/ml nicotine did not support VSA, while both 6 and 12 mg/ml nicotine concentrations produced significant reinforcement, with 6 mg/ml yielding the most stable responding. The 6 mg/ml concentration was selected for subsequent comparisons with vanilla-flavored e-liquids. Vanilla flavor (0 mg/ml nicotine) led to maintained VSA behavior, confirming its reinforcing effects. However, the combination of vanilla and nicotine (6 mg/ml) did not alter nicotine intake or withdrawal severity, as assessed by mecamylamine-precipitated somatic signs. Blood nicotine and cotinine levels were similar between nicotine and vanilla + nicotine conditions, indicating that vanilla flavor did not affect systemic nicotine metabolism. Additionally, the PG/G vehicle induced significant somatic signs, suggesting that vapor exposure itself, independent of nicotine, contributes to these physiological responses. These findings provide critical insights into the reinforcing and physiological effects of both nicotine and non-nicotine constituents in e-cigarette aerosols, underscoring the need for future studies and regulatory strategies that consider the abuse liability of flavors and solvents, such as PG/G, particularly among adolescents.

Background Substitution between tobacco products depends on similarity in abuse liability profiles, particularly pharmacologic reinforcement. While nicotine delivery has been extensively characterized in the literature, the contribution of menthol delivery remains poorly understood. This study examined menthol exposure from a heated tobacco product (IQOS) and assessed whether plasma menthol delivery predicts willingness to substitute it for menthol cigarettes. Methods This exploratory analysis used data from a randomized, parallel-group, 14-day clinical trial of adults who smoke menthol cigarettes (N = 22). Participants used their own-brand menthol cigarettes (Week 1), then were randomized to use IQOS with Fresh Menthol (IQOS-M) or Regular/Tobacco (IQOS-T) HeatSticks (Week 2). Plasma nicotine and menthol glucuronide were measured before and after a laboratory-based 10-puff directed use bout. Substitution was assessed via the cross-price elasticity (CPE) of IQOS relative to menthol cigarettes in the Experimental Tobacco Marketplace task. Results IQOS-M boosted plasma menthol glucuronide levels more than IQOS-T (18.9 versus 0.2ng/mL, p < 0.01), while nicotine delivery did not differ significantly. The CPE of IQOS was greater in the IQOS-M group than in the IQOS-T group (0.8 versus 0.0; p = 0.03), indicating greater substitutability. Menthol glucuronide boosts were positively associated with CPE (τ=0.32, p = 0.03), whereas nicotine boosts were not significantly associated (τ=0.19, p = 0.20). Conclusions IQOS can deliver high levels of menthol, and menthol delivery predicted willingness to substitute this heated tobacco product for menthol cigarettes. These findings highlight the importance of non-nicotine constituents in shaping abuse liability and have direct implications for the regulation of tobacco products.

Abstract Adults aged ≥65 years are increasingly using cannabis products. However, controlled pharmacokinetic and pharmacodynamic data on Δ9-tetrahydrocannabinol (THC) in this population are sparse, and remain limited to oral/oromucosal formulations. To characterize the acute pharmacokinetic and pharmacodynamic effects of oral and vaporized THC in healthy adults aged ≥65, we conducted a two-arm, randomized, double-blind, placebo-controlled trial in which 20 participants (mean age 70.0, SD: 5.1 years) received oral (placebo, 5 mg, or 10 mg) or vaporized THC (placebo, 2 mg, or 4 mg) across three eight-hour sessions separated by ≥72 hours. Outcomes included plasma pharmacokinetics, subjective drug effects, reinforcement value, cognitive performance, heart rate (HR), blood pressure (BP), and adverse events (AEs). Oral THC was associated with delayed, lower THC exposure (Tmax 60-90 min; Cmax 2.6-6.2 ng/mL), with 11-OH-THC concentrations approximately matching parent-THC; slow-rising subjective effects; no change in reinforcement value; no significant change in HR or BP; and no AEs. Vaporized THC was associated with rapid, THC-dominant exposure (Tmax 3 min; Cmax 24.6-53.8 ng/mL) and minimal 11-OH-THC concentrations; rapid-onset subjective effects; increased reinforcement value at 4 mg; and significant HR elevation peaking within 5 min, without significant BP change. Cognitive performance did not differ from placebo at any oral or vaporized THC dose. At vaporized THC 4 mg, two participants experienced five AEs. Oral and vaporized THC produce route-specific pharmacokinetic and pharmacodynamic profiles in adults aged ≥65, including an increase in reinforcement value only after vaporization, and should therefore not be treated as interchangeable in risk assessment for older adults.

