bluuebunny/crossref_metadata_2025 huggingface dataset:
Segregation of Different GABAAReceptors to Synaptic and Extrasynaptic Membranes of Cerebellar Granule Cells
Authors: Nusser Zoltan , Sieghart Werner , Somogyi Peter
Published: April 2018

Two types of GABAAreceptor-mediated inhibition (phasic and tonic) have been described in cerebellar granule cells, although these cells receive GABAergic input only from a single cell type, the Golgi cell. In adult rats, granule cells express six GABAAreceptor subunits abundantly (α1, α6, β2, β3, γ2, and δ), which are coassembled into at least four to six distinct GABAAreceptor subtypes. We tested whether a differential distribution of GABAAreceptors on the surface of granule cells could play a role in the different forms of inhibition, assuming that phasic inhibition originates from the activation of synaptic receptors, whereas tonic inhibition is provided mainly by extrasynaptic receptors. The α1, α6, β2/3, and γ2 subunits have been found by immunogold localizations to be concentrated in GABAergic Golgi synapses and also are present in the extrasynaptic membrane at a lower concentration. In contrast, immunoparticles for the δ subunit could not be detected in synaptic junctions, although they were abundantly present in the extrasynaptic dendritic and somatic membranes. Gold particles for the α6, γ2, and β2/3, but not the α1 and δ, subunits also were concentrated in some glutamatergic mossy fiber synapses, where their colocalization with AMPA-type glutamate receptors was demonstrated. The exclusive extrasynaptic presence of the δ subunit-containing receptors, together with their kinetic properties, suggests that tonic inhibition could be mediated mainly byextrasynapticα6β2/3δ receptors, whereas phasic inhibition is attributable to the activation ofsynapticα1β2/3γ2, α6β2/3γ2, and α1α6β2/3γ2receptors.
Steroid and Neuronal Regulation of Ecdysone Receptor Expression during Metamorphosis of Muscle in the Moth,Manduca sexta
Authors: Hegstrom Carol D. , Riddiford Lynn M. , Truman James W.
Published: April 2018

Ecdysteroids regulate the remodeling of the dorsal external oblique 1 (DEO1) muscle during metamorphosis inManduca sexta(Hegstrom and Truman, 1996a). We show that the temporal and spatial patterning of the A and B1 isoforms of the ecdysone receptor (EcR) within muscle DEO1 corresponds with the developmental fates of the fibers. Using antibodies directed to specific isoforms of EcR, we show that the expression of various EcR isoforms in myonuclei differ among the five fibers of DEO1 and correspond with the developmental response of the muscle to the changing steroid titers and to the pattern of innervation. Muscle degeneration and apoptosis of myonuclei in all fibers are correlated with the expression of only EcR-A just before pupal ecdysis and then with the expression of low levels of both EcR-A and EcR-B1 shortly after pupation. Only the first fiber of muscle DEO1 participates in the regrowth of the adult muscle, and only this fiber shows an upregulation of EcR-B1 that is evident at 3 d after pupal ecdysis. Denervation of the muscle prevents both the upregulation of EcR-B1 and myoblast proliferation. We conclude that the developmental fate of muscle DEO1 during metamorphosis is orchestrated by interactions between rising and falling ecdysteroid titers, the pattern of expression of EcR isoforms by the muscle, and interactions with other cells in the local environment.
Cochlear Electrically Evoked Emissions Modulated by Mechanical Transduction Channels
Authors: Yates Graeme K. , Kirk Desmond L.
Published: April 2018

Cochlear outer hair cells are capable of both mechanical-to-electrical and electrical-to-mechanical transduction. Vibration of their stereocilia by sound is believed to stimulate somatic motility via a receptor potential developed across the basolateral membrane, thereby enhancing the mechanical vibration and increasing the sensitivity and frequency selectivity of the ear. Extrinsic electrical currents, applied at the tops of the cells, also appear to activate motilityin vivo, presumably after entering the cell. Earlier experiments suggested such currents might enter through the transduction channels themselves, but an alternative shunt pathway through the membrane capacitance seems more likely on physical grounds. We therefore recorded electrically evoked oto-acoustic emissions while modulating the transduction channels by driving them with low-frequency sound. Recordings of the low-frequency cochlear microphonic provided a measure of the mean electrical conductance through the channels during sound stimulation. Emissions increased during displacement of the basilar membrane toward scala vestibuli, when the channels were biased open, and decreased on the opposite phase, and the modulation of the emission was in direct proportion to the cochlear microphonic. The results are the strongest evidence yet that electrically evoked emissions are generated directly by mechanisms related to cochlear transduction and lead to the surprising conclusion that, for frequencies up to at least 12 kHz, extrinsic electrical currents enter the hair cell predominantly by the resistive pathway through the transduction channels. Alternatively, the results might be consistent with direct modulation of a motility source driven by capacitive currents but whose output depends on the state of the channels.
A Point Mutation (D79N) of the α2A Adrenergic Receptor Abolishes the Antiepileptogenic Action of Endogenous Norepinephrine
Authors: Janumpalli Sridevi , Butler Linda S. , MacMillan Leigh B. , Limbird Lee E. , McNamara James O.
Published: April 2018

