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1.  DNA-Based MRI Probes for Specific Detection of Chronic Exposure to Amphetamine in Living Brains 
We designed phosphorothioate-modified DNA probes linked to superparamagnetic iron oxide nanoparticles (SPION) for in vivo magnetic resonance imaging (MRI) of fosB and ΔfosB mRNA after amphetamine (AMPH) exposure in mice. Specificity of both the fosB and ΔfosB probes was verified by in vitro reverse transcriptase-PCR amplification to a single fragment of total cDNA obtained from acutely AMPH-exposed mouse brains. We confirmed time-dependent uptake and retention profiles of both probes in neurons of GAD67-green fluorescent protein knock-in mice. MRI signal of SPION-labeled fosB probe delivered via intracerebroventricular route was elevated in both acutely and chronically AMPH-exposed mice; the signal was suppressed by dopaminergic receptor antagonist pretreatment. SPION-labeled ΔfosB probe signal elevation occurred only in chronically AMPH-exposed mice. The in vivo target specificity of these probes permits reliable MRI visualization of AMPH-induced differential elevations of fosB and ΔfosB mRNA in living brains.
PMCID: PMC2746375  PMID: 19710318
2.  Diffusion-Weighted Magnetic Resonance Imaging Reversal by Gene Knockdown of Matrix Metalloproteinase-9 Activities in Live Animal Brains 
The involvement of matrix metalloproteinase-9 (MMP-9) activities in the development of abnormal water diffusion in the brain after cardiac arrest is not fully understood. We used magnetic resonance imaging to determine the correlation between MMP-9 activity and the mechanism of abnormal water diffusion after global cerebral ischemia (GCI)-induced brain damage in C57black6 mice. We induced GCI in mice by occluding both carotid arteries for 60 min, then allowing reperfusion. We labeled a short DNA that targets mmp-9 mRNA activity [phosphorothioate-modified oligodeoxynucleotide (sODN)-mmp9] or a control probe without intracellular target (sODN-Ran) with iron-based MR contrast agent [superparamagnetic iron oxide nanoparticle (SPION)-mmp9 or SPION-Ran] or fluorescein isothiocyanate (FITC)-sODN-mmp9 or FITC-sODN-Ran; we then delivered these probes by intracerebroventricular infusion or intraperitoneal injection with in 3 h of reperfusion. At low dose (120 pmol/kg) the SPION-mmp9 probe was retained at significant levels in the striatum and cortex of living brains 10 h after GCI. Probe retention was validated by similar elevation of mmp-9 mRNA and antigens in postmortem samples taken from regions that exhibited GCI-induced hyperintensity in diffusion-weighted imaging, and a significant reduction in apparent diffusion coefficient (rADC, p = 0.0006, n = 12). At a higher dose (120 nmol/kg), the FITC-sODN-mmp9 probe revealed significant knockdown of MMP-9 activity, per zymography, and a reversal of striatal rADC (p = 0.004, n = 6). These observations were not duplicated in the control group. We conclude that expression of mmp-9 mRNA is associated with abnormal ADC after GCI.
PMCID: PMC2726707  PMID: 19295156
3.  Unesterified Cholesterol Accumulation in Late Endosomes/Lysosomes Causes Neurodegeneration and is Prevented by Driving Cholesterol Export from this Compartment 
While unesterified cholesterol (C) is essential for remodeling neuronal plasma membranes, its role in certain neurodegenerative disorders remains poorly defined. Uptake of sterol from pericellular fluid requires processing that involves two lysosomal proteins, lysosomal acid lipase (LAL) that hydrolyzes C esters and NPC1. In systemic tissues, inactivation of either protein led to sterol accumulation and cell death, but in the brain, inactivation of only NPC1 caused C sequestration and neurodegeneration. When injected into the CNS of the npc1-/- mouse, HP-β-CD, a compound known to prevent this C accumulation, diffused throughout the brain and was excreted with a T½ of 6.5 h. This agent caused suppression of C synthesis, elevation of C esters, suppression of SREBP2 target genes, and activation of LXR controlled genes. These findings indicated that HP-β-CD promoted movement of the sequestered C from lysosomes to the metabolically active pool of C in the cytosolic compartment of cells in the CNS. The ED50 for this agent in the brain was ∼0.5 mg/kg, and the therapeutic effect lasted more than 7 days. Continuous infusion of HP-β-CD into the ventricular system of npc1-/- animals between 3 and 7 weeks of age normalized the biochemical abnormalities and completely prevented the expected neurodegeneration. These studies support the concept that neurons continuously acquire C from interstitial fluid to permit plasma membrane turnover and remodeling. Inactivation of NPC1 leads to lysosomal C sequestration and neurodegeneration, but this is prevented by the continuous, direct administration of HP-β-CD into the CNS.
