We introduce optogenetic investigation of neurotransmission (OptIoN) for time-resolved and quantitative assessment of synaptic function via behavioral and electrophysiological analyses. We photo-triggered release of acetylcholine or gamma-aminobutyric acid at Caenorhabditis elegans neuromuscular junctions using targeted expression of Chlamydomonas reinhardtii Channelrhodopsin-2. In intact Channelrhodopsin-2 transgenic worms, photostimulation instantly induced body elongation (for gamma-aminobutyric acid) or contraction (for acetylcholine), which we analyzed acutely, or during sustained activation with automated image analysis, to assess synaptic efficacy. In dissected worms, photostimulation evoked neurotransmitter-specific postsynaptic currents that could be triggered repeatedly and at various frequencies. Light-evoked behaviors and postsynaptic currents were significantly (P

The ability to optically excite or silence specific cells using optogenetics has provided a powerful tool to interrogate the nervous system. Optogenetic experiments in small organisms have mostly been performed using whole-field illumination and genetic targeting, but these strategies do not always provide adequate cellular specificity. Targeted illumination can be a valuable alternative but to date it has only been shown in non-moving animals without the ability to observe behavior output. We present a real-time multimodal illumination technology that allows both tracking and recording the behavior of freely moving Caenorhabditis elegans while stimulating specific cells that express Channelrhodopsin-2 or MAC. We use this system to optically manipulate nodes within the C. elegans touch circuit and study the roles of sensory and command neurons and the ultimate behavioral output. This technology significantly enhances our ability to control, alter, observe, and investigate how neurons, muscles, and circuits ultimately produce behavior in animals using optogenetics.

BackgroundA growing body of evidence suggests that the plasma concentration of the neurofilament light chain (NfL) might be considered a plasma biomarker for the screening of neurodegeneration in Alzheimer’s disease (AD).MethodsWith a single molecule array method (Simoa, Quanterix), plasma NfL concentrations were measured in 99 subjects with AD at the stage of mild cognitive impairment (MCI-AD; n = 25) or at the stage of early dementia (ADD; n = 33), and in nondemented controls (n = 41); in all patients, the clinical diagnoses were in accordance with the results of the four core cerebrospinal fluid (CSF) biomarkers (amyloid β (Aβ)1–42, Aβ42/40, Tau, and pTau181), interpreted according to the Erlangen Score algorithm. The influence of preanalytical storage procedures on the NfL in plasma was tested on samples exposed to six different conditions.ResultsNfL concentrations significantly increased in the samples exposed to more than one freezing/thawing cycle, and in those stored for 5 days at room temperature or at 4 °C. Compared with the control group of nondemented subjects (22.0 ± 12.4 pg/mL), the unadjusted plasma NfL concentration was highly significantly higher in the MCI-AD group (38.1 ± 15.9 pg/mL, p < 0.005) and even further elevated in the ADD group (49.1 ± 28.4 pg/mL; p < 0.001). A significant association between NfL and age (ρ = 0.65, p < 0.001) was observed; after correcting for age, the difference in NfL concentrations between AD and controls remained significant (p = 0.044). At the cutoff value of 25.7 pg/mL, unconditional sensitivity, specificity, and accuracy were 0.84, 0.78, and 0.82, respectively. Unadjusted correlation between plasma NfL and Mini Mental State Examination (MMSE) across all patients was moderate but significant (r = −0.49, p < 0.001). We observed an overall significant correlation between plasma NfL and the CSF biomarkers, but this correlation was not observed within the diagnostic groups.ConclusionsThis study confirms increased concentrations of plasma NfL in patients with Alzheimer’s disease compared with nondemented controls.