Norepinephrine serves as a neurotransmitter for a population of neurons the cell bodies of which reside in a brainstem nucleus and the axons of which project widely to discrete subsets of forebrain neurons. Norepinephrine powerfully inhibits epileptogenesis in the kindling model. Pharmacological methods have demonstrated that the antiepileptogenic actions of norepinephrine are exerted via α2 adrenergic receptors residing on targets of noradrenergic neurons. The existence of three α2 adrenergic receptor subtypes together with the lack of subtype-specific ligands has precluded understanding the role of individual α2 adrenergic receptor subtypes in the antiepileptogenic actions of norepinephrine. Gene targeting was used to introduce a point mutation into the α2A adrenergic subtype in the mouse genome. The mutation produced a marked enhancement of epileptogenesis and abolished the proepileptogenic actions of the α2 adrenergic receptor antagonist idazoxan. These studies reveal the crucial contribution of the α2A receptor subtype in suppression of epileptogenesis. Development of agents that promote selective activation of the α2A receptor subtype may provide novel therapeutic strategies for the prophylaxis of epilepsy.
Functional Evidence that BDNF Is an Anterograde Neuronal Trophic Factor in the CNS
Authors: Fawcett James P. , Bamji Shernaz X. , Causing Carrie G. , Aloyz Raquel , Ase Ariel R. , Reader Tomas A. , McLean John H. , Miller Freda D.
Published: April 2018

In this report, we have tested the hypothesis that brain-derived neurotrophic factor (BDNF) is an anterograde neurotrophic factor in the CNS and have focused on central noradrenergic neurons that synthesize BDNF. Double-label immunocytochemistry for BDNF and dopamine-β-hydroxylase (DBH), a marker for noradrenergic neurons, demonstrated that BDNF is partially localized to noradrenergic nerve fibers and terminals in the adult rat brain. To test the functional importance of this anterograde BDNF, we analyzed transgenic mice carrying a DBH–BDNF minigene. Increased synthesis of BDNF in noradrenergic neurons of DBH–BDNF mice caused elevated TrkB tyrosine kinase activation throughout postnatal life in the neocortex, a noradrenergic target region. This afferently regulated increase in TrkB receptor activity led to long-lasting alterations in cortical morphology. To determine whether noradrenergic neuron-expressed BDNF also anterogradely regulated neuronal survival, we examined a second noradrenergic target, neonatal facial motoneurons. One week after axotomy, 72% of facial motoneurons were lost in control animals, whereas only 30–35% were lost in DBH–BDNF transgenic mice. Altogether, these results indicate that BDNF is anterogradely transported to fibers and terminals of noradrenergic neurons, that anterogradely secreted BDNF causes activation of TrkB in target regions, and that this secretion has functional consequences for target neuron survival and differentiation. This presynaptic secretion of BDNF may provide a cellular mechanism for modulating neural circuitry, in either the developing or mature nervous system.
Determinants of Voltage Attenuation in Neocortical Pyramidal Neuron Dendrites
Authors: Stuart Greg , Spruston Nelson
Published: April 2018

How effectively synaptic and regenerative potentials propagate within neurons depends critically on the membrane properties and intracellular resistivity of the dendritic tree. These properties therefore are important determinants of neuronal function. Here we use simultaneous whole-cell patch-pipette recordings from the soma and apical dendrite of neocortical layer 5 pyramidal neurons to directly measure voltage attenuation in cortical neurons. When combined with morphologically realistic compartmental models of the same cells, the data suggest that the intracellular resistivity of neocortical pyramidal neurons is relatively low (∼70 to 100 Ωcm), but that voltage attenuation is substantial because of nonuniformly distributed resting conductances present at a higher density in the distal apical dendrites. These conductances, which were largely blocked by bath application of CsCl (5 mm), significantly increased steady-state voltage attenuation and decreased EPSP integral and peak in a manner that depended on the location of the synapse. Together these findings suggest that nonuniformly distributed Cs-sensitive and -insensitive resting conductances generate a “leaky” apical dendrite, which differentially influences the integration of spatially segregated synaptic inputs.
Functional Analysis of the C2A Domain of Synaptotagmin 1: Implications for Calcium-Regulated Secretion
Authors: Thomas David M. , Elferink Lisa A.
Published: April 2018

Synaptotagmin 1 is proposed to function as a low affinity calcium sensor for calcium-triggered exocytosis from neural and neuroendocrine cells. Because of the calcium-binding properties of the C2A domain of synaptotagmin 1, calcium-dependent interactions through this domain may modulate neurotransmitter release. We addressed this question by using alanine-scanning mutagenesis to generate a series of mutations within the C2A domain of synaptotagmin 1. The effects of these mutations on synaptotagmin 1 C2A function were analyzed for (1) calcium-dependent phospholipid binding, (2) calcium-dependent binding to syntaxin 1A, a plasma membrane protein critical for vesicle docking or fusion, and (3) calcium-regulated secretion after microinjection into neuroendocrine pheochromocytoma (PC12) cells. Our analyses reveal that a polylysine motif at residues 189–192 confers an inhibitory effect on secretion by recombinant synaptotagmin C2A fragments. The synaptotagmin 1 C2A polylysine motif functions independently of calcium-mediated interactions with phospholipids and syntaxin 1A. Furthermore, α-latrotoxin reverses the inhibitory effect of injected recombinant C2A fragments, suggesting that they perturb the cellular calcium-sensing machinery by interfering with synaptotagmin 1 activityin vivo. Our results indicate that novel calcium-independent interactions mediated through the C2A polylysine motif of synaptotagmin 1 function to modulate neurotransmitter release.
Pore Mutation in a G-Protein-Gated Inwardly Rectifying K+Channel Subunit Causes Loss of K+-Dependent Inhibition inweaverHippocampus
Authors: Jarolimek Wolfgang , Bäurle Jörg , Misgeld Ulrich
Published: April 2018

