Dendritic spines are the primary sites of contact with presynaptic axons on excitatory hippocampal and cortical neurons. During development and plasticity spines undergo marked changes in structure that directly affect the functional communication between neurons. Elucidating the cytoskeletal events that induce these structural changes is fundamental to understanding synaptic biology. Actin plays a central role in the spine cytoskeleton, however the role of microtubules in spine function has been studied little. Although microtubules have a prominent role in transporting material throughout the dendrite that is destined for spines, they are not thought to directly influence spine structure or function. Using total internal reflectance fluorescent microscopy we discovered that microtubules rapidly invade dendritic protrusions of mature CNS neurons (up to 63 d in vitro), occasionally being associated with marked changes in spine morphology in the form of transient spine head protrusions (tSHPs). Two microtubules can occupy a spine simultaneously and microtubule targeting can occur from both the proximal and distal dendrite. A small percentage of spines are targeted at a time and all targeting events are transient, averaging only a few minutes. Nevertheless, over time many spines on a dendrite are targeted by microtubules. Importantly, we show that increasing neuronal activity enhances both the number of spines invaded by microtubules and the duration of these invasions. This study provides new insight into the dynamics of the neuronal cytoskeleton in mature CNS neurons and suggests that microtubules play an important, direct role in spine morphology and function.

Microtubules (MTs) are capable of entering dendritic spines in mature hippocampal neurons through dynamic polymerization. Although these MT invasions are directly associated with neuronal activity, their function remains unknown. Here we demonstrate in mouse hippocampal neurons that MT entries into spines regulate the increase in post-synaptic density protein-95 (PSD-95) after brain-derived neurotrophic factor (BDNF) treatment. Using multi-wavelength total internal reflectance fluorescence microscopy (TIRFM) we show that BDNF prolonged the average MT dwell time in spines and this effect was dependent on TrkB receptor activation. Further examination revealed that peaks of MT polymerization into spines corresponded to rapid PSD-95 increases in the spine head. Over time, spines targeted by MTs after BDNF application, but not before, showed a robust increase in PSD-95. Conversely, spines completely devoid of MT invasions showed no significant change in the level of PSD-95. Pharmacological inhibition of MT dynamics abolished the BDNF-induced increase in PSD-95. Together these results support the hypothesis that the well known increase in PSD-95 within spines after BDNF treatment is dependent on MT invasions of dendritic spines. Thus, our study provides a direct link between dynamic MTs and the post-synaptic structure, and provides a functional role for MT invasion of dendritic spines.

Most excitatory synaptic terminals in the brain impinge on dendritic spines. We and others have recently shown that dynamic microtubules (MTs) enter spines from the dendritic shaft. However, a direct role for MTs in long-lasting spine plasticity has yet to be demonstrated and it remains unclear whether MT-spine invasions are directly influenced by synaptic activity. Lasting changes in spine morphology and synaptic strength can be triggered by activation of synaptic NMDA receptors (NMDARs) and are associated with learning and memory processes. To determine whether MTs are involved in NMDAR-dependent spine plasticity, we imaged MT dynamics and spine morphology in live mouse hippocampal pyramidal neurons before and after acute activation of synaptic NMDARs. Synaptic NMDAR activation promoted MT-spine invasions and lasting increases in spine size, with invaded spines exhibiting significantly faster and more growth than non-invaded spines. Even individual MT invasions triggered rapid increases in spine size that persisted longer following NMDAR activation. Inhibition of either NMDARs or dynamic MTs blocked NMDAR-dependent spine growth. Together these results demonstrate for the first time that MT-spine invasions are positively regulated by signaling through synaptic NMDARs, and contribute to long-lasting structural changes in targeted spines.

Summary Neurite formation is a seminal event in the early development of neurons. However, little is known about the mechanisms by which neurons form neurites. F-BAR proteins function in sensing and inducing membrane curvature [1, 2]. Cdc42-interacting protein 4 (CIP4), a member of the F-BAR family, regulates endocytosis in a variety of cell types [3–9]. However, there is little data on how CIP4 functions in neurons [10, 11]. Here we show that CIP4 plays a novel role in neuronal development by inhibiting neurite formation. Remarkably, CIP4 exerts this effect not through endocytosis, but by producing lamellipodial protrusions. In primary cortical neurons CIP4 is concentrated specifically at the tips of extending lamellipodia and filopodia, instead of endosomes as in other cell types. Overexpression of CIP4 results in lamellipodial protrusions around the cell body, subsequently delaying neurite formation and enlarging growth cones. These effects depend on the F-BAR and SH3 domains of CIP4 and on its ability to multimerize. Conversely, cortical neurons from CIP4-null mice initiate neurites twice as fast as controls. This is the first study to demonstrate that an F-BAR protein functions differently in neuronal vs. non-neuronal cells and induces lamellipodial protrusions instead of invaginations or filopodia-like structures.

No abstract

Understanding network development in the brain is of tremendous fundamental importance, but it is immensely challenging because of the complexity of both its architecture and function. The mechanisms of axonal navigation to target regions and the specific interactions with guidance factors such as membrane-bound proteins, chemical gradients, mechanical guidance cues, etc., are largely unknown. A current limitation for the study of neural network formation is the ability to control precisely the connectivity of small groups of neurons. A first step in designing such networks is to understand the "rules" central nervous system (CNS) neurons use to form functional connections with one another. Here we begin to delineate novel rules for growth and connectivity of small numbers of neurons patterned on Au substrates in simplified geometries. These studies yield new insights into the mechanisms determining the organizational features present in intact systems. We use a previously reported atomic force microscopy (AFM) nanolithography method to control precisely the location and growth of neurons on these surfaces. By examining a series of systems with different geometrical parameters, we quantitatively and systematically analyze how neuronal growth depends on these parameters.

Clotrimazole troche discontinuation at 3 months after transplantation may cause significant tacrolimus trough level reductions. In addition, when trough levels are below 6 ng/ml, these fluctuations may contribute to the occurrence of allograft rejection.

Patients receiving tigecycline were less likely to achieve optimal clinical outcomes and had more adverse events. Alternative regimens should be selected over tigecycline for the treatment of polymicrobial IAIs in abdominal SOT recipients until additional studies are completed to examine its role in this population.