Edward S. Ruthazer

Dendrite growth increased by visual activity requires NMDA receptor and Rho GTPases

Previous studies suggest that neuronal activity may guide the development of synaptic connections in the central nervous system through mechanisms involving glutamate receptors and GTPase-dependent modulation of the actin cytoskeleton. Here we demonstrate by in vivo time-lapse imaging of optic tectal cells in Xenopus laevis tadpoles that enhanced visual activity driven by a light stimulus promotes dendritic arbor growth. The stimulus-induced dendritic arbor growth requires glutamate-receptor-mediated synaptic transmission, decreased RhoA activity and increased Rac and Cdc42 activity. The results delineate a role for Rho GTPases in the structural plasticity driven by visual stimulation in vivo.

Stabilization of Axon Branch Dynamics by Synaptic Maturation

The developmental refinement of topographic projections in the brain is reflected in the dynamic sculpting of axonal arbors that takes place as connections between CNS structures form and mature. To examine the role of synaptogenesis and synaptic maturation in the structural development of axonal projections during the formation of the topographic retinotectal projection, we coexpressed cytosolic fluorescent protein (FP) and FP-tagged synaptophysin (SYP) in small numbers of retinal ganglion cells in living albino Xenopus laevis tadpoles to reveal the distribution and dynamics of presynaptic sites within labeled retinotectal axons. Two-photon time-lapse observations followed by quantitative analysis of tagged SYP levels at individual synapses demonstrated the time course of synaptogenesis: increases in presynaptic punctum intensity are detectable within minutes of punctum emergence and continue over many hours. Puncta lifetimes correlate with their intensities. Furthermore, we found that axon arbor dynamics are affected by synaptic contacts. Axon branches retract past faintly labeled puncta but are locally stabilized at intensely labeled SYP puncta. Visual stimulation for 4 h enhanced the stability of the arbor at intense presynaptic puncta while concurrently inducing the retraction of exploratory branches with only faintly labeled or no synaptic sites.

Relationship between the Ocular Dominance and Orientation Maps in Visual Cortex of Monocularly Deprived Cats

The significance of functional maps for cortical plasticity was investigated by imaging of intrinsic optical signals together with single-unit recording in kittens. After even a brief period of monocular deprivation during the height of the critical period, only isolated patches of visual cortex continued to respond strongly to the closed eye. These deprived-eye patches were located on the pinwheel center singularities of the orientation map and consisted of neurons that were poorly selective for stimulus orientation. Neurons in regions surrounding the deprived-eye patches responded only weakly to the deprived eye but were well tuned for the same stimulus orientation that optimally excited them when presented to the open, nondeprived eye. The coincidence of deprived-eye patches with pinwheel center singularities, and the selective loss of orientation tuning within the deprived-eye patches, indicate that the orientation and ocular dominance maps are functionally linked and provide compelling evidence that pinwheel center singularities are important for cortical plasticity.

On and off domains of geniculate afferents in cat primary visual cortex

On- and off-center geniculate afferents form two major channels of visual processing that are thought to converge in the primary visual cortex. However, humans with severely reduced on responses can have normal visual acuity when tested in a white background, which indicates that off channels can function relatively independently from on channels under certain conditions. Consistent with this functional independence of channels, we demonstrate here that on- and off-center geniculate afferents segregate in different domains of the cat primary visual cortex and that off responses dominate the cortical representation of the area centralis. On average, 70% of the geniculate afferents converging at the same cortical domain had receptive fields of the same contrast polarity. Moreover, off-center afferents dominated the representation of the area centralis in the cortex, but not in the thalamus, indicating that on- and off-center afferents are balanced in number, but not in the amount of cortical territory that they cover.

Activity Dependence of Cortical Axon Branch Formation: A Morphological and Electrophysiological Study Using Organotypic Slice Cultures

The influence of neuronal activity on cortical axon branching was studied by imaging axons of layer 2/3 neurons in organotypic slice cultures of rat visual cortex. Upper layer neurons labeled by electroporation of plasmid encoding yellow fluorescent protein were observed by confocal microscopy. Time-lapse observation of single-labeled axons showed that axons started to branch after 8-10 d in vitro. Over the succeeding 7-10 d, branch complexity gradually increased by both growth and retraction of branches, resulting in axon arbors that morphologically resembled those observed in 2- to 3-week-old animals. Electrophysiological recordings of neuronal activity in the upper layers, made using multielectrode dishes, showed that the frequency of spontaneous firing increased dramatically ∼10 d in vitro and remained elevated at later stages. To examine the involvement of spontaneous firing and synaptic activity in branch formation, various blockers were applied to the culture medium. Cultures were silenced by TTX or by a combination of APV and DNQX but exhibited a homeostatic recovery of spontaneous activity over several days in the presence of blockers of either NMDA-type or non-NMDA-type glutamate receptors alone. Axonal branching was suppressed by TTX and AMPA receptor blockade but not by NMDA receptor blockade. We conclude that cortical axon branching is highly dynamic and that neural activity regulates the early developmental branching of upper layer cortical neurons through the activation of AMPA-type glutamate receptors.