Weaver(wv) mice carry a point mutation in the pore region of a G-protein-gated inwardly rectifying K+channel subunit (Kir3.2).wvKir3.2 conducts inward currents that may cause the loss of neurons in the cerebellum and substantia nigra. Although Kir3.2 is widely expressed in the CNS, significant morphological or physiological changes have not been reported for other brain areas. We studied the role ofwvKir3.2 in hippocampal slices of young [postnatal day (P) 4–18] and adultwv/wv(≥P24) mice, because protein levels of Kir 3.1 and Kir3.2 appear to be normal in the first 3 postnatal weeks and only decrease thereafter. In disinhibited slices, the GABABreceptor agonistR-baclofen reduced burst activity inwv/wvmice but was much more potent in wild-type mice. Mean resting membrane potential, slope input resistance, and membrane time constant of CA3 neurons of adultwv/wvand wild-type mice were indistinguishable. However,R-baclofen or chloroadenosine did not induce K+currents or any other conductance change inwv/wvmice. Moreover, electrical or chemical stimulation of inhibitory neurons did not evoke slow IPSPs in adultwv/wvmice. Only in a few cells of youngwv/wvmice did GABABreceptor activation byR-baclofen or presynaptic stimulation induce small inward currents, which were likely caused by a Na+ion influx throughwvKir3.2 channels. The data show that the pore mutation inwvKir3.2 channels results in a hippocampal phenotype resembling Kir3.2-deficient mutants, although it is not associated with the occurrence of seizures.
Neuronal Nicotinic Acetylcholine Receptors Are Blocked by Intracellular Spermine in a Voltage-Dependent Manner
Authors: Haghighi Ali Pejmun , Cooper Ellis
Published: April 2018

A common feature of neuronal nicotinic acetylcholine receptors (nAChRs) is that they conduct inward current at negative membrane potentials but little outward current at positive membrane potentials, a property referred to as inward rectification. Physiologically, inward rectification serves important functions, and the main goal of our study was to investigate the mechanisms underlying the rectification of these receptors. We examined recombinant α3β4and α4β2neuronal nAChR subtypes expressed inXenopusoocytes and native nAChRs expressed on superior cervical ganglion (SCG) neurons. Whole-cell ACh-evoked currents recorded from these receptors exhibited strong inward rectification. In contrast, we showed that single-channel currents from these neuronal nAChRs measured in outside-out patches outwardly rectify. On the basis of recent findings that spermine, a ubiquitous intracellular polyamine, confers rectification to glutamate receptors and inwardly rectifying potassium channels, we investigated whether spermine causes neuronal nAChRs to inwardly rectify. When spermine was added to the patch electrode in outside-out recordings, it caused a concentration- and voltage-dependent block of ACh-evoked single-channel currents. Using these single-channel data and physiological concentrations of intracellular spermine, we could account for the inward rectification of macroscopic whole-cell ACh-evoked conductance–voltage relationships. Therefore, we conclude that the voltage-dependent block by intracellular spermine underlies inward rectification of neuronal nAChRs. We also found that extracellular spermine blocks both α3β4and α4β2receptors; this finding points to a mechanism whereby increases in extracellular spermine, perhaps during pathological conditions, could selectively block these receptors.
G Protein-Mediated Inhibition of Neuronal Migration Requires Calcium Influx
Authors: Horgan Angela M. , Copenhaver Philip F.
Published: April 2018

Neuronal migration is an essential feature of the developing nervous system, but the intracellular signaling mechanisms that regulate this process are poorly understood. During the formation of the enteric nervous system (ENS) in the mothManduca sexta, the migration of an identified set of neurons (the EP cells) is regulated in part by the heterotrimeric guanyl-nucleotide binding protein (G protein) Goα. Using anin vivoculture preparation for developing embryos that allows direct access to the ENS, we have shown that EP cell migration is similarly regulated by intracellular Ca2+; treatments that increased intracellular Ca2+inhibited the migratory process, whereas buffering intracellular Ca2+induced aberrant migration onto inappropriate pathways. Imaging the spontaneous changes in intracellular Ca2+within individual EP cells showed that actively migrating neurons exhibited only small fluctuations in intracellular Ca2+. In contrast, neurons that had reached the end of migration displayed large, transient Ca2+spikes. Similar Ca2+spikes were induced in the EP cells by G protein stimulation, an effect that was reversed by removal of external Ca2+. Stimulation of Goin individual EP cells (by injection of either activated Goαsubunits or mastoparan) also inhibited migration in a Ca2+-dependent manner. These results suggest that the regulation of neuronal migration by G proteins involves a Ca2+-dependent process requiring Ca2+influx.
Role of Dopamine Transporter in Methamphetamine-Induced Neurotoxicity: Evidence from Mice Lacking the Transporter
Authors: Fumagalli Fabio , Gainetdinov Raul R. , Valenzano Kenneth J. , Caron Marc G.
Published: April 2018

The role of the dopamine transporter (DAT) in mediating the neurotoxic effects of methamphetamine (METH) was tested in mice lacking DAT. Dopamine (DA) and serotonin (5-HT) content, glial fibrillary acidic protein (GFAP) expression, and free radical formation were assessed as markers of METH neurotoxicity in the striatum and/or hippocampus of wild-type, heterozygote, and homozygote (DAT −/−) mice. Four injections of METH (15 mg/kg, s.c.), each given 2 hr apart, produced 80 and 30% decreases in striatal DA and 5-HT levels, respectively, in wild-type animals 2 d after administration. In addition, GFAP mRNA and protein expression levels, extracellular DA levels, and free radical formation were increased markedly. Hippocampal 5-HT content was decreased significantly as well (43%). Conversely, no significant changes were observed in total DA content, GFAP expression, extracellular DA levels, or free radical formation in the striatum of DAT −/− mice after METH administration. However, modest decreases were observed in striatal and hippocampal 5-HT levels (10 and 17%, respectively). These observations demonstrate that DAT is required for, and DA is an essential mediator of, METH-induced striatal dopaminergic neurotoxicity, whereas serotonergic deficits are only partially dependent on DAT.
Electrophysiological Characterization of a Putative Supporting Cell Isolated from the Frog Taste Disk
Authors: Bigiani Albertino , Sbarbati Andrea , Osculati Francesco , Pietra Pierangelo
Published: April 2018

