The polarized trafficking of axonal and dendritic proteins is essential for the structure and function of neurons. Cyclin-dependent kinase-5 (CDK-5) and its activator CDKA-1/p35 regulate diverse aspects of nervous system development and function. Here, we show that CDK-5 and CDKA-1/p35 are required for the polarized distribution of neuropeptide-containing dense-core vesicles (DCVs) in C. elegans cholinergic motor neurons. In cdk-5 or cdka-1/p35 mutants, the predominantly axonal localization of DCVs containing INS-22 neuropeptides was disrupted and DCVs accumulated in dendrites. Time-lapse microscopy in DB class motor neurons revealed decreased trafficking of DCVs in axons and increased trafficking and accumulation of DCVs in cdk-5 mutant dendrites. The polarized distribution of several axonal and dendritic markers, including synaptic vesicles, was unaltered in cdk-5 mutant DB neurons. We found that microtubule polarity is plus-end out in axons and predominantly minus-end out in dendrites of DB neurons. Surprisingly, cdk-5 mutants had increased amounts of plus-end-out microtubules in dendrites, suggesting that CDK-5 regulates microtubule orientation. However, these changes in microtubule polarity are not responsible for the increased trafficking of DCVs into dendrites. Genetic analysis of cdk-5 and the plus-end-directed axonal DCV motor unc-104/KIF1A suggest that increased trafficking of UNC-104 into dendrites cannot explain the dendritic DCV accumulation. Instead, we found that mutations in the minus-end-directed motor cytoplasmic dynein, completely block the increased DCVs observed in cdk-5 mutant dendrites without affecting microtubule polarity. We propose a model where CDK-5 regulates DCV polarity by both promoting DCV trafficking in axons and preventing dynein-dependent DCV trafficking into dendrites.

Sleep pressure and sleep depth are key regulators of wake and sleep. Current methods of measuring these parameters in Drosophila melanogaster have low temporal resolution and/or require disrupting sleep. Here we report analysis tools for high-resolution, noninvasive measurement of sleep pressure and depth from movement data. Probability of initiating activity, P(Wake), measures sleep depth while probability of ceasing activity, P(Doze), measures sleep pressure. In vivo and computational analyses show that P(Wake) and P(Doze) are largely independent and control the amount of total sleep. We also develop a Hidden Markov Model that allows visualization of distinct sleep/wake substates. These hidden states have a predictable relationship with P(Doze) and P(Wake), suggesting that the methods capture the same behaviors. Importantly, we demonstrate that both the Doze/Wake probabilities and the sleep/wake substates are tied to specific biological processes. These metrics provide greater mechanistic insight into behavior than measuring the amount of sleep alone.

The landscape of scientific research and funding is in flux as a result of tight budgets, evolving models of both publishing and evaluation, and questions about training and workforce stability. As future leaders, junior scientists are uniquely poised to shape the culture and practice of science in response to these challenges. A group of postdocs in the Boston area who are invested in improving the scientific endeavor, planned a symposium held on October 2 and 3 , 2014, as a way to join the discussion about the future of US biomedical research. Here we present a report of the proceedings of participant-driven workshops and the organizers' synthesis of the outcomes.

Regulation of both excitatory and inhibitory synaptic transmission is critical for proper nervous system function. Aberrant synaptic signaling, including altered excitatory to inhibitory balance, is observed innumerous neurological diseases. The ubiquitin enzyme system controls the abundance of many synaptic proteins and thus plays a key role in regulating synaptic transmission. The Anaphase-Promoting Complex (APC) is a multi-subunit ubiquitin ligase that was originally discovered as a key regulator of protein turnover during the cell cycle. More recently, the APC has been shown to function in postmitotic neurons, where it regulates diverse processes such as synapse development and synaptic transmission at glutamatergic synapses. Here we report that the APC regulates synaptic GABA signaling by acting in motor neurons to control the balance of excitatory (acetylcholine) to inhibitory (GABA) transmission at the Caenorhabditis elegans neuromuscular junction (NMJ). Loss-of-function mutants in multiple APC subunits have increased muscle excitation at the NMJ; this phenotype is rescued by expression of the missing subunit in GABA neurons. Quantitative imaging and electrophysiological analyses indicate that APC mutants have decreased GABA release but normal cholinergic transmission. Consistent with this, APC mutants exhibit convulsions in a seizure assay sensitive to reductions in GABA signaling. Previous studies in other systems showed that the APC can negatively regulate the levels of the active zone protein SYD-2 Liprin-α. Similarly, we found that SYD-2 accumulates in APC mutants at GABAergic presynaptic sites. Finally, we found that the APC subunit EMB-27 CDC16 can localize to presynapses in GABA neurons. Together, our data suggest a model in which the APC acts at GABAergic presynapses to promote GABA release and inhibit muscle excitation. These findings are the first evidence that the APC regulates transmission at inhibitory synapses and have implications for understanding nervous system pathologies, such as epilepsy, that are characterized by misregulated GABA signaling.

The polarized trafficking of axonal and dendritic components is essential for the development and maintenance of neuronal structure and function. Neuropeptide-containing dense-core (DCVs) vesicles are trafficked in a polarized manner from the cell body to their sites of release; however, the molecules involved in this process are not well defined. Here we show that the scaffolding protein SYD-2/Liprin-α is required for the normal polarized localization of Venus-tagged neuropeptides to axons of cholinergic motor neurons in C. elegans. In syd-2 loss of function mutants, the normal polarized localization of INS-22 neuropeptide-containing DCVs in motor neurons is disrupted, and DCVs accumulate in the cell body and dendrites. Time-lapse microscopy and kymograph analysis of mobile DCVs revealed that syd-2 mutants exhibit decreased numbers of DCVs moving in both anterograde and retrograde directions, and a corresponding increase in stationary DCVs in both axon commissures and dendrites. In addition, DCV run lengths and velocities were decreased in both axon commissures and dendrites of syd-2 mutants. This study shows that SYD-2 promotes bi-directional mobility of DCVs and identifies SYD-2 as a novel regulator of DCV trafficking and polarized distribution.