Wanhua Shen

Contribution of astrocytes to hippocampal long-term potentiation through release of d-serine

Repetitive correlated activation of pre- and postsynaptic neurons induced long-term potentiation (LTP) of synaptic transmission among hippocampal neurons grown on a layer of astrocytes (mixed cultures) but not among neurons cultured in glial conditioned medium. Supplement of d-serine, an agonist for the glycine-binding site of N-methyl-d-aspartate (NMDA) receptors, enhanced NMDA receptor activation and enabled LTP induction in glial conditioned medium cultures. The induction of LTP in both mixed cultures and hippocampal slices was suppressed by NMDA receptor antagonists, glycine-binding-site blockers of NMDA receptors, or an enzyme that degrades endogenous d-serine. By providing extracellular d-serine that facilitates activation of NMDA receptors, astrocytes thus play a key role in long-term synaptic plasticity.

Acute and gradual increases in BDNF concentration elicit distinct signaling and functions in neurons

Extracellular factors may act on cells in two distinct modes: an acute increase in concentration as a result of regulated secretion, or a gradual increase in concentration when secreted constitutively or from a distant source. We found that cellular responses to brain-derived neurotrophic factor (BDNF) differed markedly depending on how BDNF was delivered. In cultured rat hippocampal neurons, acute and gradual increases in BDNF elicited transient and sustained activation of TrkB receptor and its downstream signaling, respectively, leading to differential expression of Homer1 and Arc. Transient TrkB activation promoted neurite elongation and spine head enlargement, whereas sustained TrkB activation facilitated neurite branch and spine neck elongation. In hippocampal slices, fast and slow increases in BDNF enhanced basal synaptic transmission and LTP, respectively. Thus, the kinetics of TrkB activation is critical for cell signaling and functions. This temporal dimension in cellular signaling may also have implications for the therapeutic drug design.

Long-term potentiation of neuron-glia synapses mediated by Ca2+-permeable AMPA receptors

Interactions between neurons and glial cells in the brain may serve important functions in the development, maintenance, and plasticity of neural circuits. Fast neuron-glia synaptic transmission has been found between hippocampal neurons and NG2 cells, a distinct population of macroglia-like cells widely distributed in the brain. We report that these neuron-glia synapses undergo activity-dependent modifications analogous to long-term potentiation (LTP) at excitatory synapses, a hallmark of neuronal plasticity. However, unlike the induction of LTP at many neuron-neuron synapses, both induction and expression of LTP at neuron-NG2 synapses involve Ca2+-permeable AMPA receptors on NG2 cells.

Activity-induced rapid synaptic maturation mediated by presynaptic Cdc42 signaling

Maturation of presynaptic transmitter secretion machinery is a critical step in synaptogenesis. Here we report that a brief train of presynaptic action potentials rapidly converts early nonfunctional contacts between cultured hippocampal neurons into functional synapses by enhancing presynaptic glutamate release. The enhanced release was confirmed by a marked increase in the number of depolarization-induced FM4-64 puncta in the presynaptic axon. This rapid presynaptic maturation can be abolished by treatments that interfered with presynaptic BDNF and Cdc42 signaling or actin polymerization. Activation of Cdc42 by applying BDNF or bradykinin mimicked the effect of electrical activity in promoting synaptic maturation. Furthermore, activity-induced increase in presynaptic actin polymerization, as revealed by increased concentration of actin-YFP at axon boutons, was abolished by inhibiting BDNF and Cdc42 signaling. Thus, rapid presynaptic maturation induced by neuronal activity is mediated by presynaptic activation of the Cdc42 signaling pathway.

Clathrin-dependent Endocytosis Is Required for TrkB-dependent Akt-mediated Neuronal Protection and Dendritic Growth

Endocytosis of Trk (tropomyosin-related kinase) receptors is critical for neurotrophin signal transduction and biological functions. However, the mechanism governing endocytosis of TrkB (tropomyosin-related kinase B) and the specific contributions of TrkB endocytosis to downstream signaling are unknown. In this study, we report that blocking clathrin, dynamin, or AP2 in cultured neurons of the central nervous system inhibited brain-derived neurotrophic factor (BDNF)-induced activation of Akt but not ERK. Treating neurons with the clathrin inhibitor monodansylcadaverine or a peptide that blocks dynamin function specifically abrogated Akt pathway activation in response to BDNF but did not affect the response of other downstream effectors or the up-regulation of immediate early genes neuropeptide Y and activity-regulated cytoskeleton-associated protein. Similar effects were found in neurons expressing small interfering RNA to silence AP2 or a dominant negative form of dynamin that inhibits clathrin-mediated endocytosis. In PC12 cells, ERK but not Akt activation required TrkA endocytosis following stimulation with nerve growth factor, whereas the opposite was true when TrkA-expressing neurons were stimulated with nerve growth factor in the central nervous system. Thus, the specific effects of internalized Trk receptors probably depend on the presence of cell type-specific modulators of neurotrophin signaling and not on differences inherent to Trk receptors themselves. Endocytosis-dependent activation of Akt in neurons was found to be critical for BDNF-supported survival and dendrite outgrowth. Together, these results demonstrate the functional requirement of clathrin- and dynamin-dependent endocytosis in generating the full intracellular response of neurons to BDNF in the central nervous system.