Chemosensory cells in vertebrate taste organs have two obvious specializations: an apical membrane with access to the tastants occurring in food, and synapses with sensory axons. In many species, however, certain differentiated taste cells have access to the tastants but lack any synaptic contacts with axons, and a supportive rather than chemosensory function has been attributed to them. Until now, no functional data are available for these taste cells. To begin to understand their role in taste organ physiology, we have characterized with patch-clamp recording techniques the electrophysiological properties of a putative supporting cell—the so-called wing cell—isolated from frog taste disks. Wing cells were distinguished from chemosensory elements by the presence of a typical, sheet-like apical process. Their resting potential was approximately −52 mV, and the average input resistance was 4.8 GΩ. Wing cells possessed voltage-gated Na+currents sensitive to TTX, and an inactivating, voltage-gated K+current sensitive to TEA. Current injections elicited single action potentials but not repetitive firing. We found no evidence for voltage-gated Ca2+currents under various experimental conditions. Amiloride-sensitive Na+channels, thought to be involved in Na+chemotransduction, were present in wing cells. Many of the membrane properties of wing cells have been also reported for chemosensory taste cells. The presence of ion channels in wing cells might be suggestive of a role in controlling the microenvironment inside the taste organs or the functioning of chemosensory cells or both. In addition, they might participate directly in the sensory transduction events by allowing loop currents to flow inside the taste organs during chemostimulation.
Neuromodulators Enhance Transmitter Release by Two Separate Mechanisms at the Inhibitor of Crayfish Opener Muscle
Authors: Vyshedskiy Andrey , Delaney Kerry R. , Lin Jen-Wei
Published: April 2018

A presynaptic voltage control method has been used to investigate the modulatory effects of serotonin (5-HT) and okadaic acid (OA) on the inhibitory junction of the crayfish opener muscle. Instead of using action potentials, we used 20 msec pulses depolarized to 0 mV to activate transmitter release. This approach allowed us to monitor two separate physiological parameters related to the release process. The first parameter, transmitter release kinetics, is characterized as the delay when inhibitory postsynaptic conductance reaches its half-maximum (IPSG50). The second parameter, the total area of IPSG (IPSGarea), estimates total transmitter output. We have reported previously that the F2 component of synaptic facilitation is associated with a decrease in IPSG50but without a change in IPSGarea. These results raised the possibility that IPSG50and IPSGareacould be mediated by separate mechanisms that were modulated independently. To explore this possibility, we investigated the effects of 5-HT (100–200 nm) and OA (2.5 μm) on the two parameters. 5-HT and OA enhanced IPSG neither by changing the sensitivity of postsynaptic receptors, as tested by iontophoretically ejected GABA, nor by elevating resting and action potential-activated presynaptic free calcium, as monitored by fura-2 imaging. 5-HT and OA decreased IPSG50by 3.0 ± 1.4 and 3.6 ± 1.1 msec, respectively, and increased IPSGareaby 50 ± 21 and 37 ± 6%, respectively. The ability of F2 facilitation to accelerate release kinetics was reduced in the presence of the modulators, suggesting that the mechanism underlying the accelerated release kinetics was shared by the two modes of synaptic enhancement. This report demonstrates that the acceleration in release kinetics and the increase in total release are two separate mechanisms for enhancing transmitter output and that these two mechanisms can be activated without changes in presynaptic calcium dynamics.
Patterns of Arm Muscle Activation Involved in Octopus Reaching Movements
Authors: Gutfreund Yoram , Flash Tamar , Fiorito Graziano , Hochner Binyamin
Published: April 2018

The extreme flexibility of the octopus arm allows it to perform many different movements, yet octopuses reach toward a target in a stereotyped manner using a basic invariant motor structure: a bend traveling from the base of the arm toward the tip (Gutfreund et al., 1996a). To study the neuronal control of these movements, arm muscle activation [electromyogram (EMG)] was measured together with the kinematics of reaching movements. The traveling bend is associated with a propagating wave of muscle activation, with maximal muscle activation slightly preceding the traveling bend. Tonic activation was occasionally maintained afterward. Correlation of the EMG signals with the kinematic variables (velocities and accelerations) reveals that a significant part of the kinematic variability can be explained by the level of muscle activation. Furthermore, the EMG level measured during the initial stages of movement predicts the peak velocity attained toward the end of the reaching movement. These results suggest that feed-forward motor commands play an important role in the control of movement velocity and that simple adjustment of the excitation levels at the initial stages of the movement can set the velocity profile of the whole movement. A simple model of octopus arm extension is proposed in which the driving force is set initially and is then decreased in proportion to arm diameter at the bend. The model qualitatively reproduces the typical velocity profiles of octopus reaching movements, suggesting a simple control mechanism for bend propagation in the octopus arm.
Dopamine Inactivates Tryptophan Hydroxylase and Forms a Redox-Cycling Quinoprotein: Possible Endogenous Toxin to Serotonin Neurons
Authors: Kuhn Donald M. , Arthur Robert
Published: April 2018