Continuous real-time monitoring of chemical biomarkers is essential for advancing our understanding of neurological disorders and facilitating targeted therapeutic interventions. Conditions such as epilepsy involve disturbances in the excitatory-inhibitory balance of neural circuits, which are modulated by critical chemicals, including gamma-aminobutyric acid (GABA), lactate, and glutamate. While semiconductor field-effect transistor (FET)-based sensors are an exciting development in this area, significant limitations remain, including inadequate real-time detection capabilities and inconsistent surface functionalization. To address these shortcomings, we present a complementary metal-oxide-semiconductor (CMOS)-based FET nanowire device ("nanoribbon") for rapid and real-time biosensing of GABA, lactate, and glutamate. The sensor surface was functionally engineered to support selective and simultaneous detection of these neurochemicals in both buffered solutions and complex biological matrices such as artificial cerebrospinal fluid (aCSF). Aptamer bioreceptors were employed for lactate and glutamate, while monoclonal antibodies were used for GABA, facilitating highly selective, label-free detection. The nanosensing platform provides rapid and accurate chemical quantification within 10 min, achieving limits of detection (LOD) of 80 fM for GABA, 58 fM for glutamate, and 182 fM for lactate. This technology supports continuous neurochemical monitoring and holds strong potential to improve diagnostic accuracy and therapeutic efficacy in neurological care.

IntroductionElectronic cigarette (EC) use has increased rapidly in the last decade, especially among youth. Regulating nicotine delivery from ECs could help curb youth uptake and leverage EC use in harm reduction yet is complicated by varying device and liquid variables that affect nicotine delivery. Nicotine flux, the nicotine emission rate, is a parameter that incorporates these variables and focuses on the performance rather than the design of an EC. Nicotine flux therefore could be a powerful regulatory tool if it is shown empirically to predict nicotine delivery and subjective effects related to dependence.Methods and analysisThis project consists of two complementary clinical trials. In Trial I, we will examine the relationship between nicotine flux and the rate and dose of nicotine delivery from ECs, hence, impacting abuse liability. It will also examine the extent to which this relationship is mediated by nicotine form (i.e., freebase versus protonated). At Yale School of Medicine (YSM), study participants will puff EC devices under conditions that differ by flux and form, while arterial blood is sampled in high time resolution. In Trial II, we will assess the relationship between nicotine flux, form, and subjective effects. At the American University of Beirut (AUB), participants will use EC devices with varying nicotine fluxes and forms, while dependency measures, such as the urge to use ECs, nicotine craving, and withdrawal symptoms, will be assessed. We will also monitor puffing intensity and real-time exposure to toxicants.Ethics and disseminationThe protocol of Trial I and Trial II was approved by YSM and AUB IRBs, respectively. We will disseminate study results through peer-reviewed publications and conference presentations.Trial registrationNCT05706701 for Trial I and NCT05430334 for Trial II.

Epileptogenic triggers are multifactorial and not well understood. Here we aimed to address the hypothesis that inappropriate pro-inflammatory mechanisms contribute to the pathogenesis of refractory epilepsy (non-responsiveness to antiepileptic drugs) in human patients. We used single-cell cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to reveal the immunotranscriptome of surgically resected epileptic lesion tissues. Our approach uncovered a pro-inflammatory microenvironment, including extensive activation of microglia and infiltration of other pro-inflammatory immune cells. These findings were supported by ligand-receptor (LR) interactome analysis, which demonstrated potential mechanisms of infiltration and evidence of direct physical interactions between microglia and T cells. Together, these data provide insight into the immune microenvironment in epileptic tissue, which may aid the development of new therapeutics.

