bluuebunny/crossref_metadata_2025 huggingface dataset:
Cupidin, an Isoform of Homer/Vesl, Interacts with the Actin Cytoskeleton and Activated Rho Family Small GTPases and Is Expressed in Developing Mouse Cerebellar Granule Cells
Authors: Shiraishi Yoko , Mizutani Akihiro , Bito Haruhiko , Fujisawa Kazuko , Narumiya Shuh , Mikoshiba Katsuhiko , Furuichi Teiichi
Published: April 2018

A developmentally regulated Homer/Vesl isoform, Cupidin (Homer 2a/Vesl-2Δ11), was isolated from postnatal mouse cerebellum using a fluorescent differential display strategy. The strongest expression of Cupidin was detected in the cerebellar granule cells at approximately postnatal day 7. Cupidin was enriched in the postsynaptic density fraction, and its immunoreactivity was concentrated at glomeruli of the inner granular layer when active synaptogenesis occurred. Cupidin protein could be divided into two functional domains: the N-terminal portion, which was highly conserved among Homer/Vesl family proteins, and the C-terminal portion, which consisted of a putative coiled-coil structure, including several leucine zipper motifs. The N-terminal fragment of Cupidin, which was able to associate with metabotropic glutamate receptor 1 (mGluR1), also interacted with F-actinin vitro. In keeping with this, F-actin immunocytochemically colocalized with Cupidin in cultured cerebellar granule cells, and a Cupidin–mGluR1–actin complex was immunoprecipitated from crude cerebellar lysates using an anti-Cupidin antibody. On the other hand, the C-terminal portion of Cupidin bound to Cdc42, a member of Rho family small GTPases, in a GTP-dependent mannerin vitro, and Cupidin functionally interacted with activated-Cdc42 in a heterologous expression system. Together, our findings indicate that Cupidin may serve as a postsynaptic scaffold protein that links mGluR signaling with actin cytoskeleton and Rho family proteins, perhaps during the dynamic phase of morphological changes that occur during synapse formation in cerebellar granule cells.
Neuron–Glia Signaling via α1Adrenoceptor-Mediated Ca2+Release in Bergmann Glial CellsIn Situ
Authors: Kulik Anna , Haentzsch Antje , Lückermann Mark , Reichelt Winfried , Ballanyi Klaus
Published: April 2018

Adrenoceptors were among the first neurotransmitter receptors identified in glial cells, but it is not known whether these receptors meditate glial responses during neuronal activity. We show that repetitive nerve activity evoked a rise of intracellular calcium in Bergmann glia and neighboring Purkinje neurons of cerebellar slices of mice. The glial but not the neuronal calcium transient persisted during block of ionotropic and metabotropic glutamate receptors. In contrast, the glial calcium response was abolished by cyclopiazonic acid and prazosin; however, prazosin affected neither the inward current nor the resulting depolarization that accompanied the stimulus-induced glial calcium transients. The glial depolarization was attenuated by 38% by the mixture of glutamate receptor blockers, which abolished the evoked neuronal depolarization and afterhyperpolarization. Ba2+reduced the glial currents by 66% without affecting the concomitant calcium transients. In the presence of Ba2+, the mixture of glutamate receptor blockers exerted no effect on the glial inward current or calcium rise. Furthermore, Ba2+greatly potentiated both the activity-related Purkinje cell inward current and the accompanying neuronal calcium rises. The results indicate that release of noradrenaline from afferent fibers activates a glial α1adrenoceptor that promotes calcium release from intracellular stores. Glial calcium rises are known to stimulate a diversity of processes such as transmitter release, energy metabolism, or proliferation. Thus the adrenoceptor-mediated mechanism described here is well suited for feedback modulation of neuronal function that is independent of glutamate.
Fibroblast Growth Factor-2 Activates a Latent Neurogenic Program in Neural Stem Cells from Diverse Regions of the Adult CNS
Authors: Palmer Theo D. , Markakis Eleni A. , Willhoite Andrew R. , Safar Frank , Gage Fred H.
Published: April 2018

During development of the mammalian brain, both neurons and glia are generated from multipotent neural stem cells. Although neurogenesis ceases in most areas at birth, stem cells continue to generate neurons within the subventricular zone and hippocampal dentate gyrus throughout adult life. In this work, we provide the first demonstration that precursors native to regions of the adult brain that generate only glia can also generate neurons after exposure to FGF-2in vitro. When progenitors isolated from hippocampal tissue were directly compared with cells isolated from the neocortex,both populations were able to initiate a program of proliferative neurogenesis. Genetic marking and lineage analysis showed that a majority of the cells able to generate neurons were multipotent precursors; however, progeny from these precursors acquired the competence to differentiate into neurons only after exposure to FGF-2. The recruitment of similar FGF-2-responsive cells from the adult optic nerve, a structure well isolated from the neurogenic zones within the brain, confirmed that neuron-competent precursors naturally exist in widely divergent tissues of the adult brain.
Ca2+-Permeable AMPA Receptors and Spontaneous Presynaptic Transmitter Release at Developing Excitatory Spinal Synapses
Authors: Rohrbough Jeffrey , Spitzer Nicholas C.
Published: April 2018