Exposure of tryptophan hydroxylase (TPH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, to dopamine under mild oxidizing conditions (iron + H2O2) or in the presence of tyrosinase results in a concentration-dependent inactivation of the enzyme. Dopamine, iron, H2O2, or tyrosinase alone does not alter TPH activity. Similarly,N-acetyldopamine oxidized with one equivalent of sodium periodate causes a concentration-dependent inactivation of TPH as well. TPH is protected from dopamine-induced inactivation by reduced glutathione, ascorbic acid, and dithiothreitol but not by the radical scavengers DMSO, mannitol, or superoxide dismutase. Parallel studies with [3H]dopamine reveal a high negative correlation between inhibition of catalysis and incorporation of tritium into the enzyme. Those reducing agents and antioxidants that protect TPH from inactivation are effective in preventing the labeling of TPH by [3H]dopamine. Acid hydrolysis and HPLC with electrochemical detection (HPLC-EC) analysis of inactivated TPH revealed the formation of cysteinyl-dopamine residues within the enzyme. Exposure of dopamine-modified TPH to redox-cycling staining after SDS-PAGE confirmed the formation of a quinoprotein. These results indicate that dopamine-quinones covalently modify cysteinyl residues in TPH, leading directly to the loss of catalytic activity, and establish that TPH could be a target for dopamine-quinonesin vivoafter drugs (e.g., neurotoxic amphetamines) that cause dopamine-dependent inactivation of TPH. Redox cycling of a TPH-quinoprotein could also participate in the serotonin neuronal toxicity caused by these same drugs.
Expression Cloning and Characterization of NSIST, a Novel Sulfotransferase Expressed by a Subset of Neurons and Postsynaptic Targets
Authors: Nastuk Mary A. , Davis Samuel , Yancopoulos George D. , Fallon Justin R.
Published: April 2018

Synapses are distinguished by localized concentrations of specific proteins, many of which bear the marks of posttranslational processing such as glycosylation and sulfation. One strategy to elucidate this posttranslational tailoring is to identify the enzymes that create these modifications. Monoclonal antibody 3B3 recognizes a carbohydrate-containing epitope expressed on dystroglycan and other constituents ofTorpedoelectric organ synaptic membranes. We used mAb 3B3 in an immunofluorescence-based expression-cloning method and isolated a cDNA clone conferring mAb-3B3 immunoreactivity to transfected COS cells. The deduced polypeptide has a predicted molecular weight of 51 kDa, a type II transmembrane topology, and four potential N-linked glycosylation sites. The polypeptide, which we term NSIST (nervous system involved sulfotransferase), shows extensive, although not complete, homology to a chondroitin-6-sulfotransferase and limited homology to other sulfotransferases. In NSIST-transfected COS cells,35SO4incorporation and chondroitin-sulfate-like immunoreactivity are increased.In vivo, NSIST occurs as a single 2.4 kb transcript abundant inTorpedoelectric organ, moderately expressed in spinal cord and electric lobe, and undetectable in non-neural tissues. Immunohistochemistry shows that NSIST is expressed in a punctate distribution in the innervated portion of electrocytes. In the CNS, NSIST-like immunoreactivity is localized within the somas of motor neurons and neurons of the electromotor nucleus, whereas mAb-3B3 immunostaining is associated with cell surfaces and neuropil. Neuronal NSIST is therefore likely to exert its effects extracellularly; although NSIST is synthesized by neurons, its product, the 3B3 epitope, is found outside neuronal cell bodies. Our evidence indicates that NSIST participates in nervous system specific posttranslational modifications, perhaps including those at synapses.
Perforin-Dependent Neurologic Injury in a Viral Model of Multiple Sclerosis
Authors: Murray Paul D. , McGavern Dorian B. , Lin Xiaoqi , Njenga M. Kariuki , Leibowitz Julian , Pease Larry R. , Rodriguez Moses
Published: April 2018

In this study we demonstrate perforin-mediated cytotoxic effector function is necessary for viral clearance and may directly contribute to the development of neurologic deficits after demyelination in the Theiler’s murine encephalomyelitis virus (TMEV) model of multiple sclerosis. We previously demonstrated major histocompatability complex (MHC) class I-deficient (β2m-deficient) mice with an otherwise resistant genotype develop severe demyelination with minimal neurologic disease when chronically infected with TMEV. These studies implicate CD8+T cells as the pathogenic cell in the induction of neurologic disease after demyelination. To determine which effector mechanisms of CD8+T cells, granule exocytosis or Fas ligand expression, play a role in the development of demyelination and clinical disease, we infected perforin-deficient,lpr(Fas mutation), andgld(Fas ligand mutation) mice with TMEV. Perforin-deficient mice showed viral persistence in the CNS, chronic brain pathology, and demyelination in the spinal cord white matter. Perforin-deficient mice demonstrated severely impaired MHC class I-restricted cytotoxicity against viral epitopes, but normal MHC class II-restricted delayed-type hypersensitivity responses to virus antigen. Despite demyelination, virus-infected perforin-deficient mice showed only minimal neurologic deficits as indicated by clinical disease score, activity monitoring, and footprint analysis. Perforin- and MHC class II-deficient mice (with functional CD8+T cells and perforin molecules and an H-2bhaplotype) had comparable demyelination and genotype, however, only the latter showed severe clinical disease.Gldandlprmice demonstrated normal TMEV-specific cytotoxicity and maintained resistance to TMEV-induced demyelinating disease. These studies implicate perforin release by CD8+T cells as a potential mechanism by which neurologic deficits are induced after demyelination.
Synaptic Competition during the Reformation of a Neuromuscular Map
Authors: Laskowski Michael B. , Colman Howard , Nelson Carla , Lichtman Jeff W.
Published: April 2018