Neural Activity Regulates Synaptic Properties and Dendritic Structure In Vivo through Calcineurin/NFAT Signaling

The calcium-regulated protein phosphatase Calcineurin (CaN) participates in synaptic plasticity and the regulation of transcription factors, including Nuclear Factor of Activated T cells (NFAT). To understand how CaN contributes to neuronal circuit development, whole-cell mEPSC recordings and multiphoton imaging were performed in the visual system of living Xenopus laevis tadpoles electroporated to express either a CaN phosphatase inhibitor or N-VIVIT, a nuclear localization sequence-tagged VIVIT peptide that blocks the binding of CaN to select substrates including NFAT. Both strategies increased mEPSC frequency and dendritic arbor complexity in tectal neurons over 3 days. Expression of either of two constitutively active Xenopus NFATs (CA-NFATs) restored normal synaptic properties in neurons expressing N-VIVIT. However, the morphological phenotype was only rescued by a CA-NFAT bearing an intact regulatory domain, implying that transcriptional control of morphological and electrophysiological properties of neurons is mediated by distinct NFAT interactions.

Development of Single Retinofugal Axon Arbors in Normal and β2 Knock-Out Mice

The maturation of retinal ganglion cell (RGC) axon projections in the dorsal lateral geniculate nucleus (dLGN) and the superior colliculus (SC) relies on both molecular and activity-dependent mechanisms. Despite the increasing popularity of the mouse as a mammalian visual system model, little is known in this species about the normal development of individual RGC axon arbors or the role of activity in this process. We used a novel in vivo single RGC labeling technique to quantitatively characterize the elaboration and refinement of RGC axon arbors in the dLGN and SC in wild-type (WT) and β2–nicotinic acetylcholine receptors mutant (β2−/−) mice, which have perturbed retinal waves, during the developmental period when eye-specific lamination and retinotopic refinement occurs. Our results suggest that eye-specific segregation and retinotopic refinement in WT mice are not the result of refinement of richly exuberant arbors but rather the elaboration of arbors prepositioned in the proper location combined with the elimination of inappropriately targeted sparse branches. We found that retinocollicular arbors mature ∼1 week earlier than retinogeniculate arbors, although RGC axons reach the dLGN and SC at roughly the same age. We also observed striking differences between contralateral and ipsilateral RGC axon arbors in the SC but not in the LGN. These data suggest a strong influence of target specific cues during arbor maturation. In β2−/− mice, we found that retinofugal single axon arbors are well ramified but enlarged, particularly in the SC, indicating that activity-dependent visual map development occurs through the refinement of individual RGC arbors.

D-serine as a gliotransmitter and its roles in brain development and disease

The development of new techniques to study glial cells has revealed that they are active participants in the development of functional neuronal circuits. Calcium imaging studies demonstrate that glial cells actively sense and respond to neuronal activity. Glial cells can produce and release neurotransmitter-like molecules, referred to as gliotransmitters, that can in turn influence the activity of neurons and other glia. One putative gliotransmitter, D-serine is believed to be an endogenous co-agonist for synaptic N-methyl-D-aspartate receptors (NMDARs), modulating synaptic transmission and plasticity mediated by this receptor. The observation that D-serine levels in the mammalian brain increase during early development, suggests a possible role for this gliotransmitter in normal brain development and circuit refinement. In this review we will examine the data that D-serine and its associated enzyme serine racemase are developmentally regulated. We will consider the evidence that D-serine is actively released by glial cells and examine the studies that have implicated D-serine as a critical player involved in regulating NMDAR-mediated synaptic transmission and neuronal migration during development. Furthermore, we will consider how dysregulation of D-serine may play an important role in the etiology of neurological and psychiatric diseases.

DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain

The transmembrane protein deleted in colorectal cancer (DCC) and its ligand, netrin-1, regulate synaptogenesis during development, but their function in the mature central nervous system is unknown. Given that DCC promotes cell-cell adhesion, is expressed by neurons, and activates proteins that signal at synapses, we hypothesized that DCC expression by neurons regulates synaptic function and plasticity in the adult brain. We report that DCC is enriched in dendritic spines of pyramidal neurons in wild-type mice, and we demonstrate that selective deletion of DCC from neurons in the adult forebrain results in the loss of long-term potentiation (LTP), intact long-term depression, shorter dendritic spines, and impaired spatial and recognition memory. LTP induction requires Src activation of NMDA receptor (NMDAR) function. DCC deletion severely reduced Src activation. We demonstrate that enhancing NMDAR function or activating Src rescues LTP in the absence of DCC. We conclude that DCC activation of Src is required for NMDAR-dependent LTP and certain forms of learning and memory.

Radial Glia: Progenitor, Pathway, and Partner

Radial glia (RG) are a glial cell type that can be found from the earliest stages of CNS development. They are clearly identifiable by their unique morphology, having a periventricular cell soma and a long process extending all the way to the opposite pial surface. Due to this striking morphology, RG have long been thought of as a transient substrate for neuron migration in the developing brain. In fact, RG cells, far from exclusively serving as a passive scaffold for cell migration, have a remarkably diverse range of critical functions in CNS development and function. These include serving as progenitors of neurons and glia both during development as well as in response to injury, helping to direct axonal and dendritic process outgrowth, and regulating synaptic development and function. RG also engage in extensive bidirectional signaling both with neurons and one another. This review describes the diversity of RG cell types in the CNS and discusses their many important activities.