Graphical Abstract Highlights d Presence or absence of food promotes the dwelling or dispersal behavior of C. elegans d Dopamine signals to peptidergic interneurons in response to food d Peptidergic interneurons antagonize each other to inhibit or excite motoneurons d Cholecystokinin and RFamide modulate motoneurons to generate food response behavior SUMMARY Finding food and remaining at a food source are crucial survival strategies. We show how neural circuits and signaling molecules regulate these foodrelated behaviors in Caenorhabditis elegans. In the absence of food, AVK interneurons release FLP-1 neuropeptides that inhibit motorneurons to regulate body posture and velocity, thereby promoting dispersal. Conversely, AVK photoinhibition promoted dwelling behavior. We identified FLP-1 receptors required for these effects in distinct motoneurons. The DVA interneuron antagonizes signaling from AVK by releasing cholecystokinin-like neuropeptides that potentiate cholinergic neurons, in response to dopaminergic neurons that sense food. Dopamine also acts directly on AVK via an inhibitory dopamine receptor. Both AVK and DVA couple to head motoneurons by electrical and chemical synapses to orchestrate either dispersal or dwelling behavior, thus integrating environmental and proprioceptive signals. Dopaminergic regulation of foodrelated behavior, via similar neuropeptides, may be conserved in mammals.

Local recycling of synaptic vesicles (SVs) allows neurons to sustain transmitter release. Extreme activity (e.g., during seizure) may exhaust synaptic transmission and, in vitro, induces bulk endocytosis to recover SV membrane and proteins; how this occurs in animals is unknown. Following optogenetic hyperstimulation of Caenorhabditis elegans motoneurons, we analyzed synaptic recovery by time-resolved behavioral, electrophysiological, and ultrastructural assays. Recovery of docked SVs and of evokedrelease amplitudes (indicating readily-releasable pool refilling) occurred within ∼8-20 s (τ = 9.2 s and τ = 11.9 s), whereas locomotion recovered only after ∼60 s (τ = 20 s). During ∼11-s stimulation, 50-to 200-nm noncoated vesicles ("100nm vesicles") formed, which disappeared ∼8 s poststimulation, likely representing endocytic intermediates from which SVs may regenerate. In endophilin, synaptojanin, and dynamin mutants, affecting endocytosis and vesicle scission, resolving 100nm vesicles was delayed (>20 s). In dynamin mutants, 100nm vesicles were abundant and persistent, sometimes continuous with the plasma membrane; incomplete budding of smaller vesicles from 100nm vesicles further implicates dynamin in regenerating SVs from bulk-endocytosed vesicles. Synaptic recovery after exhaustive activity is slow, and different time scales of recovery at ultrastructural, physiological, and behavioral levels indicate multiple contributing processes. Similar processes may jointly account for slow recovery from acute seizures also in higher animals.channelrhodopsin | chemical synapse | electron microscopy | synaptic vesicle recycling E fficient chemical synaptic neurotransmission requires synaptic vesicle (SV) biogenesis, transmitter loading, membrane approximation and docking, priming, fusion, and release of transmitter (1-3). These processes are followed by retrieval of membrane and proteins from the plasma membrane (PM) via endocytosis (4, 5). Particularly, sustained SV release relies on a tight coupling of exocytosis and endocytosis (5-8). During highfrequency or long-term neuronal activity, SVs need to be efficiently recycled, because, otherwise, the readily releasable pool and the (mobilized) resting pool of SVs would be depleted and transmission would seize (9, 10). After fusion, SV membranes and proteins are recycled (11). Coupling SV exocytosis with local recycling largely eliminates the dependence of chemical transmission on somatic de novo SV synthesis and transport. Thus far, these processes have been studied in dissected preparations or cultured cells and tissues; how and at which time scales this occurs within a live, nondissected animal (e.g., during seizures) is currently unclear. For example, patients suffering from a seizure often remain unconscious for minutes to hours (12, 13). Although fatigue at different levels of circuits and brain systems is likely to contribute, also physiological changes in chemical synapses may play a role in this slow recovery.Depending on the SV fusion rate, endocytosis occurs via d...