bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Domains and Amino Acids Involved in GluR7 Ion Channel Function
Authors: Strutz Nathalie , Villmann Carmen , Thalhammer Agnes , Kizelsztein Pablo , Eisenstein Miriam , Teichberg Vivian I. , Hollmann Michael
Published: April 2018

The kainate receptors GluR6 and GluR7 differ considerably in their ion channel properties, despite sharing 86% amino acid sequence identity. When expressed inXenopusoocytes GluR6 conducts large agonist-evoked currents, whereas GluR7 lacks measurable currents. In the present study, we localized the determinants that are responsible for the functional differences between GluR6 and GluR7 to the extracellular loop domain L3. In addition, we generated several GluR7 point mutants that are able to conduct currents that can be readily measured inXenopusoocytes.

In GluR6, glutamate- and kainate-evoked maximal currents are of the same magnitude when desensitization is inhibited with the lectin concanavalin A. By contrast, all functional GluR7 mutants were found to have glutamate current amplitudes significantly larger than those evoked by kainate. We localized the domain that determines the relative agonist efficacies to the C-terminal half of the L3 domain of GluR7.

Our data show that EC50values for glutamate (but not for kainate) in GluR7 mutants or chimeras tend to be increased in comparison to the EC50values in GluR6. The high EC50for wild-type GluR7 reported in the literature appears to be linked to the S1 portion of the agonist-binding domain.

Finally, we determined the C-terminal half of the L3 domain plus the far C-terminal domain of GluR7 to be responsible for the recently reported reduction of current amplitude seen when GluR7 is coexpressed with GluR6. We conclude that coexpression of GluR6 and GluR7 leads to nonstochastical assembly of heteromeric receptor complexes.
Differential Regulation of Mitogen-Activated Protein Kinases ERK1/2 and ERK5 by Neurotrophins, Neuronal Activity, and cAMP in Neurons
Authors: Cavanaugh Jane E. , Ham James , Hetman Michal , Poser Steve , Yan Chen , Xia Zhengui
Published: April 2018

Activation of the extracellular signal-regulated kinase 1 (ERK1) and ERK2 by neurotrophins, neuronal activity, or cAMP has been strongly implicated in differentiation, survival, and adaptive responses of neurons during development and in the adult brain. Recently, a new member of the mitogen-activated protein (MAP) kinase family, ERK5, was discovered. Like ERK1 and ERK2, ERK5 is expressed in neurons, and ERK5 stimulation by epidermal growth factor is blocked by the MAP kinase/ERK kinase 1 (MEK1) inhibitors PD98059 and U0126. This suggests the interesting possibility that some of the functions attributed to ERK1/2 may be mediated by ERK5. However, the regulatory properties of ERK5 in primary cultured neurons have not been reported. Here we examined the regulation of ERK5 signaling in primary cultured cortical neurons. Our data demonstrate that, similar to ERK1/2, ERK5 is activated by neurotrophins including brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4. BDNF stimulation of ERK5 required the activity of MEK5. Surprisingly, ERK5 was not stimulated by cAMP or neuronal activity induced by glutamate or membrane depolarization. In contrast to ERK1/2, ERK5 strongly activated the transcriptional activity of myocyte enhancer factor 2C (MEF2C) in pheochromocytoma 12 (PC12) cells and was required for neurotrophin stimulation of MEF2C transcription in both PC12 cells and cortical neurons. Furthermore, ERK1/2, but not ERK5, induced transcription from Elk1 and the cAMP/ Ca2+response element in PC12 cells. Our data suggest that mechanisms for regulation of ERK5 and downstream transcriptional pathways regulated by ERK5 are distinct from those of ERK1/2 in neurons. Furthermore, ERK5 is the first MAP kinase identified whose activity is stimulated by neurotrophins but not by neuronal activity.
Combining Deconvolution and Noise Analysis for the Estimation of Transmitter Release Rates at the Calyx of Held
Authors: Neher Erwin , Sakaba Takeshi
Published: April 2018

The deconvolution method has been used in the past to estimate release rates of synaptic vesicles, but it cannot be applied to synapses where nonlinear interactions of quanta occur. We have extended this method to take into account a nonlinear current component resulting from the delayed clearance of glutamate from the synaptic cleft. We applied it to the calyx of Held and verified the important assumption of constant miniature EPSC (mEPSC) size by combining deconvolution with a variant of nonstationary fluctuation analysis. We found that amplitudes of mEPSCs decreased strongly after extended synaptic activity. Cyclothiazide (CTZ), an inhibitor of glutamate receptor desensitization, eliminated this reduction, suggesting that postsynaptic receptor desensitization occurs during strong synaptic activity at the calyx of Held. Constant mEPSC sizes could be obtained in the presence of CTZ and kynurenic acid (Kyn), a low-affinity blocker of AMPA-receptor channels. CTZ and Kyn prevented postsynaptic receptor desensitization and saturation and also minimized voltage-clamp errors. Therefore, we conclude that in the presence of these drugs, release rates at the calyx of Held can be reliably estimated over a wide range of conditions. Moreover, the method presented should provide a convenient way to study the kinetics of transmitter release at other synapses.
Cytosolic Calcium Oscillations in Astrocytes May Regulate Exocytotic Release of Glutamate
Authors: Pasti Lucia , Zonta Micaela , Pozzan Tullio , Vicini Stefano , Carmignoto Giorgio
Published: April 2018