PMCID: PMC3134878  PMID: 21697390
Niemann-Pick C Disease; Wolman Disease; 24(S)-hydroxycholesterol; microglia; astroglia; blood-brain barrier; Purkinje cells; cerebellum
4.  Functional Elimination of Excitatory Feedforward Inputs Underlies Developmental Refinement of Visual Receptive Fields in Zebrafish 
In many sensory systems, receptive fields (RFs) measured by spike responses undergo progressive refinement during development. It has been proposed that elimination of excitatory synaptic inputs underlies such functional refinement. However, despite many extracellular recording and anatomical studies, direct in vivo intracellular evidence has remained limited. In this study, by cell-attached recordings in the developing optic tectum of zebrafish, we found that during a short period after the initial formation of retinotectal synapses, spike visual RFs of tectal neurons underwent a two-stage developmental modulation: from an initial expansion to a later refinement. Whole-cell voltage-clamp recordings revealed that the underlying excitatory synaptic RF exhibited a similar developmental progression with its spatial extent first increased and then reduced, and its spatial tuning profile gradually sharpened. The inhibitory RF was initially larger than the excitatory RF, but became matched with the excitatory RF at later stages. Simulation with the integrate-and-fire neuron model suggested that the developmental changes of excitatory RFs primarily accounted for the initial enlargement and later refinement of spike RFs, while inhibitory inputs generally reduced the size of the spike RF without affecting its developmental progression. In addition, spike RF of individual retinal ganglion cells (RGCs) did not significantly change in size during the same period, and the spatial extent and tuning profile of the tectal excitatory RF barely changed after intratectal excitatory connections were silenced. Together, our results demonstrate that the functional refinement of tectal visual RFs results primarily from a selective elimination of feedforward retinotectal inputs.
PMCID: PMC3095389  PMID: 21471382
visual; receptive field; development; synaptic input; retinotectal; connectivity
5.  Glutamatergic modulation of cerebellar interneuron activity is mediated by an enhancement of GABA release and requires PKA/RIM1α-signaling 
Information processing in the CNS is controlled by the activity of neuronal networks composed of principal neurons and interneurons. Activity-dependent modification of synaptic transmission onto principal neurons is well studied, but little is known about the modulation of inhibitory transmission between interneurons. However synaptic plasticity at this level has clear implications for the generation of synchronized activity. We have investigated the molecular mechanism(s) and functional consequences of an activity-induced lasting increase in GABA release that occurs between inhibitory interneurons (stellate cells) in the cerebellum. Using whole cell recording and cerebellar slices, we found that stimulation of glutamatergic inputs (parallel fibres) with a physiological-like pattern of activity triggered a lasting increase in GABA release from stellate cells. This activity also potentiated inhibitory transmission between synaptically connected interneurons. Extracellular recording revealed that the enhanced inhibitory transmission reduced the firing frequency and altered the pattern of action potential activity in stellate cells. The induction of the sustained increase in GABA release required activation of NMDA receptors (NMDARs). Using pharmacological and genetic approaches we found that presynaptic cAMP/PKA signaling and RIM1α, an active zone protein, is the critical pathway that is required for the lasting enhancement of GABA release. Thus a common mechanism can underlie presynaptic plasticity of both excitatory and inhibitory transmission. This activity-dependent regulation of synaptic transmission between inhibitory interneurons may serve as an important mechanism for interneuronal network plasticity.
PMCID: PMC2775555  PMID: 19144838
Inhibitory transmission; RIM1α; long-term potentiation; PKA; cerebellum; interneurons
6.  Pten Deletion in Adult Neural Stem/Progenitor Cells Enhances Constitutive Neurogenesis 
Here we show that conditional deletion of PTEN in a subpopulation of adult neural stem cells in the subependymal zone (SEZ) leads to persistently enhanced neural stem cell self-renewal without sign of exhaustion. These Pten null SEZ-born neural stem cells and progenies can follow the endogenous migration, differentiation, and integration pathways and contribute to the constitutive neurogenesis in the olfactory bulb. As a result, the Pten deleted animals have increased olfactory bulb mass and enhanced olfactory function. Pten null cells in the olfactory bulb can establish normal connections with peripheral olfactory epithelium and help olfactory bulb recovery from acute damage. Following a focal stroke, Pten null progenitors give rise to greater numbers of neuroblasts that migrate to peri-infarct cortex. However, contrary to olfactory bulb, no significant long-term survival and integration can be observed, indicating that additional factors are necessary for long-term survival of newly born neurons after stroke. These data suggest that manipulating PTEN-controlled signaling pathways may be a useful step in facilitating endogenous neural stem/progenitor expansion for the treatment of disorders or lesions in regions associated with constitutive neurogenesis.