Inhibition to excitation ratio regulates visual system responses and behavior in vivo

The balance of inhibitory to excitatory (I/E) synaptic inputs is thought to control information processing and behavioral output of the central nervous system. We sought to test the effects of the decreased or increased I/E ratio on visual circuit function and visually guided behavior in Xenopus tadpoles. We selectively decreased inhibitory synaptic transmission in optic tectal neurons by knocking down the γ2 subunit of the GABA(A) receptors (GABA(A)R) using antisense morpholino oligonucleotides or by expressing a peptide corresponding to an intracellular loop of the γ2 subunit, called ICL, which interferes with anchoring GABA(A)R at synapses. Recordings of miniature inhibitory postsynaptic currents (mIPSCs) and miniature excitatory PSCs (mEPSCs) showed that these treatments decreased the frequency of mIPSCs compared with control tectal neurons without affecting mEPSC frequency, resulting in an ∼50% decrease in the ratio of I/E synaptic input. ICL expression and γ2-subunit knockdown also decreased the ratio of optic nerve-evoked synaptic I/E responses. We recorded visually evoked responses from optic tectal neurons, in which the synaptic I/E ratio was decreased. Decreasing the synaptic I/E ratio in tectal neurons increased the variance of first spike latency in response to full-field visual stimulation, increased recurrent activity in the tectal circuit, enlarged spatial receptive fields, and lengthened the temporal integration window. We used the benzodiazepine, diazepam (DZ), to increase inhibitory synaptic activity. DZ increased optic nerve-evoked inhibitory transmission but did not affect evoked excitatory currents, resulting in an increase in the I/E ratio of ∼30%. Increasing the I/E ratio with DZ decreased the variance of first spike latency, decreased spatial receptive field size, and lengthened temporal receptive fields. Sequential recordings of spikes and excitatory and inhibitory synaptic inputs to the same visual stimuli demonstrated that decreasing or increasing the I/E ratio disrupted input/output relations. We assessed the effect of an altered I/E ratio on a visually guided behavior that requires the optic tectum. Increasing and decreasing I/E in tectal neurons blocked the tectally mediated visual avoidance behavior. Because ICL expression, γ2-subunit knockdown, and DZ did not directly affect excitatory synaptic transmission, we interpret the results of our study as evidence that partially decreasing or increasing the ratio of I/E disrupts several measures of visual system information processing and visually guided behavior in an intact vertebrate.

N-methyl-D-aspartate receptor and L-type voltage-gated Ca2+channel antagonists suppress the release of cytochrome C and the expression of procaspase-3 in rat hippocampus after global brain ischemia

Transient global ischemia reportedly results in glutamate receptor stimulation and harmful Ca(2+)-overloading, then activates some proteins involved in cell apoptosis in vivo and in vitro, but underlying mechanisms remain to be elucidated. Here we evaluated the role of N-methyl-D-aspartate (NMDA) receptor antagonist and L-type voltage-gated Ca(2+) channel (L-VGCC) antagonist in mediating the release of cytochrome c and the expression of caspase-3 precursor protein (procaspase-3). Cytochrome c release from mitochondria is a critical step in the cell apoptotic process. We examined whether cytochrome c was translocated from mitochondria to the cytosol by Western blot in rat hippocampus after 15 min global ischemia. Released cytochrome c interacts with apoptotic protease activating factor-1 and caspase-9, both of which play important roles in the cytochrome c-dependent mitochondrial pathway of apoptosis by activating caspase-3. Our studies demonstrated that the inactive precursor and active cleaved subunits of caspase-3 protease increased dramatically with the extent of reperfusion time. Following pretreatment with ketamine (a non-competitive NMDA receptor antagonist) and nifedipine (L-VGCC antagonist), cytosolic cytochrome c and the expression of procaspase-3 dramatically decreased, which might result in less neuron damage after ischemia.

Nuclear factor κB activation is mediated by NMDA and non-NMDA receptor and L-type voltage-gated Ca2+ channel following severe global ischemia in rat hippocampus

