Journal of Neuroscience DevelopmentPlasticityRepair

Synaptic Activity Induces Dramatic Changes in the Geometry of the Cell Nucleus: Interplay between Nuclear Structure, Histone H3 Phosphorylation, and Nuclear Calcium Signaling

Wed, 25 Nov 2009 10:03:10 PST

Synaptic activity initiates many adaptive responses in neurons. Here we report a novel form of structural plasticity in dissociated hippocampal cultures and slice preparations. Using a recently developed algorithm for three-dimensional image reconstruction and quantitative measurements of cell organelles, we found that many nuclei from hippocampal neurons are highly infolded and form unequally sized nuclear compartments. Nuclear infoldings are dynamic structures, which can radically transform the geometry of the nucleus in response to neuronal activity. Action potential bursting causing synaptic NMDA receptor activation dramatically increases the number of infolded nuclei via a process that requires the ERK-MAP kinase pathway and new protein synthesis. In contrast, death-signaling pathways triggered by extrasynaptic NMDA receptors cause a rapid loss of nuclear infoldings. Compared with near-spherical nuclei, infolded nuclei have a larger surface and increased nuclear pore complex immunoreactivity. Nuclear calcium signals evoked by cytosolic calcium transients are larger in small nuclear compartments than in the large compartments of the same nucleus; moreover, small compartments are more efficient in temporally resolving calcium signals induced by trains of action potentials in the theta frequency range (5 Hz). Synaptic activity-induced phosphorylation of histone H3 on serine 10 was more robust in neurons with infolded nuclei compared with neurons with near-spherical nuclei, suggesting a functional link between nuclear geometry and transcriptional regulation. The translation of synaptic activity-induced signaling events into changes in nuclear geometry facilitates the relay of calcium signals to the nucleus, may lead to the formation of nuclear signaling microdomains, and could enhance signal-regulated transcription.

Prostate Stem Cell Antigen Is an Endogenous lynx1-Like Prototoxin That Antagonizes {alpha}7-Containing Nicotinic Receptors and Prevents Programmed Cell Death of Parasympathetic Neurons

Wed, 25 Nov 2009 10:03:10 PST

Vertebrate -bungarotoxin-like molecules of the Ly-6 superfamily have been implicated as balancers of activity and survival in the adult nervous system. To determine whether a member of this family could be involved in the development of the avian ciliary ganglion, we identified 6 Gallus genes by their homology in structure to mouse lynx1 and lynx2. One of these genes, an ortholog of prostate stem cell antigen (psca), is barely detectable at embryonic day (E) 8, before neuronal cell loss in the ciliary ganglion, but increases >100-fold as the number of neurons begins to decline between E9 and E14. PSCA is highly expressed in chicken and mouse telencephalon and peripheral ganglia and correlates with expression of 7-containing nicotinic acetylcholine receptors (7-nAChRs). Misexpressing PSCA before cell death in the ciliary ganglion blocks 7-nAChR activation by nicotine and rescues the choroid subpopulation from dying. Thus, PSCA, a molecule previously identified as a marker of prostate cancer, is a member of the Ly-6 neurotoxin-like family in the nervous system, and is likely to play a role as a modulator of 7 signaling-induced cell death during development.

Switching Retinogeniculate Axon Laterality Leads to Normal Targeting but Abnormal Eye-Specific Segregation That Is Activity Dependent

