Most excitatory synaptic terminals in the brain impinge on dendritic spines. We and others have recently shown that dynamic microtubules (MTs) enter spines from the dendritic shaft. However, a direct role for MTs in long-lasting spine plasticity has yet to be demonstrated and it remains unclear whether MT-spine invasions are directly influenced by synaptic activity. Lasting changes in spine morphology and synaptic strength can be triggered by activation of synaptic NMDA receptors (NMDARs) and are associated with learning and memory processes. To determine whether MTs are involved in NMDAR-dependent spine plasticity, we imaged MT dynamics and spine morphology in live mouse hippocampal pyramidal neurons before and after acute activation of synaptic NMDARs. Synaptic NMDAR activation promoted MT-spine invasions and lasting increases in spine size, with invaded spines exhibiting significantly faster and more growth than non-invaded spines. Even individual MT invasions triggered rapid increases in spine size that persisted longer following NMDAR activation. Inhibition of either NMDARs or dynamic MTs blocked NMDAR-dependent spine growth. Together these results demonstrate for the first time that MT-spine invasions are positively regulated by signaling through synaptic NMDARs, and contribute to long-lasting structural changes in targeted spines.

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Introduction: Electronic cigarette (e-cigarette) aerosol is understood to provide reduced exposure to harmful toxicants compared with tobacco cigarette smoke, as it delivers nicotine and flavors without the use of tobacco. Published studies have shown that e-cigarette aerosol is chemically simple compared with tobacco smoke and corresponding reductions in toxicity in vitro have been demonstrated. However, comprehensive analytical and in vitro assessments of many widely available and currently marketed products, including pod-based systems, are limited. Materials and Methods: Here we report comparative data for aerosol emissions and in vitro toxicity, using the neutral red uptake, the bacterial reverse mutation, and in vitro micronucleus assays, for a pod system e-cigarette compared with 3R4F reference cigarette smoke. Results and Discussion: Many of the harmful and potentially harmful constituents found in cigarette smoke were not detected in e-cigarette aerosol. Using established in vitro biological tests, e-cigarette aerosol did not display any mutagenic or genotoxic activity under the conditions of test. By contrast, 3R4F cigarette smoke displayed mutagenic and genotoxic activity. E-cigarette aerosol was also found to be *300-fold less cytotoxic than cigarette smoke in the neutral red uptake assay. Conclusion: Data presented here show clear differences between a tobacco cigarette reference product and a commercially available nontobacco containing e-cigarette product in terms of emissions and in vitro toxicity profile. Our results demonstrate that high-quality e-cigarettes and e-liquids may offer the potential for substantially reduced exposure to cigarette toxicants in adult smokers who use such products as alternatives to cigarettes.

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Graphical abstract

Electronic nicotine delivery systems (ENDS) offer adult combustible cigarette smokers an alternative, potentially reduced harm, mode of nicotine delivery, attributed to fewer and reduced levels of harmful and potentially harmful constituents (HPHCs) in their aerosols compared to cigarette smoke. These two identical, randomised, open label, two-part studies aimed to compare levels of 15 biomarkers of exposure (BoE) to selected HPHCs associated with tobacco smoking in healthy US adult smoker subjects (n = 72). Following 9 days of exclusive use of a range of allocated myblu™ ENDS variants, subjects’ levels of 14 non-nicotine BoE were substantially reduced compared to baseline values (combustible cigarette use), in the range of 46–97%. BoE reductions were sustained in subjects who continued myblu use exclusively (n = 25) for a further 5 days, and returned to near baseline levels in subjects who returned to exclusive combustible cigarette use (n = 21). Dual users (n = 24) demonstrated reductions in BoE to a lesser extent than with exclusive myblu use. Measured nicotine equivalents did not significantly change throughout the study. These data suggest exclusive use of ENDS provides adult smokers seeking an alternative to combustible cigarettes with substantial reductions in HPHC exposures whilst achieving satisfying levels of nicotine delivery. Dual use involving substitution of cigarettes may also provide some of this advantage, but to lesser extent. Overall, the data contribute to the weight of evidence that ENDS are an important tool in tobacco harm reduction for adult smokers unwilling to or uninterested in quitting smoking. Study 1: NCT 04430634, study 2: NCT 04429932, clinicaltrials.gov (10-06-2020).

Rationale Tobacco harm reduction (THR) involves encouraging adult smokers who would otherwise continue to smoke to transition to less harmful forms of nicotine delivery. These products must offer adult smokers reduced exposure to chemicals associated with tobacco combustion, satisfactory blood plasma nicotine levels and serve as an acceptable alternative. The most recent THR innovation is tobacco-free oral nicotine pouches. Objectives This study aimed to compare pharmacokinetic, pharmacodynamic and safety and tolerability profiles of two nicotine pouch variants (ZoneX #2 (5.8 mg nicotine/pouch); ZoneX #3 (10.1 mg nicotine/pouch)) with cigarette to assess the pouches’ THR potential. Methods This was a controlled use, randomised, open-label, cross-over clinical study with 24 healthy adult traditional tobacco users. Pharmacokinetic (plasma nicotine levels; up to 8 h post-use), pharmacodynamic (urge to smoke, product liking; up to 4 h post-use) and short-term safety and tolerability profiles were assessed. Results Distinct nicotine pouch pharmacokinetic profiles indicated nicotine absorption via the oral mucosa. Plasma nicotine levels were lower, and time to peak slower, for the nicotine pouches compared to cigarette (Cmax cigarette: 11.6 ng/ml vs. #2: 5.2 ng/ml, p < 0.0001; #3: 7.9 ng/ml, p < 0.0003) (Tmax cigarette: 8.6 min vs. #2: 26 min; #3: 22 min). All products effectively reduced subjects’ urge to smoke and presented favourable product liking scores; nicotine pouches were also well tolerated following short-term use (no serious adverse events). Conclusions Overall, the assessed ZoneX nicotine pouches may offer an acceptable alternative for adult smokers to achieve satisfactory levels of nicotine delivery and, based on the pharmacokinetic parameters and under the study conditions, likely have a lower abuse liability and addictive potential for current adult smokers compared to continued cigarette smoking. Clinical trial identifier: NCT04891406 (clinicaltrials.gov).

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