To obtain insights into the spatiotemporal characteristics and mechanism of Ca2+-dependent glutamate release from astrocytes, we developed a new experimental approach using human embryonic kidney (HEK) 293 cells transfected with the NMDA receptor (NMDAR), which act as glutamate biosensors, plated on cultured astrocytes. We here show that oscillations of intracellular Ca2+concentration ([Ca2+]i) in astrocytes trigger synchronous and repetitive [Ca2+]ielevations in sensor HEK cells, and that these elevations are sensitive to NMDAR inhibition. By whole-cell patch-clamp recordings, we demonstrate that the activation of NMDARs in HEK cells results in inward currents that often have extremely fast kinetics, comparable with those of glutamate-mediated NMDAR currents in postsynaptic neurons. We also show that the release of glutamate from stimulated astrocytes is drastically reduced by agents that are known to reduce neuronal exocytosis, i.e., tetanus toxin and bafilomycin A1. We conclude that [Ca2+]ioscillations represent a frequency-encoded signaling system that controls a pulsatile release of glutamate from astrocytes. The fast activation of NMDARs in the sensor cells and the dependence of glutamate release on the functional integrity of both synaptobrevin and vacuolar H+ATPase suggest that astrocytes are endowed with an exocytotic mechanism of glutamate release that resembles that of neurons.
Differential Cellular and Subcellular Localization of AMPA Receptor-Binding Protein and Glutamate Receptor-Interacting Protein
Authors: Burette Alain , Khatri Latika , Wyszynski Michael , Sheng Morgan , Ziff Edward B. , Weinberg Richard J.
Published: April 2018

Excitatory synaptic currents in the mammalian brain are typically mediated by the neurotransmitter glutamate, acting at AMPA receptors. We used immunocytochemistry to investigate the distribution of AMPA receptor-binding protein (ABP) in the cerebral neocortex. ABP was most prominent in pyramidal neurons, although it was also present (at lower levels) in interneurons. ABP and its putative binding partners, the GluR2/3 subunits of the AMPA receptor, exhibited prominent cellular colocalization. Under appropriate processing conditions, colocalization could also be documented in puncta, many of which could be recognized as dendritic spines. However, a sizable minority of GluR2/3-positive puncta were immunonegative for ABP. Because glutamate receptor-interacting protein (GRIP) may also anchor GluR2, we studied the relative distribution of ABP and GRIP. There was extensive colocalization of these two antigens at the cellular level, although GRIP, unlike ABP, was strongest in nonpyramidal neurons. Different parts of a single dendrite could stain selectively for ABP or GRIP. To further characterize this heterogeneity, we investigated punctate staining of neuropil using synaptophysin and the membrane tracer DiA to identify probable synapses. Some puncta were comparably positive for both ABP and GRIP, but the majority were strongly positive for one antigen and only weakly positive or immunonegative for the other. This heterogeneity could be seen even within adjacent spines of a single dendrite. These data suggest that ABP may act as a scaffold for AMPA receptors either in concert with or independently from GRIP.
Intrahippocampal Infusions of K-ATP Channel Modulators Influence Spontaneous Alternation Performance: Relationships to Acetylcholine Release in the Hippocampus
Authors: Stefani Mark R. , Gold Paul E.
Published: April 2018

One mechanism by which administration of glucose enhances cognitive functions may be by modulating central ATP-sensitive potassium (K-ATP) channels. K-ATP channels appear to couple glucose metabolism and neuronal excitability, with channel blockade increasing the likelihood of neurosecretion. The present experiment examined the effects of glucose and the direct K-ATP channel modulators glibenclamide and lemakalim on spontaneous alternation performance and hippocampal ACh release. Rats received either artificial CSF vehicle or vehicle plus drug for two consecutive 12 min periods via microdialysis probes (3 mm; flow rate of 2.1 μl/min) implanted in the left hippocampus. During the second 12 min period, rats were tested for spontaneous alternation performance. Dialysate was simultaneously collected for later analysis of ACh content. Both glucose (6.6 mm) and glibenclamide (100 μm) significantly increased alternation scores compared with those of controls. Conversely, lemakalim (200 μm) significantly reduced alternation scores relative to those of controls. Simultaneous administration of lemakalim with either glucose or glibenclamide resulted in alternation scores not significantly different from control values. All drug treatments enhanced hippocampal ACh output relative to control values. The results demonstrate that K-ATP channel modulators influence behavior when administered directly into the hippocampus, with channel blockers enhancing and openers impairing spontaneous alternation performance, thus supporting the hypothesis that glucose enhances memory via action at central K-ATP channels. That lemakalim, as well as glibenclamide and glucose, increased hippocampal ACh output suggests a dissociation between the effects of K-ATP channel modulators on behavior and hippocampal ACh release.
Behavioral Activation in Rats Requires Endogenous Ascorbate Release in Striatum
Authors: Rebec George V. , Wang Zhongrui
Published: April 2018

Ascorbate (vitamin C) is found in high concentrations in the striatum in which it may play a role in behavioral activation. To test this hypothesis, freely behaving rats received bilateral intrastriatal infusions of ascorbate oxidase (AAO) to inactivate extracellular ascorbate. Slow-scan voltammetry was used simultaneously to assess changes in ascorbate and 3,4-dihydroxyphenylacetic acid (DOPAC), a major dopamine metabolite, near the infusion site. Intrastriatal AAO, but not saline vehicle, caused a rapid decline in both ascorbate and behavioral activation. Within 20 min, an ascorbate loss of 50–70% led to a near-total inhibition of all recorded behavior, including open-field locomotion, approach of novel objects, and social interactions with other rats. DOPAC levels remained stable, arguing against an AAO-induced disruption of dopamine transmission. Consistent with this interpretation, subsequent injection of 1.0 mg/kgd-amphetamine, an indirect dopamine agonist, quickly restored behavioral activation, which also was accompanied by a marked rise in extracellular ascorbate. Bilateral AAO infusions into dorsal hippocampus, which also has a high level of extracellular ascorbate, failed to alter behavioral activation, indicating that a loss of brain ascorbate per se does not suppress behavior. Collectively, these results implicate ascorbate in the behavioral operations of the striatum and suggest that the extracellular level of this vitamin plays a critical role in behavioral activation.
Amygdala Regulation of Nucleus Accumbens Dopamine Output is Governed by the Prefrontal Cortex
Authors: Jackson Mark E. , Moghaddam Bita
Published: April 2018