At many mature vertebrate glutamatergic synapses, excitatory transmission strength and plasticity are regulated by AMPA and NMDA receptor (AMPA-R and NMDA-R) activation and by patterns of presynaptic transmitter release. Both receptors potentially direct neuronal differentiation by mediating postsynaptic Ca2+influx during early development. However, the development of synaptic receptor expression and colocalization has been examined developmentally in only a few systems, and changes in release properties at neuronal synapses have not been characterized extensively. We recorded miniature EPSCs (mEPSCs) from spinal interneurons inXenopusembryos and larvae. In mature 5–8 d larvae, ∼70% of mEPSCs in Mg2+-free saline are composed of both a fast AMPA-R-mediated component and a slower NMDA-R-mediated decay, indicating receptor colocalization at most synapses. By contrast, in 39–40 hr embryos ∼65% of mEPSCs are exclusively fast, suggesting that these synapses initially express predominantly AMPA-R. In a physiological Mg2+concentration (1 mm), mEPSCs throughout development are mainly AMPA-R-mediated at negative potentials. Embryonic synaptic AMPA-R are highly Ca2+-permeable, mEPSC amplitude is over twofold larger than at mature synapses, and mEPSCs frequently occur in bursts consistent with asynchronous multiquantal release. AMPA-R function in this motor pathway thus appears to be independent of previous NMDA-R activation, unlike other regions of the developing nervous system, ensuring a greater reliability for embryonic excitatory transmission. Early spontaneous excitatory activity is specialized to promote AMPA-R-mediated synaptic Ca2+influx, which likely has significant roles in neuronal development.
GABABReceptors Inhibit Mechanosensitivity of Primary Afferent Endings
Authors: Page Amanda J. , Blackshaw L. Ashley
Published: April 2018

The modulatory effects of baclofen on the sensitivity of peripheral afferent endings to mechanical stimulation were investigated using anin vitroferret gastroesophageal vagal afferent preparation. Changes in sensitivity of three types of gastroesophageal vagal afferent endings previously categorized as mucosal, tension, and tension–mucosal (TM) receptors according to their mechanoreceptive field characteristics were investigated. Baclofen (30–200 μm) dose dependently reduced responses of mucosal afferents to mucosal stroking with calibrated von Frey hairs (10–1000 mg). This was reversed by the GABABreceptor antagonist SCH50911 (1 μm). TM afferent responses to mucosal stroking (10–1000 mg) were unaffected by baclofen (30–200 μm). However, baclofen (30–200 μm) significantly inhibited the response of 11 of 18 TM afferents to circumferential tension. This was reversed by SCH50911 (1 μm). Baclofen (100 and 200 μm) significantly inhibited the response of all tension receptor afferents to circumferential tension in the lower range (1–3 gm) but not in the higher range (4–7 gm). This inhibition was reversed by SCH50911 (1 μm;n= 3). This study provides the first direct evidence for the inhibitory modulation of peripheral mechanosensory endings by the G-protein-coupled GABABreceptor. Inhibition was dose-dependent, pharmacologically reversible, and selective to certain aspects of mechanosensitivity. These findings have important relevance to strategies for selective reduction of sensory input to the CNS at a peripheral site.
Reduced Synaptic Facilitation between Pyramidal Neurons in the Piriform Cortex After Odor Learning
Authors: Saar Drorit , Grossman Yoram , Barkai Edi
Published: April 2018

Learning-related cellular modifications were studied in the rat piriform cortex after operand conditioning. Rats were trained to discriminate positive cues in pairs of odors. In one experimental paradigm, rats were trained to memorize 35–50 pairs of odors (“extensive training”). In another paradigm, training was continued only until rats acquired the rule of the task, usually after learning the first two pairs of odors (“short training”). “Pseudotrained” and “naive” rats served as controls. We have previously shown that “rule learning” of this task was accompanied by reduced spike afterhyperpolarization in pyramidal neurons in brain slices of the piriform cortex. In the present study, synaptic inputs to the same cells were examined. Pairs of electrical stimuli applied to the intrinsic fibers that interconnect layer II pyramidal neurons revealed significant reduction in paired-pulse facilitation (PPF) in this pathway even after short training. PPF in shortly trained rats was reduced to the same extent as in extensively trained rats. PPF reduction did not result from modification of membrane properties in the postsynaptic cells, change in postsynaptic inhibition, or impairment of the facilitation mechanism. Extracellular field potential recordings showed enhanced synaptic transmission in these synapses. The reduction in PPF became apparent only 3 d after task acquisition and returned to control value 5 d later. PPF evoked by stimulating the afferent fibers to the same neurons was increased 1 d after training for 2 d. We suggest that the transient enhancement in connectivity in the intrinsic pathway is related to the enhanced learning capability and not to memory for specific odors, which lasts for weeks.
Circadian Rhythms in the Suprachiasmatic Nucleus are Temperature-Compensated and Phase-Shifted by Heat PulsesIn Vitro
Authors: Ruby Norman F. , Burns D. Erik , Heller H. Craig
Published: April 2018