Rebsam, A., Petros, T. J., Mason, C. A. — Wed, 25 Nov 2009 10:03:10 PST

Partial decussation of sensory pathways allows neural inputs from both sides of the body to project to the same target region where these signals will be integrated. Here, to better understand mechanisms of eye-specific targeting, we studied how retinal ganglion cell (RGC) axons terminate in their thalamic target, the dorsal lateral geniculate nucleus (dLGN), when crossing at the optic chiasm midline is altered. In models with gain- and loss-of-function of EphB1, the receptor that directs the ipsilateral projection at the optic chiasm, misrouted RGCs target the appropriate retinotopic zone in the opposite dLGN. However, in EphB1^–/– mice, the misrouted axons do not intermingle with normally projecting RGC axons and segregate instead into a distinct patch. We also revisited the role of retinal activity on eye-specific targeting by blocking correlated waves of activity with epibatidine into both eyes. We show that, in wild-type mice, retinal waves are necessary during the first postnatal week for both proper distribution and eye-specific segregation of ipsilateral axons in the mature dLGN. Moreover, in EphB1^–/– mice, refinement of ipsilateral axons is perturbed in control conditions and is further impaired after epibatidine treatment. Finally, retinal waves are required for the formation of the segregated patch of misrouted axons in EphB1^–/– mice. These findings implicate molecular determinants for targeting of eye-specific zones that are independent of midline guidance cues and that function in concert with correlated retinal activity to sculpt retinogeniculate projections.

Combining Peripheral Nerve Grafts and Chondroitinase Promotes Functional Axonal Regeneration in the Chronically Injured Spinal Cord

Wed, 25 Nov 2009 10:03:10 PST

Because there currently is no treatment for spinal cord injury, most patients are living with long-standing injuries. Therefore, strategies aimed at promoting restoration of function to the chronically injured spinal cord have high therapeutic value. For successful regeneration, long-injured axons must overcome their poor intrinsic growth potential as well as the inhibitory environment of the glial scar established around the lesion site. Acutely injured axons that regenerate into growth-permissive peripheral nerve grafts (PNGs) reenter host tissue to mediate functional recovery if the distal graft–host interface is treated with chondroitinase ABC (ChABC) to cleave inhibitory chondroitin sulfate proteoglycans in the scar matrix. To determine whether a similar strategy is effective for a chronic injury, we combined grafting of a peripheral nerve into a highly relevant, chronic, cervical contusion site with ChABC treatment of the glial scar and glial cell line-derived neurotrophic factor (GDNF) stimulation of long-injured axons. We tested this combination in two grafting paradigms: (1) a peripheral nerve that was grafted to span a chronic injury site or (2) a PNG that bridged a chronic contusion site with a second, more distal injury site. Unlike GDNF–PBS treatment, GDNF–ChABC treatment facilitated axons to exit the PNG into host tissue and promoted some functional recovery. Electrical stimulation of axons in the peripheral nerve bridge induced c-Fos expression in host neurons, indicative of synaptic contact by regenerating fibers. Thus, our data demonstrate, for the first time, that administering ChABC to a distal graft interface allows for functional axonal regeneration by chronically injured neurons.

BDNF-Hypersecreting Human Mesenchymal Stem Cells Promote Functional Recovery, Axonal Sprouting, and Protection of Corticospinal Neurons after Spinal Cord Injury

Wed, 25 Nov 2009 10:03:10 PST

Transplantation of mesenchymal stem cells (MSCs) derived from bone marrow has been shown to improve functional outcome in spinal cord injury (SCI). We transplanted MSCs derived from human bone marrow (hMSCs) to study their potential therapeutic effect in SCI in the rat. In addition to hMSCs, we used gene-modified hMSCs to secrete brain-derived neurotrophic factor (BDNF-hMSCs). After a dorsal transection lesion was induced at T9, cells were microinjected on each side of the transection site. Fluorogold (FG) was injected into the epicenter of the lesion cavity to identify transected corticospinal tract (CST) neurons. At 5 weeks after transplantation, the animals were perfused. Locomotor recovery improvement was observed for the BDNF-hMSC group, but not in the hMSC group. Structurally there was increased sprouting of injured corticospinal tract and serotonergic projections after hMSC and BDNF-hMSC transplantation. Moreover, an increased number of serotonergic fibers was observed in spinal gray matter including the ventral horn at and below the level of the lesion, indicating increased innervation in the terminal regions of a descending projection important for locomotion. Stereological quantification was performed on the brains to determine neuronal density in primary motor (M1) cortex. The number of FG backfilled cells demonstrated an increased cell survival of CST neurons in M1 cortex in both the hMSC and BDNF-hMSC groups at 5 weeks, but the increase for the BDNF-hMSC group was greater. These results indicate that transplantation of hMSCs hypersecreting BDNF results in structural changes in brain and spinal cord, which are associated with improved functional outcome in acute SCI.