Recent studies suggest that nuclear factor NF-kappaB may be involved in excitotoxin-induced cell apopotosis. To analyze the variation of NF-kappaB, levels of NF-kappaB were measured after the rats were subjected to 30 min of four-vessel occlusion and sacrificed in selected reperfusion time points. Induction of NF-kappaB consisting mainly of p65 and p50 subunits was detected by Western blot with anti p65, p50 antibodies, respectively. DNA binding activity of NF-kappaB was performed by electrophoretic mobility-shift analysis. Our studies indicate that ischemia-induced NF-kappaB nuclear translocation is time-dependent. Inductions or binding activity of NF-kappaB in nucleus increased about 10-fold from 6 to 12 h as compared with that of the control group, then gradually declined in the following 24, 72 h. To further analyze the regulation by ionotropic glutamate receptor and L-type voltage-gated Ca(2+) channel (L-VGCC) in vivo, N-methyl-D-aspartate (NMDA) receptor antagonist ketamine, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3 (1H,4H)-dione and L-VGCC antagonist nifedipine were given 20 min prior to 30 min of ischemia. The NF-kappaB nuclear translocation was completely blocked by these three antagonists in a dose-dependent manner after ischemia/reperfusion 6 h. Increased phosphorylation of the NF-kappaB regulatory unit IkappaB-alpha was detected by Western blot. Decrement of IkappaB-alpha was found after 3 h reperfusion in the cytoplasm following global ischemia, which was also blocked by such three antagonists. These results illustrate that glutamate-gated ionotropic NMDA or non-NMDA receptors and voltage-gated Ca(2+) channels are important routes to mediate NF-kappaB activation during brain ischemic injury. Active NF-kappaB may attend the excitotoxin-induced cell death in turn. Our studies also suggest that IkappaB-alpha is an important regulatory unit that controls the activation of NF-kappaB after its phosphorylation and degradation and resynthesis in rat hippocampus following global ischemia.

Type A GABA-Receptor-Dependent Synaptic Transmission Sculpts Dendritic Arbor Structure in Xenopus Tadpoles In Vivo

The emergence of dendritic arbor structure in vivo depends on synaptic inputs. We tested whether inhibitory GABAergic synaptic transmission regulates Xenopus optic tectal cell dendritic arbor development in vivo by expressing a peptide corresponding to an intracellular loop (ICL) of the γ2 subunit of type A GABA receptors (GABAAR), which is required to anchor GABAA receptors to the postsynaptic scaffold. Enhanced green fluorescent protein (EGFP)-tagged ICL (EGFP-ICL) was distributed in a punctate pattern at putative inhibitory synapses, identified by vesicular GABA transporter immunoreactive puncta. ICL expression completely blocked GABAAR-mediated transmission in 36% of transfected neurons and significantly reduced GABAAR-mediated synaptic currents relative to AMPA receptor-mediated synaptic currents in the remaining transfected neurons without altering release probability or neuronal excitability. Further analysis of ICL-expressing neurons with residual GABAAR-mediated inputs showed that the capacity of benzodiazepine to enhance GABAergic synaptic responses was reduced in ICL-expressing neurons, indicating that they were likely depleted of γ2 subunit-containing GABAAR. Neurons expressing a mutant form of ICL were comparable to controls. In vivo time-lapse images showed that ICL-expressing neurons have more sparsely branched dendritic arbors, which expand over larger neuropil areas than EGFP-expressing control neurons. Analysis of branch dynamics indicated that ICL expression affected arbor growth by reducing rates of branch addition. Furthermore, we found that decreasing GABAergic synaptic transmission with ICL expression blocked visual experience dependent dendritic arbor structural plasticity. Our findings establish an essential role for inhibitory GABAergic synaptic transmission in the regulation of dendritic structural plasticity in Xenopus in vivo.

Neurogenesis is Required for Behavioral Recovery after Injury in the Visual System of Xenopus laevis

Nonmammalian vertebrates have a remarkable capacity to regenerate brain tissue in response to central nervous system (CNS) injury. Nevertheless, it is not clear whether animals recover lost function after injury or whether injury‐induced cell proliferation mediates recovery. We address these questions using the visual system and visually‐guided behavior in Xenopus laevis tadpoles. We established a reproducible means to produce a unilateral focal injury to optic tectal neurons without damaging retinotectal axons. We then assayed a tectally‐mediated visual avoidance behavior to evaluate behavioral impairment and recovery. Focal ablation of part of the optic tectum prevents the visual avoidance response to moving stimuli. Animals recover the behavior over the week following injury. Injury induces a burst of proliferation of tectal progenitor cells based on phospho‐histone H3 immunolabeling and experiments showing that Musashi‐immunoreactive tectal progenitors incorporate the thymidine analog chlorodeoxyuridine after injury. Pulse chase experiments indicate that the newly‐generated cells differentiate into N‐β‐tubulin‐immunoreactive neurons. Furthermore, in vivo time‐lapse imaging shows that Sox2‐expressing neural progenitors divide in response to injury and generate neurons with elaborate dendritic arbors. These experiments indicate that new neurons are generated in response to injury. To test if neurogenesis is necessary for recovery from injury, we blocked cell proliferation in vivo and found that recovery of the visual avoidance behavior is inhibited by drugs that block cell proliferation. Moreover, behavioral recovery is facilitated by changes in visual experience that increase tectal progenitor cell proliferation. Our data indicate that neurogenesis in the optic tectum is critical for recovery of visually‐guided behavior after injury. J. Comp. Neurol. 521:2262–2278, 2013.