Temperature compensation and the effects of heat pulses on rhythm phase were assessed in the suprachiasmatic nucleus (SCN). Circadian neuronal rhythms were recorded from the rat SCN at 37 and 31°Cin vitro. Rhythm period was 23.9 ± 0.1 and 23.7 ± 0.1 hr at 37 and 31°C, respectively; theQ10for tau was 0.99. Heat pulses were administered at various circadian times (CTs) by increasing SCN temperature from 34 to 37°C for 2 hr. Phase delays and advances were observed during early and late subjective night, respectively, and no phase shifts were obtained during midsubjective day. Maximum phase delays of 2.2 ± 0.3 hr were obtained at CT 14, and maximum phase advances of 3.5 ± 0.2 hr were obtained at CT 20. Phase delays were not blocked by a combination of NMDA [AP-5 (100 μm)] and non-NMDA [CNQX (10 μm)] receptor antagonists or by tetrodotoxin (TTX) at concentrations of 1 or 3 μm. The phase response curve for heat pulses is similar to ones obtained with light pulses for behavioral rhythms. These data demonstrate that circadian pacemaker period in the rat SCN is temperature-compensated over a physiological range of temperatures. Phase delays were not caused by activation of ionotropic glutamate receptors, release of other neurotransmitters, or temperature-dependent increases in metabolism associated with action potentials. Heat pulses may have phase-shifted rhythms by directly altering transcriptional or translational events in SCN pacemaker cells.
Neurotrophin-3 Contributes to the Initiation of Behavioral Sensitization to Cocaine by Activating the Ras/Mitogen-Activated Protein Kinase Signal Transduction Cascade
Authors: Pierce R. Christopher , Pierce-Bancroft Audrey F. , Prasad Balakrishna M.
Published: April 2018

These experiments were designed to assess the role of neurotrophins and the Ras/mitogen-activated protein kinase (MAP) signal transduction cascade in behavioral sensitization to cocaine. The first experiments evaluated the effect of three daily intra-ventral tegmental area (VTA) microinjections of neurotrophin-3 (NT-3) or brain-derived neurotrophic factor (BDNF) on the behavioral-activating effects of a subsequent challenge injection of cocaine in rats. Results indicated that, although NT-3 did not influence behavior across the three microinjection days, animals displayed a sensitized behavioral response to the subsequent cocaine challenge injection. In contrast, BDNF microinjections resulted in a progressive increase in behavioral activity but did not influence the subsequent behavioral response to cocaine. A second series of experiments assessed the effect of inhibiting the MAP kinase signal transduction cascade on the initiation of behavioral sensitization to cocaine. The MAP kinase kinase inhibitor PD98059, or its vehicle, was microinjected into the VTA before three daily cocaine injections. Although PD98059 did not influence the acute behavioral response to cocaine, it blocked sensitization. Finally, the effects of acute and repeated cocaine injections on NT-3 and BDNF mRNA levels in the VTA, substantia nigra, and hippocampus were assessed. Results indicated that an acute cocaine injection resulted in a transient increase in NT-3 mRNA levels in the VTA. Collectively, these results suggest that NT-3 contributes to the initiation of behavioral sensitization to cocaine by activating the Ras/MAP kinase signal transduction system. The present data also indicate that BDNF itself produced a progressive augmentation in behavioral activation with repeated administration.
The Presynaptic Calcium Channel Is Part of a Transmembrane Complex Linking a Synaptic Laminin (α4β2γ1) with Non-Erythroid Spectrin
Authors: Sunderland William J. , Son Young-Jin , Miner Jeffrey H. , Sanes Joshua R. , Carlson Steven S.
Published: April 2018

Nerve regeneration studies at the neuromuscular junction (NMJ) suggest that synaptic basal lamina components tell the returning axon where to locate neurotransmitter release machinery, including synaptic vesicle clusters and active zones. Good candidates for these components are the synaptic laminins (LNs) containing α4, α5, or β2 chains. Results from a β2 laminin knockout mouse have suggested a linkage of this extracellular laminin to cytosolic synaptic vesicle clusters. Here we report such a transmembrane link at the electric organ synapse, which is homologous to the NMJ. We immunopurified electric organ synaptosomes and found on their surface two laminins of 740 and 900 kDa. The 740 kDa laminin has a composition of α4β2γ1 (laminin-9). Immunostaining reveals that as in the NMJ, α4 and β2 chains are concentrated at the electric organ synapse. Using detergent-solubilized synaptosomes, we immunoprecipitated a complex containing α4β2γ1 laminin, the voltage-gated calcium channel, and the cytoskeletal protein spectrin. Other presynaptic proteins such as 900 kDa laminin are not found in this complex. We hypothesize that α4β2γ1 laminin in the synaptic basal lamina attaches to calcium channel, which in turn is attached to cytosolic spectrin. Spectrin could then organize synaptic vesicle clusters by binding vesicle-associated proteins.
Decoding Temporal Information: A Model Based on Short-Term Synaptic Plasticity
Authors: Buonomano Dean V.
Published: April 2018

In the current paper it is proposed that short-term plasticity and dynamic changes in the balance of excitatory–inhibitory interactions may underlie the decoding of temporal information, that is, the generation of temporally selective neurons. Our initial approach was to simulate excitatory–inhibitory disynaptic circuits. Such circuits were composed of a single excitatory and inhibitory neuron and incorporated short-term plasticity of EPSPs and IPSPs and slow IPSPs. We first showed that it is possible to tune cells to respond selectively to different intervals by changing the synaptic weights of different synapses in parallel. In other words, temporal tuning can rely on long-term changes in synaptic strength and does not require changes in the time constants of the temporal properties. When the units studied in disynaptic circuits were incorporated into a larger single-layer network, the units exhibited a broad range of temporal selectivity ranging from no interval tuning to interval-selective tuning. The variability in temporal tuning relied on the variability of synaptic strengths. The network as a whole contained a robust population code for a wide range of intervals. Importantly, the same network was able to discriminate simple temporal sequences. These results argue that neural circuits are intrinsically able to process temporal information on the time scale of tens to hundreds of milliseconds and that specialized mechanisms, such as delay lines or oscillators, may not be necessary.
Regulation of Spine Calcium Dynamics by Rapid Spine Motility
Authors: Majewska Ania , Tashiro Ayumu , Yuste Rafael
Published: April 2018