A dynamic interaction between the prefrontal cortex (PFC), amygdala, and nucleus accumbens (NAc) may be fundamental to regulation of goal-directed behavior by affective and cognitive processes. This study demonstrates that a mechanism for this triadic relationship is an inhibitory control by prefrontal cortex on accumbal dopamine release during amygdala activation. In freely moving rats, microstimulation of basolateral amygdala at intensities that produced mild behavioral activation produced an expected rapid increase in glutamate efflux in the prefrontal cortex and the nucleus accumbens shell region of the ventral striatum. However, during the stimulation, dopamine release increased only in the prefrontal cortex, not in the nucleus accumbens. An increase in accumbal dopamine release was observed during the stimulation if glutamate activation in the prefrontal cortex was inhibited at either presynaptic or postsynaptic levels. Some behaviors expressed during the stimulation were intensified in animals in which prefrontal cortex glutamate activation was blocked. In addition, these animals continued to express stimulus-induced behaviors after the termination of stimulation, whereas normal poststimulus behaviors such as ambulation and grooming were not displayed as frequently. Considering that dopamine neurotransmission in the nucleus accumbens is thought to play an integral role in goal-directed motor behavior, these findings suggest that the prefrontal cortex influences the behavioral impact of amygdala activation via a concomitant active suppression of accumbal dopamine release. Absence of this cortical influence appears to result in an aberrant pattern of behavioral expression in response to amygdala activation, including behavioral perseveration after stimulus termination.
Latent Acquisition of Timed Responses in Cerebellar Cortex
Authors: Ohyama Tatsuya , Mauk Michael D.
Published: April 2018

Evidence indicates that rabbit eyelid conditioning is mediated by plasticity in the interpositus cerebellar nucleus and in cerebellar cortex. Although the relative contributions of these sites are not fully characterized, evidence suggests that plasticity in the cerebellar cortex influences conditioned response amplitude and timing, whereas plasticity in the interpositus nucleus is necessary or permissive for conditioned response expression. Recent empirical and computational analyses suggest that, during training, plasticity is initially established in the cerebellar cortex, whereas conditioned response expression begins later as plasticity is induced in the interpositus nucleus. We used the dependence of response timing on the interstimulus interval (ISI) to test this latent learning hypothesis. Rabbits were initially trained using a tone conditioned stimulus (CS) with a relatively long ISI to a low-criterion threshold. The relative absence of plasticity in the interpositus nucleus was then examined via reversible disconnection of the cerebellar cortex. Later, to induce plasticity in the interpositus nucleus, subjects were trained to robust levels of conditioned response expression using a shorter ISI. Reversible disconnection of the cerebellar cortex at this time confirmed the presence of robust interpositus nucleus plasticity after the second phase. Subsequent probe trials with the long CS alone then revealed double-peaked responses whose peaks were appropriately timed to the two ISIs. The results are consistent with the hypothesis that temporally specific learning occurs first in the cerebellar cortex before the appearance of conditioned responses. This latent learning is expressed only after plasticity is induced in the interpositus nucleus.
Disinhibition in Rat Superior Colliculus Mediated by GABACReceptors
Authors: Schmidt Matthias , Boller Mathias , Özen Gülden , Hall William C.
Published: April 2018

The stratum griseum superficiale (SGS) of the superior colliculus contains a high concentration of the recently described GABACreceptor. In a previous study, it was postulated that activation of these receptors on inhibitory interneurons functions to disinhibit projection cells that relay visual information to the thalamus and brainstem. To test this model, we usedin vitrowhole-cell patch-clamp methods to measure effects of GABA and muscimol on EPSCs and IPSCs evoked in rat SGS by electrical optic layer stimulation. The neurons were filled with biocytin for later morphological characterization. As expected, bath applications of GABA and muscimol always strongly depressed evoked PSCs at concentrations of >100 and >1 μm, respectively. However, at lower agonist concentrations, which most likely activate GABACbut not GABAAreceptors, effects were not uniform. Evoked responses were suppressed by both agonists in 48% of the neurons, whereas the remaining cells exhibited enhanced responses with increased evoked EPSCs, decreased evoked IPSCs, or both types of change. Most morphologically identified cells with suppressed responses (14 of 17 cells) had morphological characteristics of putative GABAergic interneurons, whereas almost all cells with enhanced responses (8 of 10 cells) had morphological characteristics of projection cells. Finally, all effects of GABA and muscimol at low concentrations were blocked by (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid, a specific GABACreceptor antagonist, but not by the specific GABAAreceptor antagonist bicuculline. Taken together, these results indicate that in SGS, GABACreceptors are predominantly expressed by GABAergic neurons and that activation of these receptors leads to disinhibition of SGS projection cells.
Effects of Antisense to the α2A-Adrenoceptors Administered into the Region of the Locus Ceruleus on Behaviors in Plus-Maze and Sexual Behavior Tests in Sham-Operated and Castrated Male Rats
Authors: Shishkina Galina T. , Kalinina Tatjana S. , Sournina Natalja Yu. , Dygalo Nikolai N.
Published: April 2018

