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Schwann cell-derived neuregulin-2α can function as a cell-attached activator of muscle acetylcholine receptor expression
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Evidence for muscle-dependent neuromuscular synaptic site determination in mammals
Full text linkRecent evidence has challenged the prevalent view that neural factors induce the formation of a de novo postsynaptic apparatus during development of the vertebrate neuromuscular junction. The latest experiments suggest an alternative, muscle-dependent model in which the muscle induces the nascent postsynaptic apparatus and sets the location of the future synapse. Once contacted by the incoming axons, these sites, laid out in a pre-pattern in the central area of developing muscle fibers, mature into synapses by the combined action of neural factors such as agrin and ACh. In this study, I sought to provide a test in mammals for these two models of neuromuscular synaptogenesis. Previously, our laboratory showed that continuous muscle expression of constitutively active ErbB2 (CAErbB2) during embryogenesis leads to synaptic loss, exuberant axonal sprouting and lethality at birth. Here, I transiently induced CAErbB2 during midgestation and examined the process of synapse restoration after inducer withdrawal. Centrallyenriched AChR transcription and AChR clustering were abolished as a result of transient CAErbB2 induction. After inducer withdrawal, synapses were restored but were distributed widely over the entire surface of the diaphragm. Under the nerve-dependent model, this distribution would have been explained by the wide pattern of axonal sprouting triggered by CAErbB2 expression. Yet, in the absence of the nerve, introduced in our transgenic animals by mating to Hb9+/- mice, a very similar, wide distribution of aneural AChR clusters was generated. Thus, even in a case where the central pre-pattern of AChR transcription and clustering is missing, it is the muscle, and not the nerve, that seems to set the site for synapse formation. My results support a muscle-dependent model for the induction of neuromuscular synaptogenesis in mammals.Institute for Neuroscienc
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Zinc-finger transcription factors and the response of non-myelinating Schwann cells to axonal injury
Full text linktextSchwann cell (SCs) are the glia of the peripheral nervous system. During
development, a common precursor develops into two distinct types of SCs: myelinating
SCs and non-myelinating SCs. There is little literature regarding the non-myelinating
variety of SCs, specifically a type of non-myelinating SC found at the neuromuscular
junction (NMJ), the terminal Schwann cell (tSC). Terminal SCs are critical for the
maintenance and recovery of the NMJ. Peripheral nerve injury causes tSCs to become
reactive, a state characterized by changes in gene expression and the extension of cellular
processes. It is in this state, that tSCs help to restore functionality to the denervated
junction. What drives tSCs to become reactive after injury remains largely unknown.
Previously, nine zinc-finger proteins (ZFPs), a class of transcription factors, have been
implicated in SC development and differentiation. As a result, transcription factors from
the ZFP family were considered as potential candidates that may drive the activation of
tSCs. Because tSCs are few and far between, only two-six cells cover the NMJ, I used a
largely non-myelinated nerve of the autonomic system, the cervical sympathetic trunk
(CST), to search for ZFP candidates. I created a cDNA library from both control and
denervated CST resulting in 40 unique ZFPs. Six of these genes were studied futher:
Zipro1, Zfp36, Zfp612, Zfp180, Zfp111 and Zfp629. I found a near two-fold increase in
Zipro1 mRNA and protein in denervated CST, and no change in the other five ZFPs
(results from Zfp629 remain inconclusive). Using an antibody against Zipro1, I located
this increase of Zipro1 to the SCs of the denervated CST. I also found Zipro1 expression
in tSCs and an increase of 19% in tSCs of denervated rat muscle. Upregulation of Zipro1
in non-myelinating SCs suggests that Zipro1 may have a role in the activation of tSCs.
Further study is needed in order to clarify the extent of Zipro1's involvement.Institute for Neuroscienc
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Synaptic Sculpting: Terminal Schwann Cells Participate in Neuromuscular Synapse Elimination
Full text linkImpulse transmission from the axons of motor neurons to the myofibers of our muscles is essential for virtually all aspects of our physical behavior. It ultimately drives our ability to move, to breathe and to eat, amongst other essential activities. The sites where the impulse transmission occur are termed neuromuscular junctions (NMJs). They are similar to the synapses that mediate impulse transmission in the brain, which underlies, for example, mental behavior. Due to our dependence on neuromuscular transmission for essential aspects of our vitality and its relatively convenient availability for experimentation, considerable attention has been given to understanding how the NMJ functions, how it forms during ontogenetic development, how it is affected by disease and how it reforms after injury. Most of these studies have been made in nonhuman vertebrates, where the fundamental mechanisms are highly conserved from lower organisms to humans.