Public Health Significance In young adult nondependent smokers, the threshold for positive subjective effects of nicotine was 2-4 times lower than its reinforcing threshold (0.05-0.1 mg vs. 0.2 mg) during a nicotine self-administration procedure. Additionally, higher abstinence-induced craving and faster nicotine metabolism were associated with greater reinforcement from nicotine. A four times lower threshold for the subjective pleasurable effects than the reinforcing effects of nicotine suggests that tobacco products that are deemed to be subthreshold for reinforcement should be carefully evaluated for their subjective effects, including their discriminative stimulus effects. A recent study demonstrated that during a single sampling period, 0.1 mg of intravenous (IV) nicotine (vs. placebo) was found to be the threshold for subjective and physiological drug effects. The present study is a secondary analysis evaluating whether the threshold for subjective and physiological effects is similar when the subject has repeated opportunities to choose blinded doses of nicotine versus placebo. We also examined whether cigarette craving, withdrawal, and rate of nicotine metabolism affected nicotine reinforcement, defined by a greater number of nicotine choices than placebo. Young adult (n = 34; 68% male), daily smokers had five laboratory sessions after overnight abstinence. After sampling an IV dose of nicotine (0.0125, 0.025, 0.05, 0.1, or 0.2 mg/70 kg) versus saline (placebo), participants completed a nicotine self-administration (NSA) procedure that included 10 opportunities to self-administer IV dose of nicotine or placebo. The threshold for subjective positive effects of nicotine during the NSA was equal to or lower than the sampling period, 0.05-0.1 mg versus 0.1 mg. The threshold for nicotine-induced heart rate increase was higher during the NSA than during the sampling period (0.2 mg vs. 0.1 mg). Higher baseline craving and nicotine metabolite ratio (NMR) were associated with nicotine reinforcement at 0.2 mg and 0.1 mg doses, respectively (p < .05). The results suggest that subjective effects during NSA are reported at doses lower than the sampling period. Taken together, tobacco products thought to be subthreshold for reinforcement should be carefully evaluated for their subjective effects, including their discriminative stimulus effects.

The International League Against Epilepsy/American Epilepsy Society (ILAE/AES) Joint Translational Task Force established the TASK3 working groups to create common data elements (CDEs) for various preclinical epilepsy research disciplines. This is the second in a two-part series of omics papers, with the other including genomics, transcriptomics, and epigenomics. The aim of the CDEs was to improve the standardization of experimental designs across a range of epilepsy research-related methods. We have generated CDE tables with key parameters and case report forms (CRFs) containing the essential contents of the study protocols for proteomics, lipidomics, and metabolomics of samples from rodent models and people with epilepsy. We discuss the important elements that need to be considered for the proteomics, lipidomics, and metabolomics methodologies, providing a rationale for the parameters that should be documented.

This secondary analysis sought to determine if plasma menthol glucuronide (MG) concentrations predict changes in three outcomes, subjective drug effects, urges to smoke, and heart rate, following concurrent inhaled menthol and intravenous nicotine. A total of 45 menthol and non-menthol cigarettes smokers (36 male, nine female, 20 Black, and 23 White) were included in this double-blind, placebo-controlled study. Across three test sessions, participants were assigned to a different flavor condition for each session: 0% (no menthol), 0.5%, or 3.2% menthol. In each test session, participants received in a random order one intravenous delivery of saline and two intravenous deliveries of nicotine (0.25 mg/70 kg and 0.5 mg/70 kg), each 1 h apart, concurrent with menthol delivery by e-cigarettes. The main outcomes were subjective drug effects, urges to smoke, and heart rate. The results showed that following e-cigarette inhalation, changes in plasma MG concentrations or "menthol boost" increased proportionally to the menthol concentration in the e-liquids. While changes in plasma MG concentrations were not predictive of increases in heart rate or subjective drug effects that are reflective of acute effects from nicotine (i.e., feel good effects, stimulated, aversive effects), they were predictive of cooling effect, a typical effect of menthol, but only in menthol smokers in the absence of concurrent active nicotine infusion. These findings demonstrate the utility of plasma MG as a biomarker both for acute menthol exposure by e-cigarette inhalation and for the examination of the concentration-dependent behavioral and physiological effects of menthol in humans.

