Role of pro-brain-derived neurotrophic factor (proBDNF)
to mature BDNF conversion in activity-dependent
competition at developing neuromuscular synapses
H. Shawn Jea,b,c,1, Feng Yanga,b,d,1, Yuanyuan Jia,e, Guhan Nagappana,e, Barbara L. Hempsteadf, and Bai Lua,b,e,2
aSection on Neural Development and Plasticity, National Institute of Child Health and Human Development, Bethesda, MD 20892-3714; bGenes, Cognition,
and Psychosis Program (GCAP), National Institute of Mental Health, Bethesda, MD 20892-3714; cProgram in Neuroscience and Behavioral Disorders, Duke–
National University of Singapore (Duke-NUS) Graduate Medical School, 169857, Singapore; dLieber Institute for Brain Development, The Johns Hopkins
University Medical Campus, Baltimore, MD 21205; eR&D China, GlaxoSmithKline, Pudong, Shanghai 201203, China; and fDivision of Hematology, Department
of Medicine, Weill Medical College, Cornell University, New York, NY 10021
Edited* by Richard L. Huganir, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved August 20, 2012 (received for review May
10, 2012)
Formation of specific neuronal connections often involves compe-
tition between adjacent axons, leading to stabilization of the active
terminal, while retraction of the less active ones. The underlying
molecular mechanisms remain unknown. We show that activity-
dependent conversion of pro–brain-derived neurotrophic factor
(proBDNF) to mature (m)BDNF mediates synaptic competition. Stim-
ulation of motoneurons triggers proteolytic conversion of proBDNF
to mBDNF at nerve terminals. In Xenopus nerve–muscle cocultures,
in which two motoneurons innervate one myocyte, proBDNF-
p75NTR signaling promotes retraction of the less active terminal,
whereas mBDNF–tyrosine-related kinase B (TrkB) p75NTR (p75 neu-
rotrophin receptor) facilitates stabilization of the active one. Thus,
proBDNF and mBDNF may serve as potential “punishment” and “re-
ward” signals for inactive and active terminals, respectively, and
activity-dependent conversion of proBDNF to mBDNF may regulate
synapse elimination.
neuromuscular junction | pro-neurotrophin | synapse competition
The nervous system responds to experience by altering thenumber and strength of synaptic connections (1). Activity-
dependent synaptic competition, a general process seen in many
parts of the developing nervous system, plays a critical role in
shaping patterns of neuronal connections (2–7). At the neuro-
muscular junction (NMJ), for example, multiple axons compete
for the same postsynaptic muscle cell during early postnatal life
until all but one is eliminated (8–10). Extensive experimental
data support the view that the more active terminal or “cartel”
gets stabilized, whereas less active ones withdraw, resulting in
canonical elimination of polyneuronal innervation (8, 11). It is
generally believed that this synaptic competition is mediated by
a “punishment” or “elimination” signal, produced by the post-
synaptic cell, that cau ...
Edgardo J. Arroyo is an Associate Research Scientist at Yale University School of Medicine who has extensive experience researching various aspects of myelin formation, degradation, and regeneration in the central and peripheral nervous systems. His research has focused on elucidating the cellular mechanisms and microanatomy of neuron-glial interactions using techniques such as immunohistochemistry, confocal microscopy, and biochemistry. He has studied topics such as the effects of spinal cord injury, stem cell transplantation, sodium channel expression after nerve damage, and how demyelination affects the molecular organization of nodes of Ranvier.
Schizophrenia Research Forum Live Webinar - June 28, 2017 - Rusty Gage wef
1) The document describes a study using induced pluripotent stem cells (iPSCs) derived from bipolar disorder (BD) patients to model the disease in vitro.
2) Hippocampal dentate gyrus-like neurons were differentiated from iPSCs and showed hyper-excitability at both the molecular and functional levels in BD-derived neurons.
3) Treatment with lithium rescued the hyper-excitability phenotype in neurons derived from lithium-responsive BD patients but not lithium non-responsive patients, suggesting patient-specific responses.
This study investigated the effects of repeated deep brain stimulation of the pedunculopontine tegmental nucleus (PPTg) in rats. Electrodes were implanted in the PPTg of rats which then received 6 days of stimulation. Behavioral responses and neural activation markers cFOS and GAP-43 were assessed on days 1 and 6. Results found that repeated stimulation increased the threshold current needed to elicit behavioral responses on day 6 compared to day 1, suggesting an inhibitory effect. Expression of cFOS and GAP-43 was also found near the stimulation site, indicating repeated stimulation triggers neural plasticity.
This study investigated the effects of neonatal hyperoxia (high oxygen exposure) on neurotrophin gene expression in mice. The researchers found that hyperoxia induced alterations in brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) mRNA expression levels in the isocortex and prefrontal cortex. Specifically, BDNF mRNA expression decreased in the prefrontal cortex but increased in the isocortex, while GDNF mRNA expression increased in both regions following hyperoxia. Hyperoxia also altered mRNA expression levels of DNA methyltransferases DNMT1 and DNMT3a, which regulate neurotrophin gene expression through DNA methylation. These results suggest that neonatal
POWER SPECTRAL ANALYSIS OF EEG AS A POTENTIAL MARKER IN THE DIAGNOSIS OF SPAS...ijbesjournal
The detection and diagnosis of various neurological disorders are performed using different medical
devices among which electroencephalogram (EEG) is one of the most cost effective technique. Though
significant progress had been made in the analysis of EEG for diagnosis of different neurological
disorders, yet detection of cerebral palsy (CP) is not quite clear. This study was performed to analyze the
EEG power spectrum density (PSD) of spastic CP and normal children to find if any significant EEG
patterns could be used for early detection of CP. Twenty children participated in this study out of which ten
were spastic CP and other ten were normal healthy children. EEG of all the participants was recorded
from C3 C4 and F3 F4 regions following montage 10-20 system. The artifact-free EEG signals of 15
minutes duration was extracted for spectral analysis using Fast Fourier Transformation (FFT) algorithm
in MATLAB and power density spectrum (PSD) was plotted. The PSD revealed high intensity power peak
at frequency of 50Hz and smaller at 100 Hz, which was consistent for all healthy subjects. In case of
spastic CP children, high intensity peak at 100Hz were prominent and smaller peak was observed at 50Hz.
The high intensity 100Hz peak observed in the PSD of spastic CP patients demonstrated that this tool can
be used for early detection of spastic CP.
This study tested intrathecal enzyme replacement therapy (ERT) in a mouse model of infantile Batten disease. Mice lacking the enzyme palmitoyl-protein thioesterase 1 (PPT1) received a single intrathecal injection of recombinant human PPT1 at 6 weeks of age. This prevented decline in motor function, improved survival, and reduced brain and spinal cord pathology compared to untreated mice. The effects were similar to ERT for another form of Batten disease. This suggests intrathecal ERT may help treat infantile Batten disease caused by PPT1 deficiency in humans.
Positron-emission tomography studies of cross-modality inhibition in selectiv...Dr Brendan O'Sullivan
Published in 1994, this groundbreaking paper featured the research of Professor Per Roland, Professor Ryuta Kawashima (of “Brain Training” fame) and Professor Brendan O’ Sullivan.
This landmark research was the first to prove in human brain imaging studies that visual attention is impaired when we do other attention-competing tasks such as manual tasks such as using mobile phones while driving.
Edgardo J. Arroyo is an Associate Research Scientist at Yale University School of Medicine who has extensive experience researching various aspects of myelin formation, degradation, and regeneration in the central and peripheral nervous systems. His research has focused on elucidating the cellular mechanisms and microanatomy of neuron-glial interactions using techniques such as immunohistochemistry, confocal microscopy, and biochemistry. He has studied topics such as the effects of spinal cord injury, stem cell transplantation, sodium channel expression after nerve damage, and how demyelination affects the molecular organization of nodes of Ranvier.
Schizophrenia Research Forum Live Webinar - June 28, 2017 - Rusty Gage wef
1) The document describes a study using induced pluripotent stem cells (iPSCs) derived from bipolar disorder (BD) patients to model the disease in vitro.
2) Hippocampal dentate gyrus-like neurons were differentiated from iPSCs and showed hyper-excitability at both the molecular and functional levels in BD-derived neurons.
3) Treatment with lithium rescued the hyper-excitability phenotype in neurons derived from lithium-responsive BD patients but not lithium non-responsive patients, suggesting patient-specific responses.
This study investigated the effects of repeated deep brain stimulation of the pedunculopontine tegmental nucleus (PPTg) in rats. Electrodes were implanted in the PPTg of rats which then received 6 days of stimulation. Behavioral responses and neural activation markers cFOS and GAP-43 were assessed on days 1 and 6. Results found that repeated stimulation increased the threshold current needed to elicit behavioral responses on day 6 compared to day 1, suggesting an inhibitory effect. Expression of cFOS and GAP-43 was also found near the stimulation site, indicating repeated stimulation triggers neural plasticity.
This study investigated the effects of neonatal hyperoxia (high oxygen exposure) on neurotrophin gene expression in mice. The researchers found that hyperoxia induced alterations in brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) mRNA expression levels in the isocortex and prefrontal cortex. Specifically, BDNF mRNA expression decreased in the prefrontal cortex but increased in the isocortex, while GDNF mRNA expression increased in both regions following hyperoxia. Hyperoxia also altered mRNA expression levels of DNA methyltransferases DNMT1 and DNMT3a, which regulate neurotrophin gene expression through DNA methylation. These results suggest that neonatal
POWER SPECTRAL ANALYSIS OF EEG AS A POTENTIAL MARKER IN THE DIAGNOSIS OF SPAS...ijbesjournal
The detection and diagnosis of various neurological disorders are performed using different medical
devices among which electroencephalogram (EEG) is one of the most cost effective technique. Though
significant progress had been made in the analysis of EEG for diagnosis of different neurological
disorders, yet detection of cerebral palsy (CP) is not quite clear. This study was performed to analyze the
EEG power spectrum density (PSD) of spastic CP and normal children to find if any significant EEG
patterns could be used for early detection of CP. Twenty children participated in this study out of which ten
were spastic CP and other ten were normal healthy children. EEG of all the participants was recorded
from C3 C4 and F3 F4 regions following montage 10-20 system. The artifact-free EEG signals of 15
minutes duration was extracted for spectral analysis using Fast Fourier Transformation (FFT) algorithm
in MATLAB and power density spectrum (PSD) was plotted. The PSD revealed high intensity power peak
at frequency of 50Hz and smaller at 100 Hz, which was consistent for all healthy subjects. In case of
spastic CP children, high intensity peak at 100Hz were prominent and smaller peak was observed at 50Hz.
The high intensity 100Hz peak observed in the PSD of spastic CP patients demonstrated that this tool can
be used for early detection of spastic CP.
This study tested intrathecal enzyme replacement therapy (ERT) in a mouse model of infantile Batten disease. Mice lacking the enzyme palmitoyl-protein thioesterase 1 (PPT1) received a single intrathecal injection of recombinant human PPT1 at 6 weeks of age. This prevented decline in motor function, improved survival, and reduced brain and spinal cord pathology compared to untreated mice. The effects were similar to ERT for another form of Batten disease. This suggests intrathecal ERT may help treat infantile Batten disease caused by PPT1 deficiency in humans.
Positron-emission tomography studies of cross-modality inhibition in selectiv...Dr Brendan O'Sullivan
Published in 1994, this groundbreaking paper featured the research of Professor Per Roland, Professor Ryuta Kawashima (of “Brain Training” fame) and Professor Brendan O’ Sullivan.
This landmark research was the first to prove in human brain imaging studies that visual attention is impaired when we do other attention-competing tasks such as manual tasks such as using mobile phones while driving.
1) Mice lacking the inhibitory synapse cell adhesion molecule neuroligin 2 (NL2) were found to exhibit increased anxiety-like behavior.
2) While these NL2-deficient mice appeared to have a decrease in the density of inhibitory synaptic puncta, electron microscopy revealed no actual change in inhibitory synapse numbers.
3) This suggests that NL2 deletion impairs the function of inhibitory synapses without decreasing their numbers, and this decrease in inhibitory synaptic function correlates with increased anxiety in the mice.
1. Using clonal lineage tracing in the adult mouse dentate gyrus, the study found that neuronal precursors of glutamatergic granule neurons exhibit significant tangential migration along blood vessels, followed by limited radial migration.
2. Genetic birthdating and morphological/molecular analyses identified the neuroblast stage as the main developmental window when tangential migration occurs.
3. Observations of a dense plexus of capillaries associated specifically with neuroblasts provided insight into the role of blood vessels as a substrate for neuronal migration in the adult mammalian brain.
This study examines the maturation of synaptic connections in the retinotectal system of Xenopus laevis tadpoles using electron microscopy. The results show that early in development, retinal ganglion cell axons form synapses onto filopodial processes, but later synapses form directly onto dendritic shafts and spines. Treatment with brain-derived neurotrophic factor increases the number of spine synapses and docked vesicles within 24 hours. These structural changes correlate with BDNF's role in functional synaptic maturation.
1) The researchers overexpressed Ngn2 in combination with various forebrain-specific or upper layer-specific transcription factors in human embryonic stem cells to induce upper motor neurons.
2) They found that Emx1, Otx2, Bhlhb5, and Fezf2 as secondary transcription factors generated mature excitatory neurons, while other combinations produced immature neurons.
3) Further experiments are needed to confirm the induced neurons project like deep layer cortical neurons and express excitatory neuron markers.
1) Activated microglia secrete factors that promote neurogenesis from white matter cells, whereas resting microglia do not. When neonatal optic nerve cells were cocultured with activated microglia, there was a significant increase in neurons identified by TUJ-1 labeling compared to cocultures with resting microglia.
2) Oligodendrocyte progenitor cells (OPCs) generated some neurons but were not the major source. Around 70% of neurons came from A2B5-negative cells, which included astrocytes. Activated microglia increased neurogenesis from both OPCs and A2B5-negative cells.
3) Mass spectrometry identified protease ser
The document summarizes key findings from the North American Stroke Meeting in 2003 regarding stem cell transplantation for stroke. It discusses how immortalized stem cell lines can be mass produced and derived without ethical concerns. A phase 1 trial injected cryopreserved stem cells into 12 patients with basal ganglia strokes, finding subjective improvement in functions and increased metabolism at the injury site. A phase 2 trial of 14 patients receiving different stem cell doses found some improvement on stroke scales but left questions unanswered. Future research is needed to optimize timing, locations and doses of stem cell transplantation for stroke patients.
Sex differences in brain activation elicited by humorUTPL
This fMRI study investigated sex differences in brain activation elicited by humor. The study found that while males and females activated similar brain regions involved in humor comprehension, there were some differences:
1) Females activated left prefrontal cortex more than males, suggesting greater executive processing and language-based decoding of humor.
2) Females exhibited greater activation of mesolimbic reward regions including the nucleus accumbens, implying a greater reward network response and possibly less reward expectation than males.
3) These results indicate some sex-specific differences in neural response to humor, with females showing more activation related to executive function and reward processing. This has implications for understanding sex-based disparities in integrating
This document reports on a study that tested the effects of isoxazole 9 (Isx-9), a small synthetic molecule, on adult hippocampal neurogenesis in rats. The study found that administering Isx-9 for 14 days potentiated cell proliferation and increased the number of immature neurons in the hippocampal dentate gyrus. Isx-9 treatment also completely reversed the reduction in cell proliferation and neuronal commitment observed in vehicle-treated animals that were subjected to repeated handling and injections. These findings demonstrate that Isx-9 has promising pro-neurogenic properties and could help mitigate stress-induced deficits in adult hippocampal neurogenesis.
This study identifies novel transcriptional targets of the protein PGC-1α in the brain. The researchers:
1) Conducted a microarray on cells overexpressing PGC-1α to identify upregulated genes.
2) Mined the microarray results for genes related to inhibitory neurons and measured their expression in mice with altered PGC-1α levels.
3) Found that PGC-1α regulates genes involved in synaptic function (Syt2, Cplx1), structure (Nefh), and metabolism. Conditional deletion of PGC-1α in inhibitory neurons decreased expression of these genes and impaired neuronal function and memory.