Dendritic spines receive most excitatory inputs in the CNS and compartmentalize calcium. Spines also undergo rapid morphological changes, although the function of this motility is still unclear. We have investigated the effect of spine movement on spine calcium dynamics with two-photon photobleaching of enhanced green fluorescent protein and calcium imaging of action potential-elicited transients in spines from layer 2/3 pyramidal neurons in mouse visual cortex slices. The elongation or retraction of the spine neck during spine motility alters the diffusional coupling between spine and dendrite and significantly changes calcium decay kinetics in spines. Our results demonstrate that the spine's ability to compartmentalize calcium is constantly changing.
Differential Mechanisms of Transmission at Three Types of Mossy Fiber Synapse
Authors: Toth Katalin , Suares Gregory , Lawrence J. Josh , Philips-Tansey Emily , McBain Chris J.
Published: April 2018

The axons of the dentate gyrus granule cells, the so-called mossy fibers, innervate their inhibitory interneuron and pyramidal neuron targets via both anatomically and functionally specialized synapses. Mossy fiber synapses onto inhibitory interneurons were comprised of either calcium-permeable (CP) or calcium-impermeable (CI) AMPA receptors, whereas only calcium-impermeable AMPA receptors existed at CA3 principal neuron synapses. In response to brief trains of high-frequency stimuli (20 Hz), pyramidal neuron synapses invariably demonstrated short-term facilitation, whereas interneuron EPSCs demonstrated either short-term facilitation or depression. Facilitation at all CI AMPA synapses was voltage independent, whereas EPSCs at CP AMPA synapses showed greater facilitation at −20 than at −80 mV, consistent with a role for the postsynaptic unblock of polyamines. At pyramidal cell synapses, mossy fiber EPSCs possessed marked frequency-dependent facilitation (commencing at stimulation frequencies >0.1 Hz), whereas EPSCs at either type of interneuron synapse showed only moderate frequency-dependent facilitation or underwent depression. Presynaptic metabotropic glutamate receptors (mGluRs) decreased transmission at all three synapse types in a frequency-dependent manner. However, after block of presynaptic mGluRs, transmission at interneuron synapses still did not match the dynamic range of EPSCs at pyramidal neuron synapses. High-frequency stimulation of mossy fibers induced long-term potentiation (LTP), long-term depression (LTD), or no change at pyramidal neuron synapses, interneuron CP AMPA synapses, and CI AMPA synapses, respectively. Induction of LTP or LTD altered the short-term plasticity of transmission onto both pyramidal cells and interneuron CP AMPA synapses by a mechanism consistent with changes in release probability. These data reveal differential mechanisms of transmission at three classes of mossy fiber synapse made onto distinct targets.
R-Type Ca2+Channels Are Coupled to the Rapid Component of Secretion in Mouse Adrenal Slice Chromaffin Cells
Authors: Albillos Almudena , Neher Erwin , Moser Tobias
Published: April 2018

Patch-clamp measurements of Ca2+currents and membrane capacitance were performed on slices of mouse adrenal glands, using the perforated-patch configuration of the patch-clamp technique. These recording conditions are much closer to thein vivosituation than those used so far in most electrophysiological studies in adrenal chromaffin cells (isolated cells maintained in culture and whole-cell configuration). We observed profound discrepancies in the quantities of Ca2+channel subtypes (P-, Q-, N-, and L-type Ca2+channels) described for isolated mouse chromaffin cells maintained in culture. Differences with respect to previous studies may be attributable not only to culture conditions, but also to the patch-clamp configuration used. Our experiments revealed the presence of a Ca2+channel subtype never before described in chromaffin cells, a toxin and dihydropyridine-resistant Ca2+channel with fast inactivation kinetics, similar to the R-type Ca2+channel described in neurons. This channel contributes 22% to the total Ca2+current and controls 55% of the rapid secretory response evoked by short depolarizing pulses. Our results indicate that R-type Ca2+channels are in close proximity with the exocytotic machinery to rapidly regulate the secretory process.
The Readily Releasable Pool of Vesicles in Chromaffin Cells Is Replenished in a Temperature-Dependent Manner and Transiently Overfills at 37°C
Authors: Dinkelacker Vera , Voets Thomas , Neher Erwin , Moser Tobias
Published: April 2018

Maturation of exocytic vesicles to the release-ready state is regulated by several factors, including intracellular calcium concentration ([Ca2+]int) and the state of protein phosphorylation. Here we investigated the effects of temperature on the recovery from depletion of the readily releasable pool (RRP) of vesicles in adrenal chromaffin cells. Exocytosis and [Ca2+]intwere monitored by combined membrane capacitance and fura-2 measurements. At higher temperatures, a faster pool refilling and a larger RRP size were observed. The time constants of the recovery from depletion ranged from 3.6 to 1.1 sec (22 and 37°C, respectively) yielding aQ10of 2.3. The changes of the Ca2+signal between the different temperatures could not account for the differences in recovery kinetics. At 32 and 37°C, we observed a transient overfilling of the RRP after pool depletion, which stands in clear contrast to the sustained secretory depression seen at lower temperatures. The overshoot in RRP size was very prominent in cells with lower basal [Ca2+]int, hence with a large difference between prestimulus and poststimulus [Ca2+]int. In cells with higher basal [Ca2+]int, the pool was larger under steady-state conditions but showed less overfilling on stimulation. We conclude that vesicle maturation is markedly accelerated at physiological temperature, thus allowing for a rapid adaptation of the pool size to the relatively short-lived Ca2+transient.
Quantifying the Independence of Human Finger Movements: Comparisons of Digits, Hands, and Movement Frequencies
Authors: Häger-Ross Charlotte , Schieber Marc H.
Published: April 2018