Clinical and experimental findings have implicated brain α2-adrenoceptors in the regulation of many physiological functions, including sexual activity and stress-related behavior. However, which subtypes of the three α2-adrenoceptors that have now been cloned (α2A, α2B, and α2C) are involved in these controls have yet to be established. Here, we investigated the contribution of α2A-adrenoceptors of the locus ceruleus, the principal source of brain noradrenaline, to exploratory and sexual behaviors. Using administration of antisense oligodeoxynucleotide to inhibit the receptor expression, we found that reductions in brainstem α2A-adrenoceptor mRNA levels and α2-adrenoceptor densities induced by antisense treatment were not accompanied by any changes in the major characteristics of male sexual activity, such as mount latencies and numbers of mounts. However, in sexual behavior tests, antisense-treated male rats had decreased numbers of rearings and thus have higher percentages of behaviors positively correlated with sexual activity. Besides, antisense-treated animals had decreased anxiety in plus-maze tests. The data demonstrate that inhibition of α2A-adrenoceptor expression in the region of the locus ceruleus has an anxiolytic-like effect and facilitates male's attention to female in sexual behavior test.
Environmental Novelty Differentially Affects c-fosmRNA Expression Induced by Amphetamine or Cocaine in Subregions of the Bed Nucleus of the Stria Terminalis and Amygdala
Authors: Day Heidi E. W. , Badiani Aldo , Uslaner Jason M. , Oates Matthew M. , Vittoz Nicole M. , Robinson Terry E. , Watson Stanley J. , Akil Huda
Published: April 2018

The environmental context in which amphetamine or cocaine are administered modulates both their acute psychomotor activating effects and their ability to induce sensitization. Here we report that environmental context differentially affects patterns of amphetamine- and cocaine-induced c-fosmRNA expression in the bed nucleus of the stria terminalis (BST) and amygdala of male rats.

In the medial amygdala and medial posterior BST, exposure to novelty resulted in a marked increase in c-fosmRNA. Amphetamine given at home did not induce c-fosmRNA, and when given in the novel environment, did not increase levels beyond that observed for novelty alone. In the basolateral and lateral amygdala, amphetamine or cocaine at home or exposure to novelty induced c-fosmRNA. When amphetamine or cocaine was given in a novel environment the c-fosmRNA response was significantly enhanced. In the central nucleus of the amygdala (CEA) and oval subnucleus of the BST (BSTov), amphetamine administration at home produced a robust increase in c-fosmRNA expression, whereas exposure to novelty had little effect. In contrast to other brain regions examined, the c-fosmRNA response to amphetamine in a novel versus home environment was significantly smaller. In both “home” and “novel” amphetamine groups, c-fosmRNA in the BSTov and CEA was predominantly expressed in enkephalin-containing cells; coexpression with corticotropin-releasing hormone was rare.

These data suggest that the context in which psychostimulants are given powerfully and differentially alters the response of limbic structures that have been functionally implicated in drug reinforcement and emotional behaviors.
Differential Frequency-Dependent Regulation of Transmitter Release by Endogenous Nitric Oxide at the Amphibian Neuromuscular Synapse
Authors: Thomas Sébastien , Robitaille Richard
Published: April 2018

Nitric oxide (NO) is a potent neuromodulator in the CNS and PNS. At the frog neuromuscular junction (nmj), exogenous application of NO reduces neurotransmitter release, and NO synthases (NOSs), the enzymes producing NO, are present at this synapse. This work aimed at studying the molecular mechanisms by which NO modulates synaptic efficacy at the nmj using electrophysiological recordings and Ca2+-imaging techniques. Bath application of the NO donorsS-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside decreased end plate potential (EPP) amplitude as well as the frequency of miniature EPPs but not their amplitude. Ca2+responses elicited in presynaptic terminals by single action potentials were unaffected by NO, but responses evoked by a short train of stimuli were increased. Tonic endogenous production of NO was observed as suggested by the increase in EPP amplitude by bath application of the NO scavenger hemoglobin and the neuronal NOS inhibitor 3-bromo-7-nitroindazole sodium salt. A soluble guanylate cyclase inhibitor, 6-anilino-5,8-quinolinedione (LY-83583), increased EPP amplitude and occluded the effects of the NO donor, suggesting that NO acts via a cGMP-dependent mechanism. High-frequency-induced depression was reduced in the presence of the NO scavenger but not by LY-83583. However, adenosine-induced depression was significantly reduced after bath perfusion of SNAP and in the presence of LY-83583. Our results indicate that NO regulates transmitter release and adenosine-induced depression via a cGMP-dependent mechanism that occurs after Ca2+entry and that high-frequency-induced synaptic depression is regulated by NO in a cGMP-independent manner.
Anandamide Excites Central Terminals of Dorsal Root Ganglion Neurons via Vanilloid Receptor-1 Activation
Authors: Tognetto Michele , Amadesi Silvia , Harrison Selena , Creminon Christophe , Trevisani Marcello , Carreras Mirko , Matera Mario , Geppetti Pierangelo , Bianchi Alfredo
Published: April 2018