We have been studying the mechanisms whereby pools of motor neurons establish a rostrocaudal bias in the position of their synapses in some skeletal muscles. The serratus anterior (SA) muscle of the rat displays a rostrocaudal topographic map before birth, and the topography is re-established after denervation. In this report, we explore the potential role of synaptic competition between innervating axons as a means of generating topographic specificity. We followed the progress of the reformation of this map in neonatal animals under conditions that enhanced the likelihood of observing synaptic competition. This was accomplished by forcing caudal axons to regenerate ahead of rostral axons onto a surgically reduced SA muscle. In this way, caudal (C7) motor neurons had unopposed access to vacated synaptic sites on the remaining rostral half of the SA before the return of the rostral (C6) axons. Intracellular recording revealed that 2 d after the second denervation, most of the reinnervated end plates contained only axons from the C7branch; the remaining reinnervated end plates received input from C6only or were multiply innervated by C6and C7axons. After 6 d, the pattern was reversed, with most end plates innervated exclusively by C6. After 17 d, axons from C6were the sole input to reinnervated end plates. During the transition from C7- to C6-dominated input, at end plates coinnervated by C6and C7axons, the average quantal content from C6was the same as that from C7; after 7 d, the quantal content of C6was greater than that of C7. We have thus developed an experimental situation in which the outcome of synaptic competition is predictable and can be influenced by the positional labels associated with axons from different levels in the spinal cord.
Morphologically Docked Synaptic Vesicles Are Reduced insynaptotagminMutants ofDrosophila
Authors: Reist Noreen E. , Buchanan JoAnn , Li Jing , DiAntonio Aaron , Buxton Elizabeth M. , Schwarz Thomas L.
Published: April 2018

Nerve terminal specializations include mechanisms for maintaining a subpopulation of vesicles in a docked, fusion-ready state. We have investigated the relationship between synaptotagmin and the number of morphologically docked vesicles by an electron microscopic analysis ofDrosophila synaptotagmin(syt) mutants. The overall number of synaptic vesicles in a terminal was reduced, although each active zone continued to have a cluster of vesicles in its vicinity. In addition, there was an increase in the number of large vesicles near synapses. Examining the clusters, we found that the pool of synaptic vesicles immediately adjacent to the presynaptic membrane, the pool that includes the docked population, was reduced to 24 ± 5% (means ± SEM) of control in thesytnullmutation.

To separate contributions of overall vesicle depletion and increased spontaneous release from direct effects of synaptotagmin on morphological docking, we examinedsytmutants in an altered genetic background. Recombiningsytalleles onto a second chromosome bearing an as yet uncharacterized mutation resulted in the expected decrease in evoked release but suppressed the increase in spontaneous release frequency. Motor nerve terminals in this genotype contained more synaptic vesicles than control, yet the number of vesicles immediately adjacent to the presynaptic membrane near active zones was still reduced (33 ± 4% of control).

Our findings demonstrate that there is a decrease in the number of morphologically docked vesicles seen insytmutants. The decreases in docking and evoked release are independent of the increase in spontaneous release. These results support the hypothesis that synaptotagmin stabilizes the docked state.
Requirement for Early-Generated Neurons Recognized by Monoclonal Antibody Lot1 in the Formation of Lateral Olfactory Tract
Authors: Sato Yasufumi , Hirata Tatsumi , Ogawa Masaharu , Fujisawa Hajime
Published: April 2018

During development, mitral cells, the main output neurons of the olfactory bulb, project axons into a very narrow part of the telencephalon and form an axonal bundle called the lateral olfactory tract (LOT). The present study shows that before the first mitral cell axons elongate, the LOT position is already marked with a subset of early-generated neurons that are recognized by monoclonal antibody lot1 (lot cells). Mitral cell axons choose the lot cell position for their growth pathway and maintain a close contact with the cells until LOT formation is completed. Ablation of lot cells prevented LOT formation in organotypic culture. These results suggest that lot cells are “guidepost cells” for mitral cell axons.
Spinal Cord Neuronal Precursors Generate Multiple Neuronal Phenotypes in Culture
Authors: Kalyani Anjali J. , Piper David , Mujtaba Tahmina , Lucero Mary T. , Rao Mahendra S.
Published: April 2018

Neuronal restricted precursors (NRPs) (Mayer-Proschel et al., 1997) can generate multiple neurotransmitter phenotypes during maturation in culture. Undifferentiated E-NCAM+(embryonic neural cell adhesion molecule) immunoreactive NRPs are mitotically active and electrically immature, and they express only a subset of neuronal markers. Fully mature cells are postmitotic, process-bearing cells that are neurofilament-M and synaptophysin immunoreactive, and they synthesize and respond to different subsets of neurotransmitter molecules. Mature neurons that synthesize and respond to glycine, glutamate, GABA, dopamine, and acetylcholine can be identified by immunocytochemistry, RT-PCR, and calcium imaging in mass cultures. Individual NRPs also generate heterogeneous progeny as assessed by neurotransmitter response and synthesis, demonstrating the multipotent nature of the precursor cells.