My study concerns mechanisms involved in ontogenetic development of NMJs in the mouse, a common model used by neuromuscular experimentalists. In mouse, each NMJ is formed by the terminal branches of a motor axon. The branches lie in close apposition to a specialized region of the myofiber membrane called the motor endplate. The endplate occupies about 0.1% of the myofiber surface in adult muscles. Synaptic transmission from the axon terminals to the endplate is mediated by molecules of acetylcholine (ACh). As the nerve fires an action potential, when an impulse traveling along a motor axon reaches its terminals at a neuromuscular junction, it evokes the secretion of ACh from the terminals into a narrow crevice between them and the motor endplate. Once secreted, ACh binds to aggregates of acetylcholine receptors (AChR) on the surface of the endplate, triggering an impulse in the myofiber that leads to its contraction. Capping the axon terminals are terminal Schwann cells (tSCs), the third cellular component of neuromuscular junctions. They play supporting, but essential, roles in the NMJ���s activity. Typically, in mammals there is a single endplate on a muscle fiber. In adults, each endplate is innervated by the terminals of a single axon. On the other hand, at birth, there can be terminal branches of as many as 10 or more separate axons making synaptic contact at a single endplate. Several lines of evidence indicate that the terminals of the different axons compete for sole occupation of the endplate and that the elimination of inputs requires communication between the axon terminals and muscle fiber. Although much has been learned about the nature of the communication driving this transition from poly- to single-innervation our understanding is far from complete. Even less well understood is the behavior of the tSCs during the period of synapse elimination. The aim of my study was to determine in detail the characteristics of tSCs during this period with an eye toward learning whether the tSCs play a direct role in the process
The Potential Role of Erk1/2 Map Kinases in the Regulation of Neuromuscular Junction Integrity and Bone Morphogenetic Protein Signaling
Full text linkIt is known that extracellular signal-regulated kinases 1 and 2 (ERK1/2) play important roles in development and maintenance of skeletal muscles in vitro and in vivo. However, it is not clear whether muscle ERK1/2 can regulate neuromuscular junction (NMJ) structure and function and (or) have unique substrates and downstream pathways in skeletal muscle. Previously, we generated an ERK 1/2 double knockout (DKO) mouse line using germ line Erk1 mutation and Cre-loxP deletion of Erk2. Those DKO animals were viable after birth but displayed stunted postnatal growth, muscle weakness, and shorter lifespan. Next, two typical fast-twitch muscles, the sternomastoid (STN) and the tibialis anterior (TA), were examined. A mixture of fiber loss and mild muscle atrophy was found, and STN but not TA underwent partial denervation. To explore the role of ERK1/2 in slow-twitch, type 1 muscle fibers, we studied the mutant soleus muscle (SOL) in the aspect of morphology, expression of denervation and synaptic markers, and mitochondrial function. Next, we examined the status of bone morphogenetic (BMP) signaling in mutant SOL and myotubes by measuring mRNA and phosphorylation level of several BMP components.
It is shown that young adult mutant SOL was drastically wasted, with highly atrophied type 1 fibers, denervation at most synaptic sites, induction of “fetal” acetylcholine receptor (AChRγ) subunit, reduction of “adult” AChRε subunit, and impaired mitochondrial function. In weanlings, fiber morphology and mitochondrial markers were normal, yet AChRγ upregulation and AChRε downregulation were
observed. In mutant weanlings, most of the fetal AChRs appeared at NMJs on type1 muscle fibers. These results suggest that: (1) ERK1/2 are critical for slow-twitch fiber growth; (2) a defective γ/ε-AChR subunit switch, preferentially at synapses on slow fibers, precedes denervation and wasting of mutant SOL; (3) the neuromuscular synapse is a primary subcellular target for muscle ERK1/2 function in vivo.