PMCID: PMC2754186  PMID: 19211894
SEZ; PTEN; neurogenesis; olfactory; neural repair; post-stroke
7.  Control of precerebellar neuron development by Olig3 bHLH transcription factor 
The rhombic lip (RL) is the neuroepithelium immediately adjacent to the roof plate of the fourth ventricle and it gives rise to various brainstem and cerebellar cell types. Our study shows that the basic helix-loop-helix (bHLH) transcription factor Olig3 is expressed in the progenitors of RL and ablation of Olig3 significantly affects the development of RL. In Olig3−/− caudal RL, the expression level of Math1 in the dI1 domain is reduced and the formation of four mossy-fiber nuclei is compromised; dI2–dI3 neurons are mis-specified to dI4 interneurons and the climbing-fiber neurons (inferior olive nucleus) are completely lost. In addition, the formation of brainstem (nor)adrenergic centers and first-order relay visceral sensory neurons is also dependent on Olig3. Therefore, Olig3 plays an important role in the fate specification and differentiation of caudal RL-derived neurons.
PMCID: PMC2590637  PMID: 18829970
bHLH; cell fate; rhombic lip; mossy-fiber nuclei; climbing-fiber nucleus; cerebellum
8.  Imaging Cerebral Gene Transcripts in Live Animals 
To circumvent the limitations of using postmortem brain in molecular assays, we used avidin– biotin binding to couple superparamagnetic iron oxide nanoparticles (SPIONs) (15–20 nm) to phosphorothioate-modified oligodeoxynucleotides (sODNs) with sequence complementary to c-fos and β-actin mRNA (SPION-cfos and SPION-βactin, respectively) (14 –22 nm). The Stern–Volmer constant for the complex of SPION and fluorescein isothiocyanate (FITC)-sODN is 3.1 × 106/m. We studied the feasibility of using the conjugates for in vivo magnetic resonance imaging (MRI) to monitor gene transcription, and demonstrated that these complexes at 40 μg of Fe per kilogram of body weight were retained at least 1 d after intracerebroventricular infusion into the left ventricle of C57Black6 mice. SPION retention measured by MRI as T2* or R2* maps (R2* = 1/T2*) was compared with histology of iron oxide (Prussian blue) and FITC-labeled sODN. We observed significant reduction in magnetic resonance (MR) T2* signal in the right cortex and striatum; retention of SPION-cfos and SPION-βactin positively correlated with c-fos and β-actin mRNA maps obtained from in situ hybridization. Histological examination showed that intracellular iron oxide and FITC-sODN correlated positively with in vivo MR signal reduction. Furthermore, in animals that were administered SPION-cfos and amphetamine (4 mg/kg, i.p.), retention was significantly elevated in the nucleus accumbens, striatum, and medial prefrontal cortex of the forebrain. Control groups that received SPION-cfos and saline or that received a SPION conjugate with a random-sequence probe and amphetamine showed no retention. These results demonstrated that SPION-sODN conjugates can detect active transcriptions of specific mRNA species in living animals with MRI.
PMCID: PMC2647966  PMID: 17234603
amphetamine; antisense delivery; aptamer; drug addiction; gene transcription; immediate-early genes; nanotechnology; signal transduction
9.  Anatomical Coupling between Distinct Metacognitive Systems for Memory and Visual Perception 
A recent study found that, across individuals, gray matter volume in the frontal polar region was correlated with visual metacognition capacity (i.e., how well one’s confidence ratings distinguish between correct and incorrect judgments). A question arises as to whether the putative metacognitive mechanisms in this region are also used in other metacognitive tasks involving, for example, memory. A novel psychophysical measure allowed us to assess metacognitive efficiency separately in a visual and a memory task, while taking variations in basic task performance capacity into account. We found that, across individuals, metacognitive efficiencies positively correlated between the two tasks. However, voxel-based morphometry analysis revealed distinct brain structures for the two kinds of metacognition. Replicating a previous finding, variation in visual metacognitive efficiency was correlated with volume of frontal polar regions. However, variation in memory metacognitive efficiency was correlated with volume of the precuneus. There was also a weak correlation between visual metacognitive efficiency and precuneus volume, which may account for the behavioral correlation between visual and memory metacognition (i.e., the precuneus may contain common mechanisms for both types of metacognition). However, we also found that gray matter volumes of the frontal polar and precuneus regions themselves correlated across individuals, and a formal model comparison analysis suggested that this structural covariation was sufficient to account for the behavioral correlation of metacognition in the two tasks. These results highlight the importance of the precuneus in higher-order memory processing and suggest that there may be functionally distinct metacognitive systems in the human brain.
