Glen S. Marrs

Rapid formation and remodeling of postsynaptic densities in developing dendrites

The dynamics of postsynaptic density (PSD) formation and remodeling were investigated in live developing hippocampal tissue slices. Time lapse imaging of transfected neurons expressing GFP-tagged PSD95, a prominent PSD protein, revealed that up to 40% of PSDs in developing dendrites are structurally dynamic; they rapidly (<15 min) appear or disappear, but also grow, shrink and move within shafts and spines. New spines containing PSDs were formed by conversion of dynamic filopodia-like spine precursors in which PSDs appeared de novo, or by direct extension of spines or spine precursors carrying preformed PSDs from the shaft. PSDs are therefore highly dynamic structures that can undergo rapid structural alteration within dendrite shafts, spines and spine precursors, permitting rapid formation and remodeling of synaptic connections in developing CNS tissues.

Neuronal activity regulates glutamate transporter dynamics in developing astrocytes

Glutamate transporters (GluTs) maintain a low ambient level of glutamate in the central nervous system (CNS) and shape the activation of glutamate receptors at synapses. Nevertheless, the mechanisms that regulate the trafficking and localization of transporters near sites of glutamate release are poorly understood. Here, we examined the subcellular distribution and dynamic remodeling of the predominant GluT GLT‐1 (excitatory amino acid transporter 2, EAAT2) in developing hippocampal astrocytes. Immunolabeling revealed that endogenous GLT‐1 is concentrated into discrete clusters along branches of developing astrocytes that were apposed preferentially to synapsin‐1 positive synapses. Green fluorescent protein (GFP)‐GLT‐1 fusion proteins expressed in astrocytes also formed distinct clusters that lined the edges of astrocyte processes, as well as the tips of filopodia and spine‐like structures. Time‐lapse three‐dimensional confocal imaging in tissue slices revealed that GFP‐GLT‐1 clusters were dynamically remodeled on a timescale of minutes. Some transporter clusters moved within developing astrocyte branches as filopodia extended and retracted, while others maintained stable positions at the tips of spine‐like structures. Blockade of neuronal activity with tetrodotoxin reduced both the density and perisynaptic localization of GLT‐1 clusters. Conversely, enhancement of neuronal activity increased the size of GLT‐1 clusters and their proximity to synapses. Together, these findings indicate that neuronal activity influences both the organization of GluTs in developing astrocyte membranes and their position relative to synapses.

Hippocampal mossy fibers induce assembly and clustering of PSD95-containing postsynaptic densities independent of glutamate receptor activation.

Factors that regulate the formation, spatial patterning, and maturation of CNS synapses are poorly understood. We used organotypic hippocampal slice cultures derived from developing (P5–P7) rat to test whether synaptic activity regulates the development and organization of postsynaptic structures at mossy fiber (MF) giant synapses. Antibodies to a prominent postsynaptic density (PSD) scaffold protein, PSD95, identified large (>1 μm) and irregularly shaped PSD assemblies that codistributed with synapsin‐I or metabotropic glutamate receptor 7b (mGluR7b) ‐immunolabeled MF terminals in area CA3. To investigate the spatial organization of synaptic PSDs on individual pyramidal cells, neurons in slice cultures were transfected with a vector encoding a GFP‐PSD95 fusion protein. Confocal three‐dimensional reconstructions revealed clusters of PSDs along proximal dendrites of transfected pyramidal neurons in area CA3, but not in CA1. Clusters averaged 7.6 μm in length (range, 2.2–29 μm) and contained up to 35 individual PSDs (mean, 8.3). PSD clusters failed to form when slices were cultured without MFs, indicating that MFs induce cluster assembly. Chronic blockade of N‐methyl‐D‐apartate– and AMPA/kainate‐type glutamate receptors did not disrupt MF targeting or de novo formation of PSD clusters with a normal distribution on target cells. Additionally, glutamate receptor blockers did not alter the ultrastructural development of MF giant synapses containing multiple puncta adherens‐like junctions and asymmetric synaptic junctions at dendritic shaft and spine domains, respectively. The results indicate that MF axons can induce the assembly and clustering of PSD95‐containing postsynaptic complexes, displaying a normal subcellular and tissue distribution, by mechanisms that are independent of ionotropic glutamate receptor activation. J. Comp. Neurol. 440:284–298, 2001.

