The ability to directly visualize nanoscopic cellular structures and their spatial relationship in all three dimensions will greatly enhance our understanding of molecular processes in cells. Here we demonstrated multicolor three-dimensional (3D) stochastic optical reconstruction microscopy (STORM) as a tool to quantitatively probe cellular structures and their interactions. To facilitate STORM imaging, we generated photoswitchable probes in several distinct colors by covalently linking a photoswitchable cyanine reporter and an activator molecule to assist bioconjugation. We performed 3D localization in conjunction with focal plane scanning and correction for refractive index mismatch to obtain whole-cell images with a spatial resolution of 20-30 nm and 60-70 nm in the lateral and axial dimensions, respectively. Using this approach, we imaged the entire mitochondrial network in fixed monkey kidney BS-C-1 cells, and studied the spatial relationship between mitochondria and microtubules. The 3D STORM images resolved mitochondrial morphologies as well as mitochondria-microtubule contacts that were obscured in conventional fluorescence images.

BACKGROUND: Ecological momentary assessment (EMA) offers a highly valid strategy to assess everyday functioning in people with severe mental illness. Adherence is generally good, but several questions regarding the impact of study length, daily density of sampling, and symptom severity on adherence remain. METHODS: EMA adherence in two separate studies was examined. One sampled participants with schizophrenia (n=106) and healthy controls (n=76) 7 times per day for 7 days and the other sampled participants with schizophrenia (n=104) and participants with bipolar illness (n=76) 3 times per day for 30 days. Participants were asked where they were, who they were with, what they were doing and how they were feeling in both studies. The impact of rates of very early adherence on eventual adherence was investigated across the samples, and adherence rates were examined for associations with mood state and most common location when answering surveys. RESULTS: Median levels of adherence were over 80% across the samples, and the 10th percentile for adherence was approximately 45% of surveys answered. Early adherence predicted study-long adherence quite substantially in every sample. Mood states did not correlate with adherence in the patient samples and being home correlated with adherence in only the bipolar sample. IMPLICATIONS: Adherence was quite high and was not correlated with the length of the study or the density of sampling per study day. There was a tendency for bipolar participants who were more commonly away from home to answer fewer surveys but overall adherence for the bipolar patients was quite high. These data suggest that early nonadherence is a potential predictor of eventual nonadherence and study noncompletion.

OBJECTIVES: People with schizophrenia have challenges in their self-assessments of everyday functioning and those who report no sadness also tend to overestimate their everyday functional abilities. While previous studies were cross-sectional, this study related longitudinal assessments of sadness to self-reports of abilities in domains of everyday functioning and cognitive abilities. METHODS: 71 people with bipolar illness (BPI) were compared to 102 people with schizophrenia (SCZ). Participants were sampled 3 times per day for 30 days with a smartphone-based Ecological Momentary Assessment (EMA) survey. Each survey asked where they were, with whom they were, what they were doing, and if they were sad. Performance based assessments of executive functioning, social competence, and everyday activities were collected after the EMA period, at which time the participants and observers were asked to provide ratings of three different domains of everyday functioning and neurocognitive ability. RESULTS: 18% of participants with SCZ reported that they were never sad on any one of the 90 EMA surveys. Reports of never being sad were associated with overestimated functioning compared to observers and SCZ participants who reported that they were never sad were more commonly home and alone than both SCZ participants who reported occasional sadness and participants with BPI. These participants reported being significantly happier than all people in the study. IMPLICATIONS: Reporting that you were never sad was associated with overestimation of everyday functioning and cognitive abilities. Although participants who were never sad did not perform more poorly on objective measures than those were occasionally sad, their self-assessed functioning was significantly elevated. These data suggest that negative symptoms constructs such as reduced emotional experience need to consider reduced ability to subjectively evaluate emotional experience as a feature of negative symptoms.