PMCID: PMC4696871  PMID: 23365229
10.  Molecular Mechanism of Constitutive Endocytosis of Acid-Sensing Ion Channel 1a and Its Protective Function in Acidosis-Induced Neuronal Death 
Acid-sensing ion channels (ASICs) are proton-gated cation channels widely expressed in the peripheral and CNSs, which critically contribute to a variety of pathophysiological conditions that involve tissue acidosis, such as ischemic stroke and epileptic seizures. However, the trafficking mechanisms of ASICs and the related proteins remain largely unknown. Here, we demonstrate that ASIC1a, the main ASIC subunit in the brain, undergoes constitutive endocytosis in a clathrin- and dynamin-dependent manner in both mouse cortical neurons and heterologous cell cultures. The endocytosis of ASIC1a was inhibited by either the small molecular inhibitor tyrphostin A23 or knockdown of the core subunit of adaptor protein 2 (AP2) μ2 using RNA interference, supporting a clathrin-dependent endocytosis of ASIC1a. In addition, the internalization of ASIC1a was blocked by dominant-negative dynamin1 mutation K44A and the small molecular inhibitor dynasore, suggesting that it is also dynamin-dependent. We show that the membrane-proximal residues 465LCRRG 469 at the cytoplasmic C terminus of ASIC1a are critical for interaction with the endogenous adaptor protein complex and inhibition of ASIC1a internalization strongly exacerbated acidosis-induced death of cortical neurons from wild-type but not ASIC1a knock-out mice. Together, these results reveal the molecular mechanism of ASIC1a internalization and suggest the importance of endocytic pathway in functional regulation of ASIC1a channels as well as neuronal damages mediated by these channels during neurodegeneration.
PMCID: PMC4201763  PMID: 23595764
11.  Gdf11 Facilitates Temporal Progression of Neurogenesis in the Developing Spinal Cord 
Various types of neurons and glia are generated following a precise spatial and temporal order during neurogenesis. The mechanisms that control this sequential generation of neuronal and glial cell types from the same progenitor population are not well understood. Growth differentiation factor 11 (Gdf11) belongs to the TGF-β family of proteins and is expressed transiently in newly born neurons adjacent to the progenitor domain in the developing spinal cord. We examined the phenotypes of Gdf11−/− mouse embryos and found that without Gdf11, neuronal differentiation in the spinal cord progresses at a slower rate. Higher progenitor proliferation rate, along with a delay in gliogenesis, is also observed in Gdf11−/− spinal cord but only after the peak of Gdf11 expression, indicating that Gdf11 can cause long-lasting changes in progenitor properties. These changes can be preserved in vitro, as neurospheres derived from Gdf11−/− and wild-type littermates at a stage after, but not before the onset of Gdf11 expression, exhibit differences in proliferation and differentiation potential. Moreover, these changes in progenitor properties can be induced in vitro by the addition of Gdf11. We also demonstrate that the effects of Gdf11 on progenitor cells are associated with its ability to upregulate p57 Kip2 and p27 Kip1 while downregulating Pax6 expression. These results support a model in which Gdf11 secreted by newly born neurons in the developing spinal cord facilitates the temporal progression of neurogenesis by acting as a positive feedback signal on the progenitor cells to promote cell cycle exit and decrease proliferation ability, thus changing their differentiation potential.
PMCID: PMC4112097  PMID: 21248112
12.  Interleukin-1 Mediates Pathological Effects of Microglia on Tau Phosphorylation and on Synaptophysin Synthesis in Cortical Neurons through a p38-MAPK Pathway 
The presence of tangles of abnormally phosphorylated tau is a characteristic of Alzheimer's disease (AD), and the loss of synapses correlates with the degree of dementia. In addition, the overexpression of interleukin-1 (IL-1) has been implicated in tangle formation in AD. As a direct test of the requirement for IL-1 in tau phosphorylation and synaptophysin expression, IL-1 actions in neuron–microglia cocultures were manipulated. Activation of microglia with secreted β-amyloid precursor protein or lipopolysaccharide elevated their expression of IL-1α, IL-1β, and tumor necrosis factor α (TNFα) mRNA. When such activated microglia were placed in coculture with primary neocortical neurons, a significant increase in the phosphorylation of neuronal tau was accompanied by a decline in synaptophysin levels. Similar effects were evoked by treatment of neurons with recombinant IL-1β. IL-1 receptor antagonist (IL-1ra) as well as anti-IL-1β antibody attenuated the influence of activated microglia on neuronal tau and synaptophysin, but anti-TNFα antibody was ineffective. Some effects of microglial activation on neurons appear to be mediated by activation of p38 mitogen-activated protein kinase (p38-MAPK), because activated microglia stimulated p38-MAPK phosphorylation in neurons, and an inhibitor of p38-MAPK reversed the influence of IL-1 β on tau phosphorylation and synaptophysin levels. Our results, together with previous observations, suggest that activated microglia may contribute to neurofibrillary pathology in AD through their production of IL-1, activation of neuronal p38-MAPK, and resultant changes in neuronal cytoskeletal and synaptic elements.