Faster delivery rate enhances the abuse potential of drugs of abuse, yet systematic studies on the impact of delivery rate on the acute effects of nicotine in humans are lacking. Using an intravenous (IV) nicotine infusion procedure that allows precise control of rate of delivery, we examined the impact of nicotine delivery rate on the positive subjective drug effects, smoking urges, withdrawal, heart rate, blood pressure and attention function in smokers. Twenty‐four male and female (ages 21–35) dependent smokers attended five experimental sessions, following overnight abstinence from smoking. Using a crossover design, participants attended five sessions, where they were assigned to a random sequence of saline infusion or 1 mg nicotine delivered over 1, 2.5, 5 or 10 min at rates of 1, 0.4, 0.2 or 0.1 mg/min, respectively. The positive subjective effects of nicotine were most robust under the two faster delivery rate conditions, 1‐ and 0.4‐mg nicotine/min. In contrast, all nicotine delivery rates were equally more effective than saline in alleviating urges to smoke. Likewise, nicotine‐induced heart rate increases did not vary with the rate of nicotine delivery. Lastly, the cognitive enhancing effects of nicotine were observed only under the two slowest delivery rate conditions—0.1‐ and 0.2‐mg nicotine/min. Collectively, these findings support the critical role of delivery rate in optimizing nicotine's abuse potential versus potential therapeutic effects and have timely implications for developing novel therapeutics for nicotine dependence, as well as for tobacco regulatory science.

Metabolomics is the laboratory analysis and scientific study of the metabolome—that is, the entire collection of small molecule chemicals in an organism. The metabolome represents the functional state of an organism and provides a multifaceted readout of the aggregate activity of endogenous (cellular) and exogenous (environmental) processes. In this review, we discuss how the integrative and dynamic properties of the metabolome create unique opportunities to study complex pathologies that evolve and oscillate over time, like epilepsy. We explain how the scientific progress and clinical applications of metabolomics remain hampered by biological and technical challenges, and we propose best practices to overcome these challenges so that metabolomics can be used in a rigorous and effective manner to further epilepsy research.

Thalamic Deep Brain Stimulation Modulates Cycles of Seizure Risk in Epilepsy Gregg NM, Sladky V, Nejedly P, et al. Sci Rep . 2021;11:24250. doi: 10.1101/2021.08.25.21262616 . Chronic brain recordings suggest that seizure risk is not uniform, but rather varies systematically relative to daily (circadian) and multiday (multidien) cycles. Here, one human and seven dogs with naturally occurring epilepsy had continuous intracranial EEG (median 298 days) using novel implantable sensing and stimulation devices. Two pet dogs and the human subject received concurrent thalamic deep brain stimulation (DBS) over multiple months. All subjects had circadian and multiday cycles in the rate of interictal epileptiform spikes (IES). There was seizure phase locking to circadian and multiday IES cycles in five and seven out of eight subjects, respectively. Thalamic DBS modified circadian (all 3 subjects) and multiday (analysis limited to the human participant) IES cycles. DBS modified seizure clustering and circadian phase locking in the human subject. Multiscale cycles in brain excitability and seizure risk are features of human and canine epilepsy and are modifiable by thalamic DBS.

Reprogramming brain-resident glial cells into clinically relevant induced neurons (iNs) is an emerging strategy toward replacing lost neurons and restoring lost brain functions. A fundamental question is now whether iNs can promote functional recovery in pathological contexts. We addressed this question in the context of therapy-resistant mesial temporal lobe epilepsy (MTLE), which is associated with hippocampal seizures and degeneration of hippocampal GABAergic interneurons. Using a MTLE mouse model, we show that retrovirus-driven expression of Ascl1 and Dlx2 in reactive hippocampal glia in situ, or in cortical astroglia grafted in the epileptic hippocampus, causes efficient reprogramming into iNs exhibiting hallmarks of interneurons. These induced interneurons functionally integrate into epileptic networks and establish GABAergic synapses onto dentate granule cells. MTLE mice with GABAergic iNs show a significant reduction in both the number and cumulative duration of spontaneous recurrent hippocampal seizures. Thus glia-to-neuron reprogramming is a potential disease-modifying strategy to reduce seizures in therapy-resistant epilepsy.