Distinct Muscarinic Acetylcholine Receptor Subtypes Contribute to Stability and Growth, But Not Compensatory Plasticity, of Neuromuscular Synapses

Wed, 25 Nov 2009 10:03:10 PST

Muscarinic acetylcholine receptors (mAChRs) modulate synaptic function, but whether they influence synaptic structure remains unknown. At neuromuscular junctions (NMJs), mAChRs have been implicated in compensatory sprouting of axon terminals in paralyzed or denervated muscles. Here we used pharmacological and genetic inhibition and localization studies of mAChR subtypes at mouse NMJs to demonstrate their roles in synaptic stability and growth but not in compensatory sprouting. M₂ mAChRs were present solely in motor neurons, whereas M₁, M₃, and M₅ mAChRs were associated with Schwann cells and/or muscle fibers. Blockade of all five mAChR subtypes with atropine evoked pronounced effects, including terminal sprouting, terminal withdrawal, and muscle fiber atrophy. In contrast, methoctramine, an M_2/4-preferring antagonist, induced terminal sprouting and terminal withdrawal, but no muscle fiber atrophy. Consistent with this observation, M₂^–/– but no other mAChR mutant mice exhibited spontaneous sprouting accompanied by extensive loss of parental terminal arbors. Terminal sprouting, however, seemed not to be the causative defect because partial loss of terminal branches was common even in the M₂^–/– NMJs without sprouting. Moreover, compensatory sprouting after paralysis or partial denervation was normal in mice deficient in M₂ or other mAChR subtypes. We also found that many NMJs of M₅^–/– mice were exceptionally small and reduced in proportion to the size of parental muscle fibers. These findings show that axon terminals are unstable without M₂ and that muscle fiber growth is defective without M₅. Subtype-specific muscarinic signaling provides a novel means for coordinating activity-dependent development and maintenance of the tripartite synapse.

Telomere Shortening in Neural Stem Cells Disrupts Neuronal Differentiation and Neuritogenesis

Wed, 18 Nov 2009 10:02:12 PST

Proliferation in the subependymal zone (SEZ) and neurogenesis in the olfactory bulb decline in the forebrain of telomerase-deficient mice. The present work reveals additional effects of telomere shortening on neuronal differentiation, as adult multipotent progenitors with critically short telomeres yield reduced numbers of neurons that, furthermore, exhibit underdeveloped neuritic arbors. Genetic data indicate that the tumor suppressor protein p53 not only mediates the adverse effects of telomere attrition on proliferation and self-renewal but it is also involved in preventing normal neuronal differentiation of adult progenitors with dysfunctional telomeres. Interestingly, progenitor cells with short telomeres obtained from fetal brains do not exhibit any replicative defects but also fail to acquire a fully mature neuritic arbor, demonstrating cell cycle-independent effects of telomeres on neuronal differentiation. The negative effect of p53 on neuritogenesis is mechanistically linked to its cooperation with the Notch pathway in the upregulation of small GTPase RhoA kinases, Rock1 and Rock2, suggesting a potential link between DNA damage and the Notch signaling pathway in the control of neuritogenesis. We also show that telomerase expression is downregulated in the SEZ of aging mice leading to telomere length reductions in neurosphere-forming cells and deficient neurogenesis and neuritogenesis. Our results suggest that age-related deficits could be caused partly by dysfunctional telomeres and demonstrate that p53 is a central modulator of adult neurogenesis, regulating both the production and differentiation of postnatally generated olfactory neurons.

