The signals involved in axonal trafficking and presynaptic clustering are poorly defined. Here we show that targeting of the gamma-aminobutyric acid-synthesizing enzyme glutamate decarboxylase 65 (GAD65) to presynaptic clusters is mediated by its palmitoylated 60-aa NH(2)-terminal domain and that this region can target other soluble proteins and their associated partners to presynaptic termini. A Golgi localization signal in aa 1-23 followed by a membrane anchoring signal upstream of the palmitoylation motif are required for this process and mediate targeting of GAD65 to the cytosolic leaflet of Golgi membranes, an obligatory first step in axonal sorting. Palmitoylation of a third trafficking signal downstream of the membrane anchoring signal is not required for Golgi targeting. However, palmitoylation of cysteines 30 and 45 is critical for post-Golgi trafficking of GAD65 to presynaptic sites and for its relative dendritic exclusion. Reduction of cellular cholesterol levels resulted in the inhibition of presynaptic clustering of palmitoylated GAD65, suggesting that the selective targeting of the protein to presynaptic termini is dependent on sorting to cholesterol-rich membrane microdomains. The palmitoylated NH(2)-terminal region of GAD65 is the first identified protein region that can target other proteins to presynaptic clusters.

The relative importance of environmental pathways that results in enteropathogen transmission may vary by context. However, measurement of contact events between individuals and the environment remains a challenge, especially for infants and young children who may use their mouth and hands to explore their environment. Using a mixed-method approach, we combined 1) semistructured observations to characterize key behaviors associated with enteric pathogen exposure and 2) structured observations using Livetrak, a customized software application, to quantify the frequency and duration of contacts events among infants in rural Ecuador. After developing and iteratively piloting the structured observation instrument, we loaded the final list of prompts onto a LiveTrak pallet to assess environmental exposures of 6-month infants (N = 19) enrolled in a prospective cohort study of diarrheal disease. Here we provide a detailed account of the lessons learned. For example, in our field site, 1) most mothers reported washing their hands after diaper changes (14/18, 77.8%); however only a third (4/11, 36.4%) were observed washing their hands; 2) the observers noted that animal ownership differed from observed animal exposure because animals owned by neighboring households were reported during the observation; and 3) using Livetrak, we found that infants frequently mouthed their hands (median = 1.9 episodes/hour, median duration: 1.6 min) and mouthed surroundings objects (1.8 episodes/hour, 1.9 min). Structured observations that track events in real time, can complement environmental sampling, quantitative survey data and qualitative interviews. Customizing these observations enabled us to quantify enteric exposures most relevant to our rural Ecuadorian context.

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for human applications as well. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine, making it difficult to apply in basic and translational applications. Here we report the development of two novel DREADD agonists, JHU37152 and JHU37160, and the first dedicated ¹⁸F positron emission tomography (PET) DREADD radiotracer, [¹⁸F]JHU37107. We show that JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [¹⁸F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons, and at their long-range projections, enabling noninvasive and longitudinal neuronal projection mapping.