The Liquid Phase of a Protein Is Involved in Synaptic Vesicle Clustering

What's the science?

Synaptic vesicles carry neurotransmitters and thus are critical for neuron-to-neuron communication. Synaptic vesicles cluster together at the presynaptic membrane and yet are able to move within the neuron terminal prior to release. Why might this be? One protein called synapsin is among the most abundant proteins in presynaptic neuron terminals. It binds to synaptic vesicles and therefore might be involved in their release. Furthermore, recent research suggests that some proteins and molecules within the cytoplasm of cells (including neurons) may be able to organize themselves even in the absence of a membrane, via phase separation (e.g. like how oil and water separate). This week in Science, Milovanovic and colleagues test whether synapsin demonstrates properties of phase separation and whether it is involved in cluster forming or release of synaptic vesicles.

How did they do it?

The authors tagged synapsin with green fluorescent protein and incubated synapsin molecules with a salt containing liquid buffer (resembling typical physiological conditions) to test whether synapsin phase-separates (i.e. into two liquid phases). They also tested whether synapsin can recruit (into it’s phase, ie. liquid) other proteins within the presynaptic terminal. They then performed experiments to mimic the synaptic environment more closely (which is filled with many different molecules and organelles) by adding polyethelene glycol to the buffer as a crowding reagent. They tested how synapsin might interact with vesicles and be involved in their cluster forming by incubating small lipid vesicles with synapsin. Using green fluorescent protein tagging, fluorescence microscopy, and electron microscopy, they tested whether synapsin could recruit the lipid vesicles into its phase. They further tested whether calcium dependent phosphorylation of synapsin (which is known to occur in the synapse during neuron activity) resulted in a disassembly of the synapsin/lipid vesicle phase as this would indicate the involvement of synapsin in the vesicle dispersion that occurs in stimulated synapses.

What did they find?

Synapsin was confirmed to phase-separate under physiological conditions (i.e. similar to those occurring in neurons in the human brain) as was evident by droplets of synapsin forming within the liquid buffer solution. They found that many other proteins located in the presynaptic terminal with synaptic vesicles also underwent phase separation when incubated with synapsin. Therefore, they tested how these interacted with synapsin. Two proteins known to bind synapsin, intersectin and GRB2, were found to form droplets with synapsin in the liquid buffer. After adding a crowding reagent to mimic the environment of a synapse, droplets of synapsin (either alone or with its binding partners intersectin or GRB2) formed even more efficiently. After incubating synapsin with lipid vesicles, they found that the synapsin droplets sequestered lipid vesicles which resulted in the formation of droplets containing lipid vesicles. As a control experiment, they tested whether other liquid mediums containing proteins that phase separate (but do not bind lipid vesicles) could also sequester lipid vesicles, and found that they did not. Thus, synapsin specifically allows the clustering of lipid vesicles. Upon calcium dependent phosphorylation of synapsin, droplets of synapsin alone or synapsin + lipid vesicles dispersed, supporting the hypothesis that synapsin is important for vesicle sequestering and for their dispersion during stimulation.

What's the impact?

This is the first study to show that phase-separation of a protein, synapsin, within the presynaptic terminal plays an important role in vesicle clustering. More generally, it is the first demonstration that phase-separation of a protein can organize vesicles within a cell. We now know that synapsin, and importantly it’s liquid phase, is involved in sequestering and dispersing vesicles in the presynaptic terminal. Future studies should examine how molecules within neurons may organize themselves based on phase separation.

Milavanovic et al., A liquid phase of synapsin and lipid vesicles. Science (2018). Access the original scientific publication here.

Caudate Nucleus Stimulation Induces Negative Repetitive Decision-Making

What's the science?

Some mood disorders are characterized by irrational, pessimistic, persistent thoughts. These thoughts are related to more cautious/pessimistic and inflexible decision making or evaluation. We don’t know the neural mechanisms involved in these pessimistic evaluations. However, the caudate nucleus is known to be involved in mood disorders, and is known to be involved in behavioral flexibility. This week in Neuron, Amemori and colleagues stimulated the caudate nucleus in primates to understand the role of the nucleus in persistent negative states and value evaluation.

How did they do it?

Two monkeys performed a behavioral task while one of 15-18 electrodes distributed throughout the caudate nucleus was stimulated using a microstimulation technique. Local field potentials were recorded from the other electrodes simultaneously. In the behavioral task, the monkey was shown a red bar, with the size of the bar indicating the amount of food the monkey was about to receive, and a yellow bar indicating the size of an air puff to the face (which it disliked) the monkey was about to receive. It used a joystick to indicate one of two choices: it either chose to a) accept the airpuff and food (taking into account the size of each) (‘approach’ choice) or b) to avoid the airpuff and receive a very small amount of food (‘avoidant’ choice). Decision and reaction time was recorded. 112 stimulation experiments (across both monkeys) and 74 recording-only experiments (across both monkeys) were performed (each experiment consisted of 150-250 trials). In the stimulation experiment, a threshold of 5% was set indicating a change in decision frequency in stimulation-on versus stimulation off-blocks. If the threshold was passed during stimulation (decisions tended to be either more ‘approach’ or more ‘avoidant’) the stimulation was considered effective. The effect of microstimulation on decision behavior during the task was analyzed.