It is shown that mutant SOL exhibited alterations in the BMP pathway: decreased expression of Bmpr1b, unchanged expression of the target gene Id-1, the negative regulator Noggin and the intracellular kinase Smad6, and unchanged phosphorylation level of Smad1/5/8. These experiments suggest that unlike normal muscle, BMP signaling is not activated in mutant SOL after denervation. However, Bmpr1b expression and Smad1/5/8 phosphorylation were not altered in BMP-treated myotubes subjected to pharmacological inhibition of ERK1/2 activation. These results suggest that ERK1/2 regulate BMP signaling in SOL through an indirect mechanism
Dopamine Signaling Attenuates a Recurrent Neuronal Network during C. elegans Copulation
Full text linkNeuro-modulation of self-amplifying circuits drives the execution of behaviors to their appropriate context. These recursive networks are found throughout the Caenorhabditis elegans connectome, however the mechanism that fine-tunes reciprocal neural activity during complex behaviors is unknown. Here I dissect the cellular and molecular components involved in male copulation, a goal oriented behavior that entails initiation and termination under appropriate circumstances. The C. elegans mating circuit integrates sensory-motor cues resulting in copulatory spicule insertion into the hermaphrodite vulva. As the male tail presses against the hermaphrodite���s genitalia, cholinergic and glutamatergic reciprocal innervations of post-cloaca sensilla (PCS) neurons (PCA, PCB and PCC), hook neurons (HOA, HOB) and their post-synaptic sex muscles execute rhythmic spicule thrusts. These repetitive spicule movements continue until the male shifts off the vulva or genital penetration. However, the signaling mechanism that temporally and spatially restricts intromission attempts to vulva cues was unclear.
My results suggest that dopamine (DA) neuromodulation delimits spicule insertion attempts to the hermaphrodite vulva by dampening stimulus-independent spicule circuit activity. I found that upon vulval contact, DA signaling from male specific sensory neurons stimulates D2-like receptors, DOP-2 and DOP-3, to decrease cholinergic induced sex muscle contractions. Through pharmacology and targeted optogenetics I demonstrate that D2-like pathways act coincidentally, and as a consequence of cholinergic signaling, to reduce spicule intromission attempts with non-productive mating partners.
During spicule intromission attempts, DA up-regulates gap-junctions among PCB and a hook neuron to decrease self-amplifying PCS properties. Through forward genetics I isolated a missense mutation in an UNC-7L gap-junction isoform, which perturbs DOP-2 signaling in PCB and its electrical partner, HOA. Additional pharmacogenetic analysis suggests that the AVR-14 glutamate-gated channel partially introduces chloride ions into HOA to mediate DA downmodulation of the spicule circuit. Consistently, my analysis of the unc-7(rg396) allele indicates that when DOP-2 promotes UNC-7 electrical communication, AVR-14-mediated inhibitory signals pass from HOA to PCB. Consequently, the cholinergic PCB neuron is less receptive to stimulation by its recurrent glutamatergic synaptic partner, PCA. Furthermore, behavioral observations suggest that DA neuromodulation of UNC-7 ensures attenuation of recursive intromission attempts when the male disengages or is dislodged from the hermaphrodite genitali
Individual Synaptic Vesicles from the Electroplaque of Torpedo californica, a Classic Cholinergic Synapse, Contain Multiple Neurotransmitter Transporters and Small Ribonucleic Acids (sRNAs)
Full text linkLarge-scale proteomic analyses of heterogeneous CNS preparations, such as isolated CNS synaptic vesicles, have provided a wealth of information related to synapse specific proteins and synaptic signaling pathways. However, these studies under-represent, or lack entirely, many vesicle and neurotransmitter signaling pathways. In particular, the neuromodulatory adrenergic and cholinergic pathways are often under-represented. In order to better understand the molecules and signaling pathways involved at cholinergic synapses, I have focused on the classic preparation of the electric lobe and electric organ of Torpedo californica. Many lines of evidence support the idea that in addition to acetylcholine, glutamate and ATP are also released from cholinergic synapses. Utilizing a single-vesicle imaging approach, I have found that individual synaptic vesicles from the electric ray possess neurotransmitter transporters for glutamate, ATP, and acetylcholine. These results help to explain neurotransmitter co-release at cholinergic terminals. In addition to classic neurotransmitter, I have discovered that synaptic vesicles in the peripheral nervous system (PNS) and the central nervous system (CNS) possess small ribonucleic acids, including transfer RNA fragments and microRNAs. This discovery suggests that, in addition to releasing neurotransmitters that can induce local changes in postsynaptic membrane polarization, synaptic vesicles may also release small RNAs that can directly regulate local protein synthesis at the postsynaptic cell
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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