Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pulldown assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the nonneuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the proteinprotein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.

Rationale Intensive care unit (ICU) visitation restrictions during the coronavirus disease (COVID-19) pandemic have drastically reduced family-engaged care. Understanding the impact of physical distancing on family members of ICU patients is needed to inform future policies. Objectives To understand the experiences of family members of critically ill patients with COVID-19 when physically distanced from their loved ones and to explore ways clinicians may support them. Methods This qualitative study of an observational cohort study reports data from 74 family members of ICU patients with COVID-19 at 10 United States hospitals in four states, chosen based on geographic and demographic diversity. Adult family members of patients admitted to the ICU with COVID-19 during the early phase of the pandemic (February–June 2020) were invited to participate in a phone interview. Interviews followed a semistructured guide to assess four constructs: illness narrative, stress experiences, communication experiences, and satisfaction with care. Interviews were transcribed verbatim and analyzed using an inductive approach to thematic analysis. Results Among 74 interviewees, the mean age was 53.0 years, 55% were white, and 76% were female. Physical distancing contributed to substantial stress and harms (nine themes). Participants described profound suffering and psychological illness, unfavorable perceptions of care, and weakened therapeutic relationship between family members and clinicians. Three communication principles emerged as those most valued by family members: contact, consistency, and compassion (the 3Cs). Family members offered suggestions to guide clinicians faced with communicating with physically distanced families. Conclusions Visitation restrictions impose substantial psychological harms upon family members of critically ill patients. Derived from the voics of family members, our findings warrant strong consideration when implementing visitation restrictions in the ICU and advocate for investment in infrastructure (including staffing and videoconferencing) to support communication. This study offers family-derived recommendations to operationalize the 3Cs to guide and improve communication in times of physical distancing during the COVID-19 pandemic and beyond.

Sodium appetite is an instinct that involves avid specific intention. It is elicited by sodium deficiency, stress-evoked adrenocorticotropic hormone (ACTH), and reproduction. Genome-wide microarrays in sodium-deficient mice or after ACTH infusion showed up-regulation of hypothalamic genes, including dopamine-and cAMP-regulated neuronal phosphoprotein 32 kDa (DARPP-32), dopamine receptors-1 and -2, α-2C-adrenoceptor, and striatally enriched protein tyrosine phosphatase (STEP). Both DARPP-32 and neural plasticity regulator activity-regulated cytoskeleton associated protein (ARC) were upregulated in lateral hypothalamic orexinergic neurons by sodium deficiency. Administration of dopamine D1 (SCH23390) and D2 receptor (raclopride) antagonists reduced gratification of sodium appetite triggered by sodium deficiency. SCH23390 was specific, having no effect on osmotic-induced water drinking, whereas raclopride also reduced water intake. D1 receptor KO mice had normal sodium appetite, indicating compensatory regulation. Appetite was insensitive to SCH23390, confirming the absence of off-target effects. Bilateral microinjection of SCH23390 (100 nM in 200 nL) into rats' lateral hypothalamus greatly reduced sodium appetite. Gene set enrichment analysis in hypothalami of mice with sodium appetite showed significant enrichment of gene sets previously linked to addiction (opiates and cocaine). This finding of concerted gene regulation was attenuated on gratification with perplexingly rapid kinetics of only 10 min, anteceding significant absorption of salt from the gut. Salt appetite and hedonic liking of salt taste have evolved over >100 million y (e.g., being present in Metatheria). Drugs causing pleasure and addiction are comparatively recent and likely reflect usurping of evolutionary ancient systems with high survival value by the gratification of contemporary hedonic indulgences. Our findings outline a molecular logic for instinctive behavior encoded by the brain with possible important translational-medical implications.

IMPORTANCEThe psychological symptoms associated with having a family member admitted to the intensive care unit (ICU) during the COVID-19 pandemic are not well defined.OBJECTIVE To examine the prevalence of symptoms of stress-related disorders, primarily posttraumatic stress disorder (PTSD), in family members of patients admitted to the ICU with COVID-19 approximately 90 days after admission.DESIGN, SETTING, AND PARTICIPANTS This prospective, multisite, mixed-methods observational cohort study assessed 330 family members of patients admitted to the ICU (except in New York City, which had a random sample of 25% of all admitted patients per month) between February 1 and July 31, 2020, at 8 academic-affiliated and 4 community-based hospitals in 5 US states.EXPOSURE Having a family member in the ICU with COVID-19.MAIN OUTCOMES AND MEASURES Symptoms of PTSD at 3 months, as defined by a score of 10 or higher on the Impact of Events Scale 6 (IES-6). RESULTS A total of 330 participants (mean [SD] age, 51.2 [15.1] years; 228 [69.1%] women; 150 [52.8%] White; 92 [29.8%] Hispanic) were surveyed at the 3-month time point. Most individuals were the patients' child (129 [40.6%]) or spouse or partner (81 [25.5%]). The mean (SD) IES-6 score at 3 months was 11.9 (6.1), with 201 of 316 respondents (63.6%) having scores of 10 or higher, indicating significant symptoms of PTSD. Female participants had an adjusted mean IES-6 score of 2.6 points higher (95% CI, 1.4-3.8; P < .001) than male participants, whereas Hispanic participants scored a mean of 2.7 points higher compared with non-Hispanic participants (95% CI, 1.0-4.3; P = .002). Those with graduate school experience had an adjusted mean score of 3.3 points lower (95% CI, 1.5-5.1; P < .001) compared with those with up to a high school degree or equivalent. Qualitative analyses found no substantive differences in the emotional or communication-related experiences between those with high vs low PTSD scores, but those with higher scores exhibited more distrust of practitioners. CONCLUSIONS AND RELEVANCEIn this cohort study, symptoms of PTSD among family members of ICU patients with COVID-19 were high. Hispanic ethnicity and female gender were associated with higher symptoms. Those with higher scores reported more distrust of practitioners.

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