Differentiation can be modulated by sonic hedgehog (Shh) and bone morphogenetic protein (BMP)-2/4 molecules. Shh acts as a mitogen and inhibits differentiation (including cholinergic differentiation). BMP-2 and BMP-4, in contrast, inhibit cell division and promote differentiation (including cholinergic differentiation). Thus, a single neuronal precursor cell can differentiate into multiple classes of neurons, and this differentiation can be modulated by environmental signals.
Evidence That Excitatory Amino Acid Receptors within the Temporomandibular Joint Region Are Involved in the Reflex Activation of the Jaw Muscles
Authors: Cairns Brian E. , Sessle Barry J. , Hu James W.
Published: April 2018

We have previously shown that injection of the inflammatory irritant and small-fiber excitant mustard oil (MO) into the temporomandibular joint (TMJ) region can reflexively induce a prolonged increase in the activity of both digastric and masseter muscles in rats. It is possible that peripheral excitatory amino acid (EAA) receptors play a role in this effect, because MO-evoked increases in jaw muscle activity are attenuated by preapplication of the noncompetitive NMDA receptor antagonist MK-801 into the TMJ region. In the present study the EAA receptor agonists glutamate, NMDA, kainate, and AMPA were applied locally to the TMJ region. Jaw muscle responses similar to those evoked by MO application to the TMJ region were achieved with glutamate, NMDA, AMPA, and kainate. Repeated application of glutamate, NMDA, or AMPA at intervals of 30 min evoked responses in the ipsilateral jaw muscles that were of comparable magnitude. Co-application of the NMDA receptor antagonistDL-2-amino-5-phosphonovalerate (0.5 μmol) significantly reduced the magnitude of the glutamate- and NMDA-evoked ipsilateral jaw muscle responses without affecting responses evoked by AMPA. In contrast, co-application of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (1 nmol) significantly reduced the magnitude of the glutamate- and AMPA-evoked ipsilateral jaw muscle responses without affecting responses evoked by NMDA. This evidence suggests that both NMDA and non-NMDA EAA receptor types are located within the TMJ region and may contribute to jaw muscle activity that can be reflexively evoked from the TMJ region.
A Role for Tectal Midline Glia in the Unilateral Containment of Retinocollicular Axons
Authors: Wu Da-Yu , Schneider Gerald E. , Silver Jerry , Poston Michael , Jhaveri Sonal
Published: April 2018

Retinal fibers approach close to the tectal midline but do not encroach on the other side. Just before the entry of retinal axons into the superior colliculus (SC), a group of radial glia differentiates at the tectal midline; the spatiotemporal deployment of these cells points to their involvement in the unilateral containment of retinotectal axons.

To test for such a barrier function of the tectal midline cells, we used two lesion paradigms for disrupting their radial processes in the neonatal hamster: (1) a heat lesion was used to destroy the superficial layers of the right SC, including the midline region, and (2) a horizontally oriented hooked wire was inserted from the lateral edge of the left SC toward the midline and was used to undercut the midline cells, leaving intact the retinorecipient layers in the right SC. In both cases, the left SC was denervated by removing its contralateral retinal input. Animals were killed 12 hr to 2 weeks later, after intraocular injections of anterograde tracers to label the axons from the remaining eye. Both lesions resulted in degeneration of the distal processes of the tectal raphe glia and in an abnormal crossing of the tectal midline by retinal axons, leading to an innervation of the opposite (“wrong”) tectum. The crossover occurred only where glial cell attachments were disrupted.

These results document that during normal development, the integrity of the midline septum is critical in compartmentalizing retinal axons and in retaining the laterality of the retinotectal projection.
Correlative Ultrastructural Distribution of Neurotensin Receptor Proteins and Binding Sites in the Rat Substantia Nigra
Authors: Boudin H. , Pélaprat D. , Rostène W. , Pickel V. M. , Beaudet A.
Published: April 2018

Neurotensin (NT) produces various stimulatory effects on dopaminergic neurons of the rat substantia nigra. To gain insight into the subcellular substrate for these effects, we compared by electron microscopy the distribution of immunoreactive high-affinity NT receptor proteins (NTRH) with that of high-affinity125I-NT binding sites in this region of rat brain. Quantitative analysis showed a predominant association of immunogold and radioautographic labels with somata and dendrites of presumptive dopaminergic neurons, and a more modest localization in myelinated and unmyelinated axons and astrocytic leaflets. The distributions of immunoreactive NTRH and125I-NT binding sites along somatodendritic plasma membranes were highly correlated and homogeneous, suggesting that membrane-targeted NTRH proteins were functional and predominantly extrasynaptic. Abundant immunocytochemically and radioautographically labeled receptors were also detected inside perikarya and dendrites. Within perikarya, these were found in comparable proportions over membranes of smooth endoplasmic reticulum and Golgi apparatus, suggesting that newly synthesized receptor proteins already possess the molecular and conformational properties required for effective ligand binding. By contrast, dendrites showed a proportionally higher concentration of immunolabeled than radiolabeled intracellular receptors. A fraction of these immunoreactive receptors were found in endosomes, suggesting that they had undergone ligand-induced internalization and were under a molecular conformation and/or in a physical location that precluded their recognition by and/or access to exogenous ligand. Our results provide the first evidence that electron microscopic immunocytochemistry of the NT receptor identifies sites for both the binding and trafficking of NT in the substantia nigra.
α-Conotoxin AuIB Selectively Blocks α3β4 Nicotinic Acetylcholine Receptors and Nicotine-Evoked Norepinephrine Release
Authors: Luo Siqin , Kulak Jennifer M. , Cartier G. Edward , Jacobsen Richard B. , Yoshikami Doju , Olivera Baldomero M. , McIntosh J. Michael
Published: April 2018