To determine whether other digits move when normal humans attempt to move just one digit, we asked 10 right-handed subjects to move one finger at a time while we recorded the motion of all five digits simultaneously with both a video motion analysis system and an instrumented glove. We quantified the independence of the digits to compare (1) the different digits, (2) the right versus the left hand, and (3) movements at a self-paced frequency versus externally paced movements at 3 Hz. We also quantified the degree to which motion occurred at the proximal, middle, or distal joint of each digit. Even when asked to move just one finger, normal human subjects produced motion in other digits. Movements of the thumb, index finger, and little finger typically were more highly individuated than were movements of the middle or ring fingers. Fingers of the dominant hand were not more independent than were those of the nondominant hand. Self-paced movements made at ∼2 Hz were more highly individuated than were externally paced movements at 3 Hz. Angular motion tended to be greatest at the middle joint of each digit, with increased angular motion at the proximal and distal joints during 3 Hz movements. Simultaneous motion of noninstructed digits may result in part from passive mechanical connections between the digits, in part from the organization of multitendoned finger muscles, and in part from distributed neural control of the hand.
Activation of Pontine and Medullary Motor Inhibitory Regions Reduces Discharge in Neurons Located in the Locus Coeruleus and the Anatomical Equivalent of the Midbrain Locomotor Region
Authors: Mileykovskiy Boris Y. , Kiyashchenko Lyudmila I. , Kodama Tohru , Lai Yuan-Yang , Siegel Jerome M.
Published: April 2018

Activation of the pontine inhibitory area (PIA) including the middle portion of the pontine reticular nucleus, oral part (PnO), or the gigantocellular reticular nucleus (Gi) suppresses muscle tone in decerebrate animals. The locus coeruleus (LC) and midbrain locomotor region (MLR) have been implicated in the facilitation of muscle tone. In the current study we investigated whether PIA and Gi stimulation causes changes in activity in these brainstem motor facilitatory systems. PIA stimulation evoked bilateral muscle tone suppression and inhibited 26 of 28 LC units and 33 of 36 tonically active units located in the anatomical equivalent of the MLR (caudal half of the cuneiform nucleus and the pedunculopontine tegmental nucleus). Gi stimulation evoked bilateral suppression of hindlimb muscle tone and inhibited 20 of 35 LC units and 24 of 24 neurons located in the MLR as well as facilitated 11 of 35 LC units. GABA and glycine release in the vicinity of LC was increased by 20–40% during ipsilateral PnO stimulation inducing hindlimb muscle tone suppression on the same side of the body. We conclude that activation of pontine and medullary inhibitory regions produces a coordinated reduction in the activity of the LC units and neurons located in the MLR related to muscle tone facilitation. The linkage between activation of brainstem motor inhibitory systems and inactivation of brainstem facilitatory systems may underlie the reduction in muscle tone in sleep as well as the modulation of muscle tone in the isolated brainstem.
Water Permeability of Cochlear Outer Hair Cells: Characterization and Relationship to Electromotility
Authors: Belyantseva Inna A. , Frolenkov Gregory I. , Wade James B. , Mammano Fabio , Kachar Bechara
Published: April 2018

The distinguishing feature of the mammalian outer hair cells (OHCs) is to elongate and shorten at acoustic frequencies, when their intracellular potential is changed. This “electromotility” or “electromechanics” depends critically on positive intracellular pressure (turgor), maintained by the inflow of water through yet uncharacterized water pathways. We measured the water volume flow,Jv, across the plasma membrane of isolated guinea pig and rat OHCs after osmotic challenges and estimated the osmotic water permeability coefficient,Pf, to be ∼10−2cm/sec. This value is within the range reported for osmotic flow mediated by the water channel proteins, aquaporins.Jvwas inhibited by HgCl2, which is known to block aquaporin-mediated water transport.Pfvalues that were estimated for OHCs from neonatal rats were of the order of ∼2×10−3cm/sec, equivalent to that of membranes lacking water channel proteins. In an immunofluorescence assay we showed that an anti-peptide antibody specific for aquaporins labels the lateral plasma membrane of the OHC in the region in which electromotility is generated. Using patch-clamp recording, we found that water influx into the OHC is regulated by intracellular voltage. We also found that the most pronounced increases of the electromotility-associated charge movement and of the expression of OHC water channels occur between postnatal days 8 and 12, preceding the onset of hearing function in the rat. Our data indicate that electromotility and water transport in OHCs may influence each other structurally and functionally.
Origin Sites of Calcium Release and Calcium Oscillations in Frog Sympathetic Neurons
Authors: McDonough Stefan I. , Cseresnyés Zoltán , Schneider Martin F.
Published: April 2018