Adult-Born Hippocampal Neurons Are More Numerous, Faster Maturing, and More Involved in Behavior in Rats than in Mice

Wed, 18 Nov 2009 10:02:12 PST

Neurons are born throughout adulthood in the hippocampus and show enhanced plasticity compared with mature neurons. However, there are conflicting reports on whether or not young neurons contribute to performance in behavioral tasks, and there is no clear relationship between the timing of maturation of young neurons and the duration of neurogenesis reduction in studies showing behavioral deficits. We asked whether these discrepancies could reflect differences in the properties of young neurons in mice and rats. We report that young neurons in adult rats show a mature neuronal marker profile and activity-induced immediate early gene expression 1–2 weeks earlier than those in mice. They are also twice as likely to escape cell death, and are 10 times more likely to be recruited into learning circuits. This comparison holds true in two different strains of mice, both of which show high rates of neurogenesis relative to other background strains. Differences in adult neurogenesis are not limited to the hippocampus, as the density of new neocortical neurons was 5 times greater in rats than in mice. Finally, in a test of function, we find that the contribution of young neurons to fear memory is much greater in rats than in mice. These results reveal substantial differences in new neuron plasticity and function between these two commonly studied rodent species.

The Transition from Radial Glial to Intermediate Progenitor Cell Is Inhibited by FGF Signaling during Corticogenesis

Kang, W., Wong, L. C., Shi, S.-H., Hebert, J. M. — Wed, 18 Nov 2009 10:02:12 PST

During corticogenesis, the balance between the self-renewal of radial glial stem cells and the production of their descendent progenitor cells is essential in generating the correct size and cell composition of the neocortex. How the stem-to-progenitor cell transition is regulated is poorly understood. FGFs are commonly implicated in promoting proliferation of neural precursor cells, but it is unclear how they exert their effects on stem cells, progenitor cells, or both in vivo. Here, three FGF receptor genes are simultaneously deleted during cortical neurogenesis. In these mutants, radial glia are depleted due to an increased transition from an uncommitted state to a more differentiated one, initially causing an increase in progenitors, but ultimately resulting in a smaller cortex. The proliferation rate of progenitors themselves, however, is unchanged. These results indicate that FGFs normally repress the radial glia to progenitor cell transition during corticogenesis.

Regulation of Radial Glial Motility by Visual Experience

Wed, 11 Nov 2009 10:02:14 PST

Radial glia in the developing optic tectum express the key guidance molecules responsible for topographic targeting of retinal axons. However, the extent to which the radial glia are themselves influenced by retinal inputs and visual experience remains unknown. Using multiphoton live imaging of radial glia in the optic tectum of intact Xenopus laevis tadpoles in conjunction with manipulations of neural activity and sensory stimuli, radial glia were observed to exhibit spontaneous calcium transients that were modulated by visual stimulation. Structurally, radial glia extended and retracted many filopodial processes within the tectal neuropil over minutes. These processes interacted with retinotectal synapses and their motility was modulated by nitric oxide (NO) signaling downstream of neuronal NMDA receptor (NMDAR) activation and visual stimulation. These findings provide the first in vivo demonstration that radial glia actively respond both structurally and functionally to neural activity, via NMDAR-dependent NO release during the period of retinal axon ingrowth.

Hyperdopaminergic Tone Erodes Prefrontal Long-Term Potential via a D2 Receptor-Operated Protein Phosphatase Gate