Neuronal nicotinic acetylcholine receptors (nAChRs) with putative α3β4-subunits have been implicated in the mediation of signaling in various systems, including ganglionic transmission peripherally and nicotine-evoked neurotransmitter release centrally. However, progress in the characterization of these receptors has been hampered by a lack of α3β4-selective ligands. In this report, we describe the purification and characterization of an α3β4 nAChR antagonist, α-conotoxin AuIB, from the venom of the “court cone,”Conus aulicus.We also describe the total chemical synthesis of this and two related peptides that were also isolated from the venom. α-Conotoxin AuIB blocks α3β4 nAChRs expressed inXenopusoocytes with an IC50of 0.75 μm, akonof 1.4 × 106min-1m−1, akoffof 0.48 min-1, and aKdof 0.5 μm. Furthermore, α-conotoxin AuIB blocks the α3β4 receptor with >100-fold higher potency than other receptor subunit combinations, including α2β2, α2β4, α3β2, α4β2, α4β4, and α1β1γδ. Thus, AuIB is a novel, selective probe for α3β4 nAChRs. AuIB (1–5 μm) blocks 20–35% of the nicotine-stimulated norepinephrine release from rat hippocampal synaptosomes, whereas nicotine-evoked dopamine release from striatal synaptosomes is not affected. Conversely, the α3β2-specific α-conotoxin MII (100 nm) blocks 33% of striatal dopamine release but not hippocampal norepinephrine release. This suggests that in the respective systems, α3β4-containing nAChRs mediate norepinephrine release, whereas α3β2-containing receptors mediate dopamine release.
Accumulation ofF-Spondinin Injured Peripheral Nerve Promotes the Outgrowth of Sensory Axons
Authors: Burstyn-Cohen Tal , Frumkin Ayala , Xu Yi-Tian , Scherer Steven S. , Klar Avihu
Published: April 2018

F-spondin, an extracellular matrix protein, is present in peripheral nerve during embryonic development, but its amount diminishes by birth. Axotomy of adult rat sciatic nerve, however, causes a massive upregulation of both F-spondin mRNA and protein distal to the lesion. F-spondin in the distal stump of axotomized nerve promotes neurite outgrowth of sensory neurons, as revealed by protein neutralization with F-spondin-specific antibodies. Thus, F-spondin is likely to play a role in promoting axonal regeneration after nerve injury.
Spike-and-Wave Oscillations Based on the Properties of GABABReceptors
Authors: Destexhe Alain
Published: April 2018

Neocortical and thalamic neurons are involved in the genesis of generalized spike-and-wave (SW) epileptic seizures. The cellular mechanism of SW involves complex interactions between intrinsic neuronal firing properties and multiple types of synaptic receptors, but because of the complexity of these interactions the exact details of this mechanism are unclear. In this paper these types of interactions were investigated by using biophysical models of thalamic and cortical neurons. It is shown first that, because of the particular activation properties of GABABreceptor-mediated responses, simulated field potentials can display SW waveforms if cortical pyramidal cells and interneurons generate prolonged discharges in synchrony, without any other assumptions. Here the “spike” component coincided with the synchronous firing, whereas the “wave” component was generated mostly by slow GABAB-mediated K+currents. Second, the model suggests that intact thalamic circuits can be forced into a ∼3 Hz oscillatory mode by corticothalamic feedback. Here again, this property was attributable to the characteristics of GABAB-mediated inhibition. Third, in the thalamocortical system this property can lead to generalized ∼3 Hz oscillations with SW field potentials. The oscillation consisted of a synchronous prolonged firing in all cell types, interleaved with a ∼300 msec period of neuronal silence, similar to experimental observations during SW seizures. This model suggests that SW oscillations can arise from thalamocortical loops in which the corticothalamic feedback indirectly evokes GABAB-mediated inhibition in the thalamus. This mechanism is shown to be consistent with a number of different experimental models, and experiments are suggested to test its consistency.
Regulation of Intrinsic and Synaptic Properties of Neonatal Rat Trigeminal Motoneurons by Metabotropic Glutamate Receptors
Authors: Del Negro Christopher A. , Chandler Scott H.
Published: April 2018

We studied how metabotropic glutamate receptor (mGluR) activation modifies the synaptic and intrinsic membrane properties of neonatal rat trigeminal motoneurons using the broad-spectrum mGluR agonist (1S,3R)-1-amino-1,3-cyclopentane-dicarboxylic acid [(1S,3R)-ACPD], group I/II antagonist (±)-α-methyl-4-carboxy-phenylglycine (MCPG), and group III agonistl-2-amino-4-phosphonobutanoate (l-AP4). (1S,3R)-ACPD depressed excitatory transmission to trigeminal motoneurons presynaptically and postsynaptically via presynaptic inhibition and by reducing the currents carried by ionotropic glutamate receptors selective for AMPA. (1S,3R)-ACPD also depolarized trigeminal motoneurons and increased input resistance by suppressing a Ba2+-sensitive leakage K+current. These effects were not mimicked byl-AP4 (100–200 μm). High-threshold Ca2+currents were also suppressed by (1S,3R)-ACPD. Repetitive stimulation of excitatory premotoneurons mimicked the postsynaptic effects of (1S,3R)-ACPD. The postsynaptic effects of (1S,3R)-ACPD and repetitive stimulation were both antagonized by MCPG, suggesting that mGluRs were similarly activated in both experiments. We conclude that mGluRs can be recruited endogenously by glutamatergic premotoneurons and that mGluR-mediated depression of excitatory transmission, combined with increased postsynaptic excitability, enhances thesignal-to-noise ratioof oral-related synaptic input to trigeminal motoneurons during rhythmical jaw movements.