Recently, the cannabinoid (CB) receptor agonist anandamide (AEA) has been shown to excite perivascular terminals of primary sensory neurons via activation of the vanilloid receptor-1 (VR-1). To determine whether AEA stimulates central terminals of these neurons, via VR-1 activation, we studied the release of calcitonin gene-related peptide (CGRP)- and substance P (SP)-like immunoreactivities (LI) from slices of rat dorsal spinal cord. Mobilization of Ca2+in rat dorsal root ganglion (DRG) neurons in culture was also studied. AEA (0.1–10 μm) increased the outflow of CGRP-LI and SP-LI from slices of the rat dorsal spinal cord in a Ca2+-dependent manner and increased [Ca2+]iin capsaicin-sensitive cultured DRG neurons. Both effects of AEA were abolished by capsaicin pretreatment and by the VR-1 antagonist capsazepine but not affected by the CB receptor antagonists AM281 or AM630. Both neuropeptide release and Ca2+mobilization induced by electrical field stimulation (EFS) were inhibited by a low concentration of AEA (10 nm). Inhibition by AEA of EFS-induced responses was reversed by AM281 and AM630, but was not affected by capsazepine. Results indicate that stimulation of VR-1 with high concentrations of AEA excites central terminals of capsaicin-sensitive DRG neurons, thus causing neuropeptide release in the dorsal spinal cord. This novel activity opposes the CB receptor-mediated inhibitory action of low concentrations AEA. However, only if large amounts of endogenous AEA could be produced at the level of the dorsal spinal cord, they may not inhibit, but rather activate, nociceptive sensory neurons.
Muscarinic Regulation of Dendritic and Axonal Outputs of Rat Thalamic Interneurons: A New Cellular Mechanism for Uncoupling Distal Dendrites
Authors: Zhu J. Julius , Heggelund Paul
Published: April 2018

Inhibition is crucial for sharpening the sensory information relayed through the thalamus. To understand how the interneuron-mediated inhibition in the thalamus is regulated, we studied the muscarinic effects on interneurons in the lateral posterior nucleus and lateral geniculate nucleus of the thalamus. Here, we report that activation of muscarinic receptors switched the firing pattern in thalamic interneurons from bursting to tonic. Although neuromodulators switch the firing mode in several other types of neurons by altering their membrane potential, we found that activation of muscarinic subtype 2 receptors switched the fire mode in thalamic interneurons by selectively decreasing their input resistance. This is attributable to the muscarinic enhancement of a hyperpolarizing potassium conductance and two depolarizing cation conductances. The decrease in input resistance appeared to electrotonically uncouple the distal dendrites of thalamic interneurons, which effectively changed the inhibition pattern in thalamocortical cells. These results suggest a novel cellular mechanism for the cholinergic transformation of long-range, slow dendrite- and axon-originated inhibition into short-range, fast dendrite-originated inhibition in the thalamus observedin vivo. It is concluded that the electrotonic properties of the dendritic compartments of thalamic interneurons can be dynamically regulated by muscarinic activity.
Casein Kinase 2 Determines the Voltage Dependence of the Kv3.1 Channel in Auditory Neurons and Transfected Cells
Authors: Macica Carolyn M. , Kaczmarek Leonard K.
Published: April 2018

The Kv3.1 potassium channel can be distinguished from most other delayed rectifier channels by its very high threshold of activation and lack of use-dependent inactivation. This allows neurons that express this channel to fire at very high frequencies. We have now found that this feature of the Kv3.1 channel is strongly influenced by its constitutive phosphorylation by the enzyme casein kinase II. Using stably transfected Chinese hamster ovary cells expressing Kv3.1, we show that Kv3.1 is highly phosphorylated under basal conditions. Whole-cell patch clamp recordings were used to characterize the electrophysiological consequence of dephosphorylation using alkaline phosphatase. This enzyme produced an increase in whole-cell conductance and shifted the voltage dependence of activation to more negative potentials by >20 mV. In addition, a similar shift in the voltage dependence of inactivation was observed. These findings were also confirmed in native Kv3.1 channels expressed in medial nucleus of the trapezoid body (MNTB) neurons. Furthermore, inhibitors of casein kinase 2 mimicked the effect of phosphatase treatment on voltage-dependent activation and inactivation, whereas inhibitors of protein kinase C failed to alter these parameters. The combination of biochemical and electrophysiological evidence suggests that the biophysical characteristics of Kv3.1 that are important to its role in MNTB neurons, allowing them to follow high-frequency stimuli with fidelity, are largely determined by phosphorylation of the channel.
Rocker Is a New Variant of the Voltage-Dependent Calcium Channel GeneCacna1a
Authors: Zwingman Theresa A. , Neumann Paul E. , Noebels Jeffrey L. , Herrup Karl
Published: April 2018

Rocker (gene symbolrkr), a new neurological mutant phenotype, was found in descendents of a chemically mutagenized male mouse. Mutant mice display an ataxic, unstable gait accompanied by an intention tremor, typical of cerebellar dysfunction. These mice are fertile and appear to have a normal life span. Segregation analysis reveals rocker to be an autosomal recessive trait. The overall cytoarchitecture of the young adult brain appears normal, including its gross cerebellar morphology. Golgi-Cox staining, however, reveals dendritic abnormalities in the mature cerebellar cortex characterized by a reduction of branching in the Purkinje cell dendritic arbor and a “weeping willow” appearance of the secondary branches. Using simple sequence length polymorphism markers, therockerlocus was mapped to mouse chromosome 8 within 2 centimorgans of the calcium channel α1a subunit (Cacna1a,formerly known astottering) locus. Complementation tests with theleanermutant allele (Cacna1ala) produced mutant animals, thus identifyingrockeras a new allele ofCacna1a(Cacna1arkr). Sequence analysis of the cDNA revealedrockerto be a point mutation resulting in an amino acid exchange: T1310K between transmembrane regions 5 and 6 in the third homologous domain. Important distinctions betweenrockerand the previously characterized alleles of this locus include the absence of aberrant tyrosine hydroxylase expression in Purkinje cells and the separation of the absence seizures (spike/wave type discharges) from the paroxysmal dyskinesia phenotype. Overall these findings point to an important dissociation between the seizure phenotypes and the abnormalities in catecholamine metabolism, and they emphasize the value of allelic series in the study of gene function.
The C-Terminal Domains of the GABABReceptor Subunits Mediate Intracellular Trafficking But Are Not Required for Receptor Signaling
Authors: Calver Andrew R. , Robbins Melanie J. , Cosio Christophe , Rice Simon Q. J. , Babbs Adam J. , Hirst Warren D. , Boyfield Izzy , Wood Martyn D. , Russell Robert B. , Price Gary W. , Couve Andrés , Moss Stephen J. , Pangalos Menelas N.
Published: April 2018