OBJECTIVE:The astroglial enzyme glutamine synthetase (GS) is deficient in small loci in the brain in adult patients with different types of focal epilepsy; however, the role of this deficiency in the pathogenesis of epilepsy has been difficult to assess due to a lack of sufficiently sensitive and specific animal models. The aim of this study was to develop an in vivo approach for precise and specific deletions of the GS gene in the postnatal brain. METHODS:We stereotaxically injected various adeno-associated virus (AAV)-Cre recombinase constructs into the hippocampal formation and neocortex in 22-70-week-old GSflox/flox mice to knock out the GS gene in a specific and focal manner. The mice were subjected to seizure threshold determination, continuous video-electroencephalographic recordings, advanced in vivo neuroimaging, and immunocytochemistry for GS. RESULTS:The construct AAV8-glial fibrillary acidic protein-green fluorescent protein-Cre eliminated GS in >99% of astrocytes in the injection center with a gradual return to full GS expression toward the periphery. Such focal GS deletion reduced seizure threshold, caused spontaneous recurrent seizures, and diminished functional connectivity. SIGNIFICANCE:These results suggest that small loci of GS deficiency in the postnatal brain are sufficient to cause epilepsy and impaired functional connectivity. Additionally, given the high specificity and precise spatial resolution of our GS knockdown approach, we anticipate that this model will be extremely useful for rigorous in vivo and ex vivo studies of astroglial GS function at the brain-region and single-cell levels.

An age-related decrease in hippocampal metabolism correlates with cognitive decline. Hippocampus-dependent learning and memory requires glutamatergic neurotransmission supported by glutamate-glutamine (GLU-GLN) cycling between neurons and astrocytes. We examined whether GLU-GLN cycling in hippocampal subregions (dentate gyrus and CA1) in Fischer 344 rats was altered with age and cognitive status. Hippocampal slices from young adult, aged cognitively-unimpaired (AU) and aged cognitively-impaired (AI) rats were incubated in artificial cerebrospinal fluid (aCSF) containing 1-13C-glucose to assess neural metabolism. Incorporation of 13C-glucose into glutamate and glutamine, measured by mass spectroscopy/liquid chromatography tandem mass spectroscopy, did not significantly differ between groups. However, when 13C-acetate, a preferential astrocytic metabolite, was used, a significant increase in 13C-labeled glutamate was observed in slices from AU rats. Taken together, the data suggest that resting state neural metabolism and GLU-GLN cycling may be preserved during aging when sufficient extracellular glucose is available, but that enhanced astroglial metabolism can occur under resting state conditions. This may be an aging-related compensatory change to maintain hippocampus-dependent cognitive function.

In Vivo Gamma-Aminobutyric Acid Increase as a Biomarker of the Epileptogenic Zone: An Unbiased Metabolomics Approach Hamelin S, Stupar V, Maziere L, et al. Epilepsia. 2021;62(1):163-175. Objective: Following surgery, focal seizures relapse in 20% to 50% of cases due to the difficulty of delimiting the epileptogenic zone (EZ) by current imaging or electrophysiological techniques. Here, we evaluate an unbiased metabolomics approach based on ex vivo and in vivo nuclear magnetic resonance spectroscopy (MRS) methods to discriminate the EZ in a mouse model of mesiotemporal lobe epilepsy (MTLE). Methods: Four weeks after unilateral injection of kainic acid (KA) into the dorsal hippocampus of mice (KA-MTLE model), we analyzed hippocampal and cortical samples with high-resolution magic angle spinning (HRMAS) MRS. Using advanced multivariate statistics, we identified the metabolites that best discriminate the injected dorsal hippocampus (EZ) and developed an in vivo MEGAPRESS MRS method to focus on the detection of these metabolites in the same mouse model. Results: Multivariate analysis of HRMAS data provided evidence that γ-aminobutyric acid (GABA) is largely increased in the EZ of KA-MTLE mice and is the metabolite that best discriminates the EZ when compared with sham and, more importantly, when compared with adjacent brain regions. These results were confirmed by capillary electrophoresis analysis and were not reversed by a chronic exposition to an antiepileptic drug (carbamazepine). Then, using in vivo noninvasive GABA-edited MRS, we confirmed that a high GABA increase is specific to the injected hippocampus of KA-MTLE mice. Significance: Our strategy using ex vivo MRS-based untargeted metabolomics to select the most discriminant metabolite(s), followed by in vivo MRS-based targeted metabolomics, is an unbiased approach to accurately define the EZ in a mouse model of focal epilepsy. Results suggest that GABA is a specific biomarker of the EZ in MTLE.