This study investigated differences in the density of nitric oxide synthase (NOS) interneurons in the striatum between individuals with Tourette Syndrome (TS) and normal controls (NC). Brain tissue from 5 TS and 5 NC individuals was stained using immunohistochemistry to label NOS interneurons. The staining procedure and a pilot study to determine the most effective antibody were described. Sections from each brain were analyzed under a microscope using the Optical Fractionator probe method to count and compare NOS interneuron densities in the caudate nucleus and putamen between groups. It was hypothesized that NOS interneuron density would be decreased in the striatum of individuals with TS.
Aminah Sheikh's research focuses on understanding the neural mechanisms underlying abnormal brain development that result from early brain injuries. Using a neonatal rat model of hypoxia-ischemia, she investigates how these injuries damage subplate neurons in the developing brain and alter the maturation of brain circuits. Her work aims to identify targets for therapies to prevent long-term cognitive impairment resulting from neonatal brain damage.
A disinhibitory microcircuit initiates critical period plasticity in the visu...Taruna Ikrar
Earlysensoryexperienceinstructsthematurationofneuralcircuitry in the cortex1,2. This has been studied extensively in the primary visualcortex,inwhichlossofvisiontooneeyepermanentlydegrades corticalresponsivenesstothateye3,4,aphenomenonknownasocular dominance plasticity (ODP). Cortical inhibition mediates this process4–6,butthepreciseroleofspecificclassesofinhibitoryneurons in ODP is controversial. Here we report that evoked firing rates of binocular excitatory neurons in the primary visual cortex immediatelydropbyhalfwhenvisionisrestrictedtooneeye,butgradually return to normal over the followingtwenty-four hours, despite the fact that vision remains restricted to one eye. This restoration of binocular-like excitatory firing rates after monocular deprivation resultsfromarapid,althoughtransient,reductioninthefiringrates of fast-spiking, parvalbumin-positive (PV) interneurons, which in turncanbeattributedtoadecreaseinlocalexcitatorycircuitinput onto PV interneurons.This reduction in PV-cell-evoked responses after monocular lid suture is restricted to the critical period for ODPandappearstobenecessaryforsubsequentshiftsinexcitatory ODP. Pharmacologically enhancing inhibition at the time of sight deprivation blocks ODP and, conversely, pharmacogenetic reduction of PV cell firing rates can extend the critical period for ODP. Thesefindingsdefinethemicrocircuitchangesinitiatingcompetitive
plasticityduringcriticalperiodsofcorticaldevelopment.Moreover, they show that the restoration of evoked firing rates of layer 2/3 pyramidal neurons by PV-specific disinhibition is a key step in the progression of ODP.
A disinhibitory microcircuit initiates critical period plasticity in the visu...Taruna Ikrar
Early sensory experience instructs the maturation of neural circuitry in the cortex1, 2. This has been studied extensively in the primary visual cortex, in which loss of vision to one eye permanently degrades cortical responsiveness to that eye3, 4, a phenomenon known as ocular dominance plasticity (ODP). Cortical inhibition mediates this process4, 5, 6, but the precise role of specific classes of inhibitory neurons in ODP is controversial. Here we report that evoked firing rates of binocular excitatory neurons in the primary visual cortex immediately drop by half when vision is restricted to one eye, but gradually return to normal over the following twenty-four hours, despite the fact that vision remains restricted to one eye. This restoration of binocular-like excitatory firing rates after monocular deprivation results from a rapid, although transient, reduction in the firing rates of fast-spiking, parvalbumin-positive (PV) interneurons, which in turn can be attributed to a decrease in local excitatory circuit input onto PV interneurons. This reduction in PV-cell-evoked responses after monocular lid suture is restricted to the critical period for ODP and appears to be necessary for subsequent shifts in excitatory ODP. Pharmacologically enhancing inhibition at the time of sight deprivation blocks ODP and, conversely, pharmacogenetic reduction of PV cell firing rates can extend the critical period for ODP. These findings define the microcircuit changes initiating competitive plasticity during critical periods of cortical development. Moreover, they show that the restoration of evoked firing rates of layer 2/3 pyramidal neurons by PV-specific disinhibition is a key step in the progression of ODP.
This document summarizes a study examining the role of the nuclear factor erythroid 2-related factor 2 (Nrf2) in protecting against brain injury caused by intracerebral hemorrhage (ICH) in mice. The study found that Nrf2 knockout mice exhibited larger brain injury volumes and greater neurological deficits 24 hours after ICH induction compared to wild-type mice. Additionally, Nrf2 knockout mice showed increased leukocyte infiltration, reactive oxygen species production, DNA damage, and cytochrome c release during the early post-ICH period. These results suggest that Nrf2 provides protection against ICH-induced early brain injury, likely by reducing leukocyte-mediated free radical oxidative damage.
Analysis of Human Embryonic Stem Cells with Regulatable Expression of the Cel...Christopher S Park
This document describes research on developing a human neural stem cell line with regulatable expression of the cell adhesion molecule L1 using a tetracycline-controlled gene expression system. Embryonic stem cell-derived neural stem cells were transfected with a plasmid containing the L1 gene under control of a doxycycline-regulatable promoter. In the presence of doxycycline, L1 expression was eliminated, while without doxycycline L1 was expressed. This cell line with regulatable L1 expression was then tested in a mouse model of spinal cord injury, where expression of L1 improved locomotor recovery compared to when L1 expression was turned off with doxycycline.
Prefrontal Parvalbumin Neurons in Control of AttentionXinming Wang
This study investigated the role of prefrontal parvalbumin (PV) neurons in controlling attention using electrophysiological recordings in mice performing an attention task. The key findings were:
1) PV neurons in the medial prefrontal cortex (mPFC) increased their firing uniformly and sustainedly during goal-driven attention, correlating with attention levels.
2) Successful attention was characterized by strong synchronization of PV neurons, increased gamma oscillations, and phase locking of pyramidal neurons to gamma rhythms.
3) Optogenetic silencing of PV neurons impaired attention, while synchronizing PV neurons at gamma frequencies improved goal-directed behavior.
4) These results suggest PV neurons act as a functional unit coordinating local m
Tetrahydrohyperforin (IDN5706), a derivative of the active molecule hyperforin in St. John's Wort, was examined for its ability to prevent cognitive deficits and synaptic impairment in an Alzheimer's disease mouse model. Five-month old APPswe/PSEN1DE9 mice were treated with IDN5706 for 10 weeks. IDN5706 improved memory, prevented decreases in synaptic proteins and LTP, and reduced amyloid-beta plaque burden, tau hyperphosphorylation, and astrogliosis. In cell cultures, IDN5706 decreased amyloid precursor protein processing leading to amyloid-beta peptide generation. The results suggest IDN5706 may be a potential therapeutic for treating Alzheimer's
Tetrahydrohyperforin (IDN5706), a derivative of the active molecule hyperforin in St. John's Wort, was examined for its ability to prevent cognitive deficits and synaptic impairment in an Alzheimer's disease mouse model. Five-month old APPswe/PSEN1DE9 mice were treated with IDN5706 for 10 weeks. IDN5706 improved memory, prevented decreases in synaptic proteins and LTP, and reduced amyloid-beta plaque burden, tau hyperphosphorylation, and astrogliosis. In cell cultures, IDN5706 decreased amyloid precursor protein processing leading to amyloid-beta peptide generation. The results suggest IDN5706 may be a potential therapeutic for treating Alzheimer's
1) Dopamine transmission in the basal ganglia controls motor behavior through two pathways - the direct pathway which stimulates motion, and the indirect pathway which inhibits motion.
2) CK1δ over-expression disrupts this system, possibly through dopamine deficiency caused by downregulation of D1 and D2 receptors.
3) This study found an increase in calretinin-containing neurons in the striatum of CK1δ over-expressing mice, suggesting this imbalance contributes to their ADHD-like behaviors.
Eastern European countries appear to have become dependent on Ru.docxjoellemurphey
Eastern European countries appear to have become dependent on Russian oil originally due to the country being a reliable supplier to the European countries (Bradshaw, 2014, p. 76). Though the countries were allies with the United States, they were trying to become less dependent on the Middle East and saw that Russia was a reliable source. Much of this reliance was due to the “iron curtain” as well as the fact that many of the Eastern European countries were part of the Soviet Union in some way or affiliated with them.
It appears that much of the reason as to why these countries reached energy accords with Russia is due to the convenience. There was “limited access to storage and alternative sources of gas supply” (Bradshaw, 2014, p. 77). This pushed these countries to depend more on Russia, which appeared to be an easier access to gas supplies. Another reason might have been due to fear as the Kremlin punished Ukraine for voting for an anti-Moscow government (Bradshaw, 2015, p. 77). This action shows that many of these countries may have reached these accords due to the pressure and encouragement of the Soviet Government. In the 1980’s the dependence of European countries on Russian gas resources was 50-60%. In the 1970’s, many of the Eastern European countries also became reliant on Russia due to a greater demand of oil and gas. The surrounding countries that were providing resources were not able to keep up with the demand and thus these countries sought to get their sources from Russia. It also helped that Russia’s prices appeared to be lower than that of the world market (Bradshaw, 2014, p. 87-88). Due to the price of oil and gas and the availability, Eastern European countries were able to grow and build in gas import and its infrastructure, thus in turn causing it to be dependent on Russia.
Bradshaw, M. (2014).
Global Energy Dilemmas: Energy Security, Globalization, and Climate Change
. Cambridge, UK: Polity Press.
Based on your considered review of this module’s readings as well as your reflection upon the first three modules, evaluate the questions below.
In retrospect it seems obvious, but exactly how and why did Eastern European countries come to depend on Russian oil and natural gas after World War II?
Why did the Western Europeans reach energy accords with the Russians in the 1970s and early 1980s, building large-scale natural gas import infrastructures and increasing their dependence on Russian gas?
.
EAS 209 Second Response Paper Topic Assignment Due .docxjoellemurphey
EAS 209
Second Response Paper Topic
>>>Assignment Due Date: Friday, October 12, 2018<<<
Write 350 words, excluding works cited and references, on the following topic:
Dipesh Chakrabarty cites John Stuart Mill to show one dimension of historicist
consciousness: “a recommendation to the colonized to wait.” What does Chakrabarty
mean by this phrase? Consider, e.g. why, according to Mill, “Indians, Africans, and other
‘rude’ nations” had to be consigned to what Chakrabarty called “an imaginary waiting
room of history.”
To respond to this question, you might find it helpful to consider Chakrabarty’s discussion
on historicism or “stagist theory of history.”
▪ Submit a hard copy in your Tutorial Section on Friday, October 12.
▪ Papers must be type-written, double-spaced, appearing in 12 points Times New Roman font or
its equivalent with 1” margins. Do not exceed 400 words. You are responsible for keeping an
extra copy of your own paper.
▪ The assignment does not ask you to conduct additional research. Papers that do not respond
to the given topic or do not follow the specific instructions described above will receive no
marks. No resubmission allowed.
▪ You need to present your argument logically and clearly, fully demonstrate the precise
understanding of Chakrabarty’s argument and substantiate your argument convincingly and
with details.
▪ Observe the Chicago Manual of Style referencing practice and properly cite the passages you
quote (i.e. author, title, page number, etc.). Works cited or references should not be counted
toward the 350 word limit.
▪ Any ideas or expressions that are not your own must be placed in quotation marks and
referenced with page number. Academic misconduct will not be tolerated. See:
http://www.artsci.utoronto.ca/osai/The-rules/what-is-academic-misconduct
▪ You may share notes and discuss your ideas with others for preparation. But the paper you
submit must be exclusively written by you alone and in your own words clearly distinguishable
from others’. Papers that plagiarize, replicate others, or contain identical or near-identical
passages that appear in other papers will not be accepted or credited.
▪ You must proof-read before submission. Sentences that are incomplete or unintelligible will
not be read or credited.
▪ Late submission and papers submitted via e-mail will not be accepted or credited unless
under extraordinary circumstances. ABSOLUTELY NO EXCPETION!
http://www.artsci.utoronto.ca/osai/The-rules/what-is-academic-misconduct
I N T R O D U C T I O N
The Idea of Provincializing Europe
Europe . . . since 1914 has become provincialized, . . .
only the natural sciences are able to call forth a
quick international echo.
(Hans-Georg Gadamer, 1977)
The West is a name for a subject which gathers itself in
discourse but is also an object constituted discursively;
it is, evidently, a name always associating itself with
those regions, communities, and peoples.
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2) While these NL2-deficient mice appeared to have a decrease in the density of inhibitory synaptic puncta, electron microscopy revealed no actual change in inhibitory synapse numbers.
3) This suggests that NL2 deletion impairs the function of inhibitory synapses without decreasing their numbers, and this decrease in inhibitory synaptic function correlates with increased anxiety in the mice.
1. Using clonal lineage tracing in the adult mouse dentate gyrus, the study found that neuronal precursors of glutamatergic granule neurons exhibit significant tangential migration along blood vessels, followed by limited radial migration.
2. Genetic birthdating and morphological/molecular analyses identified the neuroblast stage as the main developmental window when tangential migration occurs.
3. Observations of a dense plexus of capillaries associated specifically with neuroblasts provided insight into the role of blood vessels as a substrate for neuronal migration in the adult mammalian brain.
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Sex differences in brain activation elicited by humorUTPL
This fMRI study investigated sex differences in brain activation elicited by humor. The study found that while males and females activated similar brain regions involved in humor comprehension, there were some differences:
1) Females activated left prefrontal cortex more than males, suggesting greater executive processing and language-based decoding of humor.
2) Females exhibited greater activation of mesolimbic reward regions including the nucleus accumbens, implying a greater reward network response and possibly less reward expectation than males.
3) These results indicate some sex-specific differences in neural response to humor, with females showing more activation related to executive function and reward processing. This has implications for understanding sex-based disparities in integrating
This document reports on a study that tested the effects of isoxazole 9 (Isx-9), a small synthetic molecule, on adult hippocampal neurogenesis in rats. The study found that administering Isx-9 for 14 days potentiated cell proliferation and increased the number of immature neurons in the hippocampal dentate gyrus. Isx-9 treatment also completely reversed the reduction in cell proliferation and neuronal commitment observed in vehicle-treated animals that were subjected to repeated handling and injections. These findings demonstrate that Isx-9 has promising pro-neurogenic properties and could help mitigate stress-induced deficits in adult hippocampal neurogenesis.
This study identifies novel transcriptional targets of the protein PGC-1α in the brain. The researchers:
1) Conducted a microarray on cells overexpressing PGC-1α to identify upregulated genes.
2) Mined the microarray results for genes related to inhibitory neurons and measured their expression in mice with altered PGC-1α levels.
3) Found that PGC-1α regulates genes involved in synaptic function (Syt2, Cplx1), structure (Nefh), and metabolism. Conditional deletion of PGC-1α in inhibitory neurons decreased expression of these genes and impaired neuronal function and memory.
This study investigated differences in the density of nitric oxide synthase (NOS) interneurons in the striatum between individuals with Tourette Syndrome (TS) and normal controls (NC). Brain tissue from 5 TS and 5 NC individuals was stained using immunohistochemistry to label NOS interneurons. The staining procedure and a pilot study to determine the most effective antibody were described. Sections from each brain were analyzed under a microscope using the Optical Fractionator probe method to count and compare NOS interneuron densities in the caudate nucleus and putamen between groups. It was hypothesized that NOS interneuron density would be decreased in the striatum of individuals with TS.
Aminah Sheikh's research focuses on understanding the neural mechanisms underlying abnormal brain development that result from early brain injuries. Using a neonatal rat model of hypoxia-ischemia, she investigates how these injuries damage subplate neurons in the developing brain and alter the maturation of brain circuits. Her work aims to identify targets for therapies to prevent long-term cognitive impairment resulting from neonatal brain damage.