What did they find?

Microstimulation at most sites did not change decision behavior. However, stimulation at about a quarter of the sites increased avoidant choices, and stimulation at a few sites increased approach choices. These effective sites were distributed throughout the caudate nucleus. Sites where stimulation invoked avoidant decisions were considered to be part of a ‘generative circuit’. Neuronal spiking activity was more likely to encode avoidant decisions near these negative effective sites. To see whether stimulation had persistent effects, the authors assessed follow-up blocks without stimulation after stimulation blocks, and found that increased avoidant decisions persisted 78% of the time after effective stimulation at negative sites, while positive effect were maintained only 10% of the time following stimulation at positive sites. This suggests that stimulation at negative sites could induce persistent negative mood. A tendency for stimulation at negative effective sites to induce a repetitive pattern of avoidant decision was also found, suggesting negative stimulation resulted in repetitive avoidant behavior. Oscillatory brain activity in the beta frequency band is known to be associated with maintenance of cognitive states. Therefore, the authors tested whether changes in beta band oscillations were related to decision making. They identified electrodes where task-related beta band activity could be observed, and found that there was greater beta band power prior to avoidant decisions when stimulation was off. During follow-up following stimulation, beta band power tracked choices even more closely, suggesting stimulation enhanced this relationship.

What's the impact?

This study suggests that the caudate nucleus can influence negative or pessimistic decision making or valuation via potential generative circuits that can lead to a persistent negative state. These findings have implications for mood disorders in which persistent, inflexible, negative thinking is a common observation.

Amemori et al., Striatal Microstimulation Induces Persistent and Repetitive Negative Decision-Making Predicted by Striatal Beta-Band Oscillation. Neuron (2018). Access the original scientific publication here.

 

Sleep Deprivation is Related to Loneliness and Changes in Brain Network Activity

What’s the science?

Humans are social animals, and social belonging is related to positive outcomes like well-being, while social isolation is associated with negative outcomes like depression and a higher risk for cardiovascular disease. Recent studies have linked loneliness to poor quality of sleep, as people who are socially isolated don’t get enough sleep or good quality sleep. However, it is unclear whether the reverse is also true: Can poor sleep quality lead to greater social isolation by making people less willing to interact with others? This week in Nature Communications, Simon and Walker set out to determine if sleep deprivation results in greater social withdrawal and if so, whether this social withdrawal behavior is related to changes in brain network activation.

How did they do it?

Two studies were conducted – one was online and the other took place both online and in-person. The online study took place on the Amazon Mechanical Turk (MTurk; a website on which people can perform tasks), in which 138 participants kept a log of their sleeping patterns for two nights and completed a questionnaire about social behavior and loneliness. In the second study, eighteen participants completed two versions of a social distance task, in which the experimenter and participant take turns walking toward each other. In both cases, participants indicate when the experimenter is uncomfortably close to them. Thus, this task provides a measure of an individual’s willingness to approach someone, and their receptiveness to being approached. Participants completed this task in-person with different experimenters, as well as a computerized version of this task while undergoing a functional magnetic resonance imaging (fMRI) scan. The fMRI scan allowed the authors to observe differences in activity in two brain networks; one associated with prosocial behaviors (‘Theory of Mind network’; ToM) and the other associated with personal space with respect to other people (‘Near Space network’). These participants were also filmed while answering questions about various topics. Video clips of these participants’ answers were then shown to over a thousand online raters on MTurk, who judged the participants in the videos on how lonely they seemed, whether they would interact and collaborate with those participants, and how lonely the participants made the raters feel.

What did they find?

Sleep-deprived participants exhibited greater social distancing behavior compared to when they were well-rested. This trend was further corroborated with findings from the online MTurk study, wherein poorer quality of sleep was associated with increased self-reports of loneliness and social isolation the following day. Sleep-deprived participants also showed greater activation in the Near Space network and decreased activity in the prosocial Theory of Mind network when they were sleep deprived than when they were well-rested. Interestingly, these changes in brain activity were related to their performance on the social distance task, as the increase in social distancing behavior following sleep deprivation was positively correlated with the increased activation of the Near Space network. Thus, when participants were sleep deprived, they showed a reduction in their willingness to approach others and be approached, which was related to activity in the Near Space network, associated with personal space. Sleep deprivation also influenced others’ perception of the participants, as online raters were more likely to judge sleep-deprived participants as being lonely and were less willing to interact with them. In fact, the raters reported feeling more lonely themselves after watching videos of sleep-deprived people.

                                      Brain, Servier Medical Art, image by BrainPost, CC BY-SA 3.0

What’s the impact?

This study highlighted the complex relationship between poor quality of sleep and changes in both social outcomes and brain activity. Specifically, the authors showed that sleep deprivation is related to an increase in social withdrawal tendencies, and that this behavioral change is further correlated with changes in brain activity. They also showed that others perceive sleep-deprived people more negatively than well-rested people, which further exacerbates the social isolation following poor sleep.

Simon & Walker. Sleep loss causes social withdrawal and loneliness. Nature Communications (2018). Access the original scientific publication here.