The objective of this study was to monitor the extracellular brain chemistry dynamics at baseline and in relation to spontaneous seizures in human patients with refractory epilepsy. Thirty patients with drug‐resistant focal epilepsy underwent intracranial electroencephalography and concurrent brain microdialysis for up to 8 continuous days. Extracellular brain glutamate, glutamine, and the branched‐chain amino acids (BCAAs) valine, leucine, and isoleucine were quantified in the dialysis samples by liquid chromatography–tandem mass spectrometry. Extracellular BCAAs and glutamate were chronically elevated at baseline by approximately 1.5–3‐fold in brain regions of seizure onset and propagation versus regions not involved by seizures. Moreover, isoleucine increased significantly above baseline as early as 3 h before a spontaneous seizure. BCAAs play important roles in glutamatergic neurotransmission, mitochondrial function, neurodegeneration, and mammalian target of rapamycin signaling. Because all of these processes have been implicated in epilepsy, the results suggest a novel role of BCAAs in the pathogenesis of spontaneous seizures.

Reducing nicotine content of inhaled tobacco products may prevent nicotine addiction, but the threshold for nicotine reinforcement has not been systematically evaluated in controlled human laboratory studies. The current study uses a novel double-blind placebo-controlled intravenous (IV) nicotine self-administration (NSA) model to determine threshold for subjective effects of nicotine and nicotine reinforcement using a forced choice self-administration procedure. Young adults (n = 34) had 5 laboratory sessions after overnight nicotine abstinence. In each session, participants sampled and rated the subjective effects of an IV dose of nicotine (0.0125, 0.025, 0.05, 0.1, or 0.2 mg nicotine/70 kg bodyweight) versus saline (placebo), then were given a total of 10 opportunities to self-administer either the IV dose of nicotine or placebo. Mixed effect models revealed a significant effect of nicotine dose for positive (i.e., “stimulatory” and “pleasurable”; p < .0001) effects, but not “aversive” effects during sampling period. Post hoc comparisons showed that higher doses (i.e., 0.1 and 0.2 mg) were associated with greater stimulatory, pleasurable, and physiological effects than placebo and lower doses. Mixed effect models revealed that only the highest dose (i.e., 0.2 mg) was consistently preferred over placebo. Sex differences were generally weak (p = .03–.05). Using our IV nicotine NSA model, the threshold for detecting positive effects of nicotine in young adult smokers is about 0.1 mg, but a higher dose of nicotine, 0.2 mg, is required to produce a consistent nicotine reinforcement. Regarding the regulatory impact, our findings further support the value of nicotine reinforcement threshold as a tobacco regulatory target.

The enzyme glutamine synthetase (GS), also referred to as glutamate ammonia ligase, is abundant in astrocytes and catalyzes the conversion of ammonia and glutamate to glutamine. Deficiency or dysfunction of astrocytic GS in discrete brain regions have been associated with several types of epilepsy, including medically-intractable mesial temporal lobe epilepsy (MTLE), neocortical epilepsies, and glioblastoma-associated epilepsy. Moreover, experimental inhibition or deletion of GS in the entorhinal-hippocampal territory of laboratory animals causes an MTLE-like syndrome characterized by spontaneous, recurrent hippocampal-onset seizures, loss of hippocampal neurons, and in some cases comorbid depressive-like features. The goal of this review is to summarize and discuss the possible roles of astroglial GS in the pathogenesis of epilepsy.