PMCID: PMC3833596  PMID: 12629164
Alzheimer's disease; β-amyloid precursor protein; cortical neuron; interleukin-1; microglia; mitogen-activated protein kinase; synaptophysin; phosphorylated tau
13.  The microRNA-17-92 cluster enhances axonal outgrowth in embryonic cortical neurons 
MicroRNAs (miRNAs) regulate dendritogenesis and plasticity. However, the biological function of miRNAs in axons has not been extensively investigated. Here, using rat primary cortical neurons cultured in a microfluidic chamber, we found that the distal axons of the neurons expressed the miR-17-92 cluster and that proteins which regulate production and activity of mature miRNAs, Dicer and Argonaute 2, respectively, were present in the distal axons. Overexpression of the miR-17-92 cluster in cortical neurons substantially increased axonal outgrowth, whereas distal axonal attenuation of endogenous miR-19a, a key miRNA of the miR-17-92 cluster, with the miRNA hairpin inhibitor suppressed axonal outgrowth. Moreover, overexpression of the miR-17-92 cluster reduced phosphatase and tensin homolog (PTEN) proteins and elevated phosphorylated mammalian target of rapamycin (mTOR) in the distal axons. In contrast, distal axonal attenuation of miR-19a increased PTEN proteins and inactivated mTOR in the axons, but did not affect these protein levels in the cell bodies. Overexpression of PTEN and attenuation of endogenous PTEN prevailed over the enhancement and inhibitory effects of the miR-19a on axonal outgrowth, respectively. Axonal application of LY294002, a PI3K inhibitor, or rapamycin, an mTOR inhibitor, abolished axonal outgrowth enhanced by overexpression of the miR-17-92 cluster. Collectively, these findings demonstrate that axonal alteration of miR-17-92 cluster expression regulates axonal outgrowth and that local modulation of PTEN protein levels by miR-19a likely contributes to the axonal outgrowth.
PMCID: PMC3657758  PMID: 23595747
14.  Exposure to Cocaine Regulates Inhibitory Synaptic Transmission in the Nucleus Accumbens 
Medium spiny neurons (MSNs) within the nucleus accumbens shell (NAc) function to gate and prioritize emotional/motivational arousals for behavioral output. The neuronal output NAc MSNs is mainly determined by the integration of membrane excitability and excitatory/inhibitory synaptic inputs. Whereas cocaine-induced alterations at excitatory synapses and membrane excitability have been extensively examined, the overall functional output of NAc MSNs following cocaine exposure still poorly defined because little is known about whether inhibitory synaptic input to these neurons is affected by cocaine. Here, our results demonstrate multidimensional alterations at inhibitory synapses in NAc neurons following cocaine self-administration in rats. Specifically, the amplitude of miniature (m) inhibitory postsynaptic currents (IPSCs) was decreased after 21-d withdrawal from 5-d cocaine self-administration. Upon re-exposure to cocaine after 21-day withdrawal, whereas the amplitude of mIPSCs remained down-regulated, the frequency became significantly higher. Furthermore, the reversal potential of IPSCs, which was not significantly altered during withdrawal, became more hyperpolarized upon cocaine re-exposure. Moreover, the relative weight of excitatory and inhibitory inputs to NAc MSNs was significantly decreased after 1-d cocaine withdrawal, increased after 21-d withdrawal, and returned to the basal level upon cocaine re-exposure after 21-d withdrawal. These results, taken together with previous results showing cocaine-induced adaptations at excitatory synapses and intrinsic membrane excitability of NAc MSNs, may provide a relatively thorough picture of the functional state of NAc MSNs following cocaine exposure.
PMCID: PMC3661009  PMID: 23595733
IPSC; EPSC; GABA; accumbens; cocaine; reversal potential
15.  MER5101, a novel Aβ1-15:DT conjugate vaccine, generates a robust anti-Aβ antibody response and attenuates Aβ pathology and cognitive deficits in APPswe/PS1ΔE9 transgenic mice 
Active amyloid-β (Aβ) immunotherapy is under investigation to prevent or treat early Alzheimer's disease (AD). In 2002, a Phase II clinical trial (AN1792) was halted due to meningoencephalitis in ∼6% of the AD patients, possibly caused by a T-cell-mediated immunological response. Thus, generating a vaccine that safely generates high anti-Aβ antibody levels in the elderly is required. In this study, MER5101, a novel conjugate of Aβ1-15 peptide (a B-cell epitope fragment) conjugated to an immunogenic carrier protein, diphtheria toxoid (DT), and formulated in a nanoparticular emulsion-based adjuvant, was administered to 10 mo-old APPswe/PS1ΔE9 transgenic (Tg) and wild-type (Wt) mice. High anti-Aβ antibody levels were observed in both vaccinated APPswe/PS1ΔE9 Tg and Wt mice. Antibody isotypes were mainly IgG1 and IgG2b, suggesting a Th2-biased response. Re-stimulation of splenocytes with the Aβ1-15:DT conjugate resulted in a strong proliferative response, whereas proliferation was absent after re-stimulation with Aβ1-15 or Aβ1-40/42 peptides, indicating a cellular immune response against DT while avoiding an Aβ-specific T cell response. Moreover, significant reductions in cerebral Aβ plaque burden, accompanied by attenuated microglial activation and increased synaptic density, were observed in MER5101 vaccinated APPswe/PS1ΔE9 Tg mice compared to Tg adjuvant controls. Lastly, MER5101 immunized APPswe/PS1ΔE9 Tg mice showed improvement of cognitive deficits in both Contextual Fear Conditioning (CFC) and the Morris Water Maze (MWM). Our novel, highly immunogenic Aβ conjugate vaccine, MER5101, shows promise for improving Aβ vaccine safety and efficacy and therefore, may be useful for preventing and/or treating early AD.
