Double UP: A Dual Color, Internally Controlled Platform for in utero Knockdown or Overexpression.

In utero electroporation (IUE) is a powerful tool for testing the role of genes in neuronal migration and function, but this technique suffers from high degrees of variability. Such variability can result from inconsistent surgery, developmental gradients along both rostral-caudal and medial-lateral axes, differences within littermates and from one litter to another. Comparisons between control and experimental electroporations rely on section matching, which is inherently subjective. These sources of variability are cumulative, leading to difficult to interpret data and an increased risk of both false positives and false negatives. To address these limitations, we developed two tools: (1) a new plasmid, termed Double UP, which combines LoxP-flanked reporters and limiting Cre dosages to generate internal controls, and (2) an automated program for unbiased and precise quantification of migration. In concert, these tools allow for more rigorous and objective experiments, while decreasing the mice, time, and reagents required to complete studies.

Opposing functions of F-BAR proteins in neuronal membrane protrusion, tubule formation, and neurite outgrowth.

The F-BAR family of proteins play important roles in many cellular processes by regulating both membrane and actin dynamics. The CIP4 family of F-BAR proteins is widely recognized to function in endocytosis by elongating endocytosing vesicles. However, in primary cortical neurons, CIP4 concentrates at the tips of extending lamellipodia and filopodia and inhibits neurite outgrowth. Here, we report that the highly homologous CIP4 family member, FBP17, induces tubular structures in primary cortical neurons and results in precocious neurite formation. Through domain swapping and deletion experiments, we demonstrate that a novel polybasic region between the F-BAR and HR1 domains is required for membrane bending. Moreover, the presence of a poly-PxxP region in longer splice isoforms of CIP4 and FBP17 largely reverses the localization and function of these proteins. Thus, CIP4 and FBP17 function as an antagonistic pair to fine-tune membrane protrusion, endocytosis, and neurite formation during early neuronal development.

Single-neuronal cell culture and monitoring platform using a fully transparent microfluidic DEP device.

Dielectrophoresis using multi-electrode arrays allows a non-invasive interface with biological cells for long-term monitoring of electrophysiological parameters as well as a label-free and non-destructive technique for neuronal cell manipulation. However, experiments for neuronal cell manipulation utilizing dielectrophoresis have been constrained because dielectrophoresis devices generally function outside of the controlled environment (i.e. incubator) during the cell manipulation process, which is problematic because neurons are highly susceptible to the properties of the physiochemical environment. Furthermore, the conventional multi-electrode arrays designed to generate dielectrophoretic force are often fabricated with non-transparent materials that confound live-cell imaging. Here we present an advanced single-neuronal cell culture and monitoring platform using a fully transparent microfluidic dielectrophoresis device for the unabated monitoring of neuronal cell development and function. The device is mounted inside a sealed incubation chamber to ensure improved homeostatic conditions and reduced contamination risk. Consequently, we successfully trap and culture single neurons on a desired location and monitor their growth process over a week. The proposed single-neuronal cell culture and monitoring platform not only has significant potential to realize an in vitro ordered neuronal network, but also offers a useful tool for a wide range of neurological research and electrophysiological studies of neuronal networks.

Transport of a kinesin-cargo pair along microtubules into dendritic spines undergoing synaptic plasticity.

Synaptic plasticity often involves changes in the structure and composition of dendritic spines. Vesicular cargos and organelles enter spines either by exocytosing in the dendrite shaft and diffusing into spines or through a kinesin to myosin hand-off at the base of spines. Here we present evidence for microtubule (MT)-based targeting of a specific motor/cargo pair directly into hippocampal dendritic spines. During transient MT polymerization into spines, the kinesin KIF1A and an associated cargo, synaptotagmin-IV (syt-IV), are trafficked in unison along MTs into spines. This trafficking into selected spines is activity-dependent and results in exocytosis of syt-IV-containing vesicles in the spine head. Surprisingly, knockdown of KIF1A causes frequent fusion of syt-IV-containing vesicles throughout the dendritic shaft and diffusion into spines. Taken together, these findings suggest a mechanism for targeting dendritic cargo directly into spines during synaptic plasticity and indicate that MT-bound kinesins prevent unregulated fusion by sequestering vesicular cargo to MTs.

The hibernating 13-lined ground squirrel as a model organism for potential cold storage of platelets.

Hibernating mammals have developed many physiological adaptations to extreme environments. During hibernation, 13-lined ground squirrels (Ictidomys tridecemlineatus) must suppress hemostasis to survive prolonged body temperatures of 4-8°C and 3-5 heartbeats per minute without forming lethal clots. Upon arousal in the spring, these ground squirrels must be able to quickly restore normal clotting activity to avoid bleeding. Here we show that ground squirrel platelets stored in vivo at 4-8°C were released back into the blood within 2 h of arousal in the spring with a body temperature of 37°C but were not rapidly cleared from circulation. These released platelets were capable of forming stable clots and remained in circulation for at least 2 days before newly synthesized platelets were detected. Transfusion of autologous platelets stored at 4°C or 37°C showed the same clearance rates in ground squirrels, whereas rat platelets stored in the cold had a 140-fold increase in clearance rate. Our results demonstrate that ground squirrel platelets appear to be resistant to the platelet cold storage lesions observed in other mammals, allowing prolonged storage in cold stasis and preventing rapid clearance upon spring arousal. Elucidating these adaptations could lead to the development of methods to store human platelets in the cold, extending their shelf life.