GABABreceptors are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. These receptors are heterodimers assembled from GABAB1and GABAB2subunits, neither of which is capable of producing functional GABABreceptors on homomeric expression. GABAB1,although able to bind GABA, is retained within the endoplasmic reticulum (ER) when expressed alone. In contrast, GABAB2is able to access the cell surface when expressed alone but does not couple efficiently to the appropriate effector systems or produce any detectable GABA-binding sites. In the present study, we have constructed chimeric and truncated GABAB1and GABAB2subunits to explore further GABABreceptor signaling and assembly. Removal of the entire C-terminal intracellular domain of GABAB1results in plasma membrane expression without the production of a functional GABABreceptor. However, coexpression of this truncated GABAB1subunit with either GABAB2or a truncated GABAB2subunit in which the C terminal has also been removed is capable of functional signaling via G-proteins. In contrast, transferring the entire C-terminal tail of GABAB1to GABAB2leads to the ER retention of the GABAB2subunit when expressed alone. These results indicate that the C terminal of GABAB1mediates the ER retention of this protein and that neither of the C-terminal tails of GABAB1or GABAB2is an absolute requirement for functional coupling of heteromeric receptors. Furthermore although GABAB1is capable of producing GABA-binding sites, GABAB2is of central importance in the functional coupling of heteromeric GABABreceptors to G-proteins and the subsequent activation of effector systems.
Laminar Organization of the NMDA Receptor Complex within the Postsynaptic Density
Authors: Valtschanoff Juli G. , Weinberg Richard J.
Published: April 2018

The NR2 subunit is an essential component of the NMDA receptor. Recent biochemical research has identified a number of molecules that can bind directly or indirectly to its cytoplasmic tail. These postsynaptic density (PSD) proteins play a role in intracellular signal transduction, and are implicated in synaptic plasticity and memory mechanisms. We performed systematic electron microscopic immunogold analysis in rat neocortex to determine the spatial organization of NR2, in relation to six other proteins thought to be involved in the NMDA receptor complex. Peak concentrations of each protein were within the PSD but in different “layers” of the density. In the axodendritic axis, gold particles coding for PSD-95 lay an average of 12 nm cytoplasmic to the extracellular face of the plasma membrane, very close to the C terminal of NR2. Nitric oxide synthase lay 18 nm inside the membrane; the scaffolding proteins guanylate kinase-associated protein and Shank lay 24–26 nm inside the membrane; and CRIPT and dynein light chain, proteins that may link the complex to cytoskeletal elements, lay on the cytoplasmic side of the PSD, 29–32 nm inside the plasma membrane and extending into the spine cytoplasm. The supramolecular organization of these molecules may modulate intracellular transduction of NMDA-mediated signals.
Regulation of Neuregulin Expression in the Injured Rat Brain and Cultured Astrocytes
Authors: Tokita Yoshihito , Keino Hiroomi , Matsui Fumiko , Aono Sachiko , Ishiguro Hiroshi , Higashiyama Shigeki , Oohira Atsuhiko
Published: April 2018

In this report, we investigated whether reactive astrocytes produce neuregulins (glial growth factor 2/heregulin/acetylcholine receptor-inducing activity or neu differentiation factor) and its putative receptors, ErbB2 and ErbB3 tyrosine kinases, in the injured CNSin vivo. Significant immunoreactivities with anti-neuregulin, anti-ErbB2, and anti-ErbB3 antibodies were detected on astrocytes at the injured site 4 d after injury to the adult rat cerebral cortex. To elucidate the mechanisms for the upregulation of neuregulin expression in astrocytes, primary cultured astrocytes were treated with certain reagents, including forskolin, that are known to elevate the intracellular level of cAMP and induce marked morphological changes in astrocytes. Western blot analysis showed that the expression of a 52 kDa membrane-spanning form of a neuregulin protein was enhanced in cultured astrocytes after administration of forskolin. The upregulation of glial fibrillary acidic protein was also observed in astrocytes treated with forskolin. In contrast, inactivation of protein kinase C because of chronic treatment with phorbol ester 12-O-tetradecanoyl phorbol 13-acetate downregulated the expression of the 52 kDa isoform, although other splice variants with apparent molecular sizes of 65 and 60 kDa were upregulated. These results suggest that the enhancement of neuregulin expression at injured sites is induced, at least in part, by elevation in intracellular cAMP levels and/or a protein kinase C signaling pathway. The neuregulin expressed on reactive astrocytes may stimulate their proliferation and support the survival of neurons surrounding cortical brain woundsin vivo.
Use-Dependent Effects of Amyloidogenic Fragments of β-Amyloid Precursor Protein on Synaptic Plasticity in Rat HippocampusIn Vivo
Authors: Kim Joung-Hun , Anwyl Roger , Suh Yoo-Hun , Djamgoz Mustafa B. A. , Rowan Michael J.
Published: April 2018