PMCID: PMC3675782  PMID: 23595760
16.  Deficit in Long-Term Synaptic Plasticity is Rescued by a Computationally-Predicted Stimulus Protocol 
Mutations in the gene encoding CREB-binding protein (CBP) cause deficits in long-term plasticity, learning, and memory. Here, long-term synaptic facilitation (LTF) at Aplysia sensorimotor synapses in cell culture was used as a model system to investigate methods for overcoming deficits in LTF produced by a CBP knockdown. Injecting CBP-siRNA into individual sensory neurons reduced CBP levels and impaired LTF produced by a Standard Protocol of five, 5-min pulses of serotonin (5-HT) delivered at 20 min interstimulus intervals. A computational model, which simulated molecular processes underlying LTF induction, identified a Rescue Protocol of five pulses of 5-HT at non-uniform interstimulus intervals that overcame the consequences of reduced CBP and restored LTF. These results suggest that complementary empirical and computational studies can identify methods for ameliorating impairments of learning due to molecular lesions.
PMCID: PMC3690371  PMID: 23595752
17.  Top-down regulation of default mode activity in spatial visual attention 
Dorsal anterior cingulate and bilateral anterior insula form a task control network (TCN) whose primary function includes initiating and maintaining task-level cognitive set and exerting top-down regulation of sensorimotor processing. The default mode network (DMN), comprising an anatomically distinct set of cortical areas, mediates introspection and self-referential processes. Resting-state data show that TCN and DMN interact. The functional ramifications of their interaction remain elusive. Recording fMRI data from human subjects performing a visual spatial attention task and correlating Granger causal influences with behavioral performance and blood-oxygen-level-dependent (BOLD) activity we report three main findings. First, causal influences from TCN to DMN, i.e., TCN→DMN, are positively correlated with behavioral performance. Second, causal influences from DMN to TCN, i.e., DMN→TCN, are negatively correlated with behavioral performance. Third, stronger DMN→TCN are associated with less elevated BOLD activity in TCN, whereas the relationship between TCN→DMN and DMN BOLD activity is unsystematic. These results suggest that during visual spatial attention, top-down signals from TCN to DMN regulate the activity in DMN to enhance behavioral performance, whereas signals from DMN to TCN, acting possibly as internal noise, interfere with task control, leading to degraded behavioral performance.
PMCID: PMC3670184  PMID: 23575842
18.  A Novel Growth-Promoting Pathway Formed by GDNF-Overexpressing Schwann Cells Promotes Propriospinal Axonal Regeneration, Synapse formation, and Partial Recovery of Function after Spinal Cord Injury 
Descending propriospinal neurons (DPSN) are known to establish functional relays for supraspinal signals, and they display a greater growth response after injury than do the long projecting axons. However, their regenerative response is still deficient due to their failure to depart from growth supportive cellular transplants back into the host spinal cord, which contains numerous impediments to axon growth. Here we report the construction of a continuous growth-promoting pathway in adult rats, formed by grafted Schwann cells (SCs) overexpressing glial cell line-derived neurotrophic factor (GDNF). We demonstrate that such a growth-promoting pathway, extending from the axonal cut ends to the site of innervation in the distal spinal cord, promoted regeneration of DPSN axons through and beyond the lesion gap of a spinal cord hemisection. Within the distal host spinal cord, regenerated DPSN axons formed synapses with host neurons leading to the restoration of action potentials and partial recovery of function.