In many neurons, Ca2+signaling depends on efflux of Ca2+from intracellular stores into the cytoplasm via caffeine-sensitive ryanodine receptors (RyRs) of the endoplasmic reticulum. We have used high-speed confocal microscopy to image depolarization- and caffeine-evoked increases in cytoplasmic Ca2+levels in individual cultured frog sympathetic neurons. Although caffeine-evoked Ca2+wave fronts propagated throughout the cell, in most cells the initial Ca2+release was from one or more discrete sites that were several micrometers wide and located at the cell edge, even in Ca2+-free external solution. During cell-wide cytoplasmic [Ca2+] oscillations triggered by continual caffeine application, the initial Ca2+release that began each Ca2+peak was from the same subcellular site or sites. The Ca2+wave fronts propagated with constant amplitude; the spread was mostly via calcium-induced calcium release. Propagation was faster around the cell periphery than radially inward. Local Ca2+levels within the cell body could increase or decrease independently of neighboring regions, suggesting independent action of spatially separate Ca2+stores. Confocal imaging of fluorescent analogs of ryanodine and thapsigargin, and of MitoTracker, showed potential structural correlates to the patterns of Ca2+release and propagation. High densities of RyRs were found in a ring around the cell periphery, mitochondria in a broader ring just inside the RyRs, and sarco-endoplasmic reticulum Ca2+ATPase pumps in hot spots at the cell edge. Discrete sites at the cell edge primed to release Ca2+from intracellular stores might preferentially convert Ca2+influx through a local area of plasma membrane into a cell-wide Ca2+increase.
Impaired Fast-Spiking, Suppressed Cortical Inhibition, and Increased Susceptibility to Seizures in Mice Lacking Kv3.2 K+Channel Proteins
Authors: Lau David , de Miera Eleazar Vega-Saenz , Contreras Diego , Ozaita Ander , Harvey Michael , Chow Alan , Noebels Jeffrey L. , Paylor Richard , Morgan James I. , Leonard Christopher S. , Rudy Bernardo
Published: April 2018

Voltage-gated K+channels of the Kv3 subfamily have unusual electrophysiological properties, including activation at very depolarized voltages (positive to −10 mV) and very fast deactivation rates, suggesting special roles in neuronal excitability. In the brain, Kv3 channels are prominently expressed in select neuronal populations, which include fast-spiking (FS) GABAergic interneurons of the neocortex, hippocampus, and caudate, as well as other high-frequency firing neurons. Although evidence points to a key role in high-frequency firing, a definitive understanding of the function of these channels has been hampered by a lack of selective pharmacological tools. We therefore generated mouse lines in which one of the Kv3 genes,Kv3.2, was disrupted by gene-targeting methods. Whole-cell electrophysiological recording showed that the ability to fire spikes at high frequencies was impaired in immunocytochemically identified FS interneurons of deep cortical layers (5-6) in which Kv3.2 proteins are normally prominent. No such impairment was found for FS neurons of superficial layers (2-4) in which Kv3.2 proteins are normally only weakly expressed. These data directly support the hypothesis that Kv3 channels are necessary for high-frequency firing. Moreover, we found that Kv3.2 −/− mice showed specific alterations in their cortical EEG patterns and an increased susceptibility to epileptic seizures consistent with an impairment of cortical inhibitory mechanisms. This implies that, rather than producing hyperexcitability of the inhibitory interneurons, Kv3.2 channel elimination suppresses their activity. These data suggest that normal cortical operations depend on the ability of inhibitory interneurons to generate high-frequency firing.
Dominant Role of the Cytosolic C-Terminal Domain of the Rat 5-HT1BReceptor in Axonal–Apical Targeting
Authors: Jolimay Nicolas , Franck Louis , Langlois Xavier , Hamon Michel , Darmon Michèle
Published: April 2018

The 5-HT1Aand 5-HT1Breceptors for serotonin exhibit a different membrane localization to either soma and dendrites (5-HT1AR) or axons and terminals (5-HT1BR) of neurons in the CNS. The mechanisms responsible for their differential targeting were investigated previously by transfecting various 5-HT1AR/5-HT1BR chimeras in the epithelial Lilly pork kidney (LLC-PK1) cell line. This first study suggested that a specific targeting signal is located in the C-terminal portion (comprising the last two transmembrane and the cytoplasmic C-terminal domains) of the 5-HT1Aand/or 5-HT1Breceptors. In the present study, the role of the cytosolic C-terminal tail of the receptors was further investigated by transfecting truncated receptors and 5-HT1AR/5-HT1BR chimeras in both the epithelial LLC-PK1 cells and rat hippocampal neurons in primary culture. Confocal microscopic analysis of immunofluorescence with specific anti-5-HTR antibodies and anti-microtubule-associated protein 2 or anti-neurofilament 200k antibodies showed that substitution of the cytosolic C-terminal tail of the 5-HT1BR in the 5-HT1AR addressed the resulting chimera to the axon of neurons and to the apical domain of LLC-PK1 cells. Therefore, the short tail of the 5-HT1BR presents an apical targeting signal that can also act as an axonal targeting signal. In addition, a domain within the third intracytoplasmic loop of the 5-HT1BR, responsible for its Golgi sequestration in LLC-PK1 cells, appeared to act as another axonal targeting signal in hippocampal neurons.
Specialized Synapse-Associated Structures within the Calyx of Held
Authors: Rowland Kevin C. , Irby Nancy K. , Spirou George A.
Published: April 2018

The calyx of Held exhibits fast glutamatergic neurotransmission at high rates with low temporal jitter and has adapted specialized synaptic mechanisms to support its functional demands. We report the presence in calyces of an atypical arrangement of subcellular organelles, called the mitochondria-associated adherens complex (MAC). We demonstrate that MACs are located adjacent to synapses and contain membranous elements linked with coated and uncoated vesicles. Mitochondria that form MACs have more complex geometries than other mitochondria within the calyx and can extend between clusters of synapses. We estimate that the calyx contains 1600 MACs, 2400 synapses, and 6200 readily releasable vesicles. We also identify synaptic vesicle endocytotic regions close to MACs and synapses and hypothesize that calyces are composed of multiple activity modules, each containing machinery for vesicle release and recycling.
Inhibitory Mechanism by Polysialic Acid for Lamina-Specific Branch Formation of Thalamocortical Axons
Authors: Yamamoto Nobuhiko , Inui Kayo , Matsuyama Yuki , Harada Akiko , Hanamura Kenji , Murakami Fujio , Ruthazer Edward S. , Rutishauser Urs , Seki Tatsunori
Published: April 2018