Introduction: In 2022, the American Heart Association (AHA) introduced Life’s Essential 8 (LE8) as an updated definition of cardiovascular health (CVH). Sleep health was added to the original Life’s Simple 7 (LS7), and a new scoring system was developed. We evaluated how LE8 characterizes CVH among women in the WHI. Hypothesis: We hypothesized that the average LE8 score would be moderate, with only a small group of WHI participants having high CVH. Methods: The WHI recruited 161,808 post-menopausal women from 40 clinical centers in 1993-1998. This analysis included 116,514 women without self-reported CVD, with measured height, weight, and blood pressure, and self-reported smoking history, sleep duration, physical activity, diet, diabetes, and cholesterol at baseline. CVH components were scored from 0-100 based on participants’ achievement of each health factor or behavior. Scores were assigned per AHA guidance with some adaptations made based on data availability. Lacking measured values, glucose and lipid scores were calculated based on history and treatment status of diabetes and high cholesterol. Overall LE8 scores were calculated as the unweighted mean of all components. Scores were further categorized into high (80-100), moderate (50-79), or low (0-49), with higher scores indicating positive CVH. Results: The average LE8 score among WHI participants at baseline was 71.5 (standard deviation (SD): 12.5; range 20.6-100) ( Figure ). Overall, sleep scores were high (mean: 86.5, SD 19.1). The lowest scores were found in diet (mean: 39.5, SD 31.3) and physical activity (mean: 47.7, SD: 43.5). LE8 classified 28.1% of WHI participants (32,773/116,514) as having high CVH whereas a prior analysis of LS7 found that 17.7% and 6.1% had ideal levels of 5 and 6-7 CVH components, respectively. Conclusions: Based on the new LE8 definition, most women had moderate CVH at study baseline, with approximately a quarter having high CVH. Although sleep health was high, scores for other health behaviors demonstrated considerable room for improvement.

Alcohol use disorder (AUD) exacts enormous personal, social and economic costs globally. Return to alcohol use in treatment-seeking patients with AUD is common, engendered by a cycle of repeated abstinence-relapse episodes even with use of currently available pharmacotherapies. Repeated ethanol use induces dopaminergic signaling neuroadaptations in ventral tegmental area (VTA) neurons of the mesolimbic reward pathway, and sustained dysfunction of reward circuitry is associated with return to drinking behavior. We tested this hypothesis by infusing adeno-associated virus serotype 2 vector encoding human glial-derived neurotrophic factor (AAV2-hGDNF), a growth factor that enhances dopaminergic neuron function, into the VTA of four male rhesus monkeys, with another four receiving vehicle, following induction of chronic alcohol drinking. GDNF expression ablated the return to alcohol drinking behavior over a 12-month period of repeated abstinence-alcohol reintroduction challenges. This behavioral change was accompanied by neurophysiological modulations to dopamine signaling in the nucleus accumbens that countered the hypodopaminergic signaling state associated with chronic alcohol use, indicative of a therapeutic modulation of limbic circuits countering the effects of alcohol. These preclinical findings suggest gene therapy targeting relapse prevention may be a potential therapeutic strategy for AUD.

Mesolimbic nicotinic acetylcholine receptor (nAChRs) activation is necessary for nicotine reinforcement behavior, but it is unknown whether selective activation of nAChRs in the dopamine (DA) reward pathway is sufficient to support nicotine reinforcement. In this study, we tested the hypothesis that activation of β2-containing (β2*) nAChRs on VTA neurons is sufficient for intravenous nicotine self-administration (SA). We expressed β2 nAChR subunits with enhanced sensitivity to nicotine (referred to as β2Leu9'Ser) in the VTA of male Sprague Dawley (SD) rats, enabling very low concentrations of nicotine to selectively activate β2* nAChRs on transduced neurons. Rats expressing β2Leu9'Ser subunits acquired nicotine SA at 1.5 μg/kg/infusion, a dose too low to support acquisition in control rats. Saline substitution extinguished responding for 1.5 μg/kg/inf, verifying that this dose was reinforcing. β2Leu9'Ser nAChRs also supported acquisition at the typical training dose in rats (30 μg/kg/inf) and reducing the dose to 1.5 μg/kg/inf caused a significant increase in the rate of nicotine SA. Viral expression of β2Leu9'Ser subunits only in VTA DA neurons (via TH-Cre rats) also enabled acquisition of nicotine SA at 1.5 μg/kg/inf, and saline substitution significantly attenuated responding. Next, we examined electrically-evoked DA release in slices from β2Leu9'Ser rats with a history of nicotine SA. Single-pulse evoked DA release and DA uptake rate were reduced in β2Leu9'Ser NAc slices, but relative increases in DA following a train of stimuli were preserved. These results are the first to report that β2* nAChR activation on VTA neurons is sufficient for nicotine reinforcement in rats.