Using Fluorescent Post‐Labeling To Probe the Subcellular Localization of DNA‐Targeted Platinum Anticancer Agents

Adducts in nuclear DNA are the major cause of cancer cell death triggered by platinum-based anticancer drugs.[1] Thus, cellular uptake and accumulation, distribution and trafficking between subcellular compartments and, ultimately, localization to the nucleus are crucial parameters in the mechanism of these agents. Several techniques have been used to monitor intracellular platinum. These include element-specific analytical methods and nondestructive absorption or emission-based imaging techniques, as well as electron microscopy.[2,3] Fluorophore-tagged derivatives have provided insight into uptake, distribution, and intracellular transformation of platinum.[2] Such an approach has to take into consideration the organelle selectivity of the fluorophore, which may vary widely depending on parameters such as molecular weight, partition coefficient (log P), amphiphilic character, and pKa value.[4] Thus, one drawback of modifying platinum drugs with organic fluorophores is that such conjugates may, at least in part, mimic the properties of the reporter molecule.[5] This would be an undesired feature unless the fluorescent group itself is a functionally important part of the bioactive molecule. Likewise, bulky fluorophores may interfere with the DNA binding mechanism of platinum. To circumvent these problems, we have developed a method based on bioorthogonal ligation chemistry, which allowed us to fluorescently label platinum–acridine hybrid agents in lung cancer cells. Here, we report the development of this technique and demonstrate, for the first time, that post-labeling is a powerful tool for detecting DNA-targeted platinum in subcellular structures.

Dendritic arbors of developing retinal ganglion cells are stabilized by β1-integrins

The architecture of dendritic arbors is a defining characteristic of neurons and is established through a sequential but overlapping series of events involving process outgrowth and branching, stabilization of the global pattern, and synapse formation. To investigate the roles of cadherins and beta1-integrins in maintaining the global architecture of the arbor, we used membrane permeable peptides and transfection with dominant-negative constructs to disrupt adhesion molecule function in intact chick neural retina at a stage when the architecture of the ganglion cell (RGC) arbor is established but synapse formation is just beginning. Inactivation of beta1-integrins induces rapid dendrite retraction, with loss of dynamic terminal filopodia followed by resorption of major branches. Disruption of N-cadherin-beta-catenin interactions has no effect; however, dendrites do retract following perturbation of the juxtamembrane region of N-cadherin, which disrupts N-cadherin-mediated adhesion and initiates a beta1-integrin inactivating signal. Thus, developing RGC dendritic arbors are stabilized by beta1-integrin-dependent processes.

Embryonic assembly of auditory circuits: spiral ganglion and brainstem

Key points  •  Neural activity in sensory systems is important for proper assembly of neural circuits during early development. •  In the auditory system, the timing for functional synapse formation and reliance of central circuit formation on peripherally generated activity are unknown. •  We used a novel whole‐head slice preparation and genetically labelled auditory neurons to provide the first assay of cells at embryonic ages and reveal a peripheral‐to‐central maturation sequence. •  Auditory nerve fibres generate spontaneous action potentials by embryonic day 14 and drive third order central neurons by embryonic day 17, nearly 2 weeks before hearing onset. •  This information provides a timeline for initial auditory circuit assembly and the basis to explore mechanisms for initial sensory circuit assembly.

Concepts in imaging and microscopy. Exploring biological structure and function with confocal microscopy.