During development, thalamocortical axons form arbors primarily in layer 4 of the neocortex. This lamina-specific branch formation was studied in cultures of rat thalamic explants grown next to chemically fixed cortical slices. After a weekin vitro, thalamic axons formed branches specifically in the target layer of fixed cortical slices, regardless of the orientation of the ingrowth. Thisin vitrosystem permits a direct assessment of contributions of membrane-associated molecules to thalamic axon branch formation. To this end, the present study uses three enzymatic perturbations: chondroitinase, phosphatidylinositol phospholipase C, or the polysialic acid (PSA)-specific endoneuraminidase (endo N). With endo N pretreatment of cortex, the number of branch points was increased significantly, whereas branch tip length was decreased. In addition, the localization of branch points to the target layer was weakened considerably. These features of branch formation were not altered by the other two enzymatic treatments, except that branch tips were shortened by chondroitinase treatment to the same extent as in endo N treatment. These results suggest that membrane-bound components are involved in lamina-specific branch formation of thalamocortical axons, and in particular that PSA moieties contribute to laminar specificity by inhibiting branch emergence in inappropriate layers.
DCAMKL1 Encodes a Protein Kinase with Homology to Doublecortin that Regulates Microtubule Polymerization
Authors: Lin Peter T. , Gleeson Joseph G. , Corbo Joseph C. , Flanagan Lisa , Walsh Christopher A.
Published: April 2018

Doublecortin (DCX) is a microtubule-associated protein required for neuronal migration to the cerebral cortex. DCAMKL1 consists of an N terminus that is 65% similar to DCX throughout the entire length of DCX, but also contains an additional 360 amino acid C-terminal domain encoding a putative Ca2+/calmodulin-dependent protein kinase. The homology to DCX suggested that DCAMKL1 may regulate microtubules, as well as mediate a phosphorylation-dependent signal transduction pathway. Here we show that DCAMKL1 is expressed throughout the CNS and PNS in migrating neuronal populations and overlaps in its expression with DCX and microtubules. Purified DCAMKL1 associates with microtubules and stimulates polymerization of purified tubulin and the formation of aster-like microtubule structures. Overexpressed DCAMKL1 leads to striking microtubule bundling in cell lines and cultured primary neural cells. Time-lapse imaging of cells transfected with a DCAMKL1-green fluorescent protein fusion protein shows that the microtubules associated with the protein remain dynamic. DCAMKL1 also encodes a functional kinase capable of phosphorylating myelin basic protein and itself. However, elimination of the kinase activity of DCAMKL1 has no detectable effect on its microtubule polymerization activity. Because DCAMKL1 is coexpressed with DCX, the two proteins form a potentially mutually regulatory network linking calcium signaling and microtubule dynamics.
Origin of Synchronized Oscillations Induced by Neocortical DisinhibitionIn Vivo
Authors: Castro-Alamancos Manuel A.
Published: April 2018

During disinhibition, the neocortex generates synchronous activities. Block of GABAAreceptors in neocortex transforms cortical slow-wave oscillations into large-amplitude ∼1 Hz discharges consisting of a negative spike or multiple negative spikes riding on a positive wave. Further block of GABABreceptors in neocortex slows the discharges to ∼0.5 Hz and increments the number of negative spikes forming rhythmic ∼10 Hz neocortical oscillations. Although the thalamus responds robustly to these neocortical discharges, these are unaffected by thalamic inactivation using tetrodotoxin. Thus, an important problem relates to the origin of these activities within the neocortex. Current source density analysis and intracellular recordings revealed that the first negative spike in a discharge corresponded to a current sink that reflected a paroxysmal depolarizing shift (PDS) and could originate in the lower layers or in the upper layers. Regardless of the origin (upper or lower layer), the initial current sink always spreads to the same site in upper layer V–IV. In contrast, the ∼10 Hz oscillation that follows the initial negative spike corresponds to current sinks that always originate in the lower layers but do not spread to upper layer V–IV, jumping directly to the upper layers. Each current sink in the ∼10 Hz oscillation reflects a small PDS and is followed by a current source that reflects the repolarization after each PDS.
The Nigrostriatal Dopaminergic System as a Preferential Target of Repeated Exposures to Combined Paraquat and Maneb: Implications for Parkinson's Disease
Authors: Thiruchelvam Mona , Richfield Eric K. , Baggs Raymond B. , Tank Arnold W. , Cory-Slechta Deborah A.
Published: April 2018