PMCID: PMC3664932  PMID: 23536080
19.  Olfactory bulb short axon cell release of GABA and dopamine produces a temporally biphasic inhibition-excitation response in external tufted cells 
Evidence for co-expression of two or more classic neurotransmitters in neurons has increased but less is known about co-transmission. Ventral tegmental area (VTA) neurons, co-release dopamine (DA), the excitatory transmitter glutamate and the inhibitory transmitter GABA onto target cells in the striatum. Olfactory bulb (OB) short axon cells (SACs) form interglomerular connections and co-express markers for dopamine (DA) and GABA. Using an optogenetic approach we provide evidence that mouse OB SACs release both GABA and DA onto external tufted cells (ETCs) in other glomeruli. Optical activation of channelrhodopsin specifically expressed in DAergic SACs produced a GABAA receptor-mediated monosynaptic inhibitory response followed by DA-D1-like receptor-mediated excitatory response in ETCs. The GABAA receptor-mediated hyperpolarization activates Ih current in ETCs; synaptically released DA increases Ih, which enhances post-inhibitory rebound spiking. Thus, the opposing actions of synaptically released GABA and DA are functionally integrated by Ih to generate an inhibition-to-excitation “switch” in ETCs. Consistent with the established role of Ih in ETC burst firing, we show that endogenous DA release increases ETC spontaneous bursting frequency. ETCs transmit sensory signals to mitral/tufted output neurons and drive intraglomerular inhibition to shape glomerulus output to downstream olfactory networks. GABA and DA co-transmission from SACs to ETCs may play a key role in regulating output coding across the glomerular array.
PMCID: PMC3727441  PMID: 23407950
20.  ATF4 protects against neuronal death in cellular PD models by maintaining levels of parkin 
Parkinson’s disease (PD) is a common neurodegenerative disorder, for which there are no effective disease-modifying therapies. The transcription factor ATF4 is induced by multiple PD-relevant stressors, such as ER stress and oxidative damage. ATF4 may exert either protective or deleterious effects on cell survival, depending on the paradigm. However, the role of ATF4 in the pathogenesis of PD has not been explored. We find that ATF4 levels are increased in neuromelanin-positive neurons in the substantia nigra of a subset of Parkinson’s disease patients, relative to controls. ATF4 levels are also up-regulated in neuronal PC12 cells treated with the dopaminergic neuronal toxins toxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+). To explore the role of ATF4 in cell survival in PD-relevant contexts, we either silenced or overexpressed ATF4 in cellular models of PD. In neuronal PC12 cells, silencing of ATF4 enhanced cell death in response to either 6-OHDA or MPP+. Conversely, overexpression of ATF4 reduced cell death caused by dopaminergic neuronal toxins. ATF4 was also protective against 6-OHDA-induced death of cultured mouse ventral midbrain dopaminergic neurons. We further show that parkin, a gene associated with autosomal recessive PD, plays a critical role in ATF4-mediated protection. After treatment with 6-OHDA or MPP+, parkin protein levels fall, despite an increase in mRNA levels. ATF4 silencing exacerbates the toxin-induced reduction of parkin, while ATF4 overexpression partially preserves parkin levels. Finally, parkin silencing blocked the protective capacity of ATF4. These results indicate that ATF4 plays a protective role in Parkinson’s disease though the regulation of parkin.
PMCID: PMC3711618  PMID: 23392669
21.  Doublecortin (Dcx) family proteins regulate filamentous actin structure in developing neurons 
Doublecortin (Dcx) is the causative gene for X-linked lissencephaly, which encodes a microtubule (MT) binding protein. Axon tracts are abnormal in both affected individuals and in animal models. To determine the reason for the axon tract defect, we performed a semi-quantitative proteomic analysis of the corpus callosum in mice mutant for Dcx. In axons from mice mutant for Dcx, wide spread differences are found in actin-associated proteins as compared with wild type axons. Decreases in actin-binding proteins, α-actinin-1, α-actinin-4 and actin-related protein 2/3 complex subunit 3 (Arp3), are correlated with dysregulation in the distribution of filamentous actin (F-actin) in the mutant neurons with increased F-actin around the cell body and decreased F-actin in the neurites and growth cones. The actin distribution defect can be rescued, by full length Dcx, and further enhanced by Dcx S297A, the unphosphorylatable mutant, but not with the truncation mutant of Dcx missing the C terminal S/P rich domain. Thus, the C-terminal region of Dcx dynamically regulates formation of F-actin features in developing neurons, likely through interaction with spinophilin, but not through α-actinin-4 or Arp3. We show with that the phenotype of Dcx/Doublecortin Like Kinase 1 (Dclk1) deficiency is consistent with actin defect as these axons are selectively deficient in axon guidance, but not elongation.