The Alzheimer's disease-related β-amyloid precursor protein (β-APP) is metabolized to a number of potentially amyloidogenic peptides that are believed to be pathogenic. Application of relatively low concentrations of the soluble forms of these peptides has previously been shown to block high-frequency stimulation-induced long-term potentiation (LTP) of glutamatergic transmission in the hippocampus. The present experiments examined how these peptides affect low-frequency stimulation-induced long-term depression (LTD) and the reversal of LTP (depotentiation). We discovered that β-amyloid peptide (Aβ1–42) and the Aβ-containing C -terminus of β-APP (CT) facilitate the induction of LTD in the CA1 area of the intact rat hippocampus. The LTD was frequency- and NMDA receptor-dependent. Thus, although low-frequency stimulation alone was ineffective, after intracerebroventricular injection of Aβ1–42, it induced an LTD that was blocked byd-(−)-2-amino-5-phosphonopentanoic acid. Furthermore, an NMDA receptor-dependent depotentiation was induced in a time-dependent manner, being evoked by injection of CT 10 min, but not 1 hr, after LTP induction. These use- and time-dependent effects of the amyloidogenic peptides on synaptic plasticity promote long-lasting reductions in synaptic strength and oppose activity-dependent strengthening of transmission in the hippocampus. This will result in a profound disruption of information processing dependent on hippocampal synaptic plasticity.
Contribution of GABAAand GABABReceptors to Thalamic Neuronal Activity during Spontaneous Absence Seizures in Rats
Authors: Staak Rainer , Pape Hans-Christian
Published: April 2018

The contribution of GABAergic mechanisms in thalamic relay nuclei to spike and wave discharges (SWDs) during spontaneous seizures was assessed using the WAG/Rij strain of rats, an established genetic model of absence epilepsy, in combination with single-unit recordings and microiontophoretic techniques in the ventrobasal thalamic complexin vivo. Spontaneous SWDs occurring on the electroencephalogram at 5–9 Hz were associated with burst firing in thalamocortical neurons, which was phase-locked with the spike component. Microiontophoretic application of the GABAAreceptor antagonist bicuculline significantly increased the magnitude of SWD-related firing in all tested cells. Application of the GABABreceptor antagonist CGP 55845A exerted a statistically insignificant modulatory effect on neuronal activity during spontaneous SWDs but significantly attenuated the bicuculline-evoked aggravation of SWD-related firing. The data indicate that, in thalamocortical neurons, (1) GABAAreceptor-mediated events are recruited with each SWD, (2) SWD-related activity can be evoked with no significant contribution of GABABreceptors, and (3) blockade of GABAAreceptors potentiates SWD-related activity, presumably through an indirect effect mediated through GABABreceptors. These results vote against a predominant or even exclusive contribution of GABABreceptors to spontaneous SWDs in thalamic relay nuclei in the WAG/Rij strain, but rather point to a critical role of GABAAreceptor activation. This conclusion is in support of the view that the two subtypes of GABA receptors play a differential role in fast (5–10 Hz) and slow (3 Hz) spike-wave paroxysms observed during absence seizures.
p38 Activation Is Required Upstream of Potassium Current Enhancement and Caspase Cleavage in Thiol Oxidant-Induced Neuronal Apoptosis
Authors: McLaughlin BethAnn , Pal Sumon , Tran Minhnga P. , Parsons Andrew A. , Barone Frank C. , Erhardt Joseph A. , Aizenman Elias
Published: April 2018

Oxidant-induced neuronal apoptosis has been shown to involve potassium and zinc dysregulation, energetic dysfunction, activation of stress-related kinases, and caspase cleavage. The temporal ordering and interdependence of these events was investigated in primary neuronal cultures exposed to the sulfhydryl oxidizing agent 2,2′-dithiodipyridine (DTDP), a compound that induces the intracellular release of zinc. We previously observed that tetraethylammonium (TEA), high extracellular potassium, or cysteine protease inhibitors block apoptosis induced by DTDP. We now report that both p38 and extracellular signal-regulated kinase phosphorylation are evident in neuronal cultures within 2 hr of a brief exposure to 100 μmDTDP. However, only p38 inhibition is capable of blocking oxidant-induced toxicity. Cyclohexamide or actinomycin D does not attenuate DTDP-induced cell death, suggesting that posttranslational modification of existing targets, rather than transcriptional activation, is responsible for the deleterious effects of p38. Indeed, an early robust increase in TEA-sensitive potassium channel currents induced by DTDP is attenuated by p38 inhibition but not by caspase inhibition. Moreover, we found that activation of p38 is required for caspase 3 and 9 cleavage, suggesting that potassium currents enhancement is required for caspase activation. Finally, we observed that DTDP toxicity could be blocked with niacinamide or benzamide, inhibitors of poly (ADP-ribose) synthetase. Based on these findings, we conclude that oxidation of sulfhydryl groups on intracellular targets results in intracellular zinc release, p38 phosphorylation, enhancement of potassium currents, caspase cleavage, energetic dysfunction, and translationally independent apoptotic cell death.
Pyruvate Released by Astrocytes Protects Neurons from Copper-Catalyzed Cysteine Neurotoxicity
Authors: Wang Xue Feng , Cynader Max S.
Published: April 2018

We have found previously that astrocytes can provide cysteine to neurons. However, cysteine has been reported to be neurotoxic although it plays a pivotal role in regulating intracellular levels of glutathione, the major cellular antioxidant. Here, we show that cysteine toxicity is a result of hydroxyl radicals generated during cysteine autoxidation. Transition metal ions are candidates to catalyze this process. Copper substantially accelerates the autoxidation rate of cysteine even at submicromolar levels, whereas iron and other transition metal ions, including manganese, chromium, and zinc, are less efficient. The autoxidation rate of cysteine in rat CSF is equal to that observed in the presence of ∼0.2 μmcopper. In tissue culture tests, we found that cysteine toxicity depends highly on its autoxidation rate and on the total amount of cysteine being oxidized, suggesting that the toxicity can be attributed to the free radicals produced from cysteine autoxidation, but not to cysteine itself.