Experimental evidence supporting 1,1′-dimethyl-4,4′-bipyridinium [paraquat (PQ)] as a risk factor for Parkinson's disease (PD) is equivocal. Other agricultural chemicals, including dithiocarbamate fungicides such as manganese ethylenebisdithiocarbamate [maneb (MB)], are widely used in the same geographical regions as paraquat and also impact dopamine systems, suggesting that mixtures may be more relevant etiological models. This study therefore proposed that combined PQ and MB exposures would produce greater effects on dopamine (DA) systems than would either compound administered alone. Male C57BL/6 mice were treated twice a week for 6 weeks with intraperitoneal saline, 10 mg/kg paraquat, 30 mg/kg maneb, or their combination (PQ + MB). MB, but not PQ, reduced motor activity immediately after treatment, and this effect was potentiated by combined PQ + MB treatment. As treatments progressed, only the combined PQ + MB group evidenced a failure of motor activity levels to recover within 24 hr. Striatal DA and dihydroxyphenylacetic acid increased 1–3 d and decreased 7 d after injections. Only PQ + MB reduced tyrosine hydroxylase (TH) and DA transporter immunoreactivity and did so in dorsal striatum but not nucleus accumbens. Correspondingly, striatal TH protein levels were decreased only by combined PQ + MB 5 d after injection. Reactive gliosis occurred only in response to combined PQ + MB in dorsal–medial but not ventral striatum. TH immunoreactivity and cell counts were reduced only by PQ + MB and in the substantia nigra but not ventral tegmental area. These synergistic effects of combined PQ + MB, preferentially expressed in the nigrostriatal DA system, suggest that such mixtures could play a role in the etiology of PD.
Antinociceptive Action of Nitrous Oxide Is Mediated by Stimulation of Noradrenergic Neurons in the Brainstem and Activation of α2BAdrenoceptors
Authors: Sawamura Shigehito , Kingery Wade S. , Davies M. Frances , Agashe Geeta S. , Clark J. David , Kobilka Brian K. , Hashimoto Toshizaku , Maze Mervyn
Published: April 2018

Although nitrous oxide (N2O) has been used to facilitate surgery for >150 years, its molecular mechanism of action is not yet defined. Having established that N2O-induced release of norepinephrine mediates the analgesic action at α2adrenoceptors in the spinal cord, we now investigated whether activation of noradrenergic nuclei in the brainstem is responsible for this analgesic action and which α2adrenoceptor subtype mediates this property. In rats, Fos immunoreactivity was examined in brainstem noradrenergic nuclei after exposure to nitrous oxide. After selective lesioning of noradrenergic nuclei by intracerebroventricular application of the mitochondrial toxin saporin, coupled to the antibody directed against dopamine β hydroxylase (DβH–saporin), the analgesic and sedative actions of N2O were determined. Null mice for each of the three α2adrenoceptor subtypes (α2A, α2B, and α2C), and their wild-type cohorts, were tested for their antinociceptive and sedative response to N2O. Exposure to N2O increased expression of Fos immunoreactivity in each of the pontine noradrenergic nuclei (A5, locus coeruleus, and A7). DβH–saporin treatment eliminated nearly all of the catecholamine-containing neurons in the pons and blocked the analgesic but not the sedative effects of N2O. Null mice for the α2Badrenoceptor subtype exhibited a reduced or absent analgesic response to N2O, but their sedative response to N2O was intact. Our results support a pivotal role for noradrenergic pontine nuclei and α2Badrenoceptors in the analgesic, but not the sedative effects of N2O. Previously we demonstrated that the analgesic actions of α2adrenoceptor agonists are mediated by the α2Asubtype; taken together with these data we propose that exogenous and endogenous α2adrenoceptor ligands activate different α2adrenoceptor subtypes to produce their analgesic action.
Cannabinoid Withdrawal Syndrome Is Reduced in Pre-Proenkephalin Knock-Out Mice
Authors: Valverde Olga , Maldonado Rafael , Valjent Emmanuel , Zimmer Anne M. , Zimmer Andreas
Published: April 2018

The functional interactions between the endogenous cannabinoid and opioid systems were evaluated in pre-proenkephalin-deficient mice. Antinociception induced in the tail-immersion test by acute Δ9-tetrahydrocannabinol was reduced in mutant mice, whereas no difference between genotypes was observed in the effects induced on body temperature, locomotion, or ring catalepsy. During a chronic treatment with Δ9-tetrahydrocannabinol, the development of tolerance to the analgesic responses induced by this compound was slower in mice lacking enkephalin. In addition, cannabinoid withdrawal syndrome, precipitated in Δ9-tetrahydrocannabinol-dependent mice by the injection of SR141716A, was significantly attenuated in mutant mice. These results indicate that the endogenous enkephalinergic system is involved in the antinociceptive responses of Δ9-tetrahydrocannabinol and participates in the expression of cannabinoid abstinence.
Neural Responses in the Retinotopic Representation of the Blind Spot in the Macaque V1 to Stimuli for Perceptual Filling-In
Authors: Komatsu Hidehiko , Kinoshita Masaharu , Murakami Ikuya
Published: April 2018

When visual stimuli that cover the entire blind spot are presented monocularly, the color and brightness of the surrounding field are seen within the blind spot, although it receives no retinal input. Important questions about such perceptual filling-in are whether neurons in the visual system representing visual field locations within the blind spot are activated when filling-in occurs and, if so, what the properties of these neurons are. To address these questions, we recorded the activities of single neurons in the primary visual cortex (V1) of the awake monkey. We first identified the area of V1 representing the region of the blind spot and then assessed neural responses to stationary visual stimuli of various size. We found that there are neurons in layer 4 and deeper laminae, particularly layer 6, that respond to large stimuli covering the blind spot which induces perceptual filling-in. Most of these neurons had very large binocular receptive fields that extended outside the blind spot. These neurons also preferred relatively large stimuli and exhibited color selectivity. These results indicate that when a large uniform surface is presented on the blind spot, neurons at the V1 region representing the blind spot transmit signals essential for filling-in that inform of the presence of a large surface as well as the absence of smaller stimuli at the blind spot.