A disinhibitory microcircuit initiates critical period plasticity in the visu...Taruna Ikrar
Earlysensoryexperienceinstructsthematurationofneuralcircuitry in the cortex1,2. This has been studied extensively in the primary visualcortex,inwhichlossofvisiontooneeyepermanentlydegrades corticalresponsivenesstothateye3,4,aphenomenonknownasocular dominance plasticity (ODP). Cortical inhibition mediates this process4–6,butthepreciseroleofspecificclassesofinhibitoryneurons in ODP is controversial. Here we report that evoked firing rates of binocular excitatory neurons in the primary visual cortex immediatelydropbyhalfwhenvisionisrestrictedtooneeye,butgradually return to normal over the followingtwenty-four hours, despite the fact that vision remains restricted to one eye. This restoration of binocular-like excitatory firing rates after monocular deprivation resultsfromarapid,althoughtransient,reductioninthefiringrates of fast-spiking, parvalbumin-positive (PV) interneurons, which in turncanbeattributedtoadecreaseinlocalexcitatorycircuitinput onto PV interneurons.This reduction in PV-cell-evoked responses after monocular lid suture is restricted to the critical period for ODPandappearstobenecessaryforsubsequentshiftsinexcitatory ODP. Pharmacologically enhancing inhibition at the time of sight deprivation blocks ODP and, conversely, pharmacogenetic reduction of PV cell firing rates can extend the critical period for ODP. Thesefindingsdefinethemicrocircuitchangesinitiatingcompetitive
plasticityduringcriticalperiodsofcorticaldevelopment.Moreover, they show that the restoration of evoked firing rates of layer 2/3 pyramidal neurons by PV-specific disinhibition is a key step in the progression of ODP.
A disinhibitory microcircuit initiates critical period plasticity in the visu...Taruna Ikrar
Early sensory experience instructs the maturation of neural circuitry in the cortex1, 2. This has been studied extensively in the primary visual cortex, in which loss of vision to one eye permanently degrades cortical responsiveness to that eye3, 4, a phenomenon known as ocular dominance plasticity (ODP). Cortical inhibition mediates this process4, 5, 6, but the precise role of specific classes of inhibitory neurons in ODP is controversial. Here we report that evoked firing rates of binocular excitatory neurons in the primary visual cortex immediately drop by half when vision is restricted to one eye, but gradually return to normal over the following twenty-four hours, despite the fact that vision remains restricted to one eye. This restoration of binocular-like excitatory firing rates after monocular deprivation results from a rapid, although transient, reduction in the firing rates of fast-spiking, parvalbumin-positive (PV) interneurons, which in turn can be attributed to a decrease in local excitatory circuit input onto PV interneurons. This reduction in PV-cell-evoked responses after monocular lid suture is restricted to the critical period for ODP and appears to be necessary for subsequent shifts in excitatory ODP. Pharmacologically enhancing inhibition at the time of sight deprivation blocks ODP and, conversely, pharmacogenetic reduction of PV cell firing rates can extend the critical period for ODP. These findings define the microcircuit changes initiating competitive plasticity during critical periods of cortical development. Moreover, they show that the restoration of evoked firing rates of layer 2/3 pyramidal neurons by PV-specific disinhibition is a key step in the progression of ODP.
This document summarizes a study examining the role of the nuclear factor erythroid 2-related factor 2 (Nrf2) in protecting against brain injury caused by intracerebral hemorrhage (ICH) in mice. The study found that Nrf2 knockout mice exhibited larger brain injury volumes and greater neurological deficits 24 hours after ICH induction compared to wild-type mice. Additionally, Nrf2 knockout mice showed increased leukocyte infiltration, reactive oxygen species production, DNA damage, and cytochrome c release during the early post-ICH period. These results suggest that Nrf2 provides protection against ICH-induced early brain injury, likely by reducing leukocyte-mediated free radical oxidative damage.
Analysis of Human Embryonic Stem Cells with Regulatable Expression of the Cel...Christopher S Park
This document describes research on developing a human neural stem cell line with regulatable expression of the cell adhesion molecule L1 using a tetracycline-controlled gene expression system. Embryonic stem cell-derived neural stem cells were transfected with a plasmid containing the L1 gene under control of a doxycycline-regulatable promoter. In the presence of doxycycline, L1 expression was eliminated, while without doxycycline L1 was expressed. This cell line with regulatable L1 expression was then tested in a mouse model of spinal cord injury, where expression of L1 improved locomotor recovery compared to when L1 expression was turned off with doxycycline.
Prefrontal Parvalbumin Neurons in Control of AttentionXinming Wang
This study investigated the role of prefrontal parvalbumin (PV) neurons in controlling attention using electrophysiological recordings in mice performing an attention task. The key findings were:
1) PV neurons in the medial prefrontal cortex (mPFC) increased their firing uniformly and sustainedly during goal-driven attention, correlating with attention levels.
2) Successful attention was characterized by strong synchronization of PV neurons, increased gamma oscillations, and phase locking of pyramidal neurons to gamma rhythms.
3) Optogenetic silencing of PV neurons impaired attention, while synchronizing PV neurons at gamma frequencies improved goal-directed behavior.
4) These results suggest PV neurons act as a functional unit coordinating local m
Tetrahydrohyperforin (IDN5706), a derivative of the active molecule hyperforin in St. John's Wort, was examined for its ability to prevent cognitive deficits and synaptic impairment in an Alzheimer's disease mouse model. Five-month old APPswe/PSEN1DE9 mice were treated with IDN5706 for 10 weeks. IDN5706 improved memory, prevented decreases in synaptic proteins and LTP, and reduced amyloid-beta plaque burden, tau hyperphosphorylation, and astrogliosis. In cell cultures, IDN5706 decreased amyloid precursor protein processing leading to amyloid-beta peptide generation. The results suggest IDN5706 may be a potential therapeutic for treating Alzheimer's
Tetrahydrohyperforin (IDN5706), a derivative of the active molecule hyperforin in St. John's Wort, was examined for its ability to prevent cognitive deficits and synaptic impairment in an Alzheimer's disease mouse model. Five-month old APPswe/PSEN1DE9 mice were treated with IDN5706 for 10 weeks. IDN5706 improved memory, prevented decreases in synaptic proteins and LTP, and reduced amyloid-beta plaque burden, tau hyperphosphorylation, and astrogliosis. In cell cultures, IDN5706 decreased amyloid precursor protein processing leading to amyloid-beta peptide generation. The results suggest IDN5706 may be a potential therapeutic for treating Alzheimer's
1) Dopamine transmission in the basal ganglia controls motor behavior through two pathways - the direct pathway which stimulates motion, and the indirect pathway which inhibits motion.
2) CK1δ over-expression disrupts this system, possibly through dopamine deficiency caused by downregulation of D1 and D2 receptors.
3) This study found an increase in calretinin-containing neurons in the striatum of CK1δ over-expressing mice, suggesting this imbalance contributes to their ADHD-like behaviors.
Similar to Role of pro-brain-derived neurotrophic factor (proBDNF)to ma.docx (20)
Eastern European countries appear to have become dependent on Ru.docxjoellemurphey
Eastern European countries appear to have become dependent on Russian oil originally due to the country being a reliable supplier to the European countries (Bradshaw, 2014, p. 76). Though the countries were allies with the United States, they were trying to become less dependent on the Middle East and saw that Russia was a reliable source. Much of this reliance was due to the “iron curtain” as well as the fact that many of the Eastern European countries were part of the Soviet Union in some way or affiliated with them.
It appears that much of the reason as to why these countries reached energy accords with Russia is due to the convenience. There was “limited access to storage and alternative sources of gas supply” (Bradshaw, 2014, p. 77). This pushed these countries to depend more on Russia, which appeared to be an easier access to gas supplies. Another reason might have been due to fear as the Kremlin punished Ukraine for voting for an anti-Moscow government (Bradshaw, 2015, p. 77). This action shows that many of these countries may have reached these accords due to the pressure and encouragement of the Soviet Government. In the 1980’s the dependence of European countries on Russian gas resources was 50-60%. In the 1970’s, many of the Eastern European countries also became reliant on Russia due to a greater demand of oil and gas. The surrounding countries that were providing resources were not able to keep up with the demand and thus these countries sought to get their sources from Russia. It also helped that Russia’s prices appeared to be lower than that of the world market (Bradshaw, 2014, p. 87-88). Due to the price of oil and gas and the availability, Eastern European countries were able to grow and build in gas import and its infrastructure, thus in turn causing it to be dependent on Russia.
Bradshaw, M. (2014).
Global Energy Dilemmas: Energy Security, Globalization, and Climate Change
. Cambridge, UK: Polity Press.
Based on your considered review of this module’s readings as well as your reflection upon the first three modules, evaluate the questions below.
In retrospect it seems obvious, but exactly how and why did Eastern European countries come to depend on Russian oil and natural gas after World War II?
Why did the Western Europeans reach energy accords with the Russians in the 1970s and early 1980s, building large-scale natural gas import infrastructures and increasing their dependence on Russian gas?
.
EAS 209 Second Response Paper Topic Assignment Due .docxjoellemurphey
EAS 209
Second Response Paper Topic
>>>Assignment Due Date: Friday, October 12, 2018<<<
Write 350 words, excluding works cited and references, on the following topic:
Dipesh Chakrabarty cites John Stuart Mill to show one dimension of historicist
consciousness: “a recommendation to the colonized to wait.” What does Chakrabarty
mean by this phrase? Consider, e.g. why, according to Mill, “Indians, Africans, and other
‘rude’ nations” had to be consigned to what Chakrabarty called “an imaginary waiting
room of history.”
To respond to this question, you might find it helpful to consider Chakrabarty’s discussion
on historicism or “stagist theory of history.”
▪ Submit a hard copy in your Tutorial Section on Friday, October 12.
▪ Papers must be type-written, double-spaced, appearing in 12 points Times New Roman font or
its equivalent with 1” margins. Do not exceed 400 words. You are responsible for keeping an
extra copy of your own paper.
▪ The assignment does not ask you to conduct additional research. Papers that do not respond
to the given topic or do not follow the specific instructions described above will receive no
marks. No resubmission allowed.
▪ You need to present your argument logically and clearly, fully demonstrate the precise
understanding of Chakrabarty’s argument and substantiate your argument convincingly and
with details.
▪ Observe the Chicago Manual of Style referencing practice and properly cite the passages you
quote (i.e. author, title, page number, etc.). Works cited or references should not be counted
toward the 350 word limit.
▪ Any ideas or expressions that are not your own must be placed in quotation marks and
referenced with page number. Academic misconduct will not be tolerated. See:
http://www.artsci.utoronto.ca/osai/The-rules/what-is-academic-misconduct
▪ You may share notes and discuss your ideas with others for preparation. But the paper you
submit must be exclusively written by you alone and in your own words clearly distinguishable
from others’. Papers that plagiarize, replicate others, or contain identical or near-identical
passages that appear in other papers will not be accepted or credited.
▪ You must proof-read before submission. Sentences that are incomplete or unintelligible will
not be read or credited.
▪ Late submission and papers submitted via e-mail will not be accepted or credited unless
under extraordinary circumstances. ABSOLUTELY NO EXCPETION!
http://www.artsci.utoronto.ca/osai/The-rules/what-is-academic-misconduct
I N T R O D U C T I O N
The Idea of Provincializing Europe
Europe . . . since 1914 has become provincialized, . . .
only the natural sciences are able to call forth a
quick international echo.
(Hans-Georg Gadamer, 1977)
The West is a name for a subject which gathers itself in
discourse but is also an object constituted discursively;
it is, evidently, a name always associating itself with
those regions, communities, and peoples.
Earth Science LabIn what order do materials settle in waterSo t.docxjoellemurphey
Earth Science Lab:In what order do materials settle in water?
So this is my Topic:
In what order do materials settle in water? Design and carry out an experiment to determine the order in which different sized materials (e.g., sand, gravel, topsoil) settle out in water after they have been mixed up.
but i don't understand the question below:
What are some possible treatments you can use to answer your question? What are some variables that may influence your question, and are they variables that you can easily manipulate and test?
What can i write about the possible treatments?
.
EarlyIntervention Strategies Paper (15 points)The pu.docxjoellemurphey
Early
Intervention Strategies Paper (15 points)
The purpose of the presentation is to help classmates understand different types of intervention strategies for early intervention. Students will be expected to write a 5-7 page paper that is comprised of two parts. In Part I, the student will discuss the role of each of the following professionals that can comprise a treatment team in a maximum of 3 pages:
Developmental Teacher Occupational Therapist Physical Therapist Speech/Language Pathologist
Audiologist Vision Consultant Psychologist Pediatrician
Part II: Furthermore, each student will set up a site visit at a local agency or provider of services to young children and will spend some time observing a particular facility or program that cares for and provides services to infants and young children. The following list should be used to guide the observations. The student should summarize thefollowing information in at least 3 pages:
Name of the facility or program
Ages of the children served
History and philosophy of the facility or program
Structure of the facility or program (Co-Op, Pre-K, )
Services provided
Activities and routines in which the children engage
Adult to child ratio
Types of delays and disabilities of the children who attend this program
Family involvement
Type of setting: inclusive setting, provisions for inclusion
Curriculum used
Would you recommend this facility to a family with a child with a disability? Why or why not?
.
Early Hominids & Australopithecus SubscribeWhat is a too.docxjoellemurphey
Early Hominids & Australopithecus
Subscribe
What is a tool? Did
Sahelanthropus
,
Orrorin
,
Ardipithecus, or Australopithecus
use tools? What evidence shows that they used tools?
What do these groups represent for human evolution? Why are these hominids unique in human evolutionary history?
.
Early scholarly and philosophical manuscripts were in Greek. However.docxjoellemurphey
Early scholarly and philosophical manuscripts were in Greek. However, by the 5th century CE – and onward – language was mainly spread by conquests, trades, religious affiliations, technological advancements or entertainment. (Gascoigne, 2001). For example, as the geographic territory under Roman control grew, the use of Latin as a common language also spread. In areas under Roman control, Latin was the spoken and written language of the courts and commerce, as well as the language of the Christian church. As the Roman Empire expanded, Latin served as a common language that allowed for people of diverse linguistic backgrounds to be able to communicate.
Onward and by the early 14th century, the trend toward the use of vernacular language had spread throughout most of Europe. As monarchies throughout the region began to consolidate, the use of vernacular languages contributed to an increasing nationalism, or feeling of pride in one’s own nation, and in this case among people of similar linguistic backgrounds. People began to feel more connected to local leaders than they did to influences from afar. These sociopolitical shifts, along with the development of moveable type (the printing press), helped to ensure the success of the vernacular languages during the Renaissance.
Assignment:
The goal of this assignment is to research and report on the origins of vernacular language, and its spread while also providing evidence of Latin’s influence on all Western languages.
Choose one native language spoken in Europe, discuss the origins of the vernacular language and describe how the language spread.
As a whole, in what ways has Latin influenced Western language development?
Prepare a 2-page essay (not including cover page and works cited page) answering the questions stated above in APA format.
.
Early Learning & Developmental Guidelines July 2017 1 .docxjoellemurphey
Early Learning &
Developmental Guidelines
July 2017 1
Early Learning and Developmental Guidelines
This document provides current Web links to all State early learning and development guidelines (ELGs). At this
time, all 56 States and Territories have developed ELGs for preschool children, and virtually all have ELGs for
infants and toddlers. The following table provides the website for ELGs from the States.