Confocal microscopy is providing new and exciting opportunities for imaging cell structure and physiology in thick biological specimens, in three dimensions, and in time. The utility of confocal microscopy relies on its fundamental capacity to reject out-of-focus light, thus providing sharp, high-contrast images of cells and subcellular structures within thick samples. Computer controlled focusing and image-capturing features allow for the collection of through-focus series of optical sections that may be used to reconstruct a volume of tissue, yielding information on the 3-D structure and relationships of cells. Tissues and cells may also be imaged in two or three spatial dimensions over time. The resultant digital data, which encode the image, are highly amenable to processing, manipulation and quantitative analyses. In conjunction with a growing variety of vital fluorescent probes, confocal microscopy is yielding new information about the spatiotemporal dynamics of cell morphology and physiology in living tissues and organisms. Here we use mammalian brain tissue to illustrate some of the ways in which multidimensional confocal fluorescence imaging can enhance studies of biological structure and function.

Does the brain connect before the periphery can direct? A comparison of three sensory systems in mice.

The development of peripheral to central neural connections within the auditory, visual, and olfactory systems of mice is reviewed to address whether peripheral signaling may play an instructive role during initial synapse formation. For each sensory system, developmental times of histogenesis and the earliest ages of innervation and function are considered for peripheral and selected central relays. For the auditory and visual system, anatomical and functional reports indicate that central connections may form prior to synapse formation in the periphery. However, evidence from the olfactory system suggests that the peripheral olfactory sensory neurons form synaptic connections before more central olfactory connections are established. We find that significant gaps in knowledge exist for embryonic development of these systems in mice and that genetic tools have not yet been systematically directed to address these issues.

Embryonic Origins of the Mouse Superior Olivary Complex

Many areas of the central nervous system are organized into clusters of cell groups, with component cell groups exhibiting diverse but related functions. One such cluster, the superior olivary complex (SOC), is located in the ventral auditory brainstem in mammals. The SOC is an obligatory contact point for most projection neurons of the ventral cochlear nucleus and plays central roles in many aspects of monaural and binaural information processing. Despite their important interrelated functions, little is known about the embryonic origins of SOC nuclei, due in part to a paucity of developmental markers to distinguish individual cell groups. In this report, we present a collection of novel markers for the developing SOC nuclei in mice, including the transcription factors FoxP1, MafB, and Sox2, and the lineage‐marking transgenic line En1‐Cre. We use these definitive markers to examine the rhombic lip and rhombomeric origins of SOC nuclei and demonstrate that they can serve to uniquely identify SOC nuclei and subnuclei in newborn pups. The markers are also useful in identifying distinct nuclear domains within the presumptive SOC as early as embryonic day (E) 14.5, well before morphological distinction of individual nuclei is evident. These findings indicate that the mediolateral and dorsoventral position of SOC nuclei characteristic of the adult brainstem is established during early neurogenesis.

N-cadherin modulates voltage activated calcium influx via RhoA, p120-catenin, and myosin–actin interaction

N-cadherin is a transmembrane adhesion receptor that contributes to neuronal development and synapse formation through homophilic interactions that provide structural-adhesive support to contacts between cell membranes. In addition, N-cadherin homotypic binding may initiate cell signaling that regulates neuronal physiology. In this study, we investigated signaling capabilities of N-cadherin that control voltage activated calcium influx. Using whole-cell voltage clamp recording of isolated inward calcium currents in freshly isolated chick ciliary ganglion neurons we show that the juxtamembrane region of N-cadherin cytoplasmic domain regulates high-threshold voltage activated calcium currents by interacting with p120-catenin and activating RhoA. This regulatory mechanism requires myosin interaction with actin. Furthermore, N-cadherin homophilic binding enhanced voltage activated calcium current amplitude in dissociated neurons that have already developed mature synaptic contacts in vivo. The increase in calcium current amplitude was not affected by brefeldin A suggesting that the effect is caused via direct channel modulation and not by increasing channel expression. In contrast, homotypic N-cadherin interaction failed to regulate calcium influx in freshly isolated immature neurons. However, RhoA inhibitors enhanced calcium current amplitude in these immature neurons, suggesting that the inhibitory effect of RhoA on calcium entry is regulated during neuronal development and synapse maturation. These results indicate that N-cadherin modulates voltage activated calcium entry by a mechanism that involves RhoA activity and its downstream effects on the cytoskeleton, and suggest that N-cadherin provides support for synaptic maturation and sustained synaptic activity by facilitating voltage activated calcium influx.