Wed, 11 Nov 2009 10:02:14 PST

Dopamine (DA) plays crucial roles in the cognitive functioning of the prefrontal cortex (PFC), which, to a large degree, depends on lasting neural traces formed in prefrontal networks. The establishment of these permanent traces requires changes in cortical synaptic efficacy. DA, via the D₁-class receptors, is thought to gate or facilitate synaptic plasticity in the PFC, with little role recognized for the D₂-class receptors. Here we show that, when significantly elevated, DA erodes, rather than facilitates, the induction of long-term potentiation (LTP) in the PFC by acting at the far less abundant cortical D₂-class receptors through a dominant coupling to the protein phosphatase 1 (PP1) activity in postsynaptic neurons. In mice with persistently elevated extracellular DA, resulting from inactivation of the DA transporter (DAT) gene, LTP in layer V PFC pyramidal neurons cannot be established, regardless of induction protocols. Acute increase of dopaminergic transmission by DAT blockers or overstimulation of D₂ receptors in normal mice have similar LTP shutoff effects. LTP in mutant mice can be rescued by a single in vivo administration of D₂-class antagonists. Suppression of postsynaptic PP1 mimics and occludes the D₂-mediated rescue of LTP in mutant mice and prevents the acute erosion of LTP by D₂ agonists in normal mice. Our studies reveal a mechanistically unique heterosynaptic PP1 gate that is constitutively driven by background DA to influence LTP induction. By blocking prefrontal synaptic plasticity, excessive DA may prevent storage of lasting memory traces in PFC networks and impair executive functions.

The Conserved Ig Superfamily Member Turtle Mediates Axonal Tiling in Drosophila

Ferguson, K., Long, H., Cameron, S., Chang, W.-T., Rao, Y. — Wed, 11 Nov 2009 10:02:14 PST

Restriction of adjacent same-type axons/dendrites to separate single columns for specific neuronal connections is commonly observed in vertebrates and invertebrates, and is necessary for proper processing of sensory information. Columnar restriction is conceptually similar to tiling, a phenomenon referring to the avoidance of neurites from adjacent same-type neurons. The molecular mechanism underlying the establishment of columnar restriction or axonal/dendritic tiling remains largely undefined. Here, we identify Turtle (Tutl), a member of the conserved Tutl/Dasm1/IgSF9 subfamily of the Ig superfamily, as a key player in regulating the tiling pattern of R7 photoreceptor terminals in Drosophila. Tutl functions to prevent fusion between two adjacent R7 terminals, and acts in parallel to the Activin pathway. Tutl mediates homophilic cell–cell interactions. We propose that extrinsic terminal–terminal recognition mediated by Tutl, acts in concert with intrinsic Activin-dependent control of terminal growth, to restrict the connection made by each R7 axon to a single column.

Robo-2 Controls the Segregation of a Portion of Basal Vomeronasal Sensory Neuron Axons to the Posterior Region of the Accessory Olfactory Bulb

Wed, 11 Nov 2009 10:02:14 PST

The ability of sensory systems to detect and process information from the environment relies on the elaboration of precise connections between sensory neurons in the periphery and second order neurons in the CNS. In mice, the accessory olfactory system is thought to regulate a wide variety of social and sexual behaviors. The expression of the Slit receptors Robo-1 and Robo-2 in vomeronasal sensory neurons (VSNs) suggests they may direct the stereotypic targeting of their axons to the accessory olfactory bulb (AOB). Here, we have examined the roles of Robo-1 and Robo-2 in the formation of connections by VSN axons within the AOB. While Robo-1 is not necessary for the segregation of VSN axons within the anterior and posterior regions of the AOB, Robo-2 is required for the targeting of some basal VSN axons to the posterior region of the AOB but is dispensable for the fasciculation of VSN axons. Furthermore, the specific ablation of Robo-2 expression in VSNs leads to mistargeting of a portion of basal VSN axons to the anterior region of the AOB, indicating that Robo-2 expression is required on projecting VSN axons. Together, these results identify Robo-2 as a receptor that controls the targeting of basal VSN axons to the posterior AOB.