PMCID: PMC3711551  PMID: 23303949
22.  The Rett Syndrome Protein MeCP2 Regulates Synaptic Scaling 
Synaptic scaling is a form of homeostatic synaptic plasticity characterized by cell-wide changes in synaptic strength in response to changes in overall levels of neuronal activity. Here we report that bicuculline-induced increase in neuronal activity leads to a decrease in mEPSC amplitude and a decrease in expression of the AMPA receptor subunit GluR2 in rat hippocampal cultures. Bicuculline treatment also leads to an increase in the levels of the transcriptional repressor MeCP2, which binds to the GluR2 promoter along with the co-repressors HDAC1 and mSin3A. Down-regulation of MeCP2 by shRNA expression or genetic deletion blocks the bicuculline-induced decrease in GluR2 expression and mEPSC amplitude. These observations indicate that MeCP2 mediates activity-dependent synaptic scaling, and suggest that the pathophysiology of Rett syndrome, which is caused by mutations in MeCP2, may involve defects in activity-dependent regulation of synaptic currents.
PMCID: PMC3711796  PMID: 22262897
23.  SRPK2 Phosphorylates Tau and Mediates the Cognitive Defects in Alzheimer’s Disease 
Serine-arginine protein kinases 2 (SRPK2) is a cell cycle-regulated kinase that phosphorylates serine/arginine domain-containing proteins and mediates pre-mRNA splicing with unclear function in neurons. Here, we show that SRPK2 phosphorylates tau on S214, suppresses tau-dependent microtubule polymerization and inhibits axonal elongation in neurons. Depletion of SRPK2 in dentate gyrus inhibits tau phosphorylation in APP/PS1 mouse and alleviates the impaired cognitive behaviors. The defective LTP in APP/PS1 mice is also improved after SRPK2 depletion. Moreover, active SRPK2 is increased in the cortex of APP/PS1 mice and the pathological structures of human Alzheimer’s disease (AD) brain. Therefore, our study suggests SRPK2 may contribute to the formation of hyperphosphorylated tau and the pathogenesis of AD.
PMCID: PMC3518045  PMID: 23197718
24.  RIM Promotes Calcium Channel Accumulation at Active Zones of the Drosophila Neuromuscular Junction 
Synaptic communication requires the controlled release of synaptic vesicles from presynaptic axon terminals. Release efficacy is regulated by the many proteins that comprise the presynaptic release apparatus, including Ca2+ channels and proteins that influence Ca2+ channel accumulation at release sites. Here we identify Drosophila RIM and demonstrate that it localizes to active zones at the larval neuromuscular junction. In Drosophila RIM mutants, there is a large decrease in evoked synaptic transmission, due to a significant reduction in both the clustering of Ca2+ channels and the size of the readily releasable pool of synaptic vesicles at active zones. Hence, RIM plays an evolutionarily conserved role in regulating synaptic calcium channel localization and readily releasable pool size. Since RIM has traditionally been studied as an effector of Rab3 function, we investigate whether RIM is involved in the newly identified function of Rab3 in the distribution of presynaptic release machinery components across release sites. Bruchpilot (Brp), an essential component of the active zone cytomatrix T bar, is unaffected by RIM disruption, indicating that Brp localization and distribution across active zones does not require wild type RIM. In addition, larvae containing mutations in both RIM and rab3 have reduced Ca2+ channel levels and a Brp distribution that is very similar to that of the rab3 single mutant, indicating that RIM functions to regulate Ca2+ channel accumulation but is not a Rab3 effector for release machinery distribution across release sites.
PMCID: PMC3516196  PMID: 23175814
25.  RIM Controls Homeostatic Plasticity through Modulation of the Readily-Releasable Vesicle Pool 
Rab3 interacting molecules (RIMs) are evolutionarily conserved scaffolding proteins that are located at presynaptic active zones. In the mammalian nervous system, RIMs have two major activities that contribute to the fidelity of baseline synaptic transmission: they concentrate calcium channels at the active zone and facilitate synaptic vesicle docking/priming. Here we confirm that RIM has an evolutionarily conserved function at the Drosophila neuromuscular junction and then define a novel role for RIM during homeostatic synaptic plasticity. We show that loss of RIM disrupts baseline vesicle release, diminishes presynaptic calcium influx, and diminishes the size of the readily-releasable pool (RRP) of synaptic vesicles, consistent with known activities of RIM. However, loss of RIM also completely blocks the homeostatic enhancement of presynaptic neurotransmitter release that normally occurs after inhibition of postsynaptic glutamate receptors, a process termed synaptic homeostasis. It is established that synaptic homeostasis requires enhanced presynaptic calcium influx as a mechanism to potentiate vesicle release. However, despite a defect in baseline calcium influx in rim mutants, the homeostatic modulation of calcium influx proceeds normally. Synaptic homeostasis is also correlated with an increase in the size of the RRP of synaptic vesicles, although the mechanism remains unknown. Here we demonstrate that the homeostatic modulation of the RRP is blocked in the rim mutant background. Therefore, RIM-dependent modulation of the RRP is a required step during homeostatic plasticity. By extension, homeostatic plasticity appears to require two genetically separable processes, the enhancement of presynaptic calcium influx and a RIM-dependent modulation of the RRP.
PMCID: PMC3523185  PMID: 23175813

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