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RationaleReducing 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.ObjectivesThe 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.MethodsYoung 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.ResultsMixed 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).ConclusionsUsing 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.

Electronic 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. This 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. The 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. NCT05706701 for Trial I and NCT05430334 for Trial II.

The liver makes ketone bodies (3-hydroxybutyrate, acetoacetate, and acetone, the breakdown product of acetoacetate) from fat when intracellular glucose is in short supply — for example, in starvation or unregulated diabetes. The generated 3-hydroxybutyrate and acetoacetate serve as alternative energy sources for the cells. However, when the production of ketone bodies greatly exceeds cellular consumption, the potentially lethal condition of ketoacidosis can occur. How do ketone bodies get out of hepatocytes and into cells in other parts of the body to be metabolized? Ketone bodies and other monocarboxylates, such as lactate and pyruvate, are transported out of and into cells . . .

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.

Recovery from neuronal activation requires rapid clearance of potassium ions (K+)and restoration of osmotic equilibrium. The predominant water channel protein in brain, aquaporin-4 (AQP4), is concentrated in the astrocyte end-feet membranes adjacent to blood vessels in neocortex and cerebellum by association with α-syntrophin protein. Although AQP4 has been implicated in the pathogenesis of brain edema, its functions in normal brain physiology are uncertain. In this study, we used immunogold electron microscopy to compare hippocampus of WT and α-syntrophin-null mice (α-Syn-/-). We found that <10% of AQP4 immunogold labeling is retained in the perivascular astrocyte end-feet membranes of the α-Syn-/-mice, whereas labeling of the inwardly rectifying K+channel, Kir4.1, is largely unchanged. Activity-dependent changes in K+clearance were studied in hippocampal slices to test whether AQP4 and K+channels work in concert to achieve isosmotic clearance of K+after neuronal activation. Microelectrode recordings of extracellular$K^+\\>(\\lbrack K^+\\rbrack_o)$from the target zones of Schaffer collaterals and perforant path were obtained after 5-, 10-, and 20-Hz orthodromic stimulations. K+clearance was prolonged up to 2-fold in α-Syn-/-mice compared with WT mice. Furthermore, the intensity of hyperthermia-induced epileptic seizures was increased in approximately half of the α-Syn-/-mice. These studies lead us to propose that water flux through perivascular AQP4 is needed to sustain efficient removal of K+after neuronal activation.

An abnormal accumulation of extracellular K+in the brain has been implicated in the generation of seizures in patients with mesial temporal lobe epilepsy (MTLE) and hippocampal sclerosis. Experimental studies have shown that clearance of extracellular K+is compromised by removal of the perivascular pool of the water channel aquaporin 4 (AQP4), suggesting that an efficient clearance of K+depends on a concomitant water flux through astrocyte membranes. Therefore, we hypothesized that loss of perivascular AQP4 might be involved in the pathogenesis of MTLE. Whereas Western blot analysis showed an overall increase in AQP4 levels in MTLE compared with non-MTLE hippocampi, quantitative ImmunoGold electron microscopy revealed that the density of AQP4 along the perivascular membrane domain of astrocytes was reduced by 44% in area CA1 of MTLE vs. non-MTLE hippocampi. There was no difference in the density of AQP4 on the astrocyte membrane facing the neuropil. Because anchoring of AQP4 to the perivascular astrocyte endfoot membrane depends on the dystrophin complex, the localization of the 71-kDa brain-specific isoform of dystrophin was assessed by immunohistochemistry. In non-MTLE hippocampus, dystrophin was preferentially localized near blood vessels. However, in the MTLE hippocampus, the perivascular dystrophin was absent in sclerotic areas, suggesting that the loss of perivascular AQP4 is secondary to a disruption of the dystrophin complex. We postulate that the loss of perivascular AQP4 in MTLE is likely to result in a perturbed flux of water through astrocytes leading to an impaired buffering of extracellular K+and an increased propensity for seizures.

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.

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. The aim of the CDEs is to improve the standardization of experimental designs across a range of epilepsy research‐related methods. Here, we have generated CDE tables with key parameters and case report forms (CRFs) containing the essential contents of the study protocols for genomics, transcriptomics, and epigenomics in rodent models of epilepsy, with a specific focus on adult rats and mice. We discuss the important elements that need to be considered for genomics, transcriptomics, and epigenomics methodologies, providing a rationale for the parameters that should be collected. This is the first in a two‐part series of omics papers with the second installment to cover proteomics, lipidomics, and metabolomics in adult rodents.

The hippocampus of patients with mesial temporal lobe epilepsy is often hardened and shrunken, a condition known as sclerosis. Magnetic resonance imaging reveals an increase in the T2-weighted signal, while diffusion weighted imaging shows a higher apparent diffusion coefficient in sclerotic hippocampi, indicating increased water content. As water transport appears to be coupled to K+ clearance and neuronal excitability [4], the molecular basis of the perturbed water homeostasis in the sclerotic hippocampus was explored. The expression of aquaporin-4 (AQP-4), the predominant water channel in the brain, was studied with quantitative real time PCR analysis, light microscopic immunohistochemistry and high-resolution immunogold labeling. A significant increase in AQP-4 was observed in sclerotic, but not in non-sclerotic, hippocampi obtained from patients with medically intractable temporal lobe epilepsy. This increase was positively correlated with an increase in the astrocyte marker glial fibrillary acidic protein. AQP-4 was localized to the plasma membranes of astrocytes including the perivascular end-feet. Gene expression associated with increased AQP-4 was evaluated by high throughput gene expression analysis using Affymetrix GeneChip U133A and related gene networks were investigated with Ingenuity Pathways Analysis. AQP-4 expression was associated with a decrease in expression of the dystrophin gene, a protein implicated in the anchoring of AQP-4 in perivascular endfeet. The decreased expression of dystrophin may indicate a loss of polarity in the distribution of AQP-4 in astrocytes. We conclude that the perturbed expression of AQP-4 and dystrophin may be one factor underlying the loss of ion and water homeostasis in the sclerotic hippocampus and hypothesize that the reported changes may contribute to the epileptogenic properties of the sclerotic tissue.

Patients with mesial temporal lobe epilepsy (MTLE) have increased basal concentrations of extracellular glutamate in the epileptogenic versus the non-epileptogenic hippocampus. Such elevated glutamate levels have been proposed to underlie the initiation and maintenance of recurrent seizures, and a key question is what causes the elevation of glutamate in MTLE. Here, we explore the possibility that neurons in the hippocampal formation contain higher levels of the glutamate synthesizing enzyme phosphate-activated glutaminase (PAG) in patients with MTLE versus patients with other forms of temporal lobe epilepsy (non-MTLE). Increased PAG immunoreactivity was recorded in subpopulations of surviving neurons in the MTLE hippocampal formation, particularly in CA1 and CA3 and in the polymorphic layer of the dentate gyrus. Immunogold analysis revealed that PAG was concentrated in mitochondria. Double-labeling experiments indicated a positive correlation between the mitochondrial contents of PAG protein and glutamate, as well as between PAG enzyme activity and PAG protein as determined by Western blots. These data suggest that the antibodies recognize an enzymatically active pool of PAG. Western blots and enzyme activity assays of hippocampal homogenates revealed no change in PAG between MTLE and non-MTLE, despite a greatly (>50%) reduced number of neurons in the MTLE hippocampal formation compared to non-MTLE. Thus, the MTLE hippocampal formation contains an increased concentration and activity of PAG per neuron compared to non-MTLE. This increase suggests an enhanced capacity for glutamate synthesis-a finding that might contribute to the disrupted glutamate homeostasis in MTLE.