Brn3a and Nurr1 Mediate a Gene Regulatory Pathway for Habenula Development

Quina, L. A., Wang, S., Ng, L., Turner, E. E. — Wed, 11 Nov 2009 10:02:15 PST

The habenula is a dorsal diencephalic structure consisting of medial and lateral subnuclei and a principal output tract, the fasciculus retroflexus, which together form a link between the limbic forebrain and ventral midbrain. Here, we have used microarray and bioinformatic approaches in the mouse to show that the habenula is a distinctive molecular territory of the CNS, with a unique profile of neurotransmitter, ion channel, and regulatory factor expression. Neurons of the medial habenula and part of the lateral habenula express the transcription factor Brn3a/Pou4f1, and Brn3a-expressing habenular neurons project exclusively to the interpeduncular nucleus in the ventral midbrain. In Brn3a mutant embryos, the fasciculus retroflexus is directed appropriately, but habenular neurons fail to innervate their targets. Microarray analysis of Brn3a null embryos shows that this factor regulates an extensive program of habenula-enriched genes, but not generic neural properties. The orphan nuclear receptor Nurr1/Nr4a2 is coexpressed with Brn3a in the developing habenula, is downstream of Brn3a, and mediates expression of a subset of Brn3a-regulated transcripts. Together, these findings begin to define a gene regulatory pathway for habenula development in mammals.

Neuroprotective and Axon Growth-Promoting Effects following Inflammatory Stimulation on Mature Retinal Ganglion Cells in Mice Depend on Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor

Leibinger, M., Muller, A., Andreadaki, A., Hauk, T. G., Kirsch, M., Fischer, D. — Wed, 11 Nov 2009 10:02:15 PST

After optic nerve injury retinal ganglion cells (RGCs) normally fail to regenerate axons in the optic nerve and undergo apoptosis. However, lens injury (LI) or intravitreal application of zymosan switch RGCs into an active regenerative state, enabling these neurons to survive axotomy and to regenerate axons into the injured optic nerve. Several factors have been proposed to mediate the beneficial effects of LI. Here, we investigated the contribution of glial-derived ciliary neurotrophic factor (CNTF) to LI-mediated regeneration and neuroprotection using wild-type and CNTF-deficient mice. In wild-type mice, CNTF expression was strongly upregulated in retinal astrocytes, the JAK/STAT3 pathway was activated in RGCs, and RGCs were transformed into an active regenerative state after LI. Interestingly, retinal LIF expression was correlated with CNTF expression after LI. In CNTF-deficient mice, the neuroprotective and axon growth-promoting effects of LI were significantly reduced compared with wild-type animals, despite an observed compensatory upregulation of LIF expression in CNTF-deficient mice. The positive effects of LI and also zymosan were completely abolished in CNTF/LIF double knock-out mice, whereas LI-induced glial and macrophage activation was not compromised. In culture CNTF and LIF markedly stimulated neurite outgrowth of mature RGCs. These data confirm a key role for CNTF in directly mediating the neuroprotective and axon regenerative effects of inflammatory stimulation in the eye and identify LIF as an additional contributing factor.

Retinotopic Mapping Requires Focal Adhesion Kinase-Mediated Regulation of Growth Cone Adhesion

Woo, S., Rowan, D. J., Gomez, T. M. — Wed, 04 Nov 2009 10:02:51 PST

Adhesion controls growth cone motility, yet the effects of axon guidance cues on adhesion site dynamics are poorly understood. Here we show that ephrin-A1 reduces retinal ganglion cell (RGC) axon outgrowth by stabilizing existing adhesions and inhibiting new adhesion assembly. Ephrin-A1 activates focal adhesion kinase (FAK) in an integrin- and Src-dependent manner and the effects of ephrin-A1 on growth cone motility require FAK activation. We also find that FAK is expressed in a high temporal to low nasal gradient in RGCs, similar to EphA receptors, and that balanced FAK activation is necessary for optimal axon outgrowth. Last, we find that FAK is required for proper topographic positioning of retinal axons along the anterior–posterior axis of the optic tectum in both Xenopus and zebrafish, a guidance decision mediated in part by A-type ephrins. Together, our data suggest that ephrin-A1 controls growth cone advance by modulating adhesive point contacts through FAK activation and that graded FAK signaling is an important component of ephrin-A-mediated retinotopic mapping.