State ELG Name and Web Site
ELG Age
Range
Alabama Alabama Early Learning Guidelines
http://dhr.alabama.gov/large_docs/aelg.pdf
Birth to 5
years
Alaska Early Learning Guidelines (2007)
https://education.alaska.gov/publications/earlylearningguidelines.pdf
Birth to 5
years
Arizona Early Learning Standards (2013)
http://www.azed.gov/early-childhood/files/2011/11/arizona-early-learning-
standards-3rd-edition.pdf
3 to 5 years
Arizona’s Infant and Toddler Developmental Guidelines (Draft)
http://www.azftf.gov/Documents/Arizona%20Infant%20and%20Toddler%20
Developmental%20Guidelines%20DRAFT%20for%20VETTING.pdf
Birth to 3
years
Arkansas Arkansas Child Development and Early Learning Standards (2016)
http://www.arheadstart.org/Ark_Early_Learning_Standards%20(19)%20(1).p
df
Birth to 5
years
California California Infant/Toddler Learning & Development Foundations (2009)
http://www.cde.ca.gov/sp/cd/re/itfoundations.asp
Birth to 3
years
California Preschool Learning Foundations, Volumes 1-3
http://www.cde.ca.gov/sp/cd/re/psfoundations.asp
3 to 5 years
Colorado Colorado Early Learning & Development Guidelines (2013)
https://www.cde.state.co.us/early/eldgs
Birth to 5
years
Connecticut Guidelines for the Development of Infant & Toddler Early Learning
http://www.ct.gov/dss/lib/dss/dss_early_learning_guidelines.pdf
Birth to 3
years
Connecticut Early Learning and Development Standards (2014)
http://www.ct.gov/oec/lib/oec/earlycare/elds/ctelds.pdf
Birth to 5
years
Connecticut Preschool Assessment Framework (2008)
http://www.sde.ct.gov/sde/lib/sde/PDF/DEPS/Early/Preschool_Assessment_
Framework.pdf
3 to 5 years
http://dhr.alabama.gov/large_docs/aelg.pdf
https://education.alaska.gov/publications/earlylearningguidelines.pdf
http://www.azed.gov/early-childhood/files/2011/11/arizona-early-learning-standards-3rd-edition.pdf
http://www.azed.gov/early-childhood/files/2011/11/arizona-early-learning-standards-3rd-edition.pdf
http://www.azftf.gov/Documents/Arizona%20Infant%20and%20Toddler%20Developmental%20Guidelines%20DRAFT%20for%20VETTING.pdf
http://www.azftf.gov/Documents/Arizona%20Infant%20and%20Toddler%20Developmental%20Guidelines%20DRAFT%20for%20VETTING.pdf
http://www.cde.ca.gov/sp/cd/re/itfoundations.asp
http://www.cde.ca.gov/sp/cd/re/psfoundations.asp
https://www.cde.state.co.us/early/eldgs
http://www.ct.gov/dss/lib/dss/dss_early_learning_guidelines.pdf
http://www.ct.gov/oec/lib/oec/earlycare/elds/ctelds.pdf
http://www.sde.ct.gov/sde/lib/sde/PDF/DEPS/Early/Preschool.
Early Innovations and Their Impact Today Wilbur and Orville Wrig.docxjoellemurphey
Early Innovations and Their Impact Today
Wilbur and Orville Wright's innovative spirit allowed them to take their place in history. Their inventions have changed the way people live around the world. At the turn of the century, an explosion in technological achievements occurred. The same kind of energy that went into advances in aviation went into the development of automobiles, telephones, televisions, and immunizations to prevent diseases. These and other innovations and achievements continue to have an enormous impact on human life.
In this week's Discussion, you will analyze two technological innovations/achievements of the late 19th/early 20th century and describe the impact they have on life today.
To prepare for this Discussion:
Review Chapter 2 (pp.10–30) from this week's Learning Resources focusing on technological innovations and achievements around the globe.
Identify two significant technological innovations/achievements (such as the telephone, television, automobiles, and vaccinations) from the late 19th and early 20th centuries.
Consider the ways in which these technologies made an impact on society at the turn of the century.
Reflect on how these technologies continue to impact your life today.
Support your assertions by making at least 2 references, in proper APA format, to your course readings.
With these thoughts in mind:
Post by Day 3 a 2- to 3-paragraph analysis where you do the following:
Identify two significant technological innovations/achievements from the late 19th and early 20th centuries.
Describe, in your opinion, why you believe your choices were significant and created global impacts during that time period.
Explain how these two particular innovations/achievements impact the way you live today.
.
Early childhood professionals have an essential role in creating.docxjoellemurphey
Early childhood professionals have an essential role in creating and supporting stable, responsive environments that reduce and reverse the impact of adversity (Center on the Developing Child, 2015b). In this Discussion, you explore the impact of adverse experiences and the role of the early childhood professional in supporting healthy, nurturing developmental contexts.
.
Early Constitutional ControversiesIn 1788, Alexander Hamilton and .docxjoellemurphey
Early Constitutional Controversies
In 1788, Alexander Hamilton and James Madison, who had both played active roles at the Constitutional Convention, worked together to write
The Federalist Papers
, a series of articles originally published in New York newspapers to convince readers to back the ratification of the Constitution. Constitutional scholars often refer to these papers to gain an appreciation of the “original intention” of the Framers, how those men expected the federal government to operate under the Constitution, and the powers they sought to grant or deny the federal government. By the early 1790s, however, Hamilton and Madison had divided over basic constitutional questions such as whether or not the federal government could charter a national bank. The American electorate, which had ratified the Constitution, had split on the issue as well, dividing into rival Federalist and Republican parties.
For this assignment, explore
one
significant constitutional controversy, from the first two decades of the United States under the Constitution (1789 to 1821). Topics to consider include:
The incorporation of the Bank of the United States
Debt assumption
The Jay Treaty
The Alien and Sedition Acts
The Election of 1800
John Marshall’s use of judicial review
The Louisiana Purchase
The trial of Aaron Burr
Jefferson’s Embargo
Federalist opposition to the War of 1812
Missouri’s application for statehood
Describe opposing views of the topic under consideration, and explain how each side used the Constitution to support its position. Assess the validity of the two sides according to your own interpretation of the Constitution as well as according to how the Constitution and constitutional principles were understood at the time the controversy occurred.
The paper should draw from at least
one
primary source and
two
scholarly, secondary sources for a total of three sources (not including the Constitution itself). For assistance on the use of primary and secondary sources, please see sections 8.1 and 8.2 of the Ashford Writing Center. The secondary sources should be accessed through any of the academic databases available through the Ashford University library.
The paper must be three pages in length and formatted according to APA style. You must use at least three scholarly resources (at least two of which can be found in the Ashford Online Library) other than the textbook to support your claims and subclaims. Cite your resources in text and on the reference page. For information regarding APA samples and tutorials, visit the Ashford Writing Center, within the Learning Resources tab on the left navigation toolbar.
.
Early Civilizations MatrixUsing your readings and outside sour.docxjoellemurphey
Early Civilizations Matrix
Using your readings and outside sources complete the following matrix. Be sure to address the following in your matrix:
•
Provide names, titles, dates, brief descriptions of important events, and other details as necessary.
•
Note the details of key political, socioeconomic, technological, artistic, musical, architectural, philosophical, and literary developments for each civilization listed in the table, which were evidenced in the humanities.
Be sure to properly cite the sources that you use in completing this matrix.
.
Early childhood teachers need to stay connected to what is occurring.docxjoellemurphey
Early childhood teachers need to stay connected to what is occurring in the community outside the classroom politically and economically because these factors will influence their classroom. Items of recent debate include social and emotional development, as well as technology in the early childhood classrooms.
For this assignment, take on the role of an early childhood teacher. The principal of your school has placed you on a committee to create a 12-15 slide digital presentation to inform families about current trends in early childhood education. Explain the trends and discuss whether they are developmentally appropriate for young children. In addition, include a description of the effect this trend has on student outcomes. The presentation should discuss early childhood trends and influences on the early childhood classroom in the following areas:
Political (legislative and regulatory)
Economic
Social-emotional
Technological
One trend of choice (e.g., assessment, physical fitness, play in the classroom, emergent curriculums, recess, common core)
Include a title slide, reference slide, and speaker’s notes in your digital presentation.
Use 3-5 scholarly resources to support your research
.
Early and Middle Adulthood PaperPrepare a 1,050- to 1,400-word.docxjoellemurphey
Early and Middle Adulthood Paper
Prepare
a 1,050- to 1,400-word paper in which you examine the psychological adjustments to aging and lifestyle that occur within individuals during early and middle adulthood. Be sure to include the following:
Discuss how social and intimate relationships evolve and change during early and middle adulthood.
Identify various role changes that occur during early and middle adulthood.
Examine the immediate and future impact of healthy and unhealthy habits practiced during early and middle adulthood.
Use
a minimum of two peer-reviewed sources.
.
Earlier this semester, you participated in a class discussion about .docxjoellemurphey
Earlier this semester, you participated in a class discussion about the character of Bath de Chaucer's wife. You are aware of the complexity of her as a resourceful, cunning, open and ambitious woman. For this essay, I would like you to write a comparison / contrast essay in which you discuss the Wife of Bath as you compare or contrast one or more of these three well-known modern American women: Beyoncé Lil 'Kim, and / or Lady Gaga.
Think beyond and below cliches and perceptions. The comparison should not be disrespectful to these modern iconic women. Obviously, times have changed, and I am in no way suggesting that these modern women share all or even some of the qualities of the Wife of Bath, aside from her drive for independence, sovereignty, and success.
When developing the comparisons and contrasts of it, you should use AT LEAST THREE SOURCES to gather information and knowledge to support the claims and interpretations of it. These sources should be cited in the text and on a works cited page using a precise MLA documentation style.
You will write one essay of 500 - 600 words for this paper . This essay must be formatted in MLA Paper form.
Here is the reading about The character of Bath de Chaucer’s life
From The Canterbury Tales:
General Prologue
Here bygynneth the Book of the Tales of Caunterbury
Whan that Aprill, with his shoures soote
The droghte of March hath perced to the root
And he bathed every veyne in swich liquor,
Of which virtue begotten is the flour;
5 Whan Zephirus eek with his sweete breeth
Inspired hath in every holt and heeth
The tender croppes, and the yonge ring
Hath in the Ram his halfe cours yronne,
And smale foweles maken melodye,
10 That slepen al the nyght with open eye-
(So priketh hem Nature in hir corages);
Thanne longen folk to goon on pilgrimages
And palmeres for to seken straunge strondes
To ferne halwes, kowthe in probry londes;
15 And specially from every shires ende
Of Engelond, to Caunterbury they wende,
The hooly blisful martir for to seke
That hem hath holpen, whan that they were seeke.
Bifil that in that seson, on a day,
20 In Southwerk at the Tabard as I lay
Redy to wenden on my pilgrymage
To Caunterbury with ful devout courage,
At nyght was come into that hostelrye
Wel nyne and twenty in a compaignye
25 Of Sondry folk, by aventure yfalle
In felaweshipe, and pilgrimes were they alle,
That toward Caunterbury wolden ryde.
The rooms and the stables weren wyde,
And wel we weren esed att beste;
30 And shortly, whan the sonne was to rest,
So hadde I spoken with hem everichon
That I was of hir felaweshipe anon,
And made forward erly for to ryse
To take our wey, ther as I yow devyse.
35 But nathelees, whil I have tyme and space,
Er that I ferther in this tale pace,
Me thynketh it acordaunt to resoun
To tel yow to the conditio.
This document provides instruction on writing comparison and contrast essays. It discusses balancing points between topics, writing a strong thesis statement using a T-diagram, and following an effective structure. Specifically, it recommends determining if an essay will focus more on similarities or differences, and using a T-diagram to outline key points for each topic. This allows writers to logically organize their ideas and ensure equal coverage of topics in their essays. Effective introductions and conclusions are also important.
Earlean, please write these notes for me. October 01, 20181. My .docxjoellemurphey
Earlean, please write these notes for me. October 01, 2018
1. My name is Brittney, this is my first day in group, I am from Lake worth, my age is 25, Originally from California, I have been clean 83 days. She grew up Catholic. She is pregnant with her first child 6 weeks states she wants to be a good mother, she went to doctor today it is confirmed. A BOY
Brittney’s does not believe in God she believes the Universe
Tell me one positive thing about yourself? I am FUNNY.
2. Tessa, I am 20 years old, I am from Missouri, I have been clean 8 months, and I’m going home Friday. I have a sister that just relapsed 4 days a go with an overdose, beaten etc. and I am showing her tough love. I got some news that my best friend in New York overdose, so my feelings have been going back and forth. And I am supposed to be the strong one. But I’m OK.
I am Out Going and Determined to make it.
Tessa has a Buddha faith says karma is a bitch
Tessa wants to co to college in January, she stated I am the SIT, says her self esteem is high.
3. Megan, I am 20-year-old from Colorado, Arizona… I am grateful and kind.
Megan believes FLDS Mormon latter-day saints, believes in God, he is loving and caring.
4. Elizabeth, I am 19 years old from St. Louis, Missouri, I was adopted, and I am very CARING. She explained to me before group she was given her meds Seroquel, and she has not had it for 4 days, so she was in and out asleep, but when I called her name she did respond politely. Believes in God
5. She is concerned about going to jail, would like to go to culinary school but this will be her first year.
6. Julian, I am 31 years old I am a Hard Worker I work two jobs Java Juice, and Brews.
Believes in God, and she prays every morning, se shared when she relapsed she did not pray that morning. July 28.
7. Dawn, originally from New York, I have been married a long time with 3children I been living in Florida. My family does not know I have another side I am like a camelina to my family my entire life they had no idea I was smoking crack an that I am a Junky I have lost everything facing divorce
Dawn was raised Catholic and she believes in God. And she would love forgiveness from husband and children, wants a chance to be understood. Teresa stated understand yourself and be accountable to you first.
When Dawn shared her story, it detoured the SPIRITUALITY meeting because Tessa gave the first feedback. And Codependency, cross addictions, service work, was discussed between them. The director Teresa interjected and explained the meaning you are replacing one thing with something else like, going to the GYM, SHOPPING, RELATIONSHIPS, any distraction to get you outside of yourself, or to get validated by someone else. You are hurting you to help someone else.
Breaktime
.
eam Assignment 4 Teaming Across Distance and Culture..docxjoellemurphey
eam Assignment 4: Teaming Across Distance and Culture.
1. What are the major effects of the physical separation of group members? How can distance, in some cases, be beneficial to groups and teams?
2. What other areas of organizational behavior or design are impacted by information technology, and what are the implications for organizational change?
3. Brainstorm some ways to “redesign” your office space (or an office space you have previously worked in) on paper using virtual or flexible space, or flexible furniture. How would this redesign enhance successful teamwork?
4. What are some of the ways that cross-cultural teams are distinguished from other types of teams? What are some of the benefits and difficulties of building a cross-cultural team?
250 Words
.
ead the following articleMother Tongue Maintenance Among North .docxjoellemurphey
ead the following article:
Mother Tongue Maintenance Among North American Ethnic Groups
, Robert W. Shrauf
Address the following:
What are some of the factors behind both the loss and persistence of native languages?
Does losing or maintaining one's native language have any impact on one's degree of acculturation or assimilation?
.
eActivityGo to the United States Equal Employment Oppo.docxjoellemurphey
eActivity
Go to the United States Equal Employment Opportunity Commission’s website to review discrimination types, located at
http://www.eeoc.gov/laws/types
. Be prepared to discuss.
Employment Relationship and Discrimination" Please respond to the following:
From the e-activity, visit the EEOC website link provided and select any three (3) types of discrimination and discuss. What key laws are applicable to the discrimination types you selected?
.
Each year on or around June 15, communities and municipalities aroun.docxjoellemurphey
Each year on or around June 15, communities and municipalities around the world plan activities and programs to recognize World Elder Abuse Awareness Day, a day set aside to spread awareness of the abuse of the elderly (Center of Excellence on Elder Abuse & Neglect, 2013). The abuse of older adults is a growing concern and statistics suggest that the number of elders experiencing abuse is an alarmingly high number. Research suggests that close to half the people diagnosed with dementia experience some form of abuse (Cooper, C., Selwood, A., Blanchard, M., Walker, Z., Blizard, R., & Livingston, G., 2009; Wiglesworth, A., Mosqueda, L., Mulnard, R., Liao, S., Gibbs, L., & Fitzgerald, W., 2010, as cited on http://www.ncea.aoa.gov/Library/Data/index.aspx). Elder abuse takes on many forms and can include physical, emotional, psychological, and economic abuse. The legendary American actor, Mickey Rooney, spoke to the United States Senate, describing his own experiences of pain and neglect at the hands of his stepson, asking legislators to take seriously the abuse of the elderly.
Respond to colleagues by suggesting alternative strategies. The Original posts are contained in the attachement.
Support your responses with specific references to the Learning Resources. Be sure to provide full APA citations for your references.
.
A Visual Guide to 1 Samuel | A Tale of Two HeartsSteve Thomason
These slides walk through the story of 1 Samuel. Samuel is the last judge of Israel. The people reject God and want a king. Saul is anointed as the first king, but he is not a good king. David, the shepherd boy is anointed and Saul is envious of him. David shows honor while Saul continues to self destruct.