We have also explored thein vivomechanisms that protect against cysteine toxicity. Catalase and pyruvate were each found to inhibit the production of hydroxyl radicals generated by cysteine autoxidation. In tissue culture, they both protected primary neurons against cysteine toxicity catalyzed by copper. This protection is attributed to their ability to react with hydrogen peroxide, preventing the formation of hydroxyl radicals. Pyruvate, but not catalase or glutathione peroxidase, was detected in astrocyte-conditioned medium and CSF. Our data therefore suggest that astrocytes can prevent cysteine toxicity by releasing pyruvate.
High-Fidelity Transmission Acquired via a Developmental Decrease in NMDA Receptor Expression at an Auditory Synapse
Authors: Futai Kensuke , Okada Masayoshi , Matsuyama Kyoko , Takahashi Tomoyuki
Published: April 2018

Central auditory relay synapses in mature animals follow high-frequency inputs for computation of sound localization. In immature mice, however, transmission at the calyx of Held synapse in auditory brainstem was inaccurate for high-frequency inputs because the summed slow synaptic potential components caused aberrant firings or blocked action potentials. As the mice matured, synaptic potentials became shorter, with smaller and faster NMDA receptor components, thereby establishing the precise one-to-one transmission for high-frequency inputs. Developmental acquisition of this high-fidelity transmission could be mimicked experimentally in immature mice by blocking NMDA receptors withd(−)2-amino-5-phosphonovaleric acid (d-APV). Furthermore, bilateral cochlear ablations at postnatal day 7 (P7) attenuated the developmental decrease of NMDA receptor expression and prevented the acquisition of high-fidelity transmission. We suggest that auditory activity, which begins at P10–P12 in mice, downregulates the expression of postsynaptic NMDA receptors, thereby contributing to the establishment of high-fidelity synaptic transmission.
Neuron-Specific Expression of Mutant Superoxide Dismutase 1 in Transgenic Mice Does Not Lead to Motor Impairment
Authors: Pramatarova Albéna , Laganière Janet , Roussel Julie , Brisebois Katéri , Rouleau Guy A.
Published: April 2018

Mutations were identified in the Cu/Zn superoxide dismutase gene (SOD1) in ∼15% of patients with familial amyotrophic lateral sclerosis. Transgenic animals expressing mutant SOD1 in all tissues develop an ALS-like phenotype. To determine whether neuron-specific expression of mutant SOD1 is sufficient to produce such a phenotype, we generated transgenic animals carrying the G37R mutation that is associated with the familial form of ALS (FALS), which is driven by the neurofilament light chain promoter. The transgenic animals express high levels of the human SOD1 protein in neuronal tissues, especially in the large motor neurons of the spinal cord, but they show no apparent motor deficit at up to 1.5 years of age. Our animal model suggests that neuron-specific expression of ALS-associated mutant human SOD1 may not be sufficient for the development of the disease in mice.
Nerve Growth Factor Rapidly Induces Prolonged Acetylcholine Release from Cultured Basal Forebrain Neurons: Differentiation between Neuromodulatory and Neurotrophic Influences
Authors: Auld Daniel S. , Mennicken Françoise , Quirion Rémi
Published: April 2018

Long-term exposure to nerve growth factor (NGF) is well established to have neurotrophic effects on basal forebrain cholinergic neurons, but its potential actions as a fast-acting neuromodulator are not as well understood. We report that NGF (0.1–100 ng/ml) rapidly (<60 min) and robustly enhanced constitutive acetylcholine (ACh) release (148–384% of control) from basal forebrain cultures without immediate persistent increases in choline acetyltransferase activity. More ACh was released in response to NGF when exposure was coupled with a higher depolarization level, suggesting activity dependence. In a long-term potentiation-like manner, brief NGF exposure (10 ng/ml; 60 min) induced robust and prolonged increases in ACh release, a capacity that was shared with the other neurotrophins. K252a (10–100 nm), BAPTA-AM (25 μm), and Cd2+(200 μm) prevented NGF enhancement of ACh release, suggesting the involvement of TrkA receptors, Ca2+, and voltage-gated Ca2+channels, respectively. Forskolin (10 μm), a cAMP generator, enhanced constitutive ACh release but did not interact synergistically with NGF. Tetrodotoxin (1 μm) and cycloheximide (2 μm) did not prevent NGF-induced ACh release, indicative of action at the level of the cholinergic nerve terminal and that new protein synthesis is not required for this neurotransmitter-like effect, respectively. In contrast, after a 24 hr NGF treatment, distinct protein synthesis-dependent and independent effects on choline acetyltransferase activity and ACh release were observed. These results indicate that neuromodulator/neurotransmitter-like (protein synthesis-independent) and neurotrophic (translation-dependent) actions likely make distinct contributions to the enhancement of cholinergic activity by NGF.