A Free 200-Page eBook ~ Brain and Mind Exercise.pptxOH TEIK BIN
(A Free eBook comprising 3 Sets of Presentation of a selection of Puzzles, Brain Teasers and Thinking Problems to exercise both the mind and the Right and Left Brain. To help keep the mind and brain fit and healthy. Good for both the young and old alike.
Answers are given for all the puzzles and problems.)
With Metta,
Bro. Oh Teik Bin 🙏🤓🤔🥰
Temple of Asclepius in Thrace. Excavation resultsKrassimira Luka
The temple and the sanctuary around were dedicated to Asklepios Zmidrenus. This name has been known since 1875 when an inscription dedicated to him was discovered in Rome. The inscription is dated in 227 AD and was left by soldiers originating from the city of Philippopolis (modern Plovdiv).
THE SACRIFICE HOW PRO-PALESTINE PROTESTS STUDENTS ARE SACRIFICING TO CHANGE T...indexPub
The recent surge in pro-Palestine student activism has prompted significant responses from universities, ranging from negotiations and divestment commitments to increased transparency about investments in companies supporting the war on Gaza. This activism has led to the cessation of student encampments but also highlighted the substantial sacrifices made by students, including academic disruptions and personal risks. The primary drivers of these protests are poor university administration, lack of transparency, and inadequate communication between officials and students. This study examines the profound emotional, psychological, and professional impacts on students engaged in pro-Palestine protests, focusing on Generation Z's (Gen-Z) activism dynamics. This paper explores the significant sacrifices made by these students and even the professors supporting the pro-Palestine movement, with a focus on recent global movements. Through an in-depth analysis of printed and electronic media, the study examines the impacts of these sacrifices on the academic and personal lives of those involved. The paper highlights examples from various universities, demonstrating student activism's long-term and short-term effects, including disciplinary actions, social backlash, and career implications. The researchers also explore the broader implications of student sacrifices. The findings reveal that these sacrifices are driven by a profound commitment to justice and human rights, and are influenced by the increasing availability of information, peer interactions, and personal convictions. The study also discusses the broader implications of this activism, comparing it to historical precedents and assessing its potential to influence policy and public opinion. The emotional and psychological toll on student activists is significant, but their sense of purpose and community support mitigates some of these challenges. However, the researchers call for acknowledging the broader Impact of these sacrifices on the future global movement of FreePalestine.
🔥🔥🔥🔥🔥🔥🔥🔥🔥
إضغ بين إيديكم من أقوى الملازم التي صممتها
ملزمة تشريح الجهاز الهيكلي (نظري 3)
💀💀💀💀💀💀💀💀💀💀
تتميز هذهِ الملزمة بعِدة مُميزات :
1- مُترجمة ترجمة تُناسب جميع المستويات
2- تحتوي على 78 رسم توضيحي لكل كلمة موجودة بالملزمة (لكل كلمة !!!!)
#فهم_ماكو_درخ
3- دقة الكتابة والصور عالية جداً جداً جداً
4- هُنالك بعض المعلومات تم توضيحها بشكل تفصيلي جداً (تُعتبر لدى الطالب أو الطالبة بإنها معلومات مُبهمة ومع ذلك تم توضيح هذهِ المعلومات المُبهمة بشكل تفصيلي جداً
5- الملزمة تشرح نفسها ب نفسها بس تكلك تعال اقراني
6- تحتوي الملزمة في اول سلايد على خارطة تتضمن جميع تفرُعات معلومات الجهاز الهيكلي المذكورة في هذهِ الملزمة
واخيراً هذهِ الملزمة حلالٌ عليكم وإتمنى منكم إن تدعولي بالخير والصحة والعافية فقط
كل التوفيق زملائي وزميلاتي ، زميلكم محمد الذهبي 💊💊
🔥🔥🔥🔥🔥🔥🔥🔥🔥
This presentation was provided by Racquel Jemison, Ph.D., Christina MacLaughlin, Ph.D., and Paulomi Majumder. Ph.D., all of the American Chemical Society, for the second session of NISO's 2024 Training Series "DEIA in the Scholarly Landscape." Session Two: 'Expanding Pathways to Publishing Careers,' was held June 13, 2024.
Level 3 NCEA - NZ: A Nation In the Making 1872 - 1900 SML.pptHenry Hollis
The History of NZ 1870-1900.
Making of a Nation.
From the NZ Wars to Liberals,
Richard Seddon, George Grey,
Social Laboratory, New Zealand,
Confiscations, Kotahitanga, Kingitanga, Parliament, Suffrage, Repudiation, Economic Change, Agriculture, Gold Mining, Timber, Flax, Sheep, Dairying,
This presentation was provided by Rebecca Benner, Ph.D., of the American Society of Anesthesiologists, for the second session of NISO's 2024 Training Series "DEIA in the Scholarly Landscape." Session Two: 'Expanding Pathways to Publishing Careers,' was held June 13, 2024.
Philippine Edukasyong Pantahanan at Pangkabuhayan (EPP) CurriculumMJDuyan
(𝐓𝐋𝐄 𝟏𝟎𝟎) (𝐋𝐞𝐬𝐬𝐨𝐧 𝟏)-𝐏𝐫𝐞𝐥𝐢𝐦𝐬
𝐃𝐢𝐬𝐜𝐮𝐬𝐬 𝐭𝐡𝐞 𝐄𝐏𝐏 𝐂𝐮𝐫𝐫𝐢𝐜𝐮𝐥𝐮𝐦 𝐢𝐧 𝐭𝐡𝐞 𝐏𝐡𝐢𝐥𝐢𝐩𝐩𝐢𝐧𝐞𝐬:
- Understand the goals and objectives of the Edukasyong Pantahanan at Pangkabuhayan (EPP) curriculum, recognizing its importance in fostering practical life skills and values among students. Students will also be able to identify the key components and subjects covered, such as agriculture, home economics, industrial arts, and information and communication technology.
𝐄𝐱𝐩𝐥𝐚𝐢𝐧 𝐭𝐡𝐞 𝐍𝐚𝐭𝐮𝐫𝐞 𝐚𝐧𝐝 𝐒𝐜𝐨𝐩𝐞 𝐨𝐟 𝐚𝐧 𝐄𝐧𝐭𝐫𝐞𝐩𝐫𝐞𝐧𝐞𝐮𝐫:
-Define entrepreneurship, distinguishing it from general business activities by emphasizing its focus on innovation, risk-taking, and value creation. Students will describe the characteristics and traits of successful entrepreneurs, including their roles and responsibilities, and discuss the broader economic and social impacts of entrepreneurial activities on both local and global scales.
Role of pro-brain-derived neurotrophic factor (proBDNF)to ma.docx
1. Role of pro-brain-derived neurotrophic factor (proBDNF)
to mature BDNF conversion in activity-dependent
competition at developing neuromuscular synapses
H. Shawn Jea,b,c,1, Feng Yanga,b,d,1, Yuanyuan Jia,e, Guhan
Nagappana,e, Barbara L. Hempsteadf, and Bai Lua,b,e,2
aSection on Neural Development and Plasticity, National
Institute of Child Health and Human Development, Bethesda,
MD 20892-3714; bGenes, Cognition,
and Psychosis Program (GCAP), National Institute of Mental
Health, Bethesda, MD 20892-3714; cProgram in Neuroscience
and Behavioral Disorders, Duke–
National University of Singapore (Duke-NUS) Graduate
Medical School, 169857, Singapore; dLieber Institute for Brain
Development, The Johns Hopkins
University Medical Campus, Baltimore, MD 21205; eR&D
China, GlaxoSmithKline, Pudong, Shanghai 201203, China; and
fDivision of Hematology, Department
of Medicine, Weill Medical College, Cornell University, New
York, NY 10021
Edited* by Richard L. Huganir, The Johns Hopkins University
School of Medicine, Baltimore, MD, and approved August 20,
2012 (received for review May
10, 2012)
Formation of specific neuronal connections often involves
compe-
tition between adjacent axons, leading to stabilization of the
active
terminal, while retraction of the less active ones. The
underlying
2. molecular mechanisms remain unknown. We show that activity-
dependent conversion of pro–brain-derived neurotrophic factor
(proBDNF) to mature (m)BDNF mediates synaptic competition.
Stim-
ulation of motoneurons triggers proteolytic conversion of
proBDNF
to mBDNF at nerve terminals. In Xenopus nerve–muscle
cocultures,
in which two motoneurons innervate one myocyte, proBDNF-
p75NTR signaling promotes retraction of the less active
terminal,
whereas mBDNF–tyrosine-related kinase B (TrkB) p75NTR
(p75 neu-
rotrophin receptor) facilitates stabilization of the active one.
Thus,
proBDNF and mBDNF may serve as potential “punishment” and
“re-
ward” signals for inactive and active terminals, respectively,
and
activity-dependent conversion of proBDNF to mBDNF may
regulate
synapse elimination.
neuromuscular junction | pro-neurotrophin | synapse
competition
The nervous system responds to experience by altering
thenumber and strength of synaptic connections (1). Activity-
dependent synaptic competition, a general process seen in many
parts of the developing nervous system, plays a critical role in
shaping patterns of neuronal connections (2–7). At the neuro-
muscular junction (NMJ), for example, multiple axons compete
for the same postsynaptic muscle cell during early postnatal life
until all but one is eliminated (8–10). Extensive experimental
data support the view that the more active terminal or “cartel”
gets stabilized, whereas less active ones withdraw, resulting in
3. canonical elimination of polyneuronal innervation (8, 11). It is
generally believed that this synaptic competition is mediated by
a “punishment” or “elimination” signal, produced by the post-
synaptic cell, that causes the retraction of the inactive
terminals,
as well as a “protective” or “reward” signal that stabilizes the
active terminal (10–12). Despite significant efforts over
decades,
the identity of the punishment or reward signals remains un-
known (4, 13). This is due at least in part, to the experimental
difficulties in manipulating gene expression selectively in one
of
the competing axons.
Brain-derived neurotrophic factor (BDNF) has been recog-
nized as a key regulator of synapse development and plasticity
(14, 15). This is because BDNF is the only neurotrophin in-
disputably secreted in an activity-dependent manner (15). In-
deed, activity-dependent secretion of BDNF has been shown to
be critical for hippocampus-dependent memory in human (16,
17). Like all neurotrophins, BDNF is initially synthesized as
a precursor (proBDNF), which is subsequently cleaved to
generate mature (m)BDNF. proBDNF interacts preferentially
with the pan-neurotrophin receptor p75 (p75NTR), whereas
mBDNF selectively binds and activates the receptor tyrosine
kinase TrkB (18, 19). Cumulative evidence supports a “yin-yang
hypothesis,” in which pro- and mBDNF elicit opposite
biological
effects by activating two distinct receptor systems (20). For ex-
ample, proBDNF, if not processed, promotes long-term de-
pression (LTD) through the activation of p75NTR in the
hippocampus (21, 22). In contrast, mBDNF-TrkB signaling is
essential for the early phase of long-term potentiation (E-LTP)
(23–25). Moreover, recent studies indicate that a significant
proportion of BDNF in the brain is secreted in the proform (26–
4. 28), and extracellular conversion of proBDNF to mBDNF by the
tissue plasminogen activator (tPA)/plasmin protease system is
critical for late-phase LTP (29). The expression of proBDNF
and
p75NTR in rodents is developmentally regulated, with the
highest
levels in the first and second postnatal week, correlating well
with
the timings of synapse formation (28). Therefore, proteolytic
cleavage of proBDNF represents an important mechanism by
which the opposing cellular actions of proBDNF and mBDNF
may be regulated (20).
The opposing nature of proBDNF and mBDNF prompted us to
hypothesize that proBDNF and mBDNF might serve as punish-
ment and reward signals, respectively, during synaptic
competition
at the developing NMJs. In this study, we developed a triplet
sys-
tem that allows alteration of gene function in one of two
distinctly
labeled axons that innervate a single, unlabeled myocyte. Our
study suggests that proBDNF serves as a general “punishment
signal” that causes p75NTR-expressing motor terminals to
retract,
whereas at the active terminal, secretion/activation of
extracellular
protease(s) converts proBDNF to mBDNF, which serves as a re-
ward signal to stabilize the terminal.
Results
Activity-Dependent Synaptic Competition in Xenopus Nerve–
Muscle
Cocultures. Xenopus nerve–muscle coculture system was used
to
study the activity-dependent synaptic competition. We
5. developed
a cell-culture system, in which an unlabeled myocyte was inner-
vated by two spinal neurons (one labeled in green and the other
in red, respectively; Fig. 1A). This was accomplished by
injecting
either FITC-dextran (green) or rhodamine-dextran (red) into a
single dorsal animal blastomere at the 8- or 16-cell stage and
mixing the neural tubes from embryos that were injected with
two different fluorophores to prepare dissociated nerve–muscle
Author contributions: H.S.J., F.Y., and B.L. designed research;
H.S.J., F.Y., Y.J., and G.N.
performed research; B.L.H. contributed new reagents/analytic
tools; H.S.J., F.Y., and Y.J.
analyzed data; and H.S.J., F.Y., and B.L. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1H.S.J. and F.Y. contributed equally to this work.
2To whom correspondence should be addressed. E-mail:
[email protected]
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1207767109/-/DCSupplemental.
15924–15929 | PNAS | September 25, 2012 | vol. 109 | no. 39
www.pnas.org/cgi/doi/10.1073/pnas.1207767109
mailto:[email protected]
http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.120776710
9/-/DCSupplemental
http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.120776710
9/-/DCSupplemental
www.pnas.org/cgi/doi/10.1073/pnas.1207767109
6. cocultures (30). Instead of employing electrical stimulation
using
a glass electrode, which might cause mechanical damages on
neurons, we stimulated one of the spinal neurons by local pho-
tolysis of caged glutamate (MNI-glutamate; 50 μM) using
a multiphoton microscope (31). Photo-uncaging of caged gluta-
mate at a neuronal soma induced a marked potentiation of
synaptic transmission, which lasted for more than 60 min (Fig.
S1A). This synaptic potentiation was completely blocked by the
sodium channel blocker tetrodotoxin (TTX) (1 μM), demon-
strating the requirement of action potentials (Fig. S1A). Fur-
thermore, synaptic potentiation was only observed when a laser
beam was applied within 25 μm from the neuronal cell body,
suggesting that the photo-uncaging occurred locally (Fig. S1B).
A brief episode (250 ms) of photolysis of MNI-glutamate was
applied to one of the two neurons, and the resulting morpho-
logical changes in synaptic terminals from both neurons, which
were innervating a single myocyte, were monitored by dual-
color,
time-lapse confocal imaging. Upon stimulation of a red neuron,
the axon terminal of the unstimulated neuron (green terminal)
gradually withdrew from the previously innervated muscle,
whereas the terminal of the stimulated neuron (red terminal)
remained stable and occasionally extended (Fig. 1B and Movie
S1). Conversely, stimulation of a green neuron triggered the
retraction of the red terminal (Fig. S2). Because stimulation of
one neuron always resulted in the retraction of the unstimulated
neuron, regardless of whether it was red or green, we randomly
stimulated neurons in subsequent experiments based on the
convenience of photo-uncaging. We performed 11 experiments
involving preferential photolysis of one neuron. In all 11 cases,
the axon terminals of the unstimulated neurons retracted to
varying degrees. In six cases, axon terminals of stimulated neu-
7. rons showed slight expansion or elongation (Fig. 1 B and C).
These results suggest that a postsynaptically derived local pun-
ishment signal may trigger synaptic retraction.
Activity-Dependent Cleavage of Secreted proBDNF at NMJ.
Given
that proBDNF and mBDNF could elicit opposite effects, we
hypothesized that proBDNF and mBDNF might serve as pun-
ishment and reward signals, respectively. Furthermore, we
hypoth-
esized that the proteolytic conversion of proBDNF to mBDNF
might be essential for synaptic competition and elimination at
the developing NMJs. First, we tested whether neuronal activity
could activate proteases to process proBDNF to mBDNF using a
fluorogenic proteolytic beacon assay. The fluorogenic beacon
consisted of synthetic peptides, harboring the propeptide cleav-
age sequences (MSMRVRR↓HSD) within proBDNF, and these
synthetic peptides were flanked by a fluorophore at the N
terminus
and a quencher at its C terminus (Fig. 2A). Small size peptides
allowed both a fluorophore and a quencher within proximity,
thereby quenching the fluorescence via fluorescence resonance
energy transfer (FRET). Upon cleavage of peptides by a pro-
tease(s), which specifically recognizes the proBDNF cleavage
sequence, the fluorogenic beacon would emit the fluorescence
signal because of the separation of a fluorophore from a
quencher
(Fig. 2A). In addition, we immobilized the fluorogenic beacon
to
polystyrene beads to minimize diffusion in culture medium.
Next, we investigated whether stimulation of neurons could
activate proteases at the nerve terminals. The fluorogenic
beacons
were placed on the axonal process or termini of spinal neurons,
and neurons were subsequently stimulated either by an
8. electrical
stimulation or by photo-uncaging of MNI-glutamate (Fig. 2B,
bright field). After stimulation, the fluorescence intensity of the
beads on the axon terminals or axonal processes (e.g., yellow
arrows in Fig. 2B) greatly increased (Fig. 2 B and C),
suggesting
cleavage of the peptide by proteases secreted from the axon. In
contrast, no fluorescence change was observed from beads,
which
were not in contact with the axonal process or termini (Fig. 2B,
“free beads,” white arrow). Time-lapse imaging indicated that
the
increase in fluorescence occurred relatively quickly: the first
surge
came within minutes, and the florescence increase reached its
peak
within 20 min (Fig. 2C). Pretreatment with a mixture of
protease
inhibitors prevented the increase in fluorescence from beads on
axon terminals (Fig. 2D). Remarkably, inhibitors for tPA and
furin, the extracellular and intracellular proteases known to
cleave
proBDNF in the brain (29, 32), did not inhibit the fluorescence
increase (Fig. 2D). In contrast, matrix metalloprotease (MMP)
inhibitors successfully blocked the stimulus-induced increase in
fluorescence. Further analyses indicated that MMP3 and MMP9,
but not MMP2, MMP8, or MMP13, could increase fluorescence
signal on the fluorogenic beacons (Fig. 2D). Finally, when the
beacon-containing beads were placed on muscle cells,
stimulation
of muscle cells did not cause an increase in fluorescence (Fig.
2D),
N
N
10. Elongation Retraction
M
er
ge
d
30 60 90 1200
Fig. 1. Activity-dependent synaptic competition in culture. (A)
Schematic
diagram (Left) and confocal image (Right) showing a triplet in
which
a spherical myocyte (M) (indicated by white dotted lines) is
innervated by
two spinal neurons (N), one labeled with FITC (green) and one
labeled in
rhodamine (red), in nerve–muscle coculture. (B) Time course of
synaptic
competition. A higher magnification of A is shown. Stimulation
of the red
neuron, by photo-uncaging of MNI-glutamate in the soma area
using a two-
photon laser, caused the unstimulated (green) axon terminal to
retract from
the synaptic target (indicated by a yellow arrow, upper row). In
contrast,
the axon terminal (red) from the stimulated neuron did not
retract, but
elongated a little (white arrows, middle row). The phase and
two-color
fluorescence images of the triplet at multiple time points (lower
row). (Scale
bar: 10 μm.) The phase (Bottom) and two-color fluorescence
11. images (Top
and Middle) of the triplet at “0” and “120” min are shown. (C)
Quantifi-
cation of axonal retraction and elongation measured 120 min
after stimu-
lation of one of the neurons in the triplets. *P < 0.01.
Je et al. PNAS | September 25, 2012 | vol. 109 | no. 39 | 15925
N
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http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.120776710
9/-
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http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.120776710
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http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.120776710
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http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.120776710
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http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.120776710
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F2
indicating that muscle cells do not secrete proteases that could
process proBDNF.
We further examined whether postsynaptic muscle cells se-
crete proBDNF upon stimulation. Because of unavailability of
a proBDNF-specific ELISA and limited number of muscle cells
in the Xenopus cocultures (<1,000 myocytes), it was not
feasible
to measure proBDNF secretion from muscle cells using existing
biochemical techniques. Thus, we used cell surface immuno-
staining to measure proBDNF secretion, given that proBDNF is
positively charged at physiological pH and could be associated
with a negatively charged cell membrane upon secretion (27,
33).
The muscle cell cultures were depolarized by high-K+ treatment
(50 mM) for 5 min, fixed, and processed for cell surface immu-
nofluorescence staining under membrane impermeable condi-
tions. A proBDNF-specific monoclonal antibody was used for
cell-
surface staining of secreted proBDNF (27). Although proBDNF
was barely detectable on the muscle cell surface at rest
(control),
the proBDNF immunoreactivity increased dramatically (88%)
upon depolarization (Fig. S3A). This increase was even more
pronounced when extracellular cleavage of proBDNF was
13. blocked
by general MMPs inhibitors (indicated as MMP-In. in Fig.
S3A).
To measure the protease-mediated conversion of endogenous
proBDNF to mBDNF at the neuromuscular synapses, we per-
formed cell-surface immunostaining using a specific antibody
against mBDNF. We applied glutamate, instead of high K+, to
selectively depolarize neurons, because muscle cells do not ex-
press glutamate receptors (34). After neuronal depolarization,
cultures were subsequently fixed and processed for surface im-
munostaining using an mBDNF-specific antibody (27). In
control
conditions, there was no mBDNF staining (Fig. S3B, Left).
Upon
glutamate application (10 mM; 5 min), we observed a dramatic,
threefold increase in surface mBDNF staining at the synaptic
junction (Fig. S3B, Center). This increase in mBDNF immuno-
reactivity was observed only in myocytes that were innervated
by a spinal neuron but not in the neighboring noninnervated
myocytes (Fig. S3B, Center). This suggested that the release
and/
or activation of proteases at the nerve terminal converted
proBDNF to mBDNF at the active synaptic terminals. Further-
more, when the cultures were pretreated with the MMP inhib-
itors, the glutamate-induced increase in mBDNF staining was
completely abolished (Fig. S3B, Right). Taken together, these
results supported the notion that activity-dependent conversion
of proBDNF to mBDNF occurred at the developing Xenopus
NMJ in situ.
Synaptic Stabilization During Competition Mediated by
mBDNF/TrkB.
Previously, we have shown that exogenous proBDNF triggers
synaptic depression and subsequent retraction of axon terminal
14. through p75NTR in Xenopus neuromuscular synapses (35).
Based
on robust secretion of proBDNF upon synaptic depolarization,
we reasoned that active terminals might cleave proBDNF to
mBDNF, which protects these active terminals from proBDNF-
mediated synaptic depression and retraction. To test this, we
knocked down endogenous TrkB in one of neurons in our triplet
system using a morpholino (Fig. S4A; also see ref. 36). Control
experiments indicated that the expression of the TrkB morpho-
lino in presynaptic neurons blocked the mBDNF-mediated syn-
aptic potentiation (Fig. S4 B and C). When the TrkB morpholino
was selectively expressed in one of the spinal neurons in our
triplet system, stimulation of the TrkB morpholino-expressing
neuron (green) resulted in retraction of both green and red
terminals (Fig. 3A, yellow arrows). Quantitative analysis of 10
triplets indicated that both stimulated and unstimulated termi-
nals retracted ∼12 μm within 120 min (Fig. 3C).
Next, we reasoned that if the activation of TrkB by mBDNF was
critical to prevent synaptic retraction, supply of exogenous
BDNF
would prevent synaptic competition. Indeed, in a nerve–muscle
culture treated with mBDNF (25 ng/mL) for 2 h, stimulation of
the
red neuron did not cause retraction of the green terminal (Fig.
3B
and Movie S2). Moreover, application of exogenous mBDNF
even
elicited axonal elongation from the stimulated neurons (Fig. 3 B
and C, white arrowheads). These results suggest that mBDNF
plays an active role in preventing synaptic retraction of the
active
terminals through TrkB signaling during synaptic competition.
Synaptic Retraction During Competition Mediated by
proBDNF/p75NTR.
15. To determine whether this activity-based synaptic retraction
was
mediated through proBDNF/p75NTR signaling, we knocked
down
endogenous p75NTR by using FITC-conjugated p75NTR siRNA
that
were targeted to all Xenopus p75NTR isoforms (p75NTRa and
p75NTRb) (37). The p75NTR siRNA was introduced into a
single
neuron in our triplet system using embryo-injection techniques.
A B
C D
ROX-MSM RVRR /HSD-QXL610
Fluorophore Quencher
Proteolytic cleavage
Emit fluorescence
proBDNF cleavage sequences
0 min 6 min
Bright field Before stim. After stim.
0.8
1
1.2
1.4
16. 1.6
1.8
0 5 10 15 20 25 30 min
N
or
m
al
iz
ed
fl
uo
re
sc
en
ce
in
te
ns
ity
(F
/F
o)
21. P
3
In
.
M
M
P
8
In
.
M
M
P
9
In
.
Fig. 2. Activity-dependent cleavage of proBDNF
detected by fluorogenic probes. (A) Schematic dia-
gram depicting the design of a fluorogenic in-
dicator of proBDNF cleavage. A peptide containing
the proBDNF cleavage site was placed between
a fluorophore and a quencher. Upon proteolytic
cleavage, the quencher was dissociated from the
fluorophore, leading to emission of fluorescence.
(B) Sample images of fluorogenic probes in phase
(Left) and fluorescence (Right) before and after
neuronal stimulation. Polystyrene beads soaked
with fluorogenic probes were positioned on (yellow
22. arrows) or near (white arrows) an axonal process
(indicated by white dotted lines), respectively, by
micromanipulation. A neuronal cell body was
stimulated by photolysis of caged-glutamate (MNI-
glutamate) with a UV laser. Note that after stimu-
lation, only beads on, but not near, the axonal
processes showed an increase in fluorescence. (C)
Time course of cleavage-dependent increase in
fluorescence intensity upon stimulation of spinal
neurons. Fluorescence intensities measured on
contacted beads or free beads were normalized to
that at “0” time point and presented as ratios (F/F0)
over time. (D) Quantification of relative fluorescence intensities
in beads 30 min after stimulation of neurons under various
conditions. Concentrations
of protease inhibitors (In.): general protease inhibitor mixture,
50 μM; pan MMP inhibitor, 60 μM; tPA inhibitor, 10 μM; furin
inhibitor, 40 μM; MMP2 inhibitor,
60 μM; MMP3 inhibitor, 50 nM; MMP8 inhibitor, 20 nM;
MMP9 inhibitor, 50 μM; MMP13 inhibitor, 80 nM. B, beads; M,
muscle cell; N, neuron.
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24. period
(Fig. 4A). In seven triplets, four showed no retraction, whereas
the
other three showed reduced retraction of the unstimulated termi-
nals after 2hrs (Fig. 4 A and C). Furthermore, an axon
expressing
scrambled siRNA still retracted when the competing axon was
stimulated (Fig. S5). Because p75NTR siRNA and p75NTR mor-
pholino target different regions of p75NTR, this experiment
con-
firmed that blockade of p75NTR signaling abolished synaptic
re-
traction. Taken together, these data support the hypothesis that
synaptic retraction was mediated by presynaptic p75NTR
signaling
during synaptic competition.
The results above, together with the finding that neuronal
stimulation triggered protease secretion/activation at nerve ter-
minals (Fig. 2), prompted us to speculate that active terminals
were spared from synaptic retraction because of proteolytic
cleavage of proBDNF. If this were true, then blockade of
proBDNF cleavage would trigger retraction of all axon
terminals
during synaptic competition. To test this hypothesis, we treated
nerve–muscle cocultures with a mixture of protease inhibitors
for
10 min before neuronal stimulation. These inhibitors were able
to
block proteases that could cleave proBDNF (Fig. 2D). Remark-
ably, axon terminals from red and green spinal neurons
retracted
simultaneously from the synaptic site (Movie S3). Quantitative
analysis indicated that both stimulated and unstimulated
terminals
25. retracted ∼10 μm (Fig. 4C). Because MMP inhibitors,
particularly
MMP3/9 inhibitors, blocked activity-induced proBDNF
cleavage
at nerve terminals (Fig. 2D), we further tested whether
inhibition
of MMPs could perturb synapse elimination. Indeed, pretreat-
ment with a combination of both MMP3 and MMP9 inhibitors
triggered synaptic retraction upon stimulation of the competing
axon, indicating that MMP3/9 are the candidate proteases that
convert proBDNF to mBDNF at the active synaptic terminal
(Fig.
S6 and Movie S4). Taken together, these data supported the hy-
pothesis that the activation of presynaptic p75NTR by muscle-
de-
rived proBDNF triggered the retraction of less active axonal
terminals during synaptic competition.
Discussion
For over a half century, activity-dependent synaptic
competition/
elimination has been one of the central issues in developmental
neurobiology. However, the underlying mechanisms remain un-
known. Because the competition depends on activity and occurs
locally, the experimental challenge has been to selectively acti-
vate one of the competing terminals and to perform molecular
manipulation selectively in competing axons (38). In the present
study, we modified the Xenopus embryo-injection protocol, so
that we could reliably obtain triplets, in which an unlabeled
myocyte is innervated by two distinctly labeled axons. Confocal
live-cell imaging and local glutamate uncaging using a multi-
photon laser enabled us to visualize and trigger synaptic re-
traction of individual axons in triplets. To demonstrate activity-
dependent secretion of proBDNF and its conversion to mBDNF,
we performed cell surface immunofluorescence staining on
cultured cells, using specific antibodies against proBDNF and
26. mBDNF. FRET-based fluorogenic probes were used to monitor
the activation of protease activities locally at axonal terminals
in
real time. Moreover, embryo injection techniques were used to
selectively inhibit TrkB or p75NTR in one neuron, but not in
others,
in triplets. By combining these techniques, we provided the evi-
dence for a local and activity-dependent activation of specific
proteases that could convert proBDNF to mBDNF in neuromus-
cular synapses. Given that proBDNF and mBDNF often elicit
opposite biological effects (20, 28), this finding may help us
un-
derstand how proBDNF-to-mBDNF conversion is regulated via
neuronal activity. Moreover, we found that the blockade of
p75NTR signaling attenuated synapse elimination, whereas the
blockade of TrkB signaling, or inhibition of proBDNF cleavage
by metalloproteases, promoted synaptic retraction of both in-
nervated axon terminals in triplets. Taken together, these
findings
suggest a model for synapse elimination in which the activity-
dependent conversion of proBDNF to mBDNF selectively sta-
bilized active terminals, whereas inactive terminals were elimi-
nated in response to proBDNF and subsequent activation of
p75NTR signaling (Fig. S7).
Activity-Dependent proBDNF Cleavage Converts a
“Synaptotoxin” to
a “Synaptotrophin.” Two theories have been proposed to explain
activity-dependent synaptic competition and elimination. The
“synaptotoxin” theory suggests that the postsynaptic cell
produces
destabilizing or “toxic” signals such as proteases that
retrogradely
punish and remove presynaptic terminals. This theory (11) was
based largely on the observation that inhibition of protease ac-
tivity (39, 40), particularly thrombin, by the naturally occurring
27. protease inhibitor nexin I (41, 42), attenuated synapse elimina-
tion. However, specific target(s) of the proteases have not been
identified yet. More importantly, selective protection of the
active
terminal by local secretion of protease inhibitors has not been
demonstrated. It is also unclear what axons are competing for, if
the fate of an axon is dependent only on its intrinsic activity
(11).
An alternative is the “synaptotrophin” hypothesis, which
suggests
that axons compete with each other for a limited supply of
trophic
A
B
C
-10 -5 0 5 10 15 20
Elongation Retraction
unstimulated neuron
stimulated neuron
BDNF (4)
TrkB morpholino (10)
Distance (µm)
unstimulated neuron
stimulated neuron
B
29. tim
.
Tr
K
B
-m
or
p.
Fig. 3. Synaptic stabilization mediated by mBDNF/TrkB. (A)
Down-regula-
tion of TrkB by TrkB-morpholino (TrkB-morp.) led to retraction
of both
stimulated and unstimulated axon terminals. In a triplet system,
stimulation
of a neuron expressing TrkB morpholino (green) resulted in
retraction of the
red axon as well as the green axon. (Scale bar: 10 μm.) (B)
Treatment with
mBDNF prevented activity-dependent synaptic retraction. The
culture was
treated with mBDNF before stimulating the soma of a red
neuron. The
terminals from both red and green neurons remained in the
synaptic site
without any retraction. (Scale bar: 20 μm.) (C) Quantification of
axonal re-
traction and elongation measured 120 min after stimulation of
one of the
neurons in the triplets.
Je et al. PNAS | September 25, 2012 | vol. 109 | no. 39 | 15927
31. fails to explain: (i) how the trophic factor is secreted locally
from
the postsynaptic cell only near the active terminal, but not at the
inactive terminal; and (ii) how the active terminal preferentially
binds, uptakes, or signals the trophic factor, if any. Neither
local
secretion nor preferential signaling has been reported at NMJs
to
support the synaptotrophin hypothesis. Moreover, synaptic elim-
ination is an active process that requires a punishment signal,
ultimately resulting in the loss of all, but one protected terminal
(13). Thus, the synaptotrophin theory fails to explain how
inactive
terminals initiate withdrawal and why the absence of trophic
support leads to synaptic depression and retraction.
Our data support a model that both the punishment and re-
ward signals are originated from the same molecule, namely
BDNF (Fig. S7). In this model, presynaptic activity drives the
secretion of proBDNF from postsynaptic muscle cells, which
serves as a default “punishment signal” to actively retract af-
ferent terminals through p75NTR. mBDNF, on the other hand,
serves as a reward signal for which all terminals compete. A
major conceptual breakthrough to the model is the activity-de-
pendent conversion of proBDNF to mBDNF by active axon
terminals. Locally secreted proteases at the active terminal not
only block the punishment signal (proBDNF), but also generate
a reward signal (mBDNF), which stabilizes the terminal by
activating TrkB. Further work is necessary to substantiate this
model in vivo.
proBDNF-mBDNF As Punishment–Reward Signals in Synapse
Elimination.
Using cultured neurons, computational modeling, and knockout
animals, two recent studies reported mechanisms for synaptic
32. competition and axonal pruning in the sympathetic nervous
system (43, 44). In this system, target-derived NGF, through
TrkA, provides protection and prosurvival of active axons,
which
are further strengthened and stabilized by increasing TrkA
transcription. On the other hand, NGF-induced synthesis and
secretion of BDNF and neurotrophin-4 (NT-4), through
p75NTR,
facilitate axonal degeneration and pruning of the competing
axons (43–45). Although these experiments were largely carried
out in cell culture, the basic premise is quite similar: TrkA
mediates reward, whereas p75NTR mediates punishment. There
are several important differences between the present study and
those published works. First, activity-dependent secretion of
the reward (NGF) or punishment signal (BDNF) in the superior
cervical ganglia (SCG) neurons was not demonstrated. In the
Xenopus neuromuscular system, we showed that BDNF is se-
creted from postsynaptic muscle cells. Thus, the target cell
(myocyte) is an important player in synaptic (as opposed to ax-
onal) competition. Second, it is widely accepted that
motoneurons
express only TrkB, but TrkC and TrkA expression is
controversial.
In addition, negligible levels of NGF are detectable in the neu-
romuscular system (46) and TrkB expression in sympathetic
neurons is extremely low (47). Therefore, application of exces-
sive BDNF, even with low affinity, can only bind to p75NTR,
trig-
gering axonal pruning. Third, and most importantly, in SCG,
two
separate ligands (NGF and BDNF) are needed for reward and
punishment signals, respectively. At the NMJ, the reward and
punishment signals are derived from the same molecule,
depending
on proteolytic cleavage. Our model suggests that the punish-
ment signal (proBDNF) acts on all competing axons to promote
33. elimination, and the active axon is spared because it activates
ex-
tracellular protease(s) that convert the punishment (proBDNF)
signal to a reward (mBDNF) signal.
Several issues require further investigation. First, what is the
identity and source of the protease(s)? Our imaging experiments
(Figs. 2 and 3) point to metalloproteases, particularly MMP3
and
MMP9, as candidates that cleave proBDNF at the active
terminal
during synaptic competition in Xenopus NMJ. Fluorogenic bea-
con experiments suggest that the MMPs are derived from axonal
terminal, but not from muscle cells (Fig. 2D). Indeed, these pro-
teases are expressed in motor neurons and are highly enriched at
the NMJs (48–50). Further work is necessary to identify the
spe-
cific proteases required at mouse NMJs in vivo. Second,
contrary
to our findings, several previous studies have shown that
inhibition
of protease activity attenuates synaptic elimination (39, 40) and
even increases numbers of acetylcholine receptor (AChR) in the
postsynaptic muscle (51, 52). One way to reconcile these two
sets
of data is that inhibition of a protease “X,” the function of
which
is to degrade MMPs, may result in an enhanced conversion of
proBDNF to mBDNF by MMPs, leading to attenuation of syn-
aptic elimination. Moreover, our results suggest that proBDNF
is
derived from postsynaptic muscle cells but not presynaptic
motor
neurons. It remains to be established whether muscle cells are
the only source of proBDNF or whether other cell types, such as
Schwann cells, also produce and secrete proBDNF at the NMJ
34. in
vivo. With the currently available technologies, it is not
possible to
demonstrate the secretion of BDNF (more specifically
proBDNF)
at the NMJs in vivo. An important future experiment is to dem-
onstrate that proBDNF is both secreted and cleaved locally at
the
active terminal but not at the inactive terminal in vivo. Finally,
activation of p75NTR may be a general mechanism for activity-
dependent synapse retraction. Indeed, in sympathetic neurons,
BDNF (possibly proBDNF) acts through p75NTR to suppress
A
B
C
Distance (µm)-5 0 5 10 15 20
p75NTR siRNA(7)
Protease
inhibitors (7)
Elongation Retraction
unstimulated neuron
stimulated neuron
unstimulated neuron
stimulated neuron
p7
5N
36. un
st
im
.
st
im
.
0 30 60 90 120
Fig. 4. Synaptic retraction mediated by proBDNF/p75NTR. (A)
Down-regu-
lation of p75NTR by siRNA prevents activity-dependent
synaptic retraction.
Fluorescence images show a double-innervated myocyte (white
dotted lines)
by a neuron expressing p75NTR siRNAs (green) and a
rhodamine-labeled
control neuron (red). The soma of the red neuron (outside the
field) was
stimulated by photo-uncaging and the axon terminals from both
neurons
were monitored by time-lapse microscopy. (Scale bar: 20 μm.)
Even after
120 min, the axon terminals (white arrow) of both red and green
neurons
remained unchanged at the synaptic site. (B) Time-lapse images
showing
retraction of both stimulated (red) and unstimulated (green)
axons in the
presence of a mixture of protease inhibitors (50 μM). (Scale
bar: 20 μm.) (C)
37. Quantification of axonal retraction and elongation measured 120
min after
stimulation of one of the neurons in the triplets.
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inactive axons, whereas active axons (depolarized by high K+)
of
the same neurons are spared (45). It will be extremely important
to test whether protease-mediated conversion of proBDNF to
mBDNF also contributes to activity-dependent synaptic compe-
tition in the central nervous system (CNS), an example being
ocular dominance formation within the visual cortex.
Experimental Procedures
Full details are contained in SI Experimental Procedures. Use
and care of
animals in this study abided by the guidelines of the
institutional Animal
Care and Use Committee at the National Institutes of Health
NIH.
Embryo Injection and Xenopus Nerve–Muscle Coculture.
Xenopus nerve–mus-
cle cocultures were prepared as described (53). Morpholinos,
siRNAs, or cDNAs
were injected into one of the blastomeres at the 2- to 4-cell or
8- to 16-cell
38. stage as described (53).
Immunocytochemistry, Confocal Microscopy Image Analysis,
and Statistics.
Xenopus nerve–muscle cocultures were immunostained,
maintained, and
analyzed as described previously (35). See SI Experimental
Procedures for
details. Image analysis was performed by investigators who
were blinded to
experimental conditions.
ACKNOWLEDGMENTS. We thank Drs. Phillip Nelson, Eugene
Zaitsev, Keri
Martinowitch, Jay Chang, and Newton Woo for thoughtful
comments
and suggestions and Regeneron Pharmaceuticals for providing
recombi-
nant BDNF. We also thank Drs. Bruce Carter, Mark Bothwell,
Moses Chao,
and Phil Barker for antibodies to p75NTR and Louis Reichardt
and Moses
Chao for antibodies to TrkB. Microscopy imaging was
performed at the
Porter Neuroscience Center Light Imaging Facility with the
assistance of
Dr. Carolyn Smith (NIH). This work was supported by the
National In-
stitute of Mental Health (NIMH) and National Institute of Child
Health
and Human Development (NICHD) intramural research
programs (B.L.)
and grants from the NIH and Muscular Dystrophy Association
(MDA)
(to B.H.).
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9/-
/DCSupplemental/pnas.201207767SI.pdf?targetid=nameddest=S
TXT
Supporting Information
Je et al. 10.1073/pnas.1207767109
SI Experimental Procedures
DNA Constructs, Embryo Injection, and Xenopus Nerve–Muscle
Coculture. Xenopus egg laying was induced by injecting female
Xenopus with human chronic gonadotrophin (hCG) (Sigma). Re-
sulting eggs were fertilized with sperm derived from male
testis.
Morpholinos, siRNA, or cDNAs (1 μg/μL) were mixed with
fluorescence dye (FITC or rhodamine) or GFP mRNA (1 μg/mL)
in a 1:1 ratio, injected into one of the blastomeres at the 2- to 4-
cell or 8- to 16-cell stage using the Picospritzer pressure ejector
(Parker Hannifin). One day after injection, the neural tube and
associated myotomal tissues were dissected, dissociated in
Ca2+-
Mg2+-free medium [58.2 mM NaCl, 0.7 mM KCl, 0.3 mM
EDTA
(pH 7.4)] for 15–20 min, and plated on glass coverslips. Cells
were grown for 1 d in culture medium consisting (vol/vol) of
50%
(vol/vol) L-15 medium, 1% (vol/vol) FCS, and 49% (vol/vol)
Ringer’s solution [117.6 mM NaCl, 2 mM CaCl2, 2.5 mM KCl,
47. 10 mM Hepes (pH 7.6)].
Western Blot Analysis. Xenopus embryos at stage 22 were
quickly
homogenized in extraction buffer I of “native membrane protein
extraction kit” according to manufacturer’s instructions (pro-
teoExtact; Calbiochem) in the presence of protease inhibitor
mixture (set III; Calbiochem). After high-speed centrifugation
(14,000 × g), the supernatants were transferred to a fresh tube
containing 300 μL of Freon (1,1,2-trichlorotrifluoroethane;
Sigma) vortexed for 1 min, incubated on ice for 5 min, and
centrifuged again to remove yolk protein. To measure proBDNF
expression, immunoprecipitation was performed. The super-
natants (cytoplasmic fraction) were measured for protein con-
centration, and an equal amount of protein was then precleared
and immunoprecipitated by chicken anti-human BDNF (de-
scribed in ref. 1; 2 μg for 500 μg of total protein) and chicken
IgY
agarose (Chemicon). The precipitated materials were subject to
Western blotting using polyclonal anti-BDNF (N-20; Santa
Cruz;
1:1,500). For membrane proteins (p75NTR and TrkB), the in-
soluble pellets after cytoplasmic extraction were solubilized in
modified RIPA buffer containing 1% SDS. Following centrifu-
gation at 10,000 × g to remove insoluble material, the superna-
tant was measured for protein concentration and 30 μg of
protein was separated on SDS/PAGE and blotted onto Im-
mobilon-P membrane (Millipore). The blots were probed with
the following primary antibodies: polyclonal antibody against
mouse intracellular domain of p75NTR (kindly provided by
Bruce
Carter, Vanderbilt University, Nashville, TN; 1:3,000); chicken
polyclonal anti-human TrkB (kindly provided by Louis Reich-
ardt, University of California, San Francisco; 1:1,000); and
polyclonal rabbit anti-tubulin (Abcam; 1:5,000). After thorough
washes, the blots were reacted with a secondary antibody con-
48. jugated with HRP. Signals were detected by ECL-plus kit (GE
Healthcare) exposed on HyperFilm for band intensities in the
linear range (Amersham Biosciences), scanned, and quantified
using ImageJ version 1.37.
To determine the expression of proBDNF in muscle cells, 10-
to 50-mg muscle tissues from the hindlimbs of neonatal mice or
Xenopus were homogenized in modified RIPA buffer with pro-
tease inhibitor mixture, sonicated, and extracted for 15 min by
increasing SDS and Triton X-100 to 1% and then centrifuged to
remove the insoluble material. The supernatant was estimated
for protein concentration and 30 μg of protein was separated on
4–12% Bis-Tris (NuPAGE), transferred to Immobilon-P mem-
brane, blocked using 5% BSA in TBST, probed overnight with
chicken polyclonal proBDNF antibody (Chemicon) in 3% BSA/
Tris-buffered saline (TBS) with 1% Triton X-100 (TBS-T),
washed, and developed using anti-chicken secondary antibody
conjugated to HRP (IgY-HRP; 1:5,000; Promega) and an ECL-
plus kit (GE Healthcare).
Morpholino and siRNA. Position 45–67 relative to the start
codon
of the ORF, which is shared between the two Xenopus p75NTR
genes [p75NTRa and p75NTRb (2)], was selected (5′-AGAGC-
CAUGUUGGUCAGGGCA-3′) to generate p75NTR siRNA.
The 23-nt sense and 23-nt antisense strands with two base
overhangs (AA) were chemically synthesized by Dharmacon in
deprotected and desalted form. Scrambled p75NTR siRNA (5′-
GCUGGCCGAGGCUUAAGAGAU-3′) was used as a control.
Antisense morpholino oligonucleotides, which achieve their ef-
fects by inhibiting translation initiation through its binding to
the
5′ UTR of the target mRNA, were used to down-regulate the
expression of Xenopus genes. The Xenopus p75NTR morpholino
49. sequence was 5′-CCATGCTGATCCTAGAAAGCTGATG-3′.
Its scrambled control was 5′-CTACTGCAAACATGGTACT-
GCTAGG-3′ (invert antisense). Anti-Xenopus TrkB morpholino
was generated as described previously (5′-CCACTGGAT-
CCCCCCTAGAATGGAG-3′) (3). Its scrambled control was
5′-CACCTAACATGGGGGGTACCTGAGG-3′ (invert anti-
sense). Anti-Xenopus BDNF morpholino (5′-CTCACCTGAT-
GGAACTTATTTTAGC-3′), which is specific to Xenopus
BDNF,
and the control morpholino oligonucleotides with a scrambled
sequence (5′-CCTCTTACCTCAGTTACAATGTATA-3′) were
synthesized by Gene Tools. To visualize their distributions, all
morpholino oligonucleotides were tagged with a fluorophore
(FITC). The effectiveness of p75NTR siRNA, TrkB morpholino,
and BDNF morpholino was confirmed by Western blotting (Fig.
S4 and see ref. 4). All of the electrophysiological and imaging
studies on synaptic depression and retraction were done in a
blind manner such that the experimenter did not know whether
or not the fluorescent cells expressed siRNAs/morpholinos for
scrambled or target genes.
Dual Color Labeling of Spinal Neurons for Synaptic
Competition. To
selectively label spinal neurons, a single dorsal, animal
blastomere
at the 8- or 16-cell stage was injected with fluorescent dyes
(FITC-
or rhodamine-conjugated dextran, 10,000 kDa). This protocol
has
been shown to selectively label spinal neurons but not other
nonneuronal cells (5). Down-regulation of gene expression in a
subset of Xenopus spinal neurons or muscle cells was achieved
by
embryo-injection techniques (6). FITC-conjugated TrkB mor-
pholino (3), FITC-conjugated BDNF morpholino, or p75NTR
50. siRNA was mixed with GFP mRNA (1 μg/mL) in a 1:1 ratio and
injected into the Xenopus embryos at the two-cell stage. One
day
after injection, the neural tube and associated myotomal tissues
were dissected from differently injected embryos and mixed,
and
nerve–muscle cocultures were prepared as described (7).
Time-Lapse Microscopy. Confocal imaging was performed
using an
inverted Zeiss LSM 510META laser scanning microscope with
a ×25 (1.0 NA) or ×40 (1.3 NA) oil immersion objective
(Zeiss).
For dual- or triple-color imaging, excitation lines of an argon
laser of 488 nm and two helium lasers of 543 nm and 640 nm
were used. Fluorescence was detected using a 458-/514-nm di-
chroic beam splitter and a 530- to 560-nm bandpass filter for
FITC,
a 580- to 620-nm bandpass filter for rhodamine, and a 650-nm
long-pass filter for Cy5. With the narrow band-pass filters, any
crossover or bleed-through of fluorescence was eliminated.
Time-
Je et al. www.pnas.org/cgi/content/short/1207767109 1 of 8
www.pnas.org/cgi/content/short/1207767109
lapse scanning was performed using Zeiss LSM 510 imaging
system software. Postacquisition images were processed with
the LSM 5 Image Browser (Zeiss) and Adobe Photoshop 7.0
software. One phase-contrast image and subsequent fluorescent
images were recorded every 5 or 10 min with Z-series stack at
1.0-μm intervals. Expression of fluorescent dyes or siRNA did
not affect axon morphology compared with uninjected controls.
Axons were reconstructed 3D. For time-lapse confocal imaging,
51. identical settings (acquisition speed, pinhole size, laser in-
tensities, dichroics, filters, size of uncaging region of interest
(ROI), uncaging iterations, etc.) were used except for the gain,
because of the heterogeneity of fluorescence signals. We also
tested the photobleaching effects. Even after 6 h, the level of
photobleaching was less than 10% of fluorescence signal. Fur-
thermore, to circumvent focal point changes during long-term
imaging, several Z-stacks (7–10 stacks) were acquired and later
projected to 2D with maximum intensity. After image acquisi-
tion, threshold was adjusted for better visualization. Finally,
we used high-powered differential interference contrast (DIC)
optics to trace the remnant of the axon terminals to ensure the
entire axon arbor was efficiently visualized.
Two-Photon Uncaging. Two-photon laser-scanning microscope
was
directly coupled to a Mira Ti:sapphire laser (100-fs pulses; 76
MHz, pumped by a 5-W Verdi laser; Coherent). For maximum
uncaging of MNI-glutamate (50 μM; Tocris), the Ti:sapphire
laser (725 nm) was beamed at the soma of a single neuron, with
a laser power of 5 mW and duration of 250 ms. The x-y
scanning
of a ROI was comprised of 25 by 25 pixels (1 pixel, 0.45 μm)
and
was performed at single z-axis planes 25 times using Zeiss
LSM510 imaging system software. Scanning speed of each pixel
was 2.5 μs.
FRET-Based Molecular Beacons. The secretion/activation of
proBDNF-
cleaving proteases was detected by a FRET-based fluorescence
beacon, which is a peptide containing the cleavage sequences
(MSMRVRRHSD) of proBDNF, flanked by a fluorophore [rho-
damine (ROX)] at the N terminus and a quencher at its C
terminus.
In the intact FRET peptide, the fluorescence of ROX is
52. quenched
by QXL610. Upon cleavage, the fluorophore is dissociated from
the quencher, generating fluorescence, which can be monitored
at excitation/emission wavelengths of 567/591 nm. To facilitate
the detection of proteolytic cleavage locally on axonal
terminals,
polystyrene beads containing the fluorogenic peptide beacons
were prepared. Polystyrene beads (10 μm in diameter;
Polysciences)
were rinsed in 50 volumes of PBS four to six times, incubated
in
5mg/mL heparin for 1 h at room temperature, and soaked in
peptide beacons (final concentration: 250 μg/mL, from a 1
mg/mL
stock in DMSO, 0.5% BSA in PBS) for 2 h at room temperature
with gentle agitation. The beads were then rinsed in PBS three
times and applied gently near neurons in the culture dishes. The
beads were manipulated into contact with neuronal axons by a
glass
pipette controlled by a micromanipulator. Control beads were
soaked in 0.5% BSA in PBS in the same manner. Various
protease
inhibitors (Sigma) were applied 10 min before stimulation of
spinal neurons by photo-uncaging. Fluorescence changes in
beads
were monitored using Olympus IX70 inverted microscope
equip-
ped with a Hamamatsu ORCA-ER cooled CCD camera, and data
were processed off-line with the IPLab software (Scanalytics).
Quantification of Axon Elongation and Retraction. Using an
ROI
tool from the IPLab software, the optical center of mass (COM)
of a given axon terminal was identified. For single muscle-in-
nervating axons, the distances between the COM of the AChR
53. fluorescence signal and the COM of axon terminal were deter-
mined at different time points after proBDNF application. For
dually innervated axons, the distances between the COM of a
single
muscle cell and the COM of the two competing terminals were
measured. The extent of retraction (or elongation) was
calculated
by subtracting the distance at a given time point after neuronal
stimulation with that at before stimulation. The values for mul-
tiple axons in the same condition were pooled and averaged.
Immunofluorescence Staining of Xenopus Cultures. Xenopus
neurons
and muscle cells grown on coverslips were washed with PBS
(PBS), fixed with 4% paraformaldehyde in PBS for 30 min at
room temperature, incubated in 0.1% NaBH4 (sodium borohy-
drate) in PBS to reduce auto-fluorescence, and blocked using
5% nonfat milk in PBS for 60 min. For proBDNF surface
staining, the cultures were incubated with proBDNF antibody
(Chemicon; 1:200) in 5% nonfat milk overnight at 4 °C. For
mBDNF surface staining, cells were reacted with a newly gen-
erated antibody specific for mBDNF (1:250) in 5% BSA. The
cultures were then reacted with secondary antibody (Alexa
Fluor
antibodies from Molecular Probes) for 1 h. Following PBS
washes, the coverslips were rinsed with water and then mounted
on medium containing Mowiol 4-88 and the antifade medium
DABCO (1,4-diazabicyclo[2.2.2]octane). Images were acquired
using a 63× oil plan Apochromat lens (1.4 NA) and multitrack
option in the Zeiss confocal LSM 510 Meta for all samples on
the same day under identical conditions (laser power, pinhole,
gain, and offset for two different colors). For quantitative anal-
ysis of proBDNF or mBDNF fluorescence signals, we first sub-
tracted background and adjusted the threshold to 50% over the
background fluorescence intensity by averaging the numbers
obtained from three nonfluorescent areas. The dynamic range of
54. fluorescence intensity values were confined to arbitral fluores-
cence unit in 8 bits [in pixels; 0 (minimum) – 255 (maximum)]
to
normalize fluorescent signals from differently treated groups.
Average intensities from all positively stained spots along syn-
aptic area in a myocyte were obtained using the ROI tool in
IPLab software. Then, the average intensity values of fluores-
cence signal in a given synapse were averaged and presented as
mean ± SEM.
For immunofluorescence staining of TrkB, p75NTR, and in-
tracellular proBDNF, similar procedures were used except that
cells were permeabilized with 0.05% Triton X-100 in PBS for
5 min. After blocking, cells were incubated with TrkB antibody
(Santa Cruz; 1:50) or p75 NTR ICD antibody (kindly provided
by
Bruce Carter; 1:100) overnight at 4 °C.
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Regulation of cell survival by secreted
proneurotrophins. Science 294:1945–1948.
2. Hutson LD, Bothwell M (2001) Expression and function of
Xenopus laevis p75(NTR)
suggest evolution of developmental regulatory mechanisms. J
Neurobiol 49:79–98.
3. Du JL, Poo MM (2004) Rapid BDNF-induced retrograde
synaptic modification in
a developing retinotectal system. Nature 429:878–883.
4. Yang F, et al. (2009) Pro-BDNF-induced synaptic depression
and retraction at
developing neuromuscular synapses. J Cell Biol 185:727–741.
5. Moody SA (1989) Quantitative lineage analysis of the origin
55. of frog primary motor and
sensory neurons from cleavage stage blastomeres. J Neurosci
9:2919–2930.
6. Wang T, Xie KW, Lu B (1995) Neurotrophins promote
maturation of developing
neuromuscular synapses. J Neurosci 15:4796–4805.
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acute and long-term synaptic potentiation. J Neurosci 25:11719–
11729.
Je et al. www.pnas.org/cgi/content/short/1207767109 2 of 8
www.pnas.org/cgi/content/short/1207767109
A
1minMNI-glutamate uncaging
60 min
Control
TTX
B
0
5 0 0
1 0 0 0
56. 1 5 0 0
0 5 0 1 0 0 1 5 0
Distance between neuronal
soma and uncaging spot ( m)
S
S
C
fr
eq
ue
nc
y
no
rm
al
iz
ed
to
un
tr
ea
te
d
57. co
nt
ro
l (
%
)
25 m
MNI-glutamate uncaging
Fig. S1. Stimulation of spinal neurons by photo-uncaging of
MNI-glutamate. Caged-glutamate compound (MNI-glutamate,
50 μM) was added to the culture
medium. Synaptic currents were recorded from a myocyte
innervated by spinal neurons. A laser spot of 10 by 10 μm was
applied to the cell body region of the
spinal neurons for 250 ms (indicated by the upward line). (A)
Glutamate uncaging on spinal neuron resulted in a dramatic
increase in spontaneous synaptic
current (SSC) frequency, which lasted for more than 1 h
(Upper). The effect of photo-uncaging is completely blocked by
inclusion of TTX (1 μM) in the medium
(Lower). (B) Relationship between percentage increase in SSC
frequency and the distance of uncaging spot from neuronal cell
body. Note that uncaging further
than 25 μm from cell body did not evoke increase in SSC
frequency (n = 15).
S
tim
.
58. U
ns
tim
.
0 30 60 90 120
M
er
ge
d
Fig. S2. Reverse case of Fig. 1, in which the soma of a green
neuron (outside the field) is stimulated. The axon terminal of a
red neuron (yellow arrow) (Lower)
retracted, whereas the terminal of a green neuron (Upper) did
not. A myocyte (also contained a small amount of red dye) was
indicated with white dotted
lines. The phase (Bottom) and two-color fluorescence images
(Top and Middle) of the triplet at “0” and “80” min are shown.
Je et al. www.pnas.org/cgi/content/short/1207767109 3 of 8
www.pnas.org/cgi/content/short/1207767109
Fl
uo
re
sc
en
62. M
M
M
M
M
N
N
N
A
Fig. S3. Quantification of cell surface staining of secreted
proBDNF and mBDNF. (A) Cultures were treated with drugs as
indicated, lightly fixed under
nonpermeable conditions, and processed for surface
immunocytochemistry using a polyclonal antibody specific for
proBDNF. Number associated with legends
represent number of myocytes. *P < 0.01. (B) Cell surface
staining of secreted mBDNF. Cultures were treated with drugs
as indicated, lightly fixed under
nonpermeable conditions, and processed for surface
immunocytochemistry using a polyclonal antibody specific for
mBDNF. mBDNF immunoreactivities are
shown as inset images on top of DIC images. Two bigger circles
(inside of a middle, lower image) indicate the enlargement of
the areas on muscle cells. Please
note more mBDNF immunoreactivity on a muscle cell,
innervated by a spinal neuron. (Scale bar: 20 μm.) (C)
Quantification of the immunofluorescence is shown
63. as bar graph on the right. Number associated with legends
represents number of myocytes. “+Glu./MMP-In” means cells
were treated with glutamate (10 mM)
in the presence of MMP inhibitors (60 μM). M, muscle cell; N,
neuron. *P < 0.01.
Je et al. www.pnas.org/cgi/content/short/1207767109 4 of 8
www.pnas.org/cgi/content/short/1207767109
B mBDNF
mBDNF
control
TrkB morpholino N+
5min
5min
50
0p
A
1min
0
1
2
64. 3
4 - mBDNF
+ mBDNF
N
or
m
al
iz
ed
S
S
C
fr
eq
ue
nc
y
N-N+ M+
TrkB morpholino
*
8 9 5
*
C
67. ph
.
TrkB
tubulin
Fig. S4. Reduction of endogenous TrkB levels by embryo
injection of morpholino. TrkB morpholinos, or its scrambled
analogs, were introduced into the
developing Xenopus by embryo injection. Neural tubes were
dissected and proteins were extracted. Western blots were
performed using TrkB specific an-
tibody. The blot was also probed with an anti-tubulin antibody
for loading controls. (A) Down-regulation of TrkB expression
by TrkB morpholino. Note
a reduction of TrkB expression in embryos injected with TrkB
morpholino but not in those with scrambled morpholino. (B and
C) Blockade of BDNF-induced
synaptic potentiation by presynaptic expression of TrkB
morpholino. BDNF (25 ng/mL) was applied directly to 1-d-old
nerve–muscle cultures expressing TrkB
morpholino either presynaptically (N+) or postsynaptically
(M+). An example (B) and the summary (C) of the effect of
TrkB morpholino on acute synaptic
potentiation induced by BDNF are shown. Each data point
represents normalized SSCs (averaged from 10 min of
recording) from a single synapse before and
after BDNF application. Note that presynaptic, but not
postsynaptic expression of TrkB morpholino prevents BDNF-
induced acute synaptic potentiation of SSC
frequency.
Je et al. www.pnas.org/cgi/content/short/1207767109 5 of 8
69. .
Fig. S5. Time course showing retraction of an axon from a
neuron expressing scrambled p75NTR siRNA. Stimulation of
the red neuron resulted in the retraction
of green terminal (yellow arrow) expressing scrambled p75NTR
siRNA but not red terminal (white arrow) expressing true
p75NTR siRNA. Phase and two-color
fluorescence images at “0” min are shown on the left.
S
tim
.
U
ns
tim
.
MMP 3 & 9 In.
Fig. S6. Synaptic retraction mediated by inhibition of MMP3
and -9 metalloproteases. Inhibition of MMP3 and -9 cleavage
leads to retraction of both axon
terminals coinnervating the same myocyte. The culture was
pretreated with both MMP3 inhibitor (50 nM) and MMP9
inhibitor (50 μM) for 15 min. After
stimulating the soma of the red neuron, both red and green
terminals (yellow arrows) retracted from their prior synaptic
sites.
Win Lose
Synaptic
70. retraction
p75NTR
Less
active
More
active
proBDNF
protease
Trk
mBDNF
proBDNF
Fig. S7. Activity-dependent conversion of proBDNF to mBDNF:
a model for activity-dependent synaptic competition and
elimination at the NMJ. During
neuromuscular development, presynaptic innervation drives the
production and secretion of proBDNF from the postsynaptic
muscle cell. At the active axonal
terminal, proteases secreted into the synaptic cleft convert
proBDNF to mBDNF, which binds TrkB and triggers a series of
downstream signaling, leading to the
stabilization of the synaptic connection. By contrast, relatively
fewer active terminals are unable to convert proBDNF, which
binds p75NTR, to facilitate synaptic
depression and eventually retraction of these terminals.
Je et al. www.pnas.org/cgi/content/short/1207767109 6 of 8
71. www.pnas.org/cgi/content/short/1207767109
Movie S1. Activity-dependent synaptic competition in culture.
Neurons were separately labeled with FITC-conjugated (green)
and rhodamine-conjugated
(red) dextran, respectively, by embryo injection. Stimulation of
the red neuron, by photo-uncaging of MNI-glutamate in the
soma area using a two photon
laser, caused the unstimulated (green) axon terminal to retract
from the synaptic target (indicated by a yellow arrow) (Upper).
In contrast, the axon terminal
(red) from the stimulated neuron sustained (indicated by a white
arrow) (Lower).
Movie S1
Movie S2. Treatment with mBDNF prevents activity-dependent
synaptic retraction. The culture was treated with mBDNF for 15
min, before stimulating the
soma of a red neuron. The terminals from both red and green
neurons remained in the synaptic site without any retraction.
Movie S2
Movie S3. Inhibition of proteolytic cleavage leads to retraction
of both axon terminals coinnervating the same myocyte. The
culture was pretreated with
protease inhibitors for 15 min. After stimulating the soma of the
red neuron, both red and green terminals (yellow arrows)
retracted from their prior
synaptic sites.
Movie S3
Je et al. www.pnas.org/cgi/